Experimental Approaches to the Development of Antianginal Drugs

Experimental Approaches to the Development of Antianginal Drugs MARTINM . WINBURY Department of Pharmacology. Warner-Lambert Research Institute. M d Plains. New Jersey I. Introduction and Definition of the Problem . . . . . . . A. Coronary Insufficiency . . . . . . . . . . . . B . Response of the Anginal Patient to Exercise . . . . . . . C . Response of the Anginal Patient to Nitroglycerin . . . . . D. Conclusion . . . . . . . . . . . . . . . I1. Physiology and Biochemistry of the Heart . . . . . . . . A . General . . . . . . . . . . . . . . . B . Hemodynamic Factors Regulating Coronary Blood Flow . . . . . . . . C: Factors Influencing Myocardial Oxygen Consumption D . Neural and Humoral Factors . . . . . . . . . . E. Myocardial Metabolism in Situ . . . . . . . . . . I11. Progrem in the Treatment of Coronary Insufficiency . . . . . . A Thyroid Inhibition . . . . . . . . . . . . . B. Monoamine Oxidase Inhibitors . . . . . . . . . . C . Adrenergic-Blocking Agents . . . . . . . . . . D . Coronary Dilators Other Than Nitrites . . . . . . . . E. Miscellaneous Agents . . . . . . . . . . . . IV . Action of Nitrites . . . . . . . . . . . . . . A. Coronary Hemodynamic Actions . . . . . . . . . B . General Hemodynamic Actions . . . . . . . . . . C . Cardiac Metabolism . . . . . . . . . . . . D. Effect on Catecholamines . . . . . . . . . . . E. Comparison of Response in Normal and Anginal Individual . . . F Effect on Collateral Circulation . . . . . . . . . . G . Conclusion . . . . . . . . . . . . . . . V. Approaches to Laboratory Evaluation of Antianginal Agents . . . . A. Coronary Dilator Action . . . . . . . . . . . B. Total Metabolic Approach . . . . . . . . . . . C . Measurement of Myocardial Oxygen Tension . . . . . . D. Experimental Coronary I d c i e n c y Induced by Coronary Occlusion or Drugs . . . . . . . . . . . . . . . E. Experimental Coronary Insufficiency Induced by Atherosclerosis . F. Prolongation of Contractile Activity during Anoxia . . . . . G. Antagonism of Effect of Catecholamines on the Heart . . . . H. Arteriographic Techniques . . . . . . . . . . . I . Use of Radioactive Tracers . . . . . . . . . . . VI . Other Potential Approaches . . . . . . . . . . . A. Hemodynamic 1

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1. Introduction and Definition of the Problem

For many years there has been tacit acceptance of the premise that coronary vasodilatation is the method of choice for the treatment of angina pectoris. This assumption originates from the early work with nitroglycerin which established the drug’s effectiveness in the management of angina pectoris in man and its coronary dilator action in animals. The “coronary vasodilator” concept has been carried over from the nitrites to other types of compounds and many non-nitrite coronary dilators have been prepared (Charlier, 1961) and studied clinically. An initial enthusiasm was reported for many of these compounds but with time negative results accumulated and, a t present, we are still left with the realization that based on objective clinical evidence, the nitrites are the only coronary vasodilators which show a beneficial effect. It is true that some of the earlier compounds developed may have produced coronary dilatation as a result of increased oxygen requirement, which we shall see is undersirable ; one of the more recently developed non-nitrite compounds, dipyridamole, increases the oxygen supply to the heart without markedly altering myocardial oxygen requirement but appears to be of little or no value in the treatment of angina pectoris (DeGraff and Lyon, 1963). By all criteria, this compound has the attributes of an agent which, theoretically, one would expect would have a beneficial effect in the treatment of angina pectoris (discussed in Section 111). It is obvious that there must be a re-evaluation of the concepts and theories regarding the mechanisms involved in angina pectoris and the approaches to the development of drugs for treatment of the disease. The use of coronary vasodilators necessitates the ability of the coronary bed to be able to dilate. Recent work has raised a question as to whether or not it is possible for drugs to increase the rate of coronary blood flow in the anginal patient (Gorlin e t al., 1959a; Rowe et al., 1961). Thus, there is a question about the exact mechanism of the antianginal effect of nitroglycerin. I n this chapter an attempt will be made to survey angina pectoris and the rational treatment from a physiological, biochemical, and pharmacological viewpoint. Methods for the laboratory evaluation of drugs will be reviewed objectively and potential new approaches considered. Angina pectoris is the syndrome associated with acute coronary insufficiency. It is merely the clinical sign of a basic pathophysiological problem resulting from atherosclerosis of the coronary arteries. There appears to be a relationship between myocardial ischemia resulting from “functional”

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coronary insufficiency and angina pectoris as well as the objective electrocardiographic abnormalities. The mechanism of the anginal pain has not been elucidated and it is not known if the pain originates in receptors in the ischemic myocardium or in the vessel wall. Present therapy is aimed at altering the circulation or obtunding the pain; however, the most desirable approach is one that improves the basic coronary insufficiency. The primary aim of this discussion is consideration of approaches to alleviate the coronary insufficiency. Therefore, when the term “antianginal” is used in this chapter, i t will refer to drugs aimed a t this objective.

A. CORONARY INSUFFICIENCY This can be defined as an imbalance between the available oxygen supply and the demand of the myocardial cells. This is a functional definition depending on the supply/demand ratio. The supply is dependent upon an adequate coronary blood flow, adequate oxygen saturation of arterial blood, effective capillary distribution and transfer of oxygen and substrate, and proper tissue utilization. The demand is related to the work load and metabolic status of the heart. Obviously, any decrease in the supply or increase in the demand will upset the balance and result in coronary insufficiency. At rest there is little difference between the coronary blood flow rate in the normal subject and in the anginal patient (Brachfeld e t al., 1959; Gorlin et al., 1959a; Gorlin, 196213). Under conditions producing an increased oxygen requirement of the heart the coronary flow in the normal patient can increase severalfold indicating a large “coronary reserve” ; however, in the anginal patient the flow can increase but little because of a limited coronary reserve and signs of coronary insufficiency may appear (pain and electrographic alterations). Thus there is nearly maximal vasodilatation a t rest in the anginal patient. The reduction in coronary reserve in the anginal patient is probably associated with a loss of the ability of the coronary vessels to dilate because of the underlying atherosclerosis. On this basis, coronary insufficiency results from a n increase in the demand for oxygen rather than a reduction in the supply. Studies in intact dogs and isolated rabbit hearts tend to confirm the fact that atherosclerosis can produce a reduction in coronary reserve (Karp e t al., 1960; Cross and Oblath, 1962; Melville and Varma, 1962). A spasm of the coronary artery would result in a decrease in the blood supply and thus produce coronary insufficiency. It seems unlikely that this is the case, but some still subscribe to this view because the nitrites can produce relaxation of an experimentally induced spasm in the animal (Modell, 1962). A recent report cites the case of one patient who had an anginal attack during coronary arteriography ; poor filling was observed

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in both the right and left coronary arteries (Gensini e t d.,1962). Administration of isosorbide dinitrate permitted good filling of the right coronary which was interpreted to be in spasm during the attack. Attempts to induce spasm of one coronary artery in the dog by occlusion of another coronary artery have been unsuccessful and it was concluded that there is no evidence for a vasoconstrictor reflex caused by occlusion (Wang et al., 1957). When embolization of the left descending or circumflex artery was produced with lycopodium spores and the caliber of the coronary vessels evaluated by arteriographic procedures, one group concluded that there was a spasm of the embolized and nonembolized artery (Guzman et al., 1962), while another group concluded that a spasm did not occur (West et al., 1962b). An impartial reviewer favored the view that a spasm did not occur (Schmidt, 1962).

B. RESPONSE OF

ANGINALPATIENT TO EXERCISE The physiological differences between the coronary circulation of the normal and anginal individual are readily demonstrated during exercise. This increases the work and oxygen requirement of the heart as a result of an increased cardiac output, elevation of blood pressure, and tachycardia. Under the stress of mild exercise the coronary blood flow, as measured by the nitrous oxide method, increased in both normal and anginal patients (Gorlin, 1962a; Messer and Neill, 1962; Wagman et al., 1962). Exercise also increased the clearance of N a P from the heart muscle of the anginal patient, indicating an increased capillary blood flow (Hollander et al., 1963). Much of the increased coronary blood flow in the anginal subject can be related to the rise in the perfusion pressure. A more sensitive parameter of the adequacy of the coronary circulation than flow alone is the myocardial oxygen extraction, as this is the only means of obtaining oxygen when flow is a limiting factor. I n normal subjects, extraction of oxygen from the arterial blood and coronary venous saturation are unchanged by exercise, but in anginal patients extraction increases progressively during exercise and coronary venous saturation falls (Gorlin, 1962a; Messer and Neill, 1962; Messer et al., 1962; Wagman et al., 1962). The increased oxygen extraction in the anginal patient is indicative of a n inadequate coronary reserve even though flow does increase. Another important difference between the two groups is the change in mechanical efficiency of the heart with a significant increase observed in the normal patients but little change in the anginal patient (Gorlin, 1962a; Messer and Neill, 1962). There is reasonably good correlation between the abnormal coronary hemodynamics (increased oxygen extraction and decreased sinus saturation) and positive electrocardioTHE

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graphic findings (STsegment depression) after exercise (Gorlin, 1962a,b; Messer and Neill, 1962; Messer et al., 1962; Wagman et al., 1962) ; an abnormal extraction was present in 82% of the anginal patients studied (Gorlin, 1962a; Messer and Neill, 1962; Messer et al., 1962). Anaerobic metabolism has been investigated in normal and anginal patients both a t rest and during exercise. The majority of studies revealed no apparent anaerobic metabolism based on “excess lactate” production (Gorlin, 1962a; Krasnow et d., 1962b; Messer and Neill, 1962; Wagman et al., 1962) or the lactate-pyruvate redox potential (Gudbjarnason et a?., 1962; Stock et al., 1962). In one study, 5 of 34 subjects produced excess lactate during exercise; 3 of these subjects had angina but several other anginal patients showed no excess lactate (Krasnow et al., 1962b). I n general, it can be concluded that anaerobic metabolism contributes only a small share of the total energy supply of the heart even during coronary insufficiency induced by exercise. The catecholamines increase the oxygen requirement of the heart by an increase in cardiac work and cardiac metabolism and may have a role in the production of coronary insdciency (Raab, 1956, 1962). Blood levels of norepinephrine and epinephrine were reported to be increased by exercise in anginal but not in normal patients (Gazes et al., 1959). Other investigators found no change in anginal or normal patients on exercise (Wagman et al., 1962). During exercise, emotional excitement, and other forms of stress there is stimulation of the sympathetic innervation of the heart resulting in release of endogenous norepinephrine which can exert a local effect on the myocardial cells, Therefore plasma levels may not be a good indicator of the role of catecholamines. ANGINALPATIENT TO NITROGLYCERIN Comparison of the responses of the normal and the anginal patient to nitroglycerin, illustrated in Fig. 1, suggests that the anginal individual has a “fixed” coronary resistance (Rowe, 1962). While the coronary flow increased 66% in the normal group the flow declined 16% in the anginal group (Brachfeld et al., 1959; Gorlin et al., 1959a). Coronary vascular resistance declined 42% in the normal group but there was no change in the anginal group. Blood pressure and cardiac work declined in both groups; cardiac oxygen consumption increased in normals but decreased in anginals. The clearance of N a P ’ did not increase after nitroglycerin in subjects with angina, indicating that capillary flow was also unchanged (Hollander et al., 1963). It has been suggested that angina induced by stress is associated with left ventricular failure. For example, during exercise there may be a rise

C. RESPONSEOF

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in pulmonary arterial pressure and left atrial pressure and an increase in diastolic heart size. Nitroglycerin will prevent or reverse these changes (Darby and Gebel, 1962; Stock et al., 1962). By means of coronary arteriographic techniques i t has been demonstrated that nitroglycerin and erythrityl tetranitrate increase the caliber

FIQ.1. Comparison of response of normal and anginal patient to nitroglycerin. MBP: mean blood pressure; CBF: coronary blood flow; CR: coronary resistance; A-V 01: arteriovenous oxygen difference; LVOz cons: left ventricular oxygen consumption; CO: cardiac output; LV work: left ventricular work; LV eff: left ventricular efficiency; HR: heart rate. Original data froin Brachfeld et al. (1959) and Gorlin et al. (1959a).

of the coronary arteries in anginal subjects (Likoff et al., 1962). However, the authors emphasize that no inference can be made that the increased caliber of the larger arteries is accompanied by an improvement in blood flow. It is important to realize that the smaller resistance vessels cannot be visualized and that these are the vessels that regulate the blood flow rate.

D. CONCLUSION The data that have been discussed above lead to the conclusion that the coronary bed in the anginal patient is adequate a t rest but is incapable of much dilatation. Therefore, the coronary reserve is seriously impaired. Any stress which increases the work and oxygen requirement of the heart will produce coronary insufficiency. It would appear that the beneficial effect of nitroglycerin is not associated with coronary dilatation but rather with the reduction in the work of the heart. However, it is conceivable that nitroglycerin may cause redistribution of blood to ischemic areas without producing a change in the total coronary blood flow rate.

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II. Physiology and Biochemistry of the Heart

A. GENERAL The heart is essentially an aerobic organ with a high rate of oxygen consumption. Myocardial arteriovenous oxygen difference (A-V 0,) is higher than that of any other organ and the oxygen extraction (A-V O,/A 0, X 100) has been estimated to be between 70 and 7576, compared with 22% for resting skeletal muscle and 25% for brain (Gorlin, 1962a). Under these circumstances, the oxygen supply to the myocardium can be considered essentially flow-limited since an increased oxygen extraction can yield only an additional 4 to 5 ~ 0 1 % .I n other organs additional oxygen can be obtained by increased extraction. During exercise, skeletal muscle will extract almost all of the available oxygen, as does cardiac muscle, but an “oxygen debt” can be contracted which is not the usual circumstance for the heart. For example, exercising skeletal muscle derives 10 to 30% of its energy anaerobically, compared with 5% for the heart during physical exercise (Gorlin, 1962a). Therefore, the factors that are involved in the regulation of coronary blood flow are of great importance in maintaining an adequate oxygen supply to the heart.

B. HEMODYNAMIC FACTORS REGULATING CORONARY BLOOD FLOW The blood flow through the coronary circulation (CBF) is related to the effective perfusion pressure divided by the resistance: CBF = effective perfusion pressure/resistance. These same physical principles apply to any vascular bed, but analysis of the interrelationship between various factors is different for the heart because the wall of the left ventricle, which develops the pressure head for perfusion of the coronary arteries, also offers phasic resistance to the coronary flow (Gregg, 1950). 1. Pressure Effective perfusion pressure is the difference between the pressure in the central coronary artery and the right atrium. A rise in perfusion pressure is invariably accompanied by a rise in coronary blood flow (Gregg, 1950; Alella et al., 1955; Berne, 1958; Rowe, 1962) ; however, the mechanism of the rise in coronary blood flow involves both physical and metabolic factors, the interrelationship of which will be discussed below. Only when the coronary arteries are perfused independently of the aorta can the direct relationship between pressure and flow be determined independently of metabolic factors (Berne, 1958) for if aortic pressure in-

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creases, the metabolic requirement of the heart increases because of an 1955; , Neil1 et al., 1963a). increased work level (Alella et a?. 2. Resistance Some of the factors which contribute to resistance are vessel tone (lumen size), extravascular support due to contraction of the myocardium, and relative duration of systole and diastole. The intrinsic portion of resistance is determined by the vessel tone regulated by the smooth muscles of the vessel walls. This is the more important portion of resistance and is involved in the autoregulation of the coronary circulation (Rowe, 1962). The arterioles on the afferent side of the capillary bed regulate vascular tone and the rate of flow out of the large arteries into the capillaries (Winsor and Hyman, 1961). Vessel tone can be influenced by endogenous and exogenous neurohumoral agents, metabolic products, nervous pathways to the coronary vessels, and by myocardial oxygen requirement (Gregg, 1950; Rowe, 1962). The latter is the most important determinant of vessel tone in the intact organism (Gorlin, 1962a). The extrinsic portion of resistance is a result of the mechanical or passive effect on flow due to the compression of the coronary vessels during ventricular systole (Gregg, 1950). Accordingly, there is a phasic increase in resistance during systole which is minimal during diastole. Approximately 75% of the flow occurs during diastole and 25% during systole (Gregg, 1962a). The phasic flow pattern emphasizes the interrelationship between the intrinsic and extrinsic portions of resistance and should be considered in analysis of drug action. With the onset of isometric contraction there is an abrupt decrease in left coronary inflow. I n open-chest dogs there may actually be backflow (Gregg, 1950, 1962a,b) but in normal unanesthetized dogs with measurement by an electromagnetic flowmeter no backflow is observed (Gregg, 1962b). After the aortic valves open and aortic pressure starts to rise, flow increases abruptly but declines somewhat during late systole. As isometric relaxation begins, flow again increases sharply to a new peak and declines gradually during diastole. On the other hand, outflow from the coronary sinus rises and falls smoothly with most flow occurring during systole. The inflow pattern is the important one since this relates to the supply to the capillaries which are compressed during systole. The importance of extravascular compression has been demonstrated in another way (Gregg and Sabiston, 1956; McKeever et al., 1958). The left coronary artery was perfused a t constant pressure from a reservoir with inflow and outflow measured continuously. During prolonged diastolic relaxation induced by vagal stimulation or ventricular fibrillation,

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the inflow and outflow invariably increased (Gregg and Sabiston, 1956; McKeever et al., 1958). It becomes obvious that any increase in systolic force or intraventricular pressure will increase the extravascular component of resistance. 3. Heart Rate

Changes in heart rate influence cardiac metabolism and affect flow indirectly (see Section 11, C) . However, changes in rate can influence the relative durations of systole and diastole; since diastolic flow accounts for 75% of the total, any alteration in the ratio systolic duration/diastolic duration will influence total coronary flow (Gregg, 1962b; Rowe, 1962). From a hemodynamic standpoint the important parameter is systolic time/minute versus diastolic time/minute. An increase in diastolic time a t the expense of systolic time should permit an increased rate of coronary flow. Other factors, such as viscosity of blood, also contribute to resistance but these are of minor importance except under unusual circumstances (Rowe, 1962). It is important to remember that resistance is a computed value relating flow to pressure under a particular circumstance and may or may not indicate changes in vascular tone. Nevertheless, the tone is the most important single variable in resistance. C. FACTORS INFLUENCING MYOCARDIAL OXYGENCONSUMPTION It has been well established that the coronary flow is correlated directly with the oxygen consumption (or requirement) of the heart (Alella et al., 1955; Katz, 1956; Berglund et al., 1957; Braunwald et al., 1958; Hashimoto et al., 1960; Gregg, 1962b; Rowe, 1962). If the oxygen supply is inadequate for the requirement there will be a reduction in vessel tone resulting in an increased flow. Thus, one can conclude that the 0, supply/02 demand ratio is the primary regulator of coronary flow from the metabolic standpoint (Gorlin, 1962a). I n fact, Katz (1956) suggested that hemodynamic factors play a minor role in the homeostasis of the coronary circulation in the absence of alterations of oxygen consumption. However, we should not lose sight of the fact that the mechanical influences involved in coronary flow, discussed previously, modify cardiac metabolism and in that way can influence coronary flow. That the coronary flow will be changed by alterations in oxygen content of the arterial blood has been demonstrated in the intact animal (Berne et al., 1957; Gregg, 1962b; Berne, 1963) and in the isolated rabbit heart (Guz et al., 1960). At normal perfusion pressure, a reduction in the oxygen content of the arterial blood is accompanied by a rise in coronary flow and a decline in the oxygen content of the coronary sinus

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blood (Berne et al., 1957; Scott et al., 1962). However, a t high perfusion pressures (greater than aortic pressure) the coronary flow is greater than normal and does not change with a decline in arterial oxygen content until the sinus content drops to less than 5.5 vol% ; below this level coronary flow increases as arterial and sinus oxygen content decline (Berne et al., 1957). Oxygen consumption does not vary except when the arterial content is exceedingly low (5 ~ 0 1 % )(Berne et al., 1957; Gregg, 1962b). Berne et al. (1957) concluded that the arterial oxygen content has no direct influence on the coronary arterioles when the oxygen supply is adequate. Only when sinus content declines below 5.5 vol% does coronary flow increase. Since the coronary venous blood reflects the changes in the cells of the myocardium it can be assumed that the oxygen tension of the tissues are of prime importance in the metabolic regulation of coronary flow (Berne et al., 1957). Katz (1956) concluded that coronary flow adjusts by a regulatory mechanism to maintain the A-V O2and venous oxygen content constant over the normal range of cardiac activity. The question arises as to whether or not myocardial hypoxia leads to the formation or release of vasodilator materials which could be involved in the autoregulation of the coronary circulation (Berne et al., 1957; Berne, 1963). Reoxygenated coronary sinus blood from normal, hypoxic, or hyperperfused hearts contained no vasoactive, inotropic, or chronotropic substance (Jelliffe et al., 1957). If any such materials were released by the tissues they were destroyed by the blood or by oxygenation. It has been suggested that adenosine may be involved in coronary autoregulation (Berne, 1963). During hypoxia of the isolated cat heart or hypoxemia of the intact dog there is a decrease in coronary vascular resistance and a release of inosine and hypoxanthine from the myocardium. These are metabolic degradation products of adenosine, which is a potent coronary dilator, and the amounts released, if considered as adenosine, could easily account for the coronary dilatation observed. The key factor in the autoregulation hypothesis of Berne (1963) is tissue oxygen tension. A decline in oxygen tension would result in breakdown of myocardial adenine nucleotides to form adenosine, which diffuses out of the cells and reaches the arterioles via interstitial fluid to produce arteriolar dilatation. Further experimental evidence is required to substantiate this hypothesis. 1. Hemodynamic Factors Influencing Oxygen Consumption.

The oxygen consumption and metabolism of the heart are adjusted to the heat production and useful work performed. Only a small part of the total energy liberated appears as useful mechanical work as evidenced by the low efficiency. The work of the heart is determined by the pressure generated (ventricular pressure) and the blood flow (stroke volume or

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cardiac output). Some have found a good correlation between oxygen consumption and cardiac work (Katz et al., 1955), whereas others have found a poor correlation (Neill et al., 1963a). Much of this divergence of correlation depends upon the type of experiment performed and the fact that the oxygen requirement for pressure work and flow work differ markedly. Oxygen consumption increases less than mechanical work when cardiac output is the predominant factor, but more than mechanical work when elevation of aortic pressure is involved (Alella et al.,1955; Braunwald et al., 1958; Sarnoff et aZ., 1958s; Katz et al., 1962; Neill et al., 1963a). Thus, when work is altered by increasing the resistance to outflow (aortic pressure) a t constant cardiac output, oxygen consumption increases markedly (175% increase in work, increase of Q4 178%) whereas increasing cardiac output at constant aortic pressure causes little change in oxygen consumption (696% increase in work, increase of Qo 53%) (Alella e t aZ., 1955; Braunwald et aZ., 1958; Sarnoff e t al., 1958a). Using the “isolated supported” heart preparation, Sarnoff et al. (1958a) found that the oxygen consumption of the heart was best correlated with the tension-time index, TTI (mean systolic pressure X duration of systole) under conditions of variations in aortic pressure and cardiac output. Recent studies by Neill et al. (1963a) in intact dogs showed that the PTI (mean systolic pressure x ejection period )( heart rate) was highly correlated with the oxygen consumption of the heart (correlation coefficient 0.93). The PTI/Qo, ratio was constant a t high and low PTI values, a t short or long ejection periods, and even under conditions of anemia in which the A-V 0, was reduced. Levine and Wagman (1962) point out that PTI is only one of the two components of myocardial tension; the other is the mean radius of the cardiac chamber. If we assume that the ventricle is a sphere, the law of Laplace applies as follows:

T =P d where T = total wall tension in dynes; P = intracavitary pressure in dynes/cm3; and r = radius in centimeters, Thus tension varies directly with pressure and with the square of the radius. At constant radius T will vary directly with PTI (and TTI) ; however, if during systole, the internal surface area (4Xr2) decreases more rapidly than P rises, T will decrease (Levine and Wagman, 1962). The importance of tension in determining oxygen consumption is more easily demonstrated in isolated cardiac muscle. As the resting tension level increases the portion of the total oxygen consumption during activity due to resting tension increases more rapidly than the portion due to activity (Lee, 1960). Under isotonic conditions (no tension) oxygen consumption did not vary with changes in the load (Whalen, 1962).

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Heart rate has an effect on coronary flow and oxygen consumption independent of changes in cardiac work (Katz, 1956; Laurent et al., 1956; Sarnoff et al., 1958a). At constant aortic pressure and cardiac output an increase in heart rate will increase oxygen consumption and thus lower mechanical efficiency (Laurent et al., 1956; Sarnoff et a,?., 1958a). However the PTI/Qo, ratio is independent of changes in heart rate (Neil1 et al., 1963a). A “stress adaptation mechanism,” described by Laurent et al. (1956) , was typified by an abnormal increase in coronary flow, a marked decline and a marked reduction in cardiac oxygen consumption even in A-V 02, though work was maintained a t a high level. This occurred when the normal oxygen supply/demand ratio was exceeded, as with a n increased load or excessive heart rate ; hypoxemia also brought on this mechanism but in this case coronary sinus blood became more and more unsaturated (Katz et al., 1955; Katz, 1956; Katz and Feinberg, 1958). After the stress adaptation mechanism became established, there was still an increase in cardiac oxygen consumption and coronary flow with an increase in heart rate, but the oxygen consumption was less at each level of heart rate than before even though work was greater. The stress adaptation mechanism lasted for more than 1 hour and was reversible. I n effect it was akin to shifting gears in a car. It is unlikely that the shift in metabolism involved a simple oxygen debt (Sarnoff et al., 1958~).Other possibilities are (1) use of storage products, (2) more efficient utilization of substrates, (3) more efficient conversion of energy to mechanical work, or (4) diversion of energy from synthesis and repair (resting state) to mechanical work (Katz and Feinberg, 1958; Sarnoff et al., 1958~).Another investigator suggested that a sudden release of anaerobic energy resulted when the oxygen supply became inadequate (Ballinger and Vollenweider, 1962). To my mind i t is necessary t o demonstrate that the stress adaptation mechanism exists in the intact animal since the results may be peculiar to the experimental design. The total oxygen consumption of the beating heart is a composite of resting oxygen consumption and activity oxygen consumption. The importance of each of these parameters was investigated in open-chest dogs with the left coronary artery perfused a t a constant pressure from an external source. The work of the heart was reduced to zero by vagal or potassium arrest, or by ventricular fibrillation (Berglund et al., 1957; McKeever et al., 1958). All of these procedures produced an initial increase in coronary flow (Berglund et al., 1957; McKeever e t al., 1958) and oxygen consumption (McKeever et al., 1958). This was rapidly followed by a decline in oxygen consumption to considerably below control (doing external work) values. During vagal or potassium arrest the oxygen

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consumption was about 25% of the activity level and during fibrillation about 50%; hemorrhage reduced oxygen consumption to about 35% of control values (McKeever et al., 1958). The basal oxygen consumption during fibrillation or hemorrhage was higher than that during vagal or potassium arrest (Berglund et al., 1957; McKeever et al., 1958). Similar results were obtained in the closed-chest dog; however, no difference was noted between arrest and fibrillation (Beuren et d.,1958). If the heart was stressed by high arterial pressure or administration of catecholamines there was a disproportionate rise in resting and active oxygen consumption with resting consumption accounting for 50% of the total (McKeever et al., 1958; Gregg, 1962a). In the potassium-arrested heart, norepinephrine decreased coronary flow but increased oxygen consumption (Berne, 1958). Presumably the resting oxygen consumption was used for maintenance of cellular integrity (selective membrane permeability) and production of a pool of high-energy phosphate which could be used for the contractile process; however, i t is difficult to explain the increase in the resting oxygen consumption produced by catecholamines or previous high work levels. In the case of the catecholamines i t is conceivable that inefficient metabolic pathways were stimulated. Computations of mechanical efficiency of the heart are usually based on the caloric equivalent of the total oxggen cmumptkm which includes both the resting and activity consumption. However, to determine true mechanical efficiency, only the energy required for the mechanical activity should be considered. When the resting metabolism is deducted from the total metabolism the efficiency of cardiac muscular activity increases to 37% for the dog and 39% for man (Gregg and Sabiston, 1956; Bing et al., 1958; Bing and Michal, 1959). Assuming that the resting metabolism does not vary greatly, it is easy to understand why the usual computed efficiency of the heart (about 25%) increases with an increased work level. This is true in the anesthetized open-chest dog whether the increased work level is due to a rise in cardiac output or in blood pressure (Katz et al., 1955). I n normal humans, the mechanical efficiency increases 30% during exercise while oxygen consumption increases 40% (Gorlin, 1962a; Messer and Neill, 1962). Similarly, the ratio of PTI/ oxygen consumption of the left ventricle increases 20% during exercise (Levine and Wagman, 1962). 2. Biochemical Factors Influencing Oxygen CmumpCion

It has been demonstrated that the utilization of certain substances by the heart is related to the level in the arterial blood (see Section 11,E, 2). Accordingly it is of interest to ascertain if oxygen consumption is related to the oxygen content of the arterial blood. Berne and co-workers (Berne

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et al., 1957; Berne, 1958) altered the oxygen supply to the heart of open-

chest dogs either by altering the rate of coronary flow or by altering the saturation of the arterial blood and found no change in oxygen consumption except in one experiment when the arterial content dropped below 5 ~ 0 1 % Using . the isolated rabbit heart, Guz et al. (1960) varied the oxygen content of the perfusion medium by using hemoglobin solutions of various concentrations that were equilibrated with a mixture of 3% CO, and 97% 02.Oxygen consumption and isometric contractile force remained constant, providing the oxygen content of the perfusion fluid did not decline below 2 ~ 0 1 % .On the other hand, Kats e t al. (1955) found that oxygen consumption was higher a t a given level of cardiac work as the arterial oxygen content increased. These conclusions were based on pooled data from individual experiments which show marked variability and the relationship between oxygen consumption and arterial supply may be spurious or a result of some other variables. I n intact dogs, Scott e t al. (1962) compared the effects of breathing 100, 10, and 5% oxygen mixtures. The 10% oxygen mixture produced an increase in cardiac work and oxygen consumption compared with 100% O2; under 5% oxygen cardiac work was again increased but oxygen consumption was reduced below that with 100% oxygen. Studies in anesthetized dogs with the left ventricular volume held constant by inflating a balloon therein suggested that cardiac oxygen consumption and left ventricular performance (heart rate x left ventricular pressure) were determined by the rate of coronary flow (Katz et al., 1962). The data available do not permit the conclusion that the oxygen consumption is related to the arterial content of oxygen or to the rate of coronary flow if the supply is adequate. Only when the supply is inadequate would a decline in cardiac oxygen consumption be expected. Raab has stressed the role of catecholamines in angina because of their effect on myocardial metabolism and oxygen consumption (Raab, 1956, 1962; Raab et al., 1962). The increased oxygen consumption is over and above that required for the additional work. Thus, the catecholamines cause wasteful metabolism with a reduction in cardiac efficiency (Raab, 1956,1962; Bing et al., 1960; Raab et al., 1962). The increase in myocardial oxygen consumption produced by catecholamines has been demonstrated by a number of different procedures. Injection of epinephrine, norepinephrine, and isoproterenol into the coronary circulation of the dog produced an increase in oxygen consumption and, usually, in coronary flow in the intact beating heart or in fibrillating or arrested heart (Eckstein et al., 1951 ; Berne, 1958; Hashimoto et al., 1960; Juhhss-Nagy and Szentivinyi, 1961 ; Winbury et al., 1962a). Similar effects were noted in intact cats following intravenous injection of epi-

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15

nephrine and norepinephrine; these changes were noted even if there was little change in cardiac work or heart rate (Popovich et al., 1956). Stimulation of the cardiac sympathetic nerves in the dog also produced an increase in oxygen consumption and coronary flow which was not always accompanied by a concomitant rise in heart rate, blood pressure, or aortic pulse (Eckstein et al., 1951; Juhhsz-Nagy and Ssentivinyi, 1961; Szentivhnyi and Juhhsz-Nagy, 1961; Gregg, 1962b). Chronic sympathectomy reduced coronary blood flow, heart rate, and myocardial oxygen consumption and was accompanied by a rise in left ventricular efficiency (Scott and Balourdas, 1959). Infusion of isoproterenol into normal man produced a rise in coronary flow and oxygen consumption together with an increase in stroke and cardiac index, heart rate, and mean systolic pressure (Krasnow et al., 1962a). The increase in oxygen consumption was proportional to the increase in PTI. Thus, PTI/Qc, was unchanged by the catecholamine. Thyroid hormone has an effect on myocardial oxygen consumption either by a direct action on myocardial function (work and heart rate) or metabolism or by sensitization of the myocardium to endogenously released catecholamines (Raab, 1956, 1962). It has been reported by Rowe et al. (1956) that therapy in patients with thyrotoxicosis caused a decrease in oxygen consumption, coronary flow, cardiac output, and left ventricular work. Gorlin (1962b), using the data of Rowe et al. (1956) to compute TTI, found that Il3' reduced this parameter but that the TTI/Qo, ratio was unchanged. Gudbjarnason et al. (1962) suggested that there is an uncoupling of oxidative phosphorylation in thyrotoxicosis.

D. NEURALAND HUMORAL FACTORS 1. F u n c t i m l Innervation of the COTOTZUTY Vessels

The heart receives both sympathetic and parasympathetic fibers which are involved in the central regulation of rate, conduction, contractility, and metabolism. The role of the sympathetic and parasympathetic systems in the regulation of the coronary circulation has been the subject of considerable controversy because many of the studies failed to consider the changes in myocardial metabolism, which can influence the coronary blood flow. Recently Szentivhnyi and Juhhsz-Nagy (Szentivhnyi and JuhhszNagy, 1959, 1961; Juhhsz-Nagy and Szentivhnyi, 1961) proposed a hypothesis for the functional innervation of the heart and coronary vessels which integrates the direct coronary and direct myocardial actions. The accelerator nerve (sympathetic) contains preganglionic fibers in addition to the postganglionic fibers. The postganglionic (C) fibers run directly to

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the muscle elements of the pacemaker system and of the myocardium, and influence heart rate, contractility, and myocardial metabolism. These postganglionic fibers do not affect the coronary arteries directly but can have an indirect effect by altering myocardial metabolism. The preganglionic ( B ) fibers of the sympathetic nerves do not form synapses in the stellate ganglion but synapse peripherally with adrenergic or cholinergic ganglia in or near the walls of the heart. The related postganglionic fibers innervate the coronary arteries directly with the sympathetic cholinergics producing dilation and sympathetic adrenergics producing constriction. These fibers do not innervate the muscle elements. The vagus nerve does not have any direct role in coronary innervation. The parasympathetic fibers synapse in the myocardium and the related postganglionic fibers go only to the pacemaker and the conducting system, influencing only rate and conduction, There is little effect on contractility, metabolism or coronary. circulation. Presumably the acetylcholine released a t these nerve terminals does not enter the coronary system but acts on specific cardiac tissue. I n some circumstances the vagus nerve contains preganglionic sympathetic fibers which innervate only the muscle cells. This hypothesis is supported by the work of Hashimoto e t al. (1960) using the fibrillating dog heart. 2. Effect of Sympathetic Stimulation and Catecholamines

Injection of catecholamines into the coronary circulation or sympathetic stimulation has been reported to produce constriction or dilatation, or both effects. The effect of sympathetic stimulation on coronary blood flow and other hemodynamic factors was analyzed by individual consecutive beats by Winbury and Green (1952). When coronary perfusion pressure was held constant there was an initial decline in coronary flow (constriction) followed in a short time by a marked increase (dilatation), but when perfusion pressure was permitted to rise with aortic pressure only an increase in coronary flow was observed. Computed coronary vascular resistance showed an initial increase followed by a decline in both types of experiments; heart rate and cardiac work increased. These effects were not blocked by atropine or ergotoxine. Eckstein et d. (1951) demonstrated that sympathetic stimulation with constant perfusion pressure produced coronary dilatation accompanied by a decline in A-V O2 and a rise in oxygen consumption. Berne (1958) noted that the dilatation was preceded by constriction as did Winbury and Green (1952). Szentivbnyi and Juhbss-Nagy (1959, 1961; Juhbsa-Nagy and Szentivbnyi, 1961) used a selective stimulation of the B and C fibers of the sympathetic nerves to separate the direct coronary effects from the in-

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17

direct metabolic effects. Stimulation of a branch of the cardiac sympathetic nerve of the dog with low voltages frequently resulted in coronary constriction without a change in heart rate, contraction or metabolism (Juhitsz-Nagy and Szentivhnyi, 1961). This effect was blocked by hexamethonium, ergotamine, or dibenamine, but not by atropine, indicating that preganglionic fibers of sympathetic adrenergic nerves were stimulated (JuhBsz-Nagy and Szentivhnyi, 1961; Szentivhyi and Juhhsz-Nagy, 1961). In the cat, stimulation of certain rami of the accelerator nerve resulted in coronary dilatation and sometimes in bradycardia; the dilatation was abolished by hexamethonium or atropine and enhanced by eserine (Szentivhnyi and Juhhsz-Nagy, 1959). These are preganglionic fibers of sympathetic cholinergic nerves. A similar pathway can be excited in some dogs by stimulation of the accelerator nerve with low parameters (JuhAss-Nagy and Szentivfinyi, 1961; Szentivhnyi and Juh fisz-Nagy, 1961) ; only vasodilatation was noted. This vasodilatation was not accompanied by any change in myocardial metabolism and, as in the cat, was abolished by hexamethonium or atropine, and enhanced by eserine. These sympathetic cholinergic nerves are interpreted to represent the true dilators of the coronaries (Szentivhnyi and Juhhsz-Nagy, 1961). When high voltages were used on the accelerator nerve in the dog there was an increase in cardiac contractility and rate, coronary flow, and oxygen consumption (Juhbz-Nagy and Szentivhnyi, 1961; Szentivhnyi and Juhhsz-Nagy, 1961). These effects were not abolished by hexamethonium, atropine, ergotamine, or dibenamine. These are postganglionic adrenergic fibers which go directly to the cardiac muscle but not to the coronaries. The vasodilation observed was secondary to the increased metabolism (oxygen consumption) (Szentivhnyi and Juhhss-Nag, 1959, 1961; Juhhsz-Nagy and Szentivhnyi, 1961). Since stimulation of these postganglionic adrenergic fibers always affects metabolism, most investigators failed to recognize the role of sympathetic preganglionic fibers which synapse with adrenergic constrictors or cholinergic dilators and have claimed that the coronaries do not have any independent vasomotor control. Catecholamines such as epinephrine, norepinephrine, and isoproterenol invariably produce coronary dilatation in the beating in situ or isolated heart (Eckstein et al., 1951; Winbury and Green, 1952; Berne, 1958; Hashimoto et al., 1960; Szentivhnyi and Juhhsz-Nagy, 1961). This is accompanied by an increase in cardiac Contractility (contractile force, stroke volume, or cardiac output), cardiac work, and oxygen consumption (Eckstein et al., 1951; Winbury and Green, 1952; Popovich et al., 1956; Berne, 1958; Hashimoto et al., 1960; Szentivhnyi and Juhfisz-Nagy, 1961). These effects are not antagonized by a-adrenergic-blocking agents (di-

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MARTIN M. WINBURY

benamine, ergotoxine, ergotamine) (Winbury and Green, 1952 ; Hashimoto e t aZ., 1960; Juhhsz-Nagy and SzentivLnyi, 1961; SzentivLnyi and Juhhsz-Nagy, 1961) ; however, Denison et al. (1956) reported that high doses of azapetine injected into the coronary artery block completely the increase in diastolic flow produced by epinephrine, norepinephrine, and isoproterenol. Berne (1958) and Hashimoto et al. (1960) demonstrated that epinephrine and norepinephrine have a direct vasoconstrictor effect in addition to the metabolic dilator action. I n the fibrillating dog heart perfused a t constant pressure, Berne (1958) observed that epinephrine and norepinephrine produce an immediate decline in coronaiy flow followed within a short time by an increase; oxygen consumption was not changed during vasoconstriction but increased during vasodilatation. When the fibrillating heart was arrested with potassium, norepinephrine produced only vasoconstriction even though oxygen consumption increased. Phasic flow measurements in the intact dog with the coronaries perfused a t constant pressure demonstrated that norepinephrine causes a temporary decline in diastolic flow before the rise (Berne, 1958). Hashimoto et al. (1960) reported that epinephrine, norepinephrine, and isoproterenol increase coronary flow and oxygen consumption in the fibrillating dog heart. After the &locking agent, dibenzylinc, the dilator effect of epinephrine and norepinephrine was increased but the metabolic effect was unchanged. After the &blocking agent, dichloroisoproterenol, isoproterenol had no effect, and epinephrine and norepinephrine produced a short constriction with no effect on metabolism. These results are in agreement with the hypothesis of SzentivLnyi and JuhBsz-Nagy (1961) that the only direct adrenergic effect on the coronaries is constriction. Preliminary studies by Winbury et al. (1962a) suggested that norepinephrine causes a reduction in “effective capillary flow” a t the time of vasodilatation and increased oxygen consumption. In these studies blood was pumped into the left coronary artery a t a constant rate with perfusion pressure as a measure of total resistance. Effective capillary flow was determined by RbS6 uptake of the myocardium; details of the procedure are given in Section V, I. Phasic coronary flow curves show that epinephrine, norepinephrine, isoproterenol, and sympathetic stimulation increase diastolic flow and decrease systolic flow (Denison et al., 1956; Berne, 1958; Denison and Green, 1958). The increase in diastolic flow is greater than the decrease in systolic flow, causing an increase in mean flow. The heart contains large catecholamine stores primarily in the form of norepinephrine (Raab, 1956) as a result of synthesis and accumulation. Sympathetic nerve stimulation will elicit discharge of norepinephrine from the adrenergic nerves directly into the myocardial effector cells (Raab,

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19

1962). Stimulation of cardiac sympathetic nerves is followed by an increase in myocardial norepinephrine and sympathetic denervation by a decrease (Raab, 1962). 3. Effect of Parasympathetic Stimulation and Acetylcholine There appears to be general agreement that stimulation of the vagus innervation of the heart has no effect on the coronary circulation independent of changes in heart rate (Winbury and Green, 1952; Schreiner et al., 1957; Denison and Green, 1958). When heart rate is controlled, vagus stimulation does not affect coronary blood flow or oxygen consumption. On the other hand, injection of acetylcholine into the coronary artery is always followed by coronary dilatation with no change in oxygen consumption (Winbury and Green, 1952; Schreiner et al., 1957; Berne, 1958; Juhhsz-Nagy and Szentivhnyi, 1961 ; Gregg, 1962b). Phasic flow measurements demonstrated a rise in both systolic and diastolic flow (Berne, 1958). It can be concluded that acetylcholine produces true coronary dilatation. The fact that vagal stimulation has no direct effect on the coronaries but that acetylcholine does is understandable in light of the hypothesis of Szentivhnyi and Juhhsz-Nagy (1959, 1961) that the parasympathetic fibers to the heart innervate only the pacemaker. Confirmation of this is provided by the fact that ventricular function is not affected by vagal stimulation but is depressed by injected acetylcholine (Schreiner et al., 1957). Injected acetylcholine mimics the vasodilator action of the preganglionic sympathetic cholinergic fibers. Coronary dilatation produced by acetylcholine and by stimulation of sympathetic cholinergics can be blocked by atropine (Winbury and Green, 1952; Szentivirnyi and JuhBszNagy, 1961).

E. MYOCARDIAL METABOLISM in Situ There are numerous studies on cardiac muscle slices, homogenates, etc., which have described enzymic pathways, substrate utilization, aerobic and anaerobic metabolism. In many cases i t is exceedingly difficult to relate the results obtained with these preparations to the results obtained on in situ intact preparations. Since the objective is to relate myocardial metabolism to the function of the heart, only in situ studies will be discussed. Substrate utilization, aerobic metabolism, and anaerobic metabolism will be considered. 1. General Considerations

It was mentioned previously that the heart has a high rate of oxygen uptake; consequently, extraction of oxygen from blood is nearly com-

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MARTIN M. WINBURY

plete. Under normal circumstances an increased oxygen requirement necessitates an increased coronary flow since the heart can contract only a small oxygen debt. When the coronary circulation is compromised as in coronary insdciency, the oxygen supply cannot keep pace with the demand and alterations in metabolism might be expected. The problem of specific substrates utilized for energy production has been studied in recent years by many groups. It has been demonstrated that the dog heart and human heart can use glucose, lactate, pyruvate, fatty acids, ketone bodies, and amino acids (Gregg and Sabiston, 1956; Bing et al., 1958, 1960; Bing, 1961; Stock et al., 1962). The utilization of these substrates is in direct relationship to the arterial level of each substance; however, there are thresholds below which a substrate will not be extracted by the heart and there is ‘(sparing” of one substrate by another (Gregg and Sabiston, 1956; Bing, 1961; Olson, 1962a,b; Shipp et al., 1962). A complication in the interpretation of the results is the fact that the uptake of a single substrate such as fatty acids or carbohydrates can account for more than 100% of t,he oxygen consumption indicating that there must be storage (Goodale and Hackel, 195313; Ballard e t al., 1960; Bing et al., 1960; Olson, 1962a,b; Shipp et al., 1962). This only serves to emphasize that uptake of a particular substrate by the heart does not indicate that the substrate is metabolized. 2. Substrate Uptake and Utilization Substrate uptake has been studied in dogs and man by determination of arteriovenous difference and coronary flow rate (A-V substrate difference, mg/ml blood X coronary flow, ml/minute = mg uptake/minute) . The share each substrate contributes to the total oxygen consumption can be computed on the basis of oxygen required for oxidation of substrate. Substrate utilization varies considerably under different dynamic circumstances (Ballard et al., 1960; Olson, 1962b). The extent to which each substrate contributes to cardiac energy production is dependent upon the arterial concentration, the state of nutrition, and the endocrine balance (Gregg and Sabiston, 1956; Bing et al., 1958; Olson, 1962b). During fasting or the postabsorptive state the heart shifts from predominantly carbohydrate utilization t o primarily fat utilization. Under postprandial conditions carbohydrates are the major substrates (Goodale and Hackel, 1953b; Gregg and Sabiston, 1956; Bing e t al., 1958; Ballard et al., 1960; Bing, 1961; Goto, 1962; Olson, 1962b). The respiratory quotient (RQ) of the fasting human or dog heart is about 0.80 and about 3/6 of the oxygen uptake can be accounted for by fatty acids and about 1/3 by carbohydrates (Bing and Michal, 1959; Bing, 1961; Olson, 1962b). On the other hand, after feeding or glucose infusion, the RQ of the heart

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is close to 1.0 and carbohydrates account for most of the oxygen utilization (Goodale and Hackel, 1953b; Olson, 1962b). According to Bing and co-workers (1958), the relative contributions of substrates to myocardial oxygen usage in the postabsorptive state is as follows: carbohydrates, 34.947% (glucose 17.9, pyruvate 0.54, and lactate 16.5) ; noncarbohydrates, 76.9% (fatty acids 67.0, amino acids 5.6, and ketones 4.3). The sum of these values accounts for more than 100%of the oxygen consumption, for the values are based on the assumption that the substrates taken up by the heart are completely oxidized. It is obvious that this is not the case and some is stored in the myocardium (Bing et al., 1958; Bing and Michal, 1959; Ballard et al., 1960). This is demonstrated by studies in which blood levels of fatty acids or glucose were raised and the contribution of each to the oxygen uptake exceeded 100% (Goodale and Hackel, 1953b; Bing et al., 1958). Olson (1962b) concluded that carbohydrates are the preferred substrates and are the determinants of the fuel used by the heart; the uptake of nonesterified fatty acids (NEFA) is regulated by the carbohydrate uptake. Glucose administration decreases the arterial level of fatty acids and cardiac extraction of NEFA (Bing et al., 1958). The interaction between palmitate, pyruvate, and acetoacetate was studied in the isolated perfused rat heart using Cl*-labeled palmitate (Olson, 1962a). When palmitate was the only substrate in the Krebs-Henseleit solution a high percentage was extracted, of which about 75% was oxidized to COz and accounted for 37% of the cardiac oxygen consumption. I n the presence of pyruvate or acetoacetate the uptake of palmitate was decreased 50% and oxidation to CO, was reduced to 25% of the value when palmitate was given alone. The uptake of pyruvate or acetoacetate was the same as if they were perfused alone. The carbohydrate contributed 7545% to the fuel of respiration and reduced the contribution of the fatty acid to 8-9%. The presence of pyruvate or acetoacetate shifted fatty acid from oxidation into a five- to sixfold increase in storage as neutral lipids in the heart. In contrast, other workers (Shipp et al., 1962), also using the perfused rat heart with labeled substrates, observed that when glucose and palmitate were present the fatty acid was oxidized in preference to glucose; the latter was converted to glycogen. There are definite threshold concentrations below which carbohydrates will not be extracted from arterial blood. In the dog these values are as follows: lactate, 2.56 mg%; pyruvate, 0.61 mg%; and glucose, 54.2 mg% (Goodale and Hackel, 1953b). The threshold for glucose in man has been estimated to be about 80 mg% (Bing et al., 1958; Bing, 1961). Above these values the extraction increases linearly with arterial concentration for lactate and pyruvate ; whereas with glucose, extraction increases linearly

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MARTIN M. WINBURY

up to about 100 mg% and then tends to become asymptotic (Goodale and Hackel, 195313; Olson, 1962b). At normal arterial concentrations the utilization of lactate is about that of glucose and is many times that of pyruvate (Goodale and Hackel, 1953b; Bing, 1961). For example, in the dog 66% of the oxygen extraction could be attributed to carbohydrates with the breakdown as follows: lactate, 34.1% ; pyruvate, 4.1% ; and glucose 27.1% (Goodale and Hackel, 1953b). However, if the arterial level of any one of these substrates is increased the proportion it contributes to the total carbohydrate uptake increases proportionately. Myocardial metabolism of fatty acids has been studied extensively in the dog and human. I n the fasting human the oxygen extraction ratio [ (0, equivalent of extracted fatty acidJmyocardial0, extraction) X 1001 for total fatty acids (TFA) averaged 145% (Ballard et al., 1960). The nonesterified fatty acids (NEFA) accounted for 42% of the total fatty acid extraction and the esterified portion made up the remaining 58% (Ballard et al., 1960; Bing, 1961). I n the fasting dog the TFA oxygen extraction ratio was 123% and NEFA accounted for 23% of the total extraction (Ballard et al., 1960; Bing, 1961). The iodine number of TFA was consistently higher in the coronary sinus blood than arterial blood, suggesting greater usage of more saturated fatty acids (Ballard et al., 1960). I n a recent study on open-chest dogs it was found that the arteriovenous differences for cholesterol, phospholipid, and TFA were quite variable and not statistically significant, but the arteriovenous difference for NEFA was significant (Goto, 1962). On the other hand, the isolated perfused rabbit heart can extract and oxidize NEFA bound to albumin as well as triglyceride fatty acids in lipoproteins (Gousios et al., 1962). The importance of the various long-chain fatty acids in the NEFA fraction has been analyzed and i t was found that the extraction of oleic acid was high compared with the other fatty acids (Bing, 1961; Carlsten et al., 1962; Goto, 1962). I n the human, extraction (in terms of moles/liter of blood) is 50 for oleic, 10 for stearic, and 2 for linoleic acids, respectively, indicating that oleic contributes considerably more to NEFA oxygen extraction ratio than stearic or lineolic (Carlsten et al., 1962). Other workers found the uptake of palmitic acid in man was also low. I n the dog oleic acid was the only long-chain fatty acid with a significant extraction; the other acids studied showed a nonsignificant positive extraction, or a negative extraction (Goto, 1962). These include lauric, myristic, palmitic, palmitoleic, stearic, linoleic, linolenic, and arachidic acids. 3. Anaerobic Metabolism There is considerable evidence that glycolysis can contribute to myocardial energy production under anaerobic conditions but there is a ques-

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tion as to whether glycolysis occurs under aerobic conditions. One group of investigators suggested that the normal dog heart has a slight “oxygen debt” on the basis of the fact that after arrest cardiac oxygen consumption did not decline for 10 to 15 seconds (Gregg and Sabiston, 1956; McKeever et al., 1958). On the other hand, another group found that when the work of the heart was reduced by lowering the outflow resistance there was a rapid readjustment of the oxygen consumption which was interpreted to indicate that an oxygen debt was ( a ) not present, ( b ) was present but that i t was repaid a t some other time, or (c) the debt remained constant despite the change in activity (Sarnoff et al., 1 9 5 8 ~ )If. an oxygen debt were present in the normal dog heart one might expect a rise in oxygen consumption with an increase in availability; however, no change in oxygen consumption was observed when arterial saturation varied between 5 and 19 vol% (Berne et al., 1957). Biochemical studies uniformly show that under aerobic conditions the heart of the dog and man utilizes lactate and certainly does not produce lactate under normal conditions (Bing and Michal, 1959; Huckabee, 1961; Gudbjarnason e t al., 1962; Stock et al., 1962; Neill et al., 1963a). Huckabee (1961) found that exercise produced by stimulating the hindlimb of the normal anesthetized dog resulted in “excess lactate” production by the heart and that anaerobic metabolism accounted for 12% of the energy production. This could not be confirmed in man since there was no increase in lactate production in normal or anginal individuals (Krasnow e t al., 1962b; Messer and Neill, 1962; Wagman et al., 1962). These investigators concluded t.hat anaerobic metabolism does not occur in the human heart even under the stress of exercise. A recent study by Neill and co-workers (1963b) in dogs showed that under control conditions the total energy output (external mechanical work and heat production) of the left ventricle (44.4 cal/min/100 gm) was approximately equal to the energy from oxidative metabolism (38.5 cal/min/100 gm).Mechanical work accounted for only about ‘/4 of the total energy output with the remainder being heat. Following injection of cyanide the total energy output (50.9 cal) was 4 times the energy available from substrate oxidation (11.4 cal) ; thus after cyanide anaerobic energy (39.5 cal) accounted for about 4/5 of the total. These changes were accompanied by excess lactate production. Mechanical energy now accounted for about ‘/z of the total energy output. Although these effects were of short duration they demonstrate that anaerobic energy production can be an important factor in maintaining cardiac activity under anaerobic conditions. I n studies on the effect of hypoxia on coronary flow the heart continued to function when the arterial oxygen saturation dropped to 5 vol% and the oxygen uptake of the heart was markedly

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MARTIN M. WINBURY

reduced (Berne et al., 1957), indicating that anaerobic mechanisms were utilized. Many investigators have demonstrated that there is a net production of lactate (venous concentration higher than arterial) by the heart under hypoxic conditions and on this basis have concluded that anaerobic metabolism has occurred (Bing and Michal, 1959; Bing, 1961). This conclusion may not always be valid, for Huckabee (1961) showed that large changes in lactate production can occur without cellular hypoxia and can be related to increased pyruvate production. The concept of excess lactate was used to distinguish between pyruvate-induced changes and those related to a shift in the DPN-DPNH (diphosphopyridine nucleotide and reduced form) system. The DPN-DPNH system reflects the state of cellular oxygenation; when cellular hypoxia occurs, there is a shift toward the reduced form. The lactate-pyruvate equilibrium is useful to indicate the state of oxidation of DPN since both substances are highly diffusible and lactate is a final metabolic step. Cyanide resulted in excess lactate production in dogs aa did exercise and hypoxia (Huckabee, 1961; Krasnow et al., 1962b). It was estimated that anaerobic metabolism of the heart accounted for 11% of the total energy production during hypoxia and 12% during exercise (Huckabee, 1961). Other studies in dogs showed that excess lactate production did not occur under hypoxic conditions until the arterial oxygen saturation declined below 9 vol% (Ballinger and Vollenweider, 1962). This was accompanied by a rise in the RQ of the heart to values in excess of 1 ; the excess lactate production paralleled the rise in RQ. After 4 minutes of total anoxia (breathing 100% nitrogen) ventilation with 100% oxygen produced a resumption of aerobic metabolism with removal of excess lactate and a reduction of the RQ to below 1. These studies demonstrate that anaerobic metabolism can occur in the heart even though all of the oxygen available in the blood is not utilized. However, there is no indication of the state of oxygenation a t the cellular level. Another approach to estimate the state of oxidation of the DPNDPNH system is the estimation of oxidation-reduction potential (redox) of the lactate-pyruvate system in cardiac muscle cells (Gudbjarnason et al., 1962; Stock et al., 1962). When the cellular oxygen supply is inadequate for metabolic needs the oxidation-reduction systems approach a more reduced state and the redox potential (Eh) decreases. Direct determination of the redox potential of the intact heart is not possible. However, the calculated lactate-pyruvate redox potentials in the arterial and coronary sinus blood bear a relationship to that in heart muscle. In dogs with adequate oxygenation the heart muscle and coronary sinus had a more positive redox potential than arterial blood (AEh positive). In the

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anoxic heart the redox potential of the heart and sinus blood was more negative than the arterial blood (AEh negative). A positive AEh indicates active oxidation and that the energy is supplied by oxidative phosphorylation. A negative AEh indicates glycolysis and that part of the energy comes from anaerobic phosphorylation. In patients with uncomplicated coronary disease AEh was positive a t rest and became more positive on exercise, indicating no over-all deficiency in aerobic metabolism. These results confirm those previously discussed with the excess lactate method. During anoxia of heart muscle, regardless of the mechanism, there was rapid disappearance of glycogen (glycogenolysis) and an increase in lactate and hexose monophosphate (Conn et al., 1959; Bing et al., 1960; Danforth et al., 1960; Bing, 1961). The rise in lactate and hexose monophosphate accounted for the decline in glycogen (Conn et al., 1959; Danforth et al., 1960; Bing, 1961). The levels of fructose lJ6-diphosphate, dehydroxyacetone, or pyruvate were unchanged (Danforth et al., 1960) or decreased (Bing et al., 1960). This suggests that phosphofructokinase waB rate-limiting in this situation. I n addition, adenosine triphosphate (ATP) and creatine phosphate disappeared rapidly and nucleotides such as D P N and T P N decreased (Bing et al., 1960; Danforth et al.,1960). I n the intact dog heart, reperfusion with oxygenated blood, after brief periods of anoxia, permitted resynthesis of glycogen and ATP (Bing, 1961). After 15 minutes of anoxia, restoration of aerobic conditions did not result in effective contractile activity ; hexose monophosphate declined to normal, ATP rose for a short time, but the decline in glycogen continued (Danforth et al., 1960). This would indicate damage to the enzymic system leading to glycogenesis a t least. I n special studies using intact dogs the heart was arrested with potassium chloride and coronary perfusion was interrupted for 0.5 to 5 hours after which the heart was reperfused with oxygenated blood (Bing et al., 1958; Michal et al., 1958). Oxygen consumption and carbohydrate metabolism were determined before and after the period of circulatory arrest. Oxygen consumption was normal after 2 hours of circulatory arrest but after this there was a rapid decline. After 4 hours of ischemia, oxygen consumption was only 25% of the preischemia control value (nonbeating) . There were severe disturbances of carbohydrate metabolism indicative of aerobic glycolysis (oxygen uptake declined more than glucose uptake). No doubt if the heart had been beating in these studies there would have been a more rapid failure of the oxidative enzyme systems. I n isolated papillary muscles which were driven electrically, anoxia (nitrogen) resulted in a rapid decline in contractile activity in the presence or absence of glucose (Winbury, 1956). Reintroduction of oxygen after 60 minutes of anoxia permitted little recovery of contractile activity

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MARTIN M. WINBURY

when glucose was absent during the anoxic period. However, when glucose was present during the entire 60 minutes of anoxia, contractile activity returned when aerobic conditions were restored. Thus glycolysis did not produce sufficient energy to support contractile activity for prolonged periods but did permit maintenance of the enzymic pathways and the contractile mechanism during anoxia. This may be related to the observation that pretreatment of dogs with glucose 2 hours before removing the heart resulted in higher glycogen levels at zero time and after 2 hours compared with control animals (Conn et al., 1959). Ill. Progress in the Treatment of Coronary Insufficiency

Many compounds have been advocated for the treatment of angina but few of these have survived a carefully controlled clinical trial. Each year sees the development of new agents in the laboratory, but little has been added to our armamentarium for the treatment of coronary insufficiency other than modified nitrites. Theoretically the pain of angina pectoris can be relieved anywhere between the heart and the central nervous system where the pain is finally perceived. Reduction in pain or increasing coronary blood flow are the two main approaches that have been used for treatment in the past (Katz, 1956). However, a recently developed P-adrenergic-blocking agent has been used with some success (Dornhorst and Robinson, 1962). Considering all of the factors, drugs could act on any of the following sites : 1. Coronary vessels a. Relief of spasm? b. Arteriolar dilatation c. Improvement of nutritional blood flow 2. Myocardium a. Reduction of metabolic requirement or improved metabolic efficiency b. Reduction in contractile effort c. Reduction in diastolic size 3. Arterial pressure Reduction of work load on heart 4. Heart rate Reduction of excessive rate decreases myocardial oxygen consumption 5. Effect m nervous system a. Interruption of nerve pathway for pain transmission b. Reduction of central awareness of pain

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Throughout the years a multitude of coronary dilators have been developed (Charlier, 1961) but few have stood the test of time. The basic mechanism of the coronary dilatation is of primary importance (see Section 11,B) and it is conceivable that coronary dilatation may not improve myocardial oxygenation. Gregg and Sabiston (1956) distinguished between “benign” and “malignant” coronary dilatation. A benign vasodilator should affect the coronary vessels directly and not myocardial metabolism, thereby resulting in an improved supply/demand ratio and an improved myocardial oxygenation. This presupposes that there is also improvement in nutritional blood flow which may not always be the case (Winbury et al., 1962a). A malignant vasodilator is one that has a primary effect on myocardial oxygen requirement and the increase in coronary blood flow is secondary to this. Under these circumstances myocardial oxygenation may not be improved. Implied in the discussion of coronary dilator action is the ability of the coronary bed to dilate. Certainly the results of Gorlin et al. (1959a), which suggest that nitroglycerin does not increase the coronary flow in the anginal patient, should cause one to reflect about the importance of coronary dilatation. A shifting of blood from nonnutritional to nutritional channels is a possibility and does not require any increase in the total rate of flow (Winbury et al., 1962a). The evidence for the possibility of spasm of the coronary is equivocal and, as indicated in Section I, spasm is unlikely to be a cause of angina. This leads to the conclusion that effective drugs such as the nitrites may act by one or more of the mechanisms in 2, 3, or 4 above. Details of the pharmacology of the nitrites will be discussed in Section IV.

A. THYROID INHIBITION This form of therapy has been successful in intractable angina, presumably because of a reduction in cardiac oxygen requirement. During hyperthyroid states there is a generalized increase in tissue metabolism including the myocardium. Coronary blood flow, heart rate, cardiac output, cardiac work, cardiac oxygen consumption, and TTI (Gorlin, 196213) therapy reduces are elevated in thyrotoxicosis (Rowe et al., 1956). these parameters and thereby reestablishes an adequate coronary reserve (Rowe et aZ., 1956). It has been suggested that uncoupling of oxidative phosphorylation may occur in hyperthyroid states but a review on this subject raises questions about the presence of such a biochemical lesion (Olson, 1962b). Suppression of thyroid function has been of some success in the treatment of angina even in euthyroid patients. Initially thyroidectomy was employed (Blumgart et al., 1933) ; a t a later time thyroid-suppressing

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agents, such as thiouracil, were used (Raab, 1945), and finally IlS1became available (Blumgart et d.,1951). The effects of IlS1are particularly dramatic in the management of patients with incapacitating angina with pain a t rest or with minimal exertion (Shelburne et al., 1962). Using an experimental model of coronary insufficiency in atherosclerotic rabbits it was found that chronic thiouracil treatment reduces the severity of the ST segment depression induced by hypoxia (Tabachnick et al., 1961). B. MONOAMINE OXIDASEINHIBITORS Various monoamine oxidase inhibitors have been shown to have palliative effect on the pain of angina without any improvement in the electrocardiogram (Cesarman, 1959; Cossio, 1959; Master and Donoso, 1959; Schweieer, 1959; Grant, 1963). The exact mechanism is unknown but i t is unlikely that there is improvement in the basic coronary insufficiency since the stress electrocardiogram is still abnormal after relief of pain (Master and Donoso, 1959; Schweieer, 1959). It has been concluded that the relief of pain may be a result of mood elevation and of increased pain threshold (Master and Donoso, 1959; Grant, 1963). I n view of this, these agents must be used with caution and overexertion be avoided since the anginal pain, which is indicative of coronary insufficiency, is obtunded (Grant, 1963). A recent study by Goldberg and co-workers (1962) has demonstrated that isocarboxazid reduced the cardiovascular response to exercise. Three patients were studied and the most prominent effect was an attenuation of the rise of blood pressure, heart rate, and cardiac output normally produced by exercise. I n two of the patients angina, which appeared during a placebo period, disappeared on drug therapy. The authors suggest that the beneficial effect may be due to the attenuation of the cardiovascular response to exercise. Such an effect should be beneficial in the patient with coronary insufficiency but objective electrocardiographic evidence for a beneficial effect is lacking. The pharmacological and cardiovascular actions of monoamine oxidase inhibitors have been explored extensively in animals in an attempt to explain the palliative action in angina. Only a few reports will be discussed. Iproniazid administration for 4 days markedly reduced the incidence of fatal ventricular fibrillation produced by coronary occlusion in unanesthetized dogs (Regelson et al., 1959). This effect was not specific since pentobarbital anesthesia, morphine, reserpine, hexamethonium, and chlorpromaeine were also effective. Perfused isolated rabbit hearts were capable of beating for longer periods under anoxic conditions when iproniazid was added to the perfusion medium or when the animals received drug for 7 days prior to the experiment (Setnikar and Ravasi, 1980).

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Pretreatment of rats with nialamide, iproniazid, isocarboxazid, or pivalybenzhydrazine reduced the severity of myocardial necrosis resulting from high doses of isoproterenol (Cahn and Herold, 1962; Zbinden, 1962). I n addition, isocarboxazid reduced the severity of the electrocardiographic changes produced by intravenous injection of vasopressin in the rat (Cahn and Herold, 1962). Other studies have indicated that iproniazid and pheniprazine are ineffective in preventing the ST segment depression induced by hypoxia in atherosclerotic rabbits (Tabachnick et al., 1961; Varma and Melville, 1962a). Furthermore, iproniazid did not prevent the ST segment depression resulting from coronary ligation in dogs or injection of picrotoxin into the lateral ventricle of rabbits (Varma and Melville, 1962a). There are conflicting reports as to the coronary dilator activity of monamine oxidase inhibitors (Charlier, 1961) ; however, in the intact animal little change in coronary flow is usually the case (Bing, 1959; Gorlin, 1962b). Since monoamine oxidase inhibitors will prevent the destruction of serotonin, which is a benign coronary vasodilator (Crumpton et al., 1959), the possibility of an indirect action might be considered. At the present time there is little evidence to support this view (Calesnick, 1963), and we cannot overlook the clinical results which fail to show an improvement in the basic coronary insufficiency. C. ADRENERGIC-BLOCKING AGENTS The fact that the catecholamines produce a marked increase in cardiac oxygen requirements has been discussed in Sections 11, C, and D. Part of the increase can be attributed to hemodynamic factors, such as increased vigor of contraction, producing a rise in the TTI, and part is due to a direct effect on myocardial metabolism, increasing oxygen consumption over and above that required for the additional work. With a limited coronary reserve as found in coronary insufficiency it is easy to understand why an increase in cardiac oxygen requirement will result in an imbalance between the supply and demand. Raab (1956, 1962) has stressed the importance of the catecholamines in angina but it was only recently that there was clinical evidence to support this hypothesis. In a carefully controlled clinical study Dornhorst and Robinson ( 1962) found that the selective 8-adrenergic-blocking agent, nethalide, increased the exercise tolerance of anginal patientsl; there was an attenuation of the tachycardia due to exercise but the increase in cardiac output or stroke volume was not altered. Studies in animals demonstrated that nethalide blocked or attenuated the positive inotropic or chronotropic effect of cardiac sympathetic stimulation, isoproterenol, or epinephrine; there was blockade of other &actions of the catecholamines

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but no effect on the a-actions (Black and Stephenson, 1962). Nethalide itself has only slight pharmacological effects, i.e., slight bradycardia in animals and man, and a slight myocardial depressant action in animals (Black and Stephenson, 1962 ; Dornhorst and Robinson, 1962). Dichloroisoproterenol (DCI) is another p-adrenergic-blocking agent that selectively blocks the positive inotropic and chronotropic actions, and the increased cardiac oxygen consumption produced by epinephrine, norepinephrine, and isoproterenol, and by cardiac sympathetic stimulation (Moran and Perkins, 1958, 1961; Hashimoto e t al., 1960; Nickerson and Chan, 1961). a-Adrenergic-blocking agents, such as dibenamine phenoxybenzamine, phentolamine, azapetine, or hydergine, did not cause a specific blockade (Hashimoto et d.,1960; Moran and Perkins, 1961; Nickerson and Chan, 1961) of these cardiac effects. D C I produces a prolonged positive inotropic and chronotropic effect which would prevent use in coronary insufficiency (Moran and Perkins, 1958).

D. CORONARY DILATORS OTHERTHANNITRITES A recent compilation of the pharmacological and clinical action of a score of coronary dilators has been prepared by Charlier (1961). Among the most recently developed compounds is dipyridamole, which has been studied extensively in both animals and man. There is no question that dipyridamole is a potent long-acting coronary dilator which increases coronary sinus oxygen saturation in dogs (Bretschneider et al., 1959; Grabner et al., 1959; Hockerts and Bogelmann, 1959; Kadatz, 1959; Jackson, 1961; Soloff e t al., 1962; West et al., 1962a). Changes in cardiac output and work were minimal ; likewise cardiac oxygen consumption and efficiency were not altered (Bretschneider e t al., 1959; Grabner et al., 1959; Kadatz, 1959; West et al., 1962a). On the basis of these findings, dipyridamole can be considered as a benign coronary dilator and should be of value in the treatment of coronary insufficiency. Clinical results in treatment of coronary insufficiency are disappointing, with the more recent studies showing little or no beneficial effect using pain, exercise tolerance, or electrocardiographic stress tests as the end point (Peel e t al., 1961; McGregor, 1962; Soloff e t al., 1962; DeGraff and Lyon, 1963). These studies utilized both the intravenous and oral routes so that poor absorption would not seem to be a factor. Yet coronary sinus catheterization studies in man demonstrated an increase in coronary blood flow and a rise in coronary sinus oxygen content after intravenous injection (Peel et al., 1961; Wendt e t al., 1962). I n fact, Wendt e t al. (1962) suggested that the action of dipyridamole on the coronary circulation of normal man is similar to that of nitroglycerin, namely, increased coronary blood flow, increased cardiac oxygen consumption, and decreased effi-

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ciency (Brachfeld et al., 1959). However, there is one difference, namely, that nitroglycerin consistently reduced cardiac work while the effect of dipyridamole was variable. These data raise the question about the importance of coronary dilatation per se in the treatment of coronary insufficiency even though the dilatation is of a benign nature. Other evidence would also suggest that dipyridamole should have a beneficial effect in coronary insufficiency. Blood flow was increased in dogs with infarcted hearts (Kiese et al., 1960) or with the lumen fixed in size (West et al., 1962a). Further there was a more rapid development of collateral circulation during gradual coronary occlusion in the dog when the animals were chronically treated with dipyridamole (Asada et al., 1962; Vineberg et al., 1962). Using the pitressin stress test in rabbits i t was found that dipyridamole reduced the electrocardiographic abnormalities normally observed (Mutti and Chiari, 1961). Also there was a reduction in the ST segment depression induced by injection of picrotoxin into the lateral ventricle of the rabbit (Varma and Melville, 1962a). Biochemical studies in animals suggest that dipyridamole may have some effect on nucleotide metabolism. The decrease in myocardial ATP levels induced by hypoxia was partially reversed by dipyridamole in intact dogs (Hockerts and Bogelmann, 1959). Using isolated atria it was observed that dipyridamole would permit recovery of the ATP and creatine phosphate levels and a return of contractile activity after digitalis arrest (Siess, 1962). In addition, when arrest was induced by hypoxia or sodium fluoride, dipyridamole caused recovery of the ATP levels (Siess, 1962). Hearts removed from rats pretreated with dipyridamole and subjected to 45 minutes of ischemia showed only an increased concentration of adenosine with no change in the total nucleotide or nucleoside content (Gerlach and Deuticke, 1963). This was probably a result of reduced breakdown of adenosine rather than increased production. It was suggested that the accumulation of adenosine may account for the coronary dilator action. This speculation is interesting in view of the recent hypothesis of Berne (1963) that adenosine is involved in the autoregulation of the coronary circulation.

E. MISCELLANEOUS AGENTS The importance of hypocholesterolemic agents in the treatment of coronary insufficiency is still questionable and many years will be required for a definitive answer. One of the agents which blocks cholesterol synthesis, triparanol, has been reported to improve coronary insufficiency in several patients after only a short period of administration (Hollander et al., 1960). This was accompanied by only minor changes in serum cholesterol so that i t seems unlikely that this improvement was associated

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M.

WINBURY

with an effect on cholesterol metabolism. Another group studied the effects of triparanol on the stress electrocardiogram in cholesterol-fed atherosclerotic rabbits (Tabachnick et al., 1961) ; chronic treatment for 1 to 2 weeks resulted in a marked reduction in the severity of the hypoxia-induced ST segment depression, After withdrawal of the triparanol the ST segment depression gradually reverted to the pretreatment value. This study suggests that triparanol may have an effect on myocardial metabolism since the coronary vessels of these rabbits were severely sclerosed. Reserpine was tested in a series of anginal patients who responded well to nitroglycerin. There was no difference between reserpine and placebo in the daily nitroglycerin requirement or exercise tolerance even though many of the patients had a lower blood pressure and heart rate while on reserpine treatment (Rosenberg and Malach, 1961). Chronic reserpine treatment of atherosclerotic rabbits intensified, rather than reduced, the electrocardiographic response to hypoxemia and produced little change in the hypercholesterolemia (Melville and Varma, 1962). IV. Action of Nitrites

The term “nitrites” may be considered a generic one referring to both nitrites and nitrates. There is still the question as to whether or not organic nitrates must be reduced to the nitrite form in the body for activity. For simplicity in this section, the term nitrite will be used to refer to all organic nitrites or nitrates that have been used for the treatment of coronary disease. These are the most useful drugs for the treatment of coronary insufficiency, with nitroglycerin somewhat of a standard because of its long successful use. There is no question that if the nitrite is absorbed i t will effectively relieve pain and improve the stress electrocardiogram. As Rineler (1962) has said, “Available evidence leads us to conclude that a nitrite is a nitrite is a nitrite.” Although nitroglycerin and other organic nitrites are the most effective agents for the relief of angina, they are not the most effective coronary dilator agents; the properties of the nitrites will be discussed in an attempt to evaluate the possible mechanisms of action. The nitrites are general smooth muscle relaxants acting on the smooth muscle of blood vessels (arteries, arterioles, and veins) and other organ systems. This action is direct (musculotropic) and not dependent on blockade of nerve pathways.

A. CORONARY HEMODYNAMIC ACTION The coronary dilator action of nitroglycerin has been demonstrated by measuring the blood flow rate in both normal animals and normal man. The increase in blood flow was not associated with an increased perfusion

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pressure indicating a reduction in coronary vascular resistance (reviewed by Charlier, 1961). Using angiocardiographic techniques in the intact dog, nitroglycerin and amyl nitrite increased the diameter of the large coronary vessels, and the small vessels were better visualized (Haight et al., 1962). Similar findings were made in anginal patients after nitroglycerin or erythrityl tetranitrate, using oral or parenteral therapy (Likoff et al., 1962). Because the smaller vessels could not be visualized, the authora concluded that one cannot imply that the increase in the caliber of the large arteries was accompanied by an improvement in coronary blood flow. Another group fortuitously studied a patient during an anginal attack and observed poor filling of the right and left coronary arteries; after isosorbide dinitrate there was relief of the angina and good filling of the right coronary artery but the left was still poorly visualized (Gensini et al., 1962). This was interpreted to indicate a spasm during the attack and its relief by the nitrite. When vasopressin was injected into the left coronary artery of the intact dog the radiopaque material did not penetrate the affected artery, indicating vasoconstriction; this was relieved by isosorbide dinitrate (Likoff et al., 1962). Studies in dogs indicate that nitroglycerin is a benign coronary dilator but this is not the case in man. I n the open-chest dog with the hemodynamic parameters permitted to vary ad Zib., injection of nitroglycerin into the coronary artery produced an increase in coronary flow accompanied by an increase in coronary sinus oxygen content, a decrease in A-V O,, and a slight increase in oxygen consumption (Eckstein et al., 1951). When the hemodynamic parameters were controlled, and cardiac work was unchanged, there was no increase in oxygen consumption (Sarnoff et al., 1958b). Others, using a fixed rate of inflow to the coronary artery, found that intracoronary injection of nitroglycerin or pentaerythritol tetranitrate reduced myocardial oxygen consumption as well as vascular resistance, but nutritional blood flow (Rbae clearance; see Section V, 1) was increased (Winbury et al., 1962a). Finally, after intravenous injection of nitroglycerin in the open-chest dog, the cardiac oxygen consumption was invariably reduced, presumably as a result of a reduction in the hemodynamic work load (blood pressure) ; changes in coronary blood flow and A-V O2were variable depending upon the degree of hypotension (Fig. 2) (Winbury and Rubin, 1961). I n normal man both coronary blood flow and cardiac oxygen consumption increased, cardiac efficiency decreased, coronary vascular resistance decreased, and the A-V 0, was unchanged after sublingual nitroglycerin (Fig. 1) (Brachfeld et al., 1959; Gorlin, 1962b). These investigators suggest that the increase in oxygen consumption may be due to uncoupling of oxidative phosphoryla-

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tion; however, one could interpret these data as indicating the existence of an oxygen debt in the normal heart and when more blood was supplied the debt was temporarily satisfied. The experimental evidence in support of either thesis is weak a t the present time (McKeever e t al., 1958; Honig et al., 1960). In the anginal patient, coronary blood flow decreased to the same extent as blood pressure, and coronary resistance was unchanged

FIQ.2. Effect of nitroglycerin (2.5 pg/kg) on coronary circulation and cardiac oxygen consumption. Nitroglycerin was given intravenously; point of injection indicated by vertical line. CBF: coronary blood flow (ml/min); A SAT and CS SAT: arterial and coronary sinus oxygen saturation ( ~ 0 1 %;) BP: blood pressure (mm Hg) : upper line systolic, lower line diastolic; O2CONS: cardiac oxygen consumption (ml/min) . Note decrease in oxygen consumption.

(Fig. 1) (Gorlin et al., 1959a; Gorlin, 1962b). Erythrityl tetranitrate did not alter coronary blood flow or cardiac oxygen consumption in the normal or anginal patient ; however, coronary vascular resistance did decline (Rowe et al., 1961). The reason for the discrepancy between these results and those previously discussed (Brachfeld et al., 1959; Gorlin et al., 1959a; Gorlin, 196213) is difficult to explain. ACTIONS B. GENERALHEMODYNAMIC In recent years many have suggested that the beneficial effect of the nitrites in the relief of angina is extracoronary and could well be associated with a reduction in the work load and oxygen requirement of the heart (Gorlin et al,, 1959a,b; Darby and Aldinger, 1960; Rowe et al., 1961; Darby and Gebel, 1962; Gorlin, 1962b; McGregor, 1962; Calesnick,

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1963). If one were to consider the number of agents that are far more effective coronary dilators than nitroglycerin but are ineffective in the treatment of angina, the above conclusion is inevitable. Further, the evidence of Gorlin et al. (1959a,b) suggests that nitroglycerin does not increase the coronary blood flow in the anginal patient; Rowe found the same to be true for erythrityl tetranitrate (Rowe et al., 1961). Some of the hemodynamic alterations produced by nitroglycerin (and other organic nitrites) are decrease in blood pressure (systolic more than diastolic) (Brachfeld et al., 1959; Gorlin e t al., 1959a; Rowe et al.,1961; Gorlin, 1962b) ; decrease in right and left atrial pressure (Brachfeld et al., 1959; Gorlin et al., 1959a; Rowe et al., 1961; Gorlin, 1962b) ; decrease in cardiac size (Darby and Aldinger, 1960; Darby and Gebel, 1962; Gorlin, 1962b) ; decrease in left ventricular work (Brachfeld et al., 1959; Gorlin et al., 1959a; Rowe et al., 1961; Gorlin, 1962b) ; decline in TTI (Gorlin, 1962b) ; increase in heart rate (Brachfeld et al., 1959; Rowe et al., 1961), but decline in systolic duration (Gorlin, 1962b) and reduction in the rise in blood pressure and pulmonary arterial pressure during exercise (Gorlin, 1962b; Stock et al., 1962). Cardiac output was either unchanged (Brachfeld et al., 1959) or reduced (Gorlin et al., 1959a; Rowe et al., 1961). Many of these changes are interrelated and will reduce TTI which, as indicated in Section 11, C, is the major determinant of cardiac oxygen consumption. Certainly the decrease in blood pressure, diastolic heart size, and systolic duration will decrease TTI and the wall tension of the heart. The classic reports of Brachfeld e t al. (1959) and of Gorlin et al. (1959a, 1962b), which show that nitroglycerin increased coronary blood flow and cardiac oxygen consumption in normal individuals but reduced both parameters in the anginal patient, clearly demonstrated the factors leading to the reduction in cardiac work and TTI. Although the general hemodynamic changes were similar on a qualitative basis in both groups, there are quantitative differences. There was a greater decrease in blood pressure, cardiac output, cardiac work, and TTI, and a smaller rise in heart rate in the anginal patient (Fig. 1).The decline in cardiac efficiency was considerably less in the anginal than normal individual. It is paradoxic that in spite of the decrease in cardiac work and wall tension, oxygen consumption increased or remained unchanged. I n the dog, nitroglycerin given intravenously has positive inotropic and positive chronotropic effects which are reflex in origin, since the effects can be blocked by spinal anesthesia (Darby et al., 1958).Administration of nitroglycerin during infusion of norepinephrine caused a reduction in end diastolic tension and length, presumably as a result of the decline in blood pressure (Darby and Aldinger, 1960). During an anginal attack induced by exercise there was increased heart size which could be reduced

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by nitroglycerin (Darby and Gebel, 1962). This, of course, results in a reduction in wall tension and cardiac oxygen requirement. Nitroglycerin produces an “unsteady state” in the circulation of normal dog and man with an initial increase in cardiac work for about 1 minute followed by a decline (Honig e t al., 1960; PoijB and Rudewald, 1962). Kinetic work (flow) accounted for almost all of the increase; pressure-volume work was relatively unchanged (Honig e t al., 1960). The authors explained the increased cardiac oxygen consumption produced by nitroglycerin in normal man on the basis of the increased cardiac work, but this does not follow, since kinetic work involves very little tension development and therefore requires little additional oxygen consumption. I n these studies (Honig e t al., 1960; Poij6 and Rudewald, 1962) there was an over-all decline in blood pressure a t the time of the increased cardiac output, which should result in a reduction of TTI. Nitroglycerin decreases the degree of rise in blood pressure, cardiac work, and TTI produced by exercise (Gorlin, 1962b). Further, i t can prevent the rise in pulmonary arterial pressure, left atrial pressure, and cardiac size that may occur in anginal patients on exercise (Darby and Gebel, 1962; Stock et al., 1962). It has been concluded that there may be left ventricular failure during angina of effort which is relieved by nitroglycerin. This beneficial effect is probably associated with the reduction in the hemodynamic work load on the heart reducing the wall tension.

C. CARDIAC METABOLISM A comprehensive study of the effects of nitroglycerin on myocardial metabolism in intact dogs by Goto (1962) indicated a general decline in cardiac metabolism with a shift toward carbohydrate utilization. Intramuscular administration of 0.5 gm/kg produced a marked decline in the utilization of oxygen, NEFA, and oleic acid but total carbohydrate utilization was only slightly reduced. Glucose and pyruvate utilization declined, but an increase in lactate utilization compensated for most of the decline. The slight rise in RQ (0.81 to 0.89) agrees with the slight shift toward carbohydrate metabolism. The general decline in metabolism was associated with a decline in arterial pressure but the studies do not permit one to determine how much of the general decline in cardiac metabolism was due to the reduced cardiac work load. A preliminary report by other investigators (McGregor, 1962) suggests that nitroglycerin does influence the uptake of substrates by the myocardium and that this may have some bearing on the manner in which i t relieves angina pectoris. The apparent “oxygen wasting” in normal man is difficult to understand. This has been explained by an uncoupling of phosphorylation and an inhibition of electron transport produced by nitroglycerin in in vitro

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studies (Brachfeld et al., 1959; Honig et al., 1960; Calesnick, 1963). Use of in vitro metabolic data for explanation of an in vivo result is not without pitfalls and a t present it would be best to assume that the oxygen wasting in normal man is unexplained. Organic nitrites and nitrates inhibit adenosine triphosphatme activity in arterial smooth muscle and it was suggested that this may be related to the vasodilator action (Krantz et al., 1951).

D. EFFECT ON CATECHOLAMINES Raab (1956) has stressed the importance of the catecholamines and the sympathetic nervous system in the precipitation of coronary insufficiency. Indeed, during exercise, plasma levels of norepinephrine and epinephrine increased significantly (Gazes et al., 1959; Chidsey et al., 1962). Raab and Lepeschkin (1950) suggested that nitroglycerin has an antiadrenergic effect on the heart and blocks the wasteful increase in oxygen consumption produced by endogenous release of catecholamines. The studies on which this conclusion was based were carried out in atropinized cats (Raab and Lepeschkin, 1950). The tachycardia and T wave depression which follow administration of epinephrine or norepinephrine were blocked by nitroglycerin. Popovich et al. (1956) were unable to confirm these results even though the same procedures and animal species (cat) were used. In addition, nitroglycerin did not prevent the increase in myocardial oxygen consumption induced by catecholamines or cardiac sympathetic nerve stimulation in the cat or dog (Eckstein et al., 1951; Popovich et al., 1956; Winbury et al., 1962a). Likewise, nitroglycerin had no apparent effect on the increase in isometric systolic tension produced by norepinephrine (Darby and Aldinger, 1960). The evidence to the present does not support the thesis that nitroglycerin has a true antiadrenergic effect. It is true that it can counteract the increased hemodynarnic work load due to norepinephrine (Darby and Aldinger, 1960), but this is merely due to an opposing physiological action on blood pressure.

E. COMPARISON OF RESPONSE IN NORMAL AND ANGINAL INDIVIDUAL There is a marked difference in the response of the coronary circulation

to nitroglycerin in the normal human and the anginal patient (Fig. 1).I n

the normal, there was a rise in coronary blood flow, a decline in coronary vascular resistance, and an increase in left ventricular oxygen consumption (Brachfeld et al., 1959; Gorlin, 1962a). On the other hand, in the anginal patient coronary flow declined, coronary resistance was unchanged, and cardiac oxygen consumption declined (Gorlin et ul.,1959a,b; Gorlin, 1962b). Other investigators found that erythrityl tetranitrate pro-

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duced no change in coronary flow or cardiac oxygen consumption in normal or anginal patients, but did decrease coronary resistance (Rowe et al., 1961). The clearance of 1131 in anginal patients following percutaneous injection into the left ventricular apex was not altered by nitroglycerin (Hollander et al., 1963), confirming other reports that coronary flow was not increased (Gorlin e t al., 1959a,b; Rowe et al., 1961; Gorlin, 196213). It can be inferred, from these observations, that the coronary blood flow rate is relatively fixed in the anginal patient (Rowe, 1962). Nitroglycerin was studied in an animal model of coronary insufficiency, the atherosclerotic rabbit, and did not prevent the S T segment depression induced by hypoxia (Winbury et al., 1961; Varma and Melville, 1962a). When isolated perfused rabbit hearts were studied, i t was found that nitroglycerin produced a greater increase in coronary blood flow in normal hearts compared with atherosclerotic (‘coronary insufficient hearts” (Karp et al., 1960; Melville and Varma, 1962). This animal model demonstrates an impaired ability of the atherosclerotic coronary vessels for dilatation.

F. EFFECT ON COLLATERAL CIRCULATION Gradual occlusion of the right coronary artery of the pig produced death in 13 of 15 pigs, Treatment of animals with pentaerythratol tetranitrate before and during the occlusion increased the survival rate significantly (Lumb e t al., 1962; Lumb and Hardy, 1963). Filling of the coronary circulation with a radiopaque mass demonstrated an extensive collateral circulation in the treated animals. A similar improvement in the collateral circulation has been reported for sodium nitrate (Zoll and Norman, 1952). G. CONCLUSION

It appears that the coronary bed of the anginal patient cannot increase the volume flow under the influence of nitrites, Therefore the beneficial effect must be associated with some other change such as a reduction in wall tension of the heart. It is conceivable that there may be a redistribution of blood with more blood going to nutritional vessels and permitting better tissue oxygenation in that way. V. Approaches to laboratory Evaluation of Antianginal Agents

The coronary circulation is a complex integrated system involving many variables which were emphasized in Section 11. An analysis of the influence of drugs on the coronary circulation, of necessity, should consider these variables in order to have a rational basis for predicting possible utility in coronary insufficiency (Katz, 1956). However, the majority of physiological and pharmacological studies have been carried out in

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animals with a normal coronary circulation and the results obtained may not predict the status of the abnormal coronary circulation. Certainly studies in man indicate that the coronary bed of the anginal patient is incapable of dilatation so that nitroglycerin produces no increase in coronary flow (Gorlin et al., 1959a,b; Gorlin, 1962b). Yet, the pharmacologist has been using coronary dilatation in the normal animal as the basis for the evaluation of drugs for coronary insufficiency (Charlier, 1961). It is possible that dilatation may play some part in the action of the nitrites but there are other actions involved, as was mentioned in Section IV. Where does the reduction in the work load of the heart fit in and how can we evaluate agents on a more logical basis? If we could have an animal replicate or procedure that takes into account the abnormal physiology, we would be on firmer ground and perhaps could increase the probability of discovering new agents or approaches to the treatment of angina. Many approaches have been used to investigate the physiology and pharmacology of the coronary circulation and over-all metabolic function of the heart. Some of these have not yet been applied to the study of new drugs and it is the purpose of this section to consider the approaches that can be drawn from the fields of physiology, pharmacology, and biochemistry, Among the topics that will be discussed are: coronary dilatation; total metabolic studies ; measurement of myocardial oxygen tension ; various attempts a t producing experimental coronary insufficiency ; prolongation of activity under hypoxia ; antagonism of catecholamines ; arteriographic techniques ; and coronary capillary circulation.

A. CORONARY DILATOR ACTION The ability of agents to dilate coronary arteries has been determined in a variety of ways, ranging from isolated strips or segments to determination of the rate of coronary blood flow or the caliber of the coronary arteries in situ (Kranta and Ling, 1958; Charlier, 1961). These procedures have all been based on the fact that nitroglycerin is a good coronary dilator and is the drug of choice for the relief of angina (McGregor, 1962). Isolated arterial strips or segments are useful as a tool for understanding the pharmacology of a compound, but because of the complexity of the integrated coronary circulation it would be difficult to predict what can be expected in the intact animal. Furthermore, there is variation among the species in the response to various agents. I n addition, the larger vessels studied may not reflect the response of the arterioles. 1. Isolated Heart The isolated heart procedure originally described by Langendorff (1895) is probably the most frequently used method for evaluating coro-

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nary dilator action. It has the advantage over isolated arterial rings or segments of permitting simultaneous evaluation of the effect of drugs on the coronary circulation and cardiac action. Although there have been many diverse technical modifications of the procedure, the basic principle of the preparation remains unchanged. The coronary arteries are perfused at a constant pressure via the aorta with a balanced salt solution (or blood) and the rate of coronary blood flow is determined as the outflow from the right heart, Drugs under study are added to the perfusion fluid. I n order to study the vasomotor effects on the coronary bed independent of the extravascular component, some have induced fibrillation of the heart (Katz et d.,1938). Although the isolated perfused heart has certain advantages over isolated rings or segments because of the intact coronary bed, it does not provide information on the over-all effect that can be anticipated in the intact animal. Certainly the predictability of what effect might be expected on the coronary circulation in man can be better obtained from the intact animal, where the heart is performing work and the coronary circulation is influenced by the numerous hemodynamic variables that are normally in operation. To my mind the intact animal would better serve for the initial evaluation of drugs, and if one then wants to explore the mechanism of the effect on the coronary circulation, the isolated heart can then be used. It is true that there is a significant correlation between the potencies of a series of compounds determined by the Langendorff procedure and those obtained in the intact dog by intracoronary injection (Winbury et al., 1950). However, the correlation is low, and there are many changes noted in contractile activity of the isolated heart which are not observed in the intact animal. Recent work by Karp et al. (1960) and by Melville and Varma (1962) showed that the response of the isolated perfused heart from atherosclerotic rabbits differs from hearts obtained from normal animals (see Section V, E) , For example there is a qualitative difference in the response to ergonovine which invariably caused coronary constriction in the atherosclerotic heart but no change or dilatation in the normal heart (Karp et al., 1960). A quantitative difference was noted in the response to nitroglycerin with the normal heart showing greater dilatation (Karp et al., 1960; Melville and Varma, 1962). 2. Heart in Situ

It is only in the intact animal that one can determine, simultaneously, the effect of an agent on the coronary circulation and other hemodynamic parameters, Since the ultimate use of an agent will be in the human, i t is critical to assess the hemodynamic actions a t an early stage, because some factor may be undesirable. It would appear that compounds should first

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be evaluated in the intact animal in order to reduce the number of false positives and t o enable early elimination of those agents which have undesirable effects that can be observed only with an intact circulation. When a drug is injected directly into the coronary artery, the situation is similar to that of the isolated perfused heart and, in fact, there is reasonable correlation between both procedures (Winbury et al., 1950). Thus, intracoronary injection in the intact animal with direct measurement of blood flow is as useful and rapid a procedure for assaying coronary dilator action as the isolated heart (Winbury et al., 1950). The added advantage is that the heart is under a relatively intact humoral, nervous, and hemodynamic control so that autoregulation is more likely to occur. I n addition, the direct chronotropic and inotropic actions can be determined. When the drug is injected intravenously, we are closer to the clinical situation for which the compound is intended, and the hemodynamic factors that are integrated in the regulation of coronary flow are in operation. Most agents that produce vasodilatation of the coronary arteries will produce a similar effect in other areas and may lead to an excessive fall in blood pressure. It is the balance between these actions that determines whether or not the blood flow will increase after intravenous injection of a vasodilator agent. Although a moderate decline in blood pressure will in effect reduce coronary perfusion pressure, this may be beneficial because of the reduction in pressure work (see Section 11, C), providing i t is not associated with a marked increase in cardiac output or heart rate. After intravenous injection, nitroglycerin may or may not produce an increase in blood flow depending on the degree of hypotension ; however, coronary vascular resistance is reduced. On the other hand, dipyridamole invariably produces a prolonged increase in blood flow after intravenous injection even when blood pressure is reduced. In view of the fact that coronary dilator agents will produce marked vasodilatation in other vascular areas, the changes in coronary flow and femoral flow produced by intravenous injection of 8 series of agents have been determined simultaneously with rotameters. It is of interest that all the compounds, including nitroglycerin, produced a greater percentage and absolute increase in femoral flow than in coronary flow. For example, 2-diethylaminoethyl dicyclohexylcarbamate caused a coronary flow (anterior descending) increase of 29% while femoral flow increased 100% (Winbury and Hambourger, 1953). Under similar circumstances nitroglycerin produced a smaller increase in coronary flow even though there was a similar increase in femoral flow. To determine which of the compounds had more selectivity for the coronary bed, the relationship of change in coronary flow per unit change in femoral blood flow was calculated for several doses of each compound, and the two compounds which showed

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better selectivity for coronary dilatation were evaluated further (Winbury and Hambourger, 1953). These compounds did not alter stroke volume, cardiac output, heart rate, or cardiac work, and did reduce coronary vascular resistance ; but when tested in humans with coronary insufficiency, no beneficial effect was observed (Winbury and Hambourger, 1953). These studies merely serve to emphasize that coronary dilatation without other hemodynamic changes does not indicate whether or not a compound will be effective in man. It may be that the dilatation was malignant (Gregg and Sabiston, 1956; Gorlin, 1962b) but that is unlikely because cardiac work was unchanged. The fact that compounds appear to show some selectivity for the coronary vasculature rather than the peripheral vasculature proves little, since a vasodilator in one bed will generally cause dilatation in another bed and specificity is unlikely. It would appear that the use of such methods for the evaluation of antianginal activity is fraught with danger, and many false positives will result. To emphasize this point further there are the data of Charlier (1961) comparing the ratio of the coronary dilator potency to the peripheral dilator potency for a series of compounds using a common standard for both procedures. The coronary dilator activity was determined in the isolated rabbit heart, and the direct peripheral dilator activity (intra-arterial) in the femoral bed of the intact dog. The index of coronary dilator potencyJfemora1 dilator potency showed many agents superior to nitroglycerin, yet none of the agents is effective in the treatment of coronary insufficiency in man. The ratio indicates the relationship between the direct vasodilator activity for the two vascular areas but does not indicate what might be expected in the intact animal or man following intravenous administration. a. Direct measurement of blood flow. These procedures are usually carried out in anesthetized open-chest animals with the dog being the most frequently used species. The rate of blood flow in the coronary bed can be estimated by measuring the outflow from the coronary sinus or the bypassed right ventricle, or by measuring the inflow to one or more of the coronary arteries. Recently it has been possible t o monitor, continuously, the inflow in the unanesthetized normal animal by means of an electromagnetic flowmeter using a permanently implanted probe on a branch of the left coronary artery (Gregg, 1962a). The original procedure for cannulation of the coronary sinus via the atrial appendage (Morawitz and Zahn, 1912) has been subjected to some criticism because the ratio between sinus outflow and total left coronary inflow can vary under different physiological circumstances (La fontant et al., 1962). I n spite of these objections it appears that the coronary sinus flow is derived almost entirely from the left ventricle (Rayford et al., 1959; Moir et al., 1963) and can serve to indicate directional changes

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in coronary flow rate (Gregg, 1950). However, caution must be observed to avoid increasing the pressure within the coronary sinus by use of too small a cannula or by resistance imposed by a flowmeter, since blood may be diverted directly into the cardiac chambers. A recent study by West et al. (1962a) described the cannulation of the coronary sinus via the external jugular vein and recording outflow with a rotameter. A branch of the left coronary artery was catheterized via the carotid artery under fluoroscopic guidance. This procedure has the advantage of a closed thorax, permitting normal respiration and more physiological hemodynamic conditions. I n spite of these advantages, one wonders about the resistance imposed by the coronary sinus catheter and rotameter, and how these affect sinus outflow. To avoid the problems in accurately determining coronary outflow, Rodbard et al. (1953) described a procedure which permits collection of total coronary outflow in the right ventricle by bypassing systemic venous return. Considerable surgery is involved in this preparation and the right ventricle is performing little work. In many experiments, arterial pressure was a t levels well below 100 mm Hg, which suggests either reduced cardiac function or peripheral dilatation. Direct measurement of the rate of coronary inflow can be effected by a variety of flowmeters (orifice meter, rotameter, bubble flowmeter, electromagnetic flowmeter, thermostromuhr) . It is necessary to cannulate the coronary arteries either externally or internally through the aorta and coronary ostia except for the electromagnetic flowmeter or thermostromuhr which can be placed around the isolated branch of an artery. Using the rotameter for measurement of inflow, a rather reproducible coronary dilator assay was described which measured other hemodynamic variables simultaneously (Winbury et al., 1950). The recent development of small electromagnetic flowmeter probes permits the use of chronic preparations (dogs) in the unanesthetized state (Gregg, 1962a). Thoracotomy alters cardiac function so that hemodynamic changes observed while the thorax and pericardium are open may differ from those in the normal animal (Rushmer, 1961). b. Indirect measurement of blood flow.The nitrous oxide technique permits estimation of the rate of coronary blood flow without thoracotomy and has been used extensively in man and animals. The majority of investigators use the desaturation method of Goodale and Hackel (1953a). The computation of the rate of coronary blood flow is based on the Fick principle and requires measurement of nitrous oxide levels in the arterial and coronary sinus blood after the heart has reached saturation. This necessitates catheterization of the coronary sinus and a systemic artery and withdrawal of blood over a 5-minute period during desaturation of the

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heart. Blood flow is expressed as m1/100 gm left ventricle. This procedure requires no major surgical intervention and minimal anesthesia so that physiological conditions are more normal than when blood flow is measured directly in the thoracotomized animal. The time required for a determination ( 5 minutes) may be a disadvantage when a compound has a short duration of action. Nevertheless, the nitrous oxide method has been a valuable tool in the study of the coronary circulation of man and animals and the effect of drugs thereon. It is the only practical method available for studies in man. 3. Conclusicms Whenever coronary dilator action is studied in animals using either the isolated heart or the heart in situ we are investigating normal coronary arteries and are assuming that similar changes will obtain in the coronary arteries of the anginal patient. This is probably not the case and may partially explain why coronary dilator procedures have been unsuccessful in predicting clinical antianginal activity. The coronary circulation of atherosclerotic animals responds differently than that of normal animals, but further physiological and pharmacological studies are required before we can assume that the response of the atherosclerotic animal is similar to the response of the anginal patient. Another objection to the study of coronary dilator action alone is that the dilatation may be malignant in nature and does not improve the oxygen supply to the myocardium. Thus it is important to evaluate the effects on myocardial oxygen consumption simultaneously with the effects on coronary circulation. None of the blood flow methods distinguish between nutritional and nonnutritional flow so that there is no index of actual blood delivery to the capillaries. This will be considered in Section V, I. B. TOTAL METABOLIC APPROACH Flow measurements alone or even in conjunction with hemodynamic factors have limited value in the evaluation of antianginal agents unless the effect on myocardial oxygen metabolism is also studied. Any agent that increases myocardial oxygen requirement, either directly or by increasing the work of the heart, will produce coronary vasodilatation which is malignant (Gregg and Sabiston, 1956; Gorlin, 1962b) in nature. This is part of the autoregulation mechanism (Berne, 1963) previously discussed (see Section 11). 1. Arteriovenous Oxygen Difference (A-V 0,)

If the coronary arteries are capable of normal vasodilatation (adequate coronary reserve) an agent which would increase coronary blood

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flow in excess of that required would be useful even if oxygen demand were increased, Such an agent would decrease A-V 0,. I n fact, on a theoretical basis, any agent that would diminish the A-V 0, should be useful since the oxygen supply would be in excess of the demand. Such a reduction could result either from an increase in blood flow or a reduction in the demand or a combination of both. However, measuring of A-V 0, alone could result in many false positives and actually missing potentially effective agents. For example, a metabolic poison such as cyanide will prevent tissue utilization of oxygen (demand reduced) and thereby diminish A-V O2 (Neil1 et al., 1963b). Also, a n agent that would increase non-nutritional blood flow (arteriovenous shunts) would have the same effect because the venous blood in the coronary sinus would be diluted with shunted arterial blood. Although intracoronary injection of nitroglycerin diminishes A-V 0, (Sarnoff et al., 1958b; Winbury et al., 1962a), there is little or no change after intravenous injection because coronary blood flow is actually reduced (Fig. 2). A compound which reduces the A-V 0, in animals because of an increase in coronary blood flow may not have the same effect in the anginal patient since the coronary reserve is limited, I n an attempt to take this factor into account we have perfused the circumflex and descending branches of the coronary artery a t a constant rate and have recorded arterial and coronary sinus oxygen saturation continuously with an oximeter, in addition to the perfusion pressure (Winbury et d.,1962a). Thus the A-V O2 can only be influenced by changes in demand and actually reflects changes in left ventricular oxygen consumption. Using such a procedure, it was found that intracoronary injection of norepinephrine and epinephrine increased A-V 0, and nitroglycerin and pentaerythritol tetranitrate diminished A-V 0, (Winbury et al., 1962a). 2. Oxygen Consumption of the Heart in Situ

Simultaneous evaluation of changes in coronary blood flow and myocardial oxygen consumption permits a more rational analysis of the mechanisms involved in the changes in coronary flow. Such an analysis is a necessary requisite before a compound should be considered for evaluation in man (Katz, 1956; Calesnick, 1963). Myocardial oxygen consumption is computed from the arteriovenous oxygen difference times the rate of coronary flow [A-V 0, ( ~ 0 1 % )X coronary flow rate (ml/min)]. To reduce the oxygen consumption to basal terms, the weight of the area perfused by the coronary artery under study .should be known and the value expressed as cm8 O,/lOO gm.This is not important for the evaluation of drugs, since the oxygen consumption after drug is usually compared with the untreated state in the same animal.

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Most investigators have determined the oxygen content of the coronary sinus and arterial blood a t intervals before and after drug administration using conventional volumetric or manometric techniques. It is critical that the coronary flow rate used in the computations correspond temporally to the time the samples were drawn from the coronary sinus and artery. The development of the oxygen electrode and oximeter has made possible continuous recording of the oxygen tension or saturation of blood so that myocardial oxygen consumption can be determined almost continuously. It will be seen that this has certain advantages since compounds can have a biphasic effect which would not be uncovered unless continuous recording were possible. Nevertheless, measurement of myocardial oxygen consumption, in conjunction with other factors that affect coronary blood flow, has provided important information about the physiology and pharmacology of the coronary circulation in animals and man. Various experimental designs have been used in animals depending upon the objective of the study. The major differences are (1) the manner in which the coronary arteries are supplied with blood-autoperfusion at arterial pressure (Popovich e t al., 1956; Schreiner et al., 1957; Berne, 1958; Crumpton e t al., 1959; Scott and Balourdas, 1959; West et al., 1962a), or constant pressure (Eckstein et al., 1951 ; Berne, 1958), or constant flow (Winbury et al., 1962a) ; (2) cardiac action-fibrillation (Berne, 1958; Beuren e t al., 1958; McKeever e t al., 1958), arrest (Beuren et al., 1958; McKeever et al., 1958), or beating heart; (3) regulation of hemodynamic parameters (Sarnoff e t al., 1958b) (aortic pressure, heart rate, and cardiac output) or permissive variation; and (4) route of injection-intracoronary or intravenous. Blood flow can be measured by direct methods or by the nitrous oxide procedure (Crumpton e t al., 1959; Scott and Balourdas, 1959; Wendt et al., 1962). Basic to the computation of the oxygen consumption of the left ventricle is the assumption that the coronary sinus represents venous blood primarily from the area supplied by the left coronary artery (left ventricle) with only a small amount from the right coronary. A recent study in which left coronary inflow and coronary sinus outflow were measured simultaneously in the open-chest dog under different physiological circumstances demonstrated that the sinus flow was derived almost entirely from left coronary inflow (Rayford et al., 1959). These data suggest that a reasonably accurate value for left ventricular oxygen consumption can be obtained from measurement of left coronary inflow and the arteriocoronary sinus oxygen difference. Another group measured separately the flow and oxygen content in the coronary sinus and that entering the empty right ventricle using a systemic venous return bypass technique (Rodbard

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e t al., 1953). The oxygen content of the two drainage systems differed significantly but the coronary sinus represented blood draining primarily from the left ventricular musculature (Lafontant et al., 1962). By direct injection of I131-albumin into the septal branch of the left coronary artery it was estimated that 80% of the septal artery outflow drains directly into the right ventricle (Moir et al., 1963) ; this accounts for 12.8% of the total left coronary inflow. This study confirmed previous work indicating that the coronary sinus receives venous blood almost entirely from the left ventricle. If the venous blood from the septal area differs markedly from that of the area supplied by the circumflex and descending branches, an error can be introduced into the value for left ventricular oxygen consumption (Moir et al., 1963). However, if only the circumflex and descending branches are perfused directly, this potential inaccuracy can be avoided since the coronary sinus primarily drains that inflow. Intracoronary injection of agents shows the direct action on the heart but compounds may alter myocardial oxygen consumption by extracardiac mechanisms such as a reduction in aortic pressure. Nevertheless, it is of interest to determine the direct action during evaluation of a compound. Of greater importance are the effects following intravenous injection. Some of our experimental results will be used to illustrate various points. These studies were carried out in anesthetized dogs under artificial respiration. A left thoracotomy was performed and the circumflex and/or descending branch of the left coronary artery cannulated. The inflow to the artery was supplied by the carotid artery and a rotameter was interposed into the circuit to measure the blood flow rate. Blood pressure was determined from a side arm connected to the carotid artery. A small catheter was placed to the coronary sinus via the right atrium or the jugular vein and samples were drawn continuously through an oximeter a t a constant rate and returned to the animal via the contralateral jugular vein. Arterial oxygen saturation was recorded continuously in the same fashion using a bypass in the carotid or femoral artery. Oxygen consumption was computed a t 10-second intervals from the records. All the drugs were injected intravenously. Norepinephrine and epinephrine invariably increased myocardial oxygen consumption and coronary flow but did not alter A-V 0,. The experiment in Fig. 3 is of particular interest because blood pressure did not increase, yet coronary flow and oxygen consumption were elevated. Angiotensin produced a marked and prolonged increase in oxygen consumption, coronary blood flow, and blood pressure; the A-V 0, was diminished (Fig. 4). This illustrates the problems of using A-V 0, alone. Histamine and acetylcholine showed a biphasic effect on oxygen consump-

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FIG.3. Effect of norepinephrine ( 1 pglkg) on coronary circulation and cardiac oxygen consumption. Norepinephrine was given intravenously. Note increase in cardiac oxygen consumption. Remainder of legend as in Fig. 2.

tion which might not have been observed if continuous recording of A-V 0, were not possible (Fig. 5 ) . The elevated oxygen consumption occurred a t the time when the A-V 0, was markedly diminished. Nitroglycerin invariably diminished oxygen consuinption and blood pressure (Fig. 2).

FIG.4. Effect of angiotensin ( 1 &kg) on coronary circulation and cardiac oxygen consumption. Angiotensin waa given intravenously. Note reduction in A-V 0,but increase in oxygen consumption. Remainder of legend aa in Fig. 2.

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FIQ.5. Effect of histamine (2 pg/kg) on coronary circulation and cardiac oxygen consumption. Histamine was given intravenously. Note biphasic change in oxygen consumption. Over-all change was an increase. Remainder of legend aa in Fig. 2.

Coronary flow usually decreased or remained unchanged and A-V O2 changed only slightly. Dipyridamole increased oxygen consumption slightly, increased coronary blood flow markedly, and narrowed A-V 02. 3. Conclusion Myocardial oxygen consumption furnishes an important parameter required in the evaluation of any antianginal agent; the A-V O2alone is not adequate. Continuous recording of saturation of arterial and sinus blood, which is possible in animals, has certain advantages in that rapid changes in oxygen consumption can be observed. These may be limiting to the use of the compound. If coronary dilatation cannot occur in the anginal patient any increase in oxygen consumption, even if for a short period, would be undesirable. The method might be improved by perfusing blood into the coronary artery a t a constant rate, thereby tending to resemble the anginal situation.

C. MEASUREMENT OF MYOCARDIAL OXYGENTENSION 1. Method In view of the fact that coronary insufllciency is believed to result from an imbalance between the oxygen supply and demand a t the tissue tension) may be an level, the determination of tissue oxygen tension (02 important approach to the evaluation of antianginal drugs. The 0, tension of the tissues (muscle cells) is dependent upon the rate of arterial blood flow (actually capillary flow), the O2tension of the plasma and the

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rate of diffusion of 0, from the plasma to the muscle cells. Diffusion rate is related to the concentration gradient between the cells and the extracellular fluid. Blood flow really gives an indication of the rate of oxygen availability per unit time (blood flow in ml/min X 0, content of arterial blood in ~ 0 1 % ) . The use of the polarographic method for the study of myocardial oxygen tension started with the work of Sayen et al. (1951). Since that time numerous reports have been published by that group, but we will only consider those related to the present discussion of the technique-the advantages and limitations. Oxygen tension is measured with an open-tip platinum electrode which is inserted to a depth of about 2 mm into the myocardium of an openchest dog. Since the electrodes are small, 0.4 to 0.6 mm in diameter, several can be placed in the heart a t one time. A limb is usually placed in a saline bath which is connected to a calomel electrode by a salt bridge thereby completing the circuit. Electrolysis is carried out a t 0.6 v and the relative oxygen tension is read from a sensitive galvanometer or recorded continuously. This is the basic procedure used by most investigators. Occlusion of a branch of a coronary artery results in an immediate reduction in the 0, tension of the dependent area (Sayen et al., 1951,1958, 1960; Miyashita, 1962). The electrocardiographic changes (ST segment elevation) in the ischemic area appear considerably later than the reduction in 0, tension (Sayen et al., 1958; Miyashita, 1962). When the animal breathes pure oxygen there is a marked increase in the 0, tension of the normal area but little change in the central zone of the ischemic area, and the electrocardiographic change is not altered (Sayen et al., 1951, 1958, 1960; Miyashita, 1962). Similarly, norepinephrine intravenously produces a rise in the 0, tension of the normal tissue with no change in the ischemic zone (Sayen et ul., 1952, 1960; Miyashita, 1962). Breathing 10% oxygen lowers 0, tension in the nonoccluded areas (Sayen e t aZ., 1960). After partial occlusion there is a decline in 0, tension but the area still shows a response to 10 and 100% oxygen inhalation or norepinephrine (Sayen et al., 1960). A recent study with norepinephrine is of particular interest in that it showed a distinction between the changes in oxygen tension and the electrocardiogram (Sayen e t uZ., 1960). I n the normal areas there was ST segment displacement, increased contractility, and a concomitant increase in O2tension. I n severely ischemic areas (central zone of occluded area), norepinephrine produced some alteration of the electrocardiogram (partial reversal of ischemic pattern), but 0,tension remained a t low levels and contractions were not altered. The authors concluded that the ST segment deviations due to norepinephrine do not indicate ischemia but are closely associated with the positive inotropic effects and an increased

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oxygen supply/demand ratio (Sayen et aZ., 1960). These results can be interpreted differently because of the limitations of the technique. The electrodes do not really reflect what is occurring a t a cellular level but rather indicate oxygen delivery to the tissues by the blood. Norepinephrine increases myocardial oxygen consumption, and it may well be that cellular hypoxia develops because of some metabolic change in spite of the plethora of oxygen. The electrocardiographic changes may be associated with cellular hypoxia and loss of electrolytes. We have carried out studies with the polarographic technique over several years in an attempt to determine if this would be a more rational approach to evaluation of antianginal agents. Theoretically, the electrodes show changes in the supply/demand ratio to the tissues, and this seems to be the case. Unfortunately, when the blood flow is permitted to change, the electrodes reflect primarily the changes in the supply and give no indication of changes in myocardial metabolism. We were able to duplicate the previously discussed effects on 0, tension of occlusion of an artery, of breathing 10 or 100% oxygen, or of injection of norepinephrine in both the dog and miniature pig. Nitroglycerin given intravenously reduced the 0, tension of the left ventricle but when given directly into the coronary artery, 0, tension rose. Direct measurement of coronary blood flow rate demonstrated a close correlation between blood flow and tissue 0, tension when flow was altered manually (screw clamp) or by drugs, indicating that when blood flow can vary, the 0, tension is primarily a reflection of tissue availability. When the animal was allowed to breathe 100% oxygen instead of room air, there was a latent period of 10 to 20 seconds before the 0, tension increased. It was felt that nitroglycerin might shorten this latent period; however, intravenous administration did not alter the delay in normal areas or areas made slightly ischemic by partial occlusion of a coronary artery. Another approach was based on the premise that nitroglycerin may promote increased collateral blood flow and thereby improve the 0, supply to a compromised area. To perform these studies, the 0,tension of a small area was reduced to about 50% of the control value by partial occlusion of the artery, and nitroglycerin was administered intravenously. The 0, tension fell in the ischemic area as well as in the normal area. Another group (Honig et aZ., 1960), using a permanently implanted oxygen electrode, studied the effect of nitroglycerin in intact dogs and found an increased 0, tension which was interpreted to indicate an increased blood flow. It would appear this technique could be a valuable tool if 0, availability was maintained a t a constant level. I n this way, the tissue 0,

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tension would be dependent upon changes in demand. This could be approached by perfusing the coronary arteries a t a constant rate, which would keep 0, availability constant. Then the changes in O2tension would reflect changes in tissue utilization. Some preliminary attempts were carried out, but because of the use of anticoagulants there was bleeding a t the site of electrode insertion so that the values for tissue 0, tension were incorrect. However, if this aspect of the problem could be solved, such an approach might be a valuable tool for evaluation of antianginal agents. 2. Conclusion

The polarographic technique for measuring tissue 0, tension by means of oxygen electrodes really measures availability of oxygen which is dependent upon blood flow and plasma 0, content. It does not reflect what is occurring within the cell. Intracellular recordings might be very valuable but such techniques have yet to be developed. If the rate of coronary blood flow were constant, the O2 tension of the tissues would be altered only by changes in oxygen requirements. This might be an approach to the study of oxygen consumption a t a tissue level.

D. EXPERIMENTAL CORONARY INSUFFICIENCY INDUCED BY CORONARY OCCLUSION OR DRUGS It may well be that the failure in transposing the laboratory pharmacology on the coronary circulation to the clinic is due to the fact that the pharmacological studies have been carried out in animals with a normal coronary circulation, which differs considerably from the coronary circulation in the patient with coronary insufficiency. Experimental coronary insufficiency can be induced in animals in a number of ways but there is always the question as to how closely the experimental procedure resembles the clinical situation. Several of the procedures have used the electrocardiogram as the basis of evaluation; the changes induced frequently resemble those that occur during an acute anginal episode in man. The similarity of the electrocardiographic changes does not indicate that the same fundamental mechanism obtains, but a t least the models have an abnormality of the coronary circulation somewhat akin to that in man. The approaches that will be considered are coronary occlusion, vasopressin-induced spasm, ergotoxine-induced ST segment alteration, and insufficiency induced by atherosclerosis. 1. Coronary Occlusion

Abrupt total occlusion of a branch of the left or right coronary artery of the dog, cat, or pig results in a marked ST segment elevation in the

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surface electrocardiogram from the ischemic area and an ST segment depression in limb leads (DiPalma, 1961; Raab et al., 1962; Varma and Melville, 1962a; Winbury et al., 1962b). Similar changes have been observed even after partial occlusion (reduction in diameter of 50 to 75% of normal) of the descending branch in the pig (Winbury et al., 1962b). I n the dog, a reduction of blood flow of 35 to 70% in the descending branch is required to produce slight ST segment changes; with a greater reduction in flow the ST segment elevation (surface lead) is marked (WQgria et al., 1949). a. Epicardial electrocardiographic recordings. DiPalma (1961) used the rate of development of the ischemic electrocardiogram after occlusion of a coronary artery for the evaluation of the coronary circulation. The rate at which the ischemic changes appear is assumed to depend mainly on the collateral circulation available to the compromised area and the state of cardiac metabolism. A marginal branch of the right coronary artery was occluded and the time required for the maximal degree of elevation of the ST segment in the ischemic area was determined (ischemia time) ; recovery time after release of occlusion was also determined. It was observed that the ischemia time varied inversely with blood pressure and heart rate, whereas recovery time varied directly with these parameters. Thus when the work of the heart was increased (increased heart rate or blood pressure), ischemia developed more rapidly and recovery took longer. For example, doses of norepinephrine which increased heart rate and blood pressure shortened ischemia time and lengthened recovery time. Hexamethonium prolonged ischemia time and did not alter recovery time. It was concluded that hexamethonium reduced the work of the heart relatively more in proportion to the reduction in coronary flow. I n order to take into account the effect of a drug on the work of the heart and be able to evaluate the supply/demand ratio, the author used the ischemia index which is ischemia time X blood pressure X heart rate. A change in the index should indicate a change in the balance between coronary blood flow and cardiac muscle metabolism. Sympathomimetic amines (norepinephrine, metaraminol, and methoxamine) did not significantly affect the ischemia index or the recovery index; likewise, angiotensin I1 and sodium nitrite did not affect the indexes. This method might provide a new approach for the evaluation of antianginal agents but I wonder if the ischemia time or ischemia index is more significant, because we may well be seeking a compound which reduces the work of the heart. This would prolong ischemia time but may not change the ischemia index. It would be of interest to study ischemia time and index a t a fixed coronary blood flow rate, which is similar to the situation in the anginal patient.

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We have used a somewhat different approach in the dog and determined the degree of reduction in coronary blood flow required to induce epicardial ST segment elevation before and after a drug. There is considerable variability within an animal over a period of time so that it is difficult to determine if a compound produces a significant effect. Perhaps if the changes in blood pressure and heart rate were taken into account, and an index similar to that proposed by DiPalma (1961) calculated, the variability would be reduced; the ischemia index has been found to be much less variable than the ischemia time. Varma and Melville (1962a) noted that the ST segment alteration was variable during the first hour after total occlusion of the anterior descending branch in the dog, but the pattern remained more consistent for the following 2 to 3 hours. Hypoxemia (breathing 10% oxygen) produced a further depression of the ST segment (lead 11). Intravenous administration of nitroglycerin, trolnitrate, aminophylline, papaverine, dipyridamole, or N - [ 3'-phenyl propyl- ('2') ] -1,l-diphenyl propyl- (3)amine (Segontin) after 1 hour of coronary occlusion enhanced the ST segment depression whether the animal was breathing room air or 10% oxygen. Thus the ST segment deviation produced by coronary occlusion was not favorably modified by coronary dilator drugs including nitroglycerin, indicating that this procedure would not be suitable for evaluation of antianginal agents. Using vagotomized cats, Raab et al. (1962) occluded the anterior descending branch to a point just short of producing ST segment displacement of the surface electrocardiogram. General hypoxia (nitrogen breathing) for 2 minutes produced maximal ST segment elevation in the animals with a restricted coronary flow, but no change in animals with normal coronary flow. Similar effects were produced by stimulation of the cardiac sympathetic nerves, intravenous injection of norepinephrine, or epinephrine, or by stimulation of the muscles of the hindlimb only when there was coronary restriction. There were no studies on nitrites or other antianginal agents since the primary objective was to demonstrate the importance of the catecholamines in the development of the electrocardiographic changes. The procedures described would be worthwhile to investigate with antianginal agents and adrenergic-blocking agents, but until this is done we must withhold judgment on the relative merits of this approach, particularly in view of the negative results with nitroglycerin following coronary occlusion and hypoxemia in the dog, which were previously discussed (Varma and Melville, 1962a). b. Chronic occlusion. Acute partial or total occlusion of a major branch of the left coronary artery of the anesthetized pig frequently results in ventricular fibrillation within 1 hour (Winbury et al., 1962b).

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Electrical defibrillation procedures were successful in a high percentage of the cases and the animals recovered after closing the chest and proper postsurgical care. I n our experiments, anesthetized dogs are not as vulnerable to ventricular fibrillation following abrupt total occlusion, and partial occlusion of the anterior descending branch has no effect. These results are at variance with those of Harris (1950), who reported a high incidence of fibrillation following abrupt total occlusion of the descending branch; partial occlusion 30 minutes prior to the total occlusion prevented the fibrillation. Regelson and co-workers (1959) studied the effect of iproniazid on the survival of conscious dogs following acute coronary occlusion. One week prior to the experiment a loose ligature was placed around the anterior descending coronary artery. Occlusion was produced by tightening the ends of the ligature that had been passed through the chest wall. I n the control group, 67% died within 1 hour after occlusion due to ventricular fibrillation, but in the group treated with iproniazid daily for 4 days prior to the occlusion only 21% succumbed to ventricular fibrillation. This effect does not appear to be specific since pentobarbital anesthesia, morphine, reserpine, hexamethonium, and chlorpromazine also reduced the incidence of fibrillation. In all probability the protective effects involve some form of autonomic blockade or depression of myocardial excitability, and it is rather unlikely that this type of procedure would be suitable for evaluation of antianginal agents. We have attempted, without much success, to induce electrocardiographic changes by stress in unanesthetized pigs and dogs with a compromised coronary circulation. Pigs subjected to hypoxemia (10% oxygen) for 30 minutes a t various times during the first 2 weeks following acute, partial or total occlusion of the anterior descending coronary artery did not show any specific alteration in the electrocardiogram. Likewise, exercise of pigs or dogs with partial or fotal occlusion did not produce diagnostic electrocardiographic changes. Further, the exercise tolerance was not significantly reduced. Asada et al. (1962) carried out some quantitative studies on the effect of hypoxemia in dogs with progressively developing coronary occulsion. A gelatin sponge saturated with dicetyl phosphate was placed around the anterior descending branch; gradual narrowing of the lumen occurred over 4 to 6 weeks and was maintained for several months to 1 year. Hypoxemia tests under pentothal anesthesia were used to evaluate the degree of coronary insdciency. The oxygen content of the inspired air was gradually lowered until some positive electrocardiographic findings appeared. At this point the oxygen saturation of the arterial blood was determined. Any of the following was considered a positive response: (1) ST segment dis-

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placement of more than 0.1 mv; (2) T flattening of more than 0.15 mv, or (3) abnormal rhythm. The oxygen saturation a t the point of a positive electrocardiographic response was 71% for dogs with coronary occlusion compared with 49% for normal dogs. Animals treated with dipyridamole for 30 to 280 days, starting on the day after coronary occlusion, had an arterial saturation of 58% a t the point of a positive response. The authors did not perform any experiments on animals to whom drug was given only after the arterial narrowing was established, This approach to the production of chronic coronary insufficiency appears to offer potential, using the degree of desaturation of the arterial blood required for positive electrocardiographic changes as the end point. Determination of the effect of drugs on an acute or chronic basis after the arterial lumen has been reduced would have similarity to the clinical situation. Theoretically, an increase in the tolerance to hypoxemia could be effected either by a hemodynamic or metabolic change or a combination of both. 2. Electrocardiographic Changes Produced by Pituitrin Posterior pituitary extract (pituitrin) and vasopressin produce coronary constriction in the isolated heart and intact animal (WBgria, 1951; Winbury and Green, 1952; Hashimoto et al., 1960; Karp et al., 1960; Winbury et al., 1962a). Studies on Rbs6uptake by the myocardium suggested a decrease in “effective capillary blood flow” (Love and Burch, 1957a,b ; Winbury et al., 1962a) ,but there appeared to be no change in the oxygen consumption of the isolated fibrillating heart (Hashimoto et al., 1960). Thus pituitrin leads to coronary insufficiency and electrocardiographic alterations because of inadequate blood flow. Lindner and co-workers (1953) described a method for evaluation of agents in the intact dog, giving pituitrin intravenously before and after the drug. The significant changes in the electrocardiogram were an elevation in the amplitude pf the T wave and extrasystoles. Recosen, an extract of fresh heart, which has coronary dilator properties, given before the pituitrin prevented the electrocardiographic changes, A similar procedure was used in anesthetized rabbits for the evaluation of dipyridamole (Mutti and Chiari, 1961) and in rats for the evaluation of monoamine oxidase inhibitors (Cahn and Herold, 1962). Both dipyridamole and isocarboxazid attenuated or prevented the electrocardiographic alterations produced by pituitrin (or vasopressin). This approach seems rather nonspecific, since any agent which is an effective coronary dilator should prevent the coronary constriction induced by pituitrin. Moreover, it is rather unlikely that spasm of the coronary artery is involved in the majority of patients with coronary insufficiency (Section I).

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3. Electrocardiographic Alterations Produced by P k o t o d n Injection into the Lateral Ventricle Varma and Melville (1962a) showed that injection of 0.1 mg of picrotoxin into the lateral cerebral ventricle of the rabbit produced ST segment depression and cardiac irregularities. The animals were anesthetized, vagotomized, curarized, and under artifical respiration. The effecb of picrotoxin usually persisted for 30 to 60 minutes. Nitroglycerin, trolnitrate, and dipyridamole reversed or reduced the ST segment depression, whereas aminophylline and Segontin enhanced the ST segment depression. Iproniazid had no effect on the ST segment depression. This method seems to have some promise on an empirical basis, but it is important to learn more about the hemodynamic changes produced by the injection of picrotoxin into the lateral ventricle before any conclusion can be reached.

E. EXPERIMENTAL CORONARY INSUFFICIENCY INDUCED BY ATHEROSCLEROSIS Atherosclerosis has been studied extensively from the standpoint of endocrinology, nutrition, biochemistry, and pathology. Few physiological studies have been carried out on atherosclerotic animals, and i t would appear a useful means of attempting to duplicate an experimental model of coronary insufficiency. This, of course, would require evidence, other than pathological, that the coronary circulation is compromised. 1. Stress Tests in Atherosclerotic Rabbits

Rinzler et al. (1955, 1956) and Karp et al. (1960) demonstrated that ergonovine maleate produced an ST segment depression in Dutch belted male rabbits on a 2% cholesterol diet. All rabbits with a positive ST segment depression had extensive coronary atherosclerosis. Studies by Stein (1949, 1963) on a large number of anginal patients clearly demonstrated that ergonovine maleate produced significant ST segment depression and pain similar to that observed during an anginal attack or induced during the exercise (Master et al., 1944) or anoxia (Levy et al., 1941) test. The pain and electrocardiographic abnormalities were relieved by sublingual administration of nitroglycerin, indicating that coronary insufficiency was involved. It has been assumed that ergonovine induced constriction of the coronary arteries in these anginal patients. At the time of the original report on the use of ergonovine (Stein, 1949) for diagnosis of angina, we studied the effect of ergonovine on normal dogs, but could find no evidence of coronary constriction (coronary flow) nor any electrocardiographic changes

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(ST segment depression) following intravenous administration of ergonovine to unanesthetized dogs with chronic partial occlusion of the anterior descending branch of the left coronary artery. It was not until the report of Karp and co-workers (1960) that there was experimental evidence suggesting that ergonovine produces coronary constriction. These studies are of particular importance because they demonstrate for the first time a difference in the pharmacological response of the normal heart and the atherosclerotic heart. I n isolated atherosclerotic rabbit hearts perfused by a modified Langendorff procedure, ergonovine frequently produced a decrease in coronary flow (coronary constriction) and did not produce a significant increase in coronary flow. On the other hand, similar doses produced virtually no change or coronary dilatation in normal hearts. Quantitative differences were noted in the response of the normal and atherosclerotic heart to other vasoactive agents. This will be discussed below. The ergonovine test in atherosclerotic rabbits appeared to be an excellent approach for the evaluation of agents for use in coronary insufficiency since the model closely duplicates the situation in the human with angina. Accordingly, investigations were initiated on the use of this technique by Winbury et al. (1961), but i t was found that less than 30% of the atherosclerotic rabbits (Dutch belted) showed a significant electrocardiographic response to ergonovine even after 6 months on the 2% cholesterol diet. Use of Levy e t al. (1941) anoxia test instead of the ergonovine test markedly increased the sensitivity of the procedure so that after approximately 4 months on the 2% cholesterol diet 74% of the rabbits showed a consistent ST segment depression when subjected to anoxia. To perform the hypoxia test the rabbits were lightly narcotized with pentobarbital sodium and a small chamber placed over the head. A mixture of 10% oxygen in nitrogen was passed through the chamber for 10 minutes and the animal then permitted to breathe room air. Electrocardiograms were recorded at, intervals before, during, and after the hypoxia test using lead I1 and a mid-sternal chest lead ( V) . ST segment depression of 0.75 mm (1 mv = 10 mm) was considered a positive response. Normal animals subjected to hypoxemia for 10 to 30 minutes did not show such changes. I n atherosclerotic animals S T segment depression occurred within 1 to 2 minutes after anoxemia was started and disappeared within 1 minute after return to room air. After a positive response to hypoxemia was established continued feeding of the 2% cholesterol diet was not essential. Animals that were returned to the control diet (no added cholesterol) continued to display a positive response to hypoxia for a t least 3 months. However, after this period responses were variable and by 5 months after withdrawal of cholesterol a positive response to hypoxemia was absent in spite of the same coronary pathology as animals continu-

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ously on the diet. All of the animals on the 2% cholesterol diet showed similar pathological changes consisting of marked atherosclerotic involvement of the coronary arteries, aorta, pulmonary arteries, and of the aortic valves and mitral valves. In addition, there was a varying degree of myocardial damage. It is important to note that there was no obvious relationship between the electrocardiographic response to hypoxemia and the pathological changes. Varma and Melville (1962b) were able to duplicate the procedure previously described and affirmed that a positive or negative response in atherosclerotic animals does not appear to be related to the degree of pathology. The sensitivity of the procedure was improved by Tabachnick and co-workers (1961) by using a gas mixture containing 4.5% CO, :89% N, (Coulshed, 1960). Another modification pro6.5% 02: posed was the use of ergonovine maleate 0.05 mg/kg intravenously while the rabbits are breathing 10% oxygen (Varma and Melville, 1962b). With this combined procedure 100% of atherosclerotic rabbits showed a positive response. Reserpine pretreatment intensified the ST depression following hypoxemia (Melville and Varma, 1962). There is no doubt that the hypoxemia procedures (Tabachnick et al., 1961; Winbury et al., 1961; Varma and Melville, 1962b) have many advantages over the ergonovine procedure (Rinzler e t al., 1955, 1956; Karp et al., 1960) because of increased sensitivity, ease of performance, and rapidity of response. Various drugs used in the treatment of coronary insufficiency were evaluated in atherosclerotic rabbits using the hypoxemia test. Nitroglycerin (5 to 40 pg/kg, intravenously), or aminophylline (10 mg/kg, intravenously) were ineffective in relieving or preventing the ST segment depression induced by hypoxemia (Winbury et al., 1961). Other agents found ineffective were trolnitrate (1 to 2 mg/kg, intravenously), papaverine (1 to 2 mg/kg, intravenously), dipyridamole (0.5 mg/kg, intravenously), and Segontin (0.25 to 1 mg/kg, intravenously) (Varma and Melville, 1962a). Thus it can be seen that agents which produce coronary dilatation do not prevent the response of atherosclerotic rabbits to hypoxemia. Likewise, monoamine oxidase inhibitors such as iproniazid or pheniprazine did not prevent the hypoxemia response (Tabachnick et al., 1961; Varma and Melville, 1962a). Concurrent feeding of propylthiouracil with the cholesterol reduced the severity of the hypoxemic ST segment depression (Tabachnick et al., 1961). Of great significance are the results of Tabachnick et al. (1961) on triparanol. Treatment of rabbits with a well-established hypoxemia response with 25 or 50 mg/kg, intraperitoneally, daily for 2 weeks reduced the ST segment depression while simultaneous vehicle controls showed no improvement. When therapy was discontinued, the hypoxemic ST segment depression gradually reverted to the pretreatment pattern. These findings correlate well with the clinical obser-

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vation that this agent prevented the ST segment alteration produced by exercise in the anginal patient (Hollander et aZ., 1960). It can be concluded that although the response of atherosclerotic rabbits to hypoxemia resembles that of the anginal patient, there must be a fundamental difference, since a nitrate such as nitroglycerin which is highly effective in man did not prevent the hypoxemic ST segment depression in atherosclerotic rabbits (Winbury et al., 1961; Varma and Melville, 1962a). Winbury et al. (1961) raised the question about the mechanism of the coronary insufficiency in these rabbits on the basis of the gross and microscopic pathology observed. The fact that there was considerable myocardial damage and valvular incompetence and insufficiency in addition to the coronary atherosclerosis would suggest an additional factor of myocardial failure. When the stress of hypoxemia increases the circulatory load the heart may not be capable of responding sufficiently to maintain homeostasis. Nevertheless, the results with triparonol indicate that some agents can prevent the electrocardiographic changes (Tabachnick et al., 1961). This method may offer an approach to the “in vivo total metabolic” evaluation of agents for the treatment of coronary insufficiency and enable the development of antianginal agents with an entirely unique mechanism of action, primarily based on a change in myocardial metabolism. 2. Response of Atherosclerotic Heart to Vmoactive Agents Pharmacological studies on atherosclerotic hearts have been limited. Certainly the results of Gorlin et al. (1959a) demonstrate that there is a difference in the response of the normal and anginal patient to nitroglycerin. This suggests that studies on atherosclerotic hearts may indicate different pharmacology. Karp et al. (1960) compared isolated hearts from normal and atherosclerotic rabbits using a modified Langendorf technique. The atherosclerotic heart had a higher basal coronary flow rate and a smaller contraction amplitude than normal hearts. Melville and Varma (1962) noted that the atherosclerotic heart was heavier than the normal heart (9.7 versus 6.3 gm) ; when the coronary flow rate was calculated on a unit weight basis (CBF/gm), the normal group was higher than the atherosclerotic group. Qualitative differences in the response of the normal and atherosclerotic heart (constriction) to ergonovine were previously discussed (Section V, E, 1). Nitroglycerin produced a greater percentage increase in coronary flow in the normal than in the atherosclerotic heart, and the maximum flow achieved was greater (Karp et al., 1960). The greater response of the normal heart was also noted after papaverine and aminophylline (Melville and Varma, 1962). However, there was no dif-

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ference in the response to trolnitrate (Melville and Varma, 1962). Likewise vasopressin (0.008-0.8 U) produced a profound decrease in coronary flow in normal and atherosclerotic rabbit hearts which could be reversed by nitroglycerin (Karp e t al., 1960). Cross and Oblath (1962) compared the pressure-flow relationship in normal dogs with dogs made atherosclerotic by a diet containing cholesterol, thiouracil, meat, and lard. For the study of coronary function the heart was placed on a bypass pump-oxygenator system and the pressureflow relationship (P/F) studied under control conditions and during infusion of Pitressin ( 5 U/liter) and norepinephrine (15 pg/liter) in blood infused. Under control conditions the P/F curve wm the same in the atherosclerotic and normal dogs. Pitressin produced a greater reduction in the slope of the normal than the atherosclerotic animals ; norepinephrine produced a greater increase in the slope in the normal. These studies lead to the conclusion that under control conditions the P/F relationship is normal in the atherosclerotic dog heart; however the vessels are less responsive to vasodilator or vasoconstrictor agents. The fact that the atherosclerotic heart may well respond differently than the normal heart has been demonstrated in rabbit, dog, and man, and emphasizes the importance of understanding the physiological basis of the disease. Further studies on atherosclerotic hearts are needed in order to better define the physiological and pharmacological differences, and i t may well be that this is a better preparation for evaluation of antianginal agents than the normal heart.

3. Conclusion-Experimental

Coronary Insufficiency

There are a variety of approaches to the production of coronary insufficiency. These include coronary occlusion, pituitrin vasospasm, and atherosclerosis. The coronary insufficiency produced by means of these procedures is based on alterations in the electrocardiogram which resemble those used for diagnostic purposes in man (ST segment or T wave changes). The model which appears t o show a close similarity to the clinical disease is the atherosclerotic rabbit. Hypoxemia stress tests in such animals produce ST segment depression ; however, prophylactic or therapeutic treatment with nitrites or other coronary vasodilators do not prevent these hypoxemia-induced changes. Nevertheless, this model could be of great value since it permits evaluation of the effect of agents of myocardial 0, metabolism. Hypoxemia reduces O2 availability to the myocardium, producing insufficiency, and an agent which would prevent the electrocardiographic changes would do so by preventing or reducing the insuflkiency.

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This beneficial effect could result from a reduction in the work of the heart or by improvement of the biochemical efficiency for energy production. Another model which warrents further investigation is the dog with progressively developing coronary narrowing as a result of local administration of dicetyl phosphate. These animals showed an increased sensitivity to the electrocardiographic disturbances produced by hypoxemia. This was quantitated by determination of the degree of arterial desaturation required to produce significant electrocardiographic changes. An agent which would permit a greater degree of hypoxemia before such electrocardiographic changes occur might have potential in the treatment of angina. Such an effect could result from a reduction in the cardiac 0, requirement, increased collateral coronary flow, or a cardiac metabolic change. These two models also have the advantage of permitting the chronic use of the animals. Other procedures which seem of interest and should be studied further are (1) determination of time to produce ischemic electrocardiogram after acute coronary occlusion in the dog; (2) anoxia-induced electrocardiographic changes in the cat with partial occlusion of a coronary artery; and (3) electrocardiographic abnormalities produced by injection of ergotoxine into the lateral cerebral ventricle.

F. PROLONGATION OF CONTRACTILE ACTIVITY DURING ANOXIA Anoxia of the isolated atria, papillary muscle, or isolated heart results in a rapid reduction in contractile activity (Winbury, 1956; Setnikar and Ravasi, 1960; Siess, 1962). Reintroduction of 0, permits full recovery of contractile amplitude, providing the period of anoxia was not prolonged and adequate substrate was present (Winbury, 1956; Setnikar and Ravasi, 1960). The biochemical consequences of anoxia have been discussed in detail in Section 11, E and will not be considered here. In any event, an agent which permits a longer duration of contractile activity under anoxia might be considered to have an influence on myocardial metabolism. Isolated rabbit hearts were used to determine the effect of iproniazid on contractile activity under anoxic conditions (Setnikar and Ravasi, 1960). With iproniazid present in the perfusion medium the period of anoxic contractions was increased. Likewise, hearts from animals treated for 7 days wit.h iproniazid were capable of more prolonged activity during anoxia compared with untreated controls. During anoxia of the electrically driven isolated atria from the guinea pig or rat, contractile activity ceases and there is little response to epinephrine or strophanthin (Siess, 1962). Pretreatment with dipyridamole does not prevent the arrest of activity during anoxia but does permit the

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full response to epinephrine and strophanthin. Dipyridamole prevents the normal decline of ATP due to anoxia and it has been assumed that this permits the response to epinephrine or strophanthin. The question arises as to whether or not these methods reflect properties of compounds that might be involved in antianginal action. Certainly we know that iproniazid may relieve the pain but does not improve the basic coronary insufficiency. Furthermore, there is question about the clinical effectiveness of dipyridamole. Until more data have been accumulated on the actions of nitrites and other drugs, no conclusions can be drawn.

0. ANTAGONISM OF EFFECT OF CATECHOLAMINES ON THE HEART The catecholamines increase myocardial oxygen consumption in excess of that required for the increased cardiac work (Section 11, C ) . Thus there is a decline in cardiac efficiency. Not only is there an increase in “active” oxygen consumption but “resting” oxygen consumption is increased as well. The increased oxygen consumption occurs if the catecholamine is released from endogenous stores in the myocardium, sympathetic nerve endings (norepinephrine), the adrenal gland (epinephrine), or is injected intravenously or into the coronary artery. Coronary flow increases in response to sympathetic stimulation or injection of catecholamines. The increase in flow frequently more than compensates for the increased oxygen consumption and oxygen availability to the myocardium rises. This results in an increase in myocardial 0, tension, an increase in coronary sinus 0, saturation, and a decrease in A-V 0,. Under these circumstances the O2supply/demand ratio actually improves and may not indicate what is taking place at the cellular level. When the coronary blood flow cannot rise, the coronary sinus 0, saturation declines and A-V 0, increases in response to catecholamines. The direct action of norepinephrine and epinephrine on the coronary vessels is vasoconstriction which becomes evident when the metabolic actions are blocked. In addition to the anoxiating action of norepinephrine there is a reduction in effective capillary blood flow. Both of these actions would lead to coronary insufficiency when the coronary circulation is compromised and vasodilatation is limited. Raab and co-workers (1950, 1956, 1962) emphasized the role of the catecholamines in angina and postulated that nitroglycerin may antagonize the catecholamine-induced increase in myocardial oxygen consumption. This could not be confirmed by others (Eckstein et al., 1951; Popovich e t d., 1956; Winbury et al., 1962a). Nevertheless, recent clinical studies have demonstrated that a p-adrenergic-blocking agent, nethalide, has a beneficial effect in anginal patients (Dornhorst and Robinson, 1962). This is

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a most important development and lends support to the role of the catecholamines in angina. It seems likely to suppose that p-adrenergic-blocking agents may provide a new and more logical approach to the treatment of coronary insufficiency than coronary dilators. This is particularly significant in view of the suggestion that the coronary bed of the anginal patient is incapable of vasodilatation and the observed rise in blood catecholamine levels in anginal patients during exercise. 1. Adrenergic Blockade

The inotropic and chronotropic actions induced by the catecholamines or by cardiac sympathetic stimulation can be antagonized by p-adrenergic-blocking agents (dichloroisoproterenol or nethalide) but not by aadrenergic-blocking agents (phenoxybenzamine, phentolamine, piperoxan, azapetine, Hydergine, or dibenamine) (Moran and Perkins, 1958, 1961 ; wickerson and Chan, 1961; Black and Stephenson, 1962). The increase in oxygen consumption produced by epinephrine, norepinephrine, and isoproterenol is antagonized by dichloroisoproterenol (Hashimoto et al., 1960). Although there are a number of ways of evaluating p-adrenergicblocking activity, i t would be most logical to use the heart for the initial screen. It is conceivable that a compound may have some greater specificity of action for the heart than another organ system, which would be most desirable (Black and Stephenson, 1962). Blockade of the inotropic or chronotropic action of the catecholamines in vitro or in vivo is a desirable parameter but more to the point is the blockade of the increased oxygen consumption (Hashimoto et al., 1960). I n addition, it would be important to determine if agents will antagonize the reduction in effective capillary flow induced by norepinephrine. 2. Myocardial Necrosis Induced by Isoproterenol or Stress

Rona et al. (1959) and Chappel et al. (1959) demonstrated that administration of large doses of isoproterenol to rats on each of two consecutive days resulted in extensive necrosis of the left ventricle. No doubt this effect is due to the intense and prolonged cardiac stimulation. This procedure was used for the investigation of a large group of compounds of diverse pharmacological action including coronary dilators, monoamine oxidase inhibitors, psychosedatives, and adrenolytics (a) (Zbinden, 1962). The monoamine oxidase inhibitors were the only agents that provided any protection. Raab et al. (1961) presented some evidence that the sympathetic nervous system is involved in the myocardial necrosis produced in rats by stress and fluorocortisol. It was suggested that reflex hypothalamic stimulation leads to general adrenergic discharge and adrenal medullary

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stimulation, causing an increase in plasma catecholamines. The degree of necrosis was reduced by dibenamine, guanethidine, reserpine, or mecamylamine, all of which produce some type of autonomic blockade (Raab et al., 1961).

3. Conclusion Blockade of the actions of the catecholamines on the heart appears to be one of the most promising new approaches to antianginal therapy. New agents which can antagonize p-adrenergic action on the heart (positive inotropic and chronotropic effects, and increased oxygen consumption) should be sought.

H. ARTERIOGRAPHIC TECHNIQUES 1. Method

Coronary arteriography permits visualization of the coronary vascular system in the intact animal. I n addition to providing a means for visualization of narrowing or obstruction of a coronary artery, which is useful in confirming clinical diagnosis, the effect of drugs on the caliber of the coronary arteries can be observed in both man and animals. There are many technical modifications but the basic procedure is the same in all cases, namely, injection of an X-ray contrast medium into the root of the aorta or into selected coronary arteries and taking frequent or serial X-ray photographs after the injection. I n some cases the contrast medium has been injected into the aorta during acetylcholine arrest or temporary occlusion of the aorta by an inflatable balloon and then flushed through the coronaries during the first few contractions; in others the injection into the aorta was given automatically during a specific phase of the cardiac cycle. The most useful injection technique is that of direct injection into specific branches, using the catheterization technique described by West and Guzman (1959). Direct injection of coronary arteries has also been used in man for evaluation of drugs (Likoff et al., 1962). All of the coronary dilator agents studied including nitroglycerin, amyl nitrite, isosorbide dinitrate, erythrityl tetranitrate, dipyridamole, aminophylline, papaverine, and Segontin produced an increase in the caliber of the coronaries of dog or man (West and Guzman, 1959; Gensini et al., 1962; Haight et al., 1962; Likoff et al., 1962; Soloff et al., 1962). The effect of other pharmacologically active agents on the caliber of the coronary arteries, as determined by direct visualization following intracoronary injection, is similar to that demonstrated by direct measurement of the changes in coronary flow rate. For example, isoproterenol, epinephrine,

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norepinephrine, acetylcholine, methacholine, hypoxia, and sodium cyanide increase the caliber of the coronary arteries while Pitressin causes a decrease in the caliber (West and Guzman, 1959). This suggests that coronary arteriographic techniques show the same properties of drugs as demonstrated by coronary blood flow measurements. This is certainly true for normal animals and man; however, there appears to be a dichotomy in the anginal patient, since nitroglycerin increases the caliber of the atherosclerotic vessel (Likoff et al., 1962) but does not increase the rate of coronary blood flow measured by the nitrous oxide technique (Gorlin et al., 1959a). One group of investigators (Likoff et al., 1962) emphasizes that an increase in the caliber of the coronary arteries does not imply an improvement in blood flow. 2. conclusion

Coronary arteriographic techniques permit visualization of the larger vessels under relatively normal conditions. However, it is questionable as to whether or not the results obtained by these techniques have any greater significance than those obtained by measurement of blood flow. The smaller vessels that are involved in the regulation of blood flow cannot be well visualized.

I. USE OF RADIOACTIVE TRACERS With the development of equipment of detection of radioactivity in the blood stream and tissues, a number of new techniques using radioactive tracers have been proposed for the measurement of regional blood flow. The tracers used have included nondiffusible substances such as P'albumin and diffusible materials such as Rbs6, RbS4,K'?, NaZ4,and P i * . External counting techniques for determination of coronary blood flow in humans and animals are being developed but there are still many problems, both theoretical and practical, in their use. Some of t.he procedures depend upon retention of the isotope in the blood, i.e., 1131-albuminor red cells labeled with C P , and estimation of dilution curves. Others depend upon the appearance or disappearance of a diffusible isotope in the heart using the activity time curve as the estimate of flow rate. Since the uptake or removal of a rapidly diffusible substance by an organ is dependent upon plasma flow through capillary beds, the use of Rbs6 and K42 has pcrmitted study of blood flow which serves an actual nutritional purpose in transport of solute from plasma to the tissues. It is such use of isotopes that may provide a new approach to the evaluation of antianginal agents since the nutrition of a tissue is dependent upon effective capillary blood flow (nutritional flow),

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1. NondiffusibleTracers

The indicator-dilution principle has been applied to the estimation of cardiac output for many years and the same concepts have been applied to the measurement of blood flow through various organs. If a nondiffusible indicator is injected into an organ system, a dilution curve will be obtained from the venous blood, the area under which will be dependent upon the amount of indicator injected and the volume of blood flow. This, of course, assumes adequate mixing and that indicator is not lost or added to in the initial passage through the organ. If the blood flow remains constant, injection of a smaller amount of indicator will yield a curve of proportionately smaller area. This principle would apply if the second curve were generated by direct injection of indicator or by indicator having recirculated through the organ as a result of the first injection (Conn, 1962). Thus, blood flow through an organ can be estimated after a single intravenous injection into man or an animal on the basis of the assumption that the amount of indicator passing through the organ on the initial circuit is related to the fraction of cardiac output passing through that organ. Although this principle seems simple enough, there are many practical problems when applied to measurement of coronary blood flow; these have been considered by Conn (1962). For measurement of the coronary flow it has been assumed that the coronary circuit is the shortest circuit between the aorta and the right ventricle, and that the first curve due to recirculation is related to coronary flow. However, it appears that this may not be the case, since blood containing indicator frequently returns to the right ventricle from the superior vena cava simultaneously with that from the coronary sinus. Thus the derived values for coronary flow exceed the true values. I n spite of the problems encountered with the direct sampling procedures, Sevelius and Johnson (1959) described a procedure using external precordial measurement of activity in right heart blood after injection of 1131-albumin. As with the direct sampling technique previously discussed, the coronary blood flow is related to cardiac output as the portion of injected indicator passing through the coronary circuit. The practical and theoretical problems in the application of this procedure to the human are many and considerably more work is required before there can be general acceptance of the technique (Conn, 1962). 2. Diffusible Tracers

a. Theory. The exchange of materials between the capillary blood and the tissues is essentially 8 flow-limited process (Love and Burch,

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1957a, 1959; Renkin, 1959; Conn, 1962). K42and Rbsa have similar kinetic movements and distribution (Love and Burch, 1957a,b; Conn, 1962) and have been used as the tracer substance. I n heart and skeletal muscle, the rate of K exchange between the interstitial fluid (ISF) and intracellular fluid (ICF) is considerably greater than the plasma-ISF exchange rate; therefore, cell membrane transport is eliminated as a rate-limiting factor (Renkin, 1959; Conn, 1962). Further, the amount of K in I C F is so great compared to that in ISF and plasma that an almost infinite sink or Rbss which leaves the capillaries. is provided for the K42 The theoretical aspect of the transport of K42from blood to tissues has been thoroughly analyzed by Renkin (1959), using the gracilis or gastrocnemius muscle of the dog which has been isolated from all nervous and vascular connections and perfused from a reservoir a t various rates with blood from the same animal, A constant arterial concentration of K42 was maintained throughout each flow rate and arterial and venous radioactivity levels were recorded continuously. The venous blood was a t a constant lower level of K42 than the arterial blood indicating constant removal of K4*a t a steady blood flow rate. The extraction ( E ) of the tracer can be computed as follows: E = A - V / A , where A and V represent arterial and venous levels of radioactivity. The product of extraction and blood flow ( Q ) represents the capillary clearance (C) of the tracer: C = QE. Extraction varies inversely with flow rate so that a t very low flows E is almost complete, but as the flow rate increases E declines. Clearance increases with flow rate but not in proportion to it and tends to plateau a t high flow rates. It is beyond the scope of this presentation to discuss the derivation of PS, the permeability-surface area product, which represents the maximal clearance of tracer theoretically attainable a t an infinite flow rate. I n a recent investigation, Laurence et nl. (1963) was able to confirm the inverse relationship between Q and E and the direct relationship between Q and C using a single-slug technique with Rbss. I n this case extraction was computed from the equation E = (total count Rbs6 injected) /(total count Rbs6 recovered). The RbB6 was injected rapidly into the arterial stream and the total RbS8output in the venous blood from the muscle was determined. b. Application to heart. Love and Burch analyzed the dynamics of Rbss uptake by the dog heart (1957a, 1959) and proposed measurement of blood flow on the basis of precordial activity (1957b). An intravenous infusion of RbB8was administered a t a continuously decreasing rate in order to maintain a constant plasma level. Based on the increase in Rbs6

DEVELOPMENT OF ANTIANGINAL DBUGS

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level of the myocardium, it was concluded that the initial clearance (C) was representative of the rate of plasma flow (Love and Burch, 1957a). Infusion of norepinephrine increased C Rbss and Pitressin decreased C RbsE (Love and Burch, 1957a,b; 1959). Extraction declined during the increase in blood flow induced by norepinephrine; likewise E declined as the specific activity of the myocardium increased (Love and Burch, 1959). A recent abstract from this group (Love and O'Meallie, 1963) presented the following conclusions: (1) extraction varied inversely with the rate of coronary flow and myocardial specific activity; (2) the isotope content of the myocardium after a known period of Rb8* infusion is determined by the total blood flow; and (3) C is influenced by differences in myocardial hemodynamic status, cellular exchange rates, and K concentration. The dependence of C on coronary flow was confirmed by another group using RbS4 (Bennish and Bing, 1962). Others have approached the study of tissue blood flow by measurefrom the myocardium ment of the rate of removal of tracer (NaZ4or P) using precordial counting techniques (Madoff and Hollander, 1961; Salisbury, et al., 1962; Hollander et al., 1963). A small sample of tracer was injected into the myocardium and the half-time (T-S)for the decline in specific activity determined. NaZ4and 1131 were removed from the normal heart a t an exponential rate. T-% was increased by coronary occlusion and decreased by a rise in coronary blood flow. Thus the removal of isotope is related to changes in coronary blood flow or, more specifically, was determined in local tissue (capillary) blood flow. The T-1/2 for normal and anginal humans by percutaneous injection of IlS1 into the left ventricular apex or injection into the myocardium during surgery (Hollander et al., 1963). T-1/2 for normal patients averaged about 1.4 minutes compared with 5.6 minutes for the anginal patients; there was a wide range in the anginal patients (0.8 to 13.5 minutes) so that the difference may not be statistically significant. I n addition, there were regional differences in T-Y2 in the anginal patients, suggesting that there are regional differences in the tissue perfusion rate. This was not the case in the normal individual. Exercise in the anginal patient reduced T-Y2 but nitroglycerin did not change the value. The absence of a decline in T-% following nitroglycerin in the anginal patient suggests that the nitrites may not augment myocardial capillary blood flow. This may have been associated with the decline in blood pressure which could have prevented a change in coronary flow even if coronary dilatation were present. There are limitations with all of these clearance procedures for the determination of the total coronary blood flow (Love and Burch, 1957b, 1959; Conn, 1962; Salisbury et al., 1962) ; however, they do give a meas-

70

MARTIN M. WINBURY

ure of effective capillary perfusion which, to my mind, is the more important parameter since this is a n indication of the availability of blood for exchange of materials with the tissues. Because C and E are related to coronary blood flow it would be difficult to analyze the direct action of drugs on effective capillary flow unless blood flow were constant. We elected to utilize the Rbss extraction approach for the study of vasoactive agents (Winbury et d.,1962a) for the following reasons: (1) measurement of total coronary blood flow does not indicate what portion of flow is available for capillary exchange with the tissues and serves a nutritional purpose; (2) an actual increase in nonnutritional flow can divert blood from the tissues and would be undesirable; and (3) the extraction of RbsGby an organ gives some indication of the relationship of effective capillary flow to total flow. A double isotope procedure using RbsE (diffusible) and P1-albumin (nondiffusible) was ut,ilieed in order to avoid the problems involved in collection of total coronary sinus outflow (Fig. 6). Blood was pumped at

To Vena Densitometsr

Collection Well Counter

1~3i-20,000 cpm

Rbs-5,000 cpm I:Rb= 1:0.25 YoRecovered:25% Rbs6 Uplake'75%

FIG.6. Double isotope procedure for estimation of myocardial Rb" extraction.

a constant rate from a carotid artery into the circumflex and anterior descending branches of the left coronary artery. Perfusion pressure was recorded continuously to indicate changes in vascular resistance (VR) . Blood was drawn continuously from the coronary sinus via a small catheter and passed through a well counter for radioactivity measurement and a densitometer for O2 saturation measurement and returned to a jugular vein. Il3l-alburnin and Rbs6 in the same syringe were injected rapidly into the arterial inflow and a sample of the coronary sinus blood was collected a t the peak of radioactivity. Comparison of the ratio of I131:RbsG injected to the same ratio in the sample of sinus blood permits computation of E a8 follows:

DEVELOPMENT OF ANTIANGINAL DRUGS

E=

(F)- (F)

(3

or 1 -

71

(&)(%)

where Ii and Rbi are counts injected and IT and Rbr are counts recovered in the sample of sinus blood. Since the rate of coronary flow is constant, changes in C (effective capillary flow) are the same as those in E. Drugs were administered into the arterial inflow (intracoronary). Rbsa extraction was determined during the height of drug action based on changes in VR and compared with values before and after the effect of the drug. These studies demonstrate that changes in E RbE6are not necessarily related to changes in VR or 0, consumption. Nitrites such as nitroglycerin and pentaerythritol tetranitrate increased E RbEs and decreased VR, suggesting arteriolar dilatation and increased effective capillary flow. Norepinephrine decreased E Rbss even though VR was reduced, suggesting arteriolar dilatation but decreased effective capillary flow. When the rate of coronary blood flow was permitted to change, as during perfusion of the coronary arteries a t a constant pressure, the results were more difficult to analyze. Graded doses of nitroglycerin produced graded increases in coronary blood flow but E Rbsa declined progressively. However, C RbsGincreased in a graded fashion. Under these experimental conditions it is difficult to determine whether or not the increase in C RbE6 produced by nitroglycerin is a result of direct action on the capillary circulation or secondary to the increased blood flow rate. Only if the relationship between coronary flow, E, and C were determined with and without drug present could this question be answered. It is of interest to compare our results using regulated coronary flow with those of Love and Burch (1957a,b, 1959) using unregulated coronary flow. Norepinephrine decreased E Rbsa using either procedure, but when blood flow was permitted to rise, C RbE6increased. When blood flow was constant, C Rbse declined. Although Love and Burch (1959) concluded that norepinephrine did not affect E RbsGother than by changes in total blood flow, it is difficult to reconcile their results with ours, for no change in E Rbss should have been observed a t a constant blood flow. In addition to the ease of analysis of results, there is another reason for evaluating antianginal agents a t a constant blood flow rate, namely, the fact that the blood flow rate in the anginal patient is relatively fixed (Section I). Comparison of effect on E Rbsa and oxygen consumption a t constant coronary flow is of interest. Nitroglycerin and pentaerythritol tetranitrate increase E Rbse but reduce oxygen consumption (Fig. 7) whereas norepinephrine decreases E Rb8" but increases oxygen consumption (Fig. 8 ) .

72

MARTIN M. WINBURY

- 7.2 cor. sinus ' 0 content VOI.

-6.4

Yo

-

'

t

j

Rb'%

.t

Sample

1 N

Flow -68 ml/min

mmHg -5'6 200-

perfusion pressure 100-

0-

Nitroglycerine 4Y

RbS6

Somple

Conlrol yo Rbe6 Uptake = 76 9%

, ,

Post Nitroglycerine o/oRb*6 Uptake=81.4%

30 sec

FIQ.7. Effect of nitroglycerin on coronary vascular resistance, Rb" extraction, and cardiac oxygen consumption. Blood flow into left coronary artery at a constant rate and nitroglycerin injected into flow stream. Rb" and I1a'-albumin injected and 2 content curve is temporally out of sample taken a t arrows. The coronary sinus 0 phase with perfusion pressure curve but arrows indicating injections correspond temporally. Vascular resistance is reflected by perfusion premre. Changes in O2 consumption are the opposite of those in coronary sinus 0,content. Rb" uptake is given at bottom of figure. Note decline in vascular resistance and O2 consumption but increase in E Rbm. Val.% cor. sinus

o2 content

-6.4

VOl..%

mmHa -5.6 perfusion

pressure 100-

-

U-

Flow i68 ml/min Control YORbasUptake = 80.1yo

Post Norepi,%Rbe6 Uptake '71.7%

~ I O ~ C ,

FIQ.8. Effect of norepinephrine on coronary vascular resistance, RbM extraction, and cardiac oxygen consumption. Legend as in Fig. 7. Note decline in vascular resistance and E Rbm but increase in oxygen consumption.

Nitroglycerin given simultaneously with norepinephrine does not prevent the changes in E RbE6 or oxygen consumption due to norepinephrine. 3. Conclusion

The use of diffusible tracers such as RbE6and K42offers a new approach to the evaluation of antianginal agents, since it is possible to study the effects on capillary or nutritional circulation, Measurement of total blood flow does not provide this type of information. There are many physiological aspects of extraction and clearance of RbE6that must be considered in order to interpret the action of drugs. However, by regula-

DEVELOPMENT OF ANTIANQINAL DBUQS

73

tion of some of these factors, i.e., blood flow rate, i t is possible to devise procedures that will be useful. VI. Other Potential Approaches

I n Section V, established procedures and techniques were discussed from the theoretical and practical standpoint. On further reflection about the physiology and biochemistry of the heart (see Section 11) , i t becomes evident that there are other potential avenues of approach. The objective of this section is to present several of these thoughts with the hope that someone will find them s d c i e n t l y logical to warrant exploration in the future.

A. HEMODYNAMIC 1. Heart Rate and Systolic/Diastolic Ratio

Since the greater portion of the coronary inflow occurs during diastole, it should be possible to increase the rate of coronary flow without vasodilatation merely by a prolongation of diastole. Such a mechanism would be effective even when the coronary vessels are incapable of vasodilatation. If the increase in diastolic duration is accompanied by an increase in the total cycle duration (bradycardia) there would be an additional advantage because a reduction in heart rate will decrease myocardial oxygen requirements. Bradycardia due to an increase in systolic duration could have disadvantages in that the systole/diastole ratio would be increased and the TTI/minute might be greater; these changes could lead to a decreased coronary flow and increased oxygen requirement. Thus, the desired feature is a decreased systolic/diastolic ratio with or without bradycardia and without much change in diastolic tension. 2. Autoregulation

Assuming that the coronary blood flow rate is under the basal control of an autoregulatory mechanism (Section 11, C), part of the problem in angina might be associated with a reduction in the sensitivity of the arterioles (or smaller vessels) to the mediator (adenosine?). An agent which has no direct vascular effect, but which would increase the sensitivity of the arterioles to autoregulation should permit a greater than normal increase in coronary flow in response to myocardial hypoxia. I n effect, this means that the basal coronary flow would be within the normal range, but under a stress such as exercise or catecholamines, which leads to tissue hypoxia, there would be an increased perfusion of the capillary beds.

74

MARTIN M. WINBVRY

B. METABOLIC 1. Resting versus Activity Oxygen Consumption

The resting oxygen requirement of the heart in vivo ranges somewhere between 25 and 35% of the total requirement during activity (beating). Catecholamines increase the resting oxygen requirement and the percentage it occupies of the total oxygen requirement. Presumably, the energy released in the resting state is used for the maintenance of cellular integrity, accumulation of ions against a diffusion gradient, and the production of high-energy phosphate and heat. One question that arises is whether or not the high-energy phosphate produced is in excess of that required for the activity period (systole) and may be dissipated in the form of heat. Can any phase of basal metabolism temporarily be suppressed without impairing cellular integrity and contractile function, and can the increase due to norepinephrine be blocked by a /I-adrenergic-blocking agent? I n this way it might be possible to reduce the oxygen requirement of the heart so that hypoxia would not develop under stress. The problem is to find a drug that would specifically affect the noncritical portion of the basal metabolism. Out of the total energy output during activity, heat accounts for 3/4 with mechanical work comprising l/. The heat produced is associated with the mechanical and osmotic work performed, the change in entropy due to energy transformation, and the chemical restitution process. Although most of the heat production is obligatory, one wonders if some could be avoided and thereby reduce energy requirements. If the highenergy phosphate produced in the resting period is in excess of the requirement for activity, is this excess degraded and the energy released as heat without production of useful work? This, of course, is related to the comments made previously about the resting energy production. 2. I n Vivo Study of D P N :DPNH System as Index of Aerobic Metabolism

The DPN:DPNH equilibrium is an indication of the state of cellular oxidative function; when hypoxia occurs the equilibrium shifts toward the reduced form ( D P N H ) . Indirect information about this system can be obtained from the lactate-pyruvate equilibrium, for when aerobic mechanisms are not available for regeneration of D P N (hypoxic conditions), pyruvate is reduced t o lactate. Huckabee (1961) devised the “excess lactate” concept for evaluation of the state of cellular oxidation ; excess lactate production occurs during anaerobic metabolism. Any reduction in the function of the aerobic enzyme systems (hypoxia or cyanide) will lead to excess lactate production.

DEVELOPMENT OF ANTIANGINAL DRUGS

75

Determination of the change in the redox (oxidation-reduction) potential of the lactate-pyruvate system as blood passes through the heart can also be used to determine the state of tissue oxidative function (Gudbjarnason et al., 1962; Stock et al., 1962). With adequate oxygenation, the difference between the redox potential of the coronary sinus blood and arterial blood (A Eh) is positive, indicating active tissue oxidation. During anoxia 4 Eh becomes negative, indicating anaerobic metabolism. In principle, the redox potential gives a measure similar to that of the excess lactate procedure, namely, the balance between aerobic and anaerobic metabolism. These procedures have not been applied to a systematic evaluation of drugs used in the treatment of angina. Such a study might be enlightening and offer a new approach to the evaluation of antianginal drugs. 3. Direct Intracellular Visualization of Oxidation-Reduction Status

Chance et al. (1962) has described a procedure which permits estimation of changes in the DPN:DPNH system of the mitochondria of intact cells by measurement of the emission spectra from an area 20 p in diameter on the surface of an organ. The excitation wavelength was 366 mp and the emission wavelength 472 mp. There was an increase in the intensity of emission in going from an aerobic to anoxic state ( D P N reduction). This procedure has been applied to the kidney and brain and i t was found that anoxia or hydrogen sulfide produced an abrupt increase in fluorescence, indicating an increase in pyridine nucleotide reduction. This procedure should permit study of the intracellular metabolic state of the heart and the effect of drugs thereon. 4. Stress-Adaptation Mechanism

During extreme cardiac work loads, tachycardia, or hypoxia a sudden reduction in myocardial oxygen consumption occurs even though the cardiac work level, tachycardia, or hypoxia is maintained (Kate, 1956; Kate et al., 1955; Laurent et al., 1956). This provides a means by which the heart can adjust to sudden and excessive stress. It has been suggested that the mechanism involves the sudden release of anaerobic energy (Ballinger and Vollenweider, 1962). Does a similar mechanism appear during an acute anginal episode or during the relief thereof by an effective nitrite? If a drug were able to induce such a change i t should prevent or reduce coronary insufficiency induced by stress. Perhaps what is required is an agent that would lower the threshold of stress required to initiate the “stress-adaptation” mechanism.

76

MARTIN M. WINBURY

5. Eficiency Efficiency can be considered from two points: (1) mechanical efficiency, which is in terms of work output in relation to oxygen consumption, and (2) biochemical efficiency, which is in terms of high-energy phosphate produced in relation to oxygen consumed. Since ( 1 ) is a measure of the over-all efficiency of the system it is, in part, determined by (2). It has been estimated that the maximal possible biochemical efficiency for the oxidation of glucose is 39% (Bing and Michal, 1959). This is based on the development of 266 kcal of high-energy phosphate (38 moles X7000 cal/mole) for the 686 kcal of free energy liberated on complete oxidation of a mole of glucose. It has not been possible to determine the level of biochemical efficiency in the intact heart, and the actual value may well be below 39%. Under normal circumstances the biochemical efficiency may be the same in the heart of the normal and anginal patient, but under stress, such as exercise, differences may appear. I n the normal heart, acceleration of metabolism may cause no change or a rise in biochemical efficiency, whereas, in the anginal heart, the ability to accelerate metabolism may be limited, or the biochemical efficiency may decline. This could be a result of a limited capacity of some phase of the metabolic machinery involved in the conversion of substrate to high-energy phosphate. Thus, tissue hypoxia would result even though an adequate oxygen supply were present. Certainly the results in man, which show a rise in over-all mechanical efficiency of the heart in normal individuals during exercise but a decline in anginal patients, are in agreement with such a concept. It is difficult to conceive of an improvement in the mechanical efficiency other than by hemodynamic changes (Section 11,C) or by alterations in the biochemical efficiency. It is true that the calculated mechanical efficiency is increased as the work level rises because the resting oxygen requirement remains relatively constant, and only the activity requirement increases; but this is not a real improvement in energy transfer. However, one does not know if the TTI/Qo, ratio could be improved by coupling a greater amount of the phosphate bond energy to useful work. REFERENCES Alella, A., Williams, F. L., Bolene-Williams, C., and Katz, L. N. (1955). Am. J. Physiol. 183, 570. Asada, S., Chiba, T., Osawa, K., Nakamura, K., and Murakawa, S. (1962). Japan. Circulation J. [English Ed.] 28, 849. Ballard, F. B., Danforth, W. H., Naegle, S., and Bing, R. J. (1960). J . Clin. Invest.

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DEVELOPMENT OF ANTIANGINAL DBUGS

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