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In vivo models of myocardial metabolism during ischemia
Journal of Pharmacological and Toxicological Methods 43 (2000) 133 ± 140
In vivo models of myocardial metabolism during ischemia Application to drug discovery and evaluation William C. Stanley* Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4970, USA Received 1 June 2000; accepted 1 July 2000
Abstract This review examines the in vivo techniques that are available for evaluation of the metabolic effects of agents intended for the treatment of myocardial ischemia. Energy substrate metabolism is complex, and requires simultaneous measurement of a variety of processes in order to obtain a thorough understanding of the biochemical mechanisms underlying any functional response. Small animals (from the mouse to the rabbit) are generally not very useful in the study of cardiac metabolism in vivo because it is not possible to sample the coronary venous drainage and measure the rate of substrate uptake or metabolic efflux. Anesthetized open-chest swine or dog models allow simultaneous serial measurement of myocardial substrate use, and repeated tissue sampling for the activities and contents of key enzymes and metabolites. The swine model is particularly good because pigs, like humans, lack innate collateral vessels, thus one can induce regional myocardial ischemia in the left anterior descending coronary artery and sample the venous effluent from the anterior interventricular vein. In this review the biochemical and physiological methods that can be used in conjunction with this preparation are described. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Angina; Animal models; Cardiac; Coronary artery disease; Heart; Metabolism; Microdialysis; Swine
1. Introduction Coronary artery disease (CAD) induces myocardial ischemia. The definition of myocardial ischemia is somewhat elusive, but can be broadly given as an insufficient blood flow to meet the normal demand for oxygen for the given mechanical power output of the myocardium. Clinically, myocardial ischemia initially presents a failure to meet the demand for myocardial blood flow and oxygen delivery in response to an increase in the demand for contractile work. When the arterial blockage is more severe, ischemia occurs even under resting conditions. Traditional pharmacological treatments for chronic stable myocardial ischemia (clinically referred to as chronic stable angina) are aimed at re-establishing the balance between oxygen delivery to the myocardium and the demand for oxygen by the tissue by (1) increasing the blood flow to the myocardium (e.g. via coronary vasodilation), or (2) decreasing the mechanical power output of the tissue through a reduction in heart rate,
* Tel.: +1-216-368-5585; fax: +1-216-368-3952. E-mail address: [email protected] (W.C. Stanley).
blood pressure, and contractility. Drugs designed for the treatment of stable angina are evaluated and approved by regulatory agencies based on their ability to improve the patient's exercise time without chest pain or classic S-T segment depression on the electrocardiogram during an incremental exercise test. More severe ischemia is caused by an acute myocardial infarction induced by formation of an intra-arterial thrombus, which is treated by rapidly restoring blood flow using thrombolytic therapy or coronary angioplasty. Thus, traditional therapies for CAD and AMI are aimed at better matching coronary flow to the demand for myocardial mechanical power. These hemodynamic and ``plumbing'' approaches to the treatment of ischemic heart disease have proven relatively effective, especially for the treatment of acute myocardial infarction. There are, however, CAD patients with severe symptoms of myocardial ischemia who have undergone revascularization procedures (i.e. angioplasty or coronary by-pass surgery) and are on maximally tolerated medications for stable angina. Additionally, there are AMI patients who die or develop infarcts despite optimal thrombolytic therapy. An adjunctive treatment for ischemia is to manipulate energy metabolism in the ischemic zone in a way that
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optimizes the metabolism of the ischemic cardiac muscle (Lewandowski, 2000; Stanley, Lopaschuk, Hall, & McCormack, 1997). This approach, termed ``metabolic modulation,'' reduces the tissue content and release of anginaproducing noxious stimuli (e.g. K + , H + , and adenosine), and could potentially increase the rate of mechanical power generated for a given rate of myocardial oxygen consumption (Stanley et al., 1997). 2. Rationale for metabolic therapies for CAD Cardiac muscle requires a constant high rate of carbon substrate oxidation to generate the reducing equivalents for the electron transport chain and drive ATP formation by oxidative phosphorylation (Liedtke, 1981; Stanley et al., 1997). ATP synthesis is matched by ATP hydrolysis, which provides the energy to fuel contractile work and Ca2 + reuptake into the sarcoplasmic reticulum for diastolic relaxation (Fig. 1). The oxidation of fatty acids in the mitochondria normally supplies approximately two-thirds of the energy for oxidative phosphorylation, and the remaining one-third is provided by the oxidation of glucose and lactate. Myocardial ischemia results in a profound disruption in myocardial energy metabolism. Reduction in coronary flow ( > 30%) results in an immediate burst of glycolysis, glycogen breakdown, a switch from lactate uptake to lactate production, lactate accumulation in the tissue, a fall in ATP concentration and a decrease in intracellular pH (Stanley et al., 1997). This coincides with severe or complete contractile dysfunction and, if the ischemia is of sufficient severity and duration, myocardial necrosis and infarction. It is important to note that during
myocardial ischemia, there is a reduction, not elimination, of blood flow, and, thus, there is continued oxidative phosphorylation and oxidation of carbon substrates, although at a reduced rate. Paradoxically, studies in pigs (Liedtke, 1981; Liedtke, Hughes, & Neely, 1975) and dogs (McNulty et al., 1996) demonstrate that the primary source of carbon substrate is fatty acids during partial myocardial ischemia (30 ± 60% reduction in coronary flow). Thus, there is lactate production and a fall in pH during ischemia despite a continued oxidation of fatty acids in the mitochondria. An alternative approach for the treatment of myocardial ischemia and post-ischemic reperfusion injury is to prevent or reduce the disruption to cardiomyocyte energy metabolism. Specifically, much effort has been placed on increasing the rate of pyruvate oxidation and thus reducing the accumulation of lactate in the cell and the associated fall in pH. Myocardial ischemia results in profound derangements in myocardial substrate utilization, particularly impaired pyruvate oxidation and an increase in anaerobic glycolysis (Stanley et al., 1997). Post-ischemic reperfused myocardium rapidly returns to a normal rate of oxygen consumption, but has an accelerated rate of anaerobic glycolysis, impaired pyruvate oxidation, and a disproportionately high rate of fatty acid oxidation, which all correspond with a decrease in contractile work (Lopaschuk, Wambolt, & Barr, 1993; Stanley et al., 1997). Pyruvate is oxidized in the mitochondrial matrix by the pyruvate dehydrogenase complex (PDH), which is strongly inhibited by its products, acetyl-CoA and NADH (Fig. 2). PDH is phosphorylated and inhibited by PDH kinase, and is dephosphorylated and activated by PDH phosphatase. PDH kinase is activated by increases in the NADH/NAD + and acetyl-CoA/CoA ratios, and inhibited by pyruvate and the
Fig. 1. Schematic depiction of myocardial metabolism. Abbreviations: GLUT, glucose transporters; G 6-P, glucose 6-phosphate; PDH, pyruvate dehydrogenase; CAC, citric acid cycle; CPT-I, carnitine palmitoyl transferase I.
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Fig. 2. Regulation of pyruvate dehydrogenase (PDH) by phosphorylation and product inhibition.
pyruvate analogue dichloroacetate (DCA) (Fig. 2). Acetyl CoA and NADH are also the products of b-oxidation of fatty acids, thus fatty acid oxidation inhibits pyruvate oxidation (Figs. 1 and 2). Pyruvate oxidation can be increased by partially inhibiting fatty acid oxidation, which removes product inhibition on PDH (Stanley et al., 1997). The rate of fatty acid oxidation in cardiomyocytes is primarily controlled by the activity of carnitine palmitoyl transferase I (CPT I). Pharmacological stimulation of PDH with the PDH kinase inhibitor DCA, or by inhibition of CPT-I (e.g. with oxfenicine or etomoxir) or fatty acid beta-oxidation results in a significant improvement in left ventricular function following ischemia without an increase in myocardial oxygen consumption (Stanley et al., 1997). The enhanced pyruvate oxidation lowers lactate accumulation and efflux from the cell, and helps maintain myocyte pH and ion homeostasis. This would allow more efficient use of ATP for contractile work by myosin ATPases and for relaxation by the sarcoplasmic Ca2 + pump, and result in higher ATP levels. At present, there is interest in developing drugs that will optimize myocardial energy metabolism during and after ischemia (Lewandowski, 2000). The partial fatty acid oxidation inhibitors trimetazidine (Kantor, Lucien, Kozak, & Lopaschuk, 2000) and ranolazine (McCormack, Stanley, & Wolff, 1998) have been used successfully for the treatment of stable angina (Stanley et al., 1997), without eliciting any of the classic anti-ischemic effects of traditional therapies (e.g. decrease heart rate, coronary vasodilation and decreased arterial blood pressure). Adjunctive treatment with infusions of glucose and insulin during an AMI reduce plasma free fatty acid concentration and presumably stimulate pyruvate oxidation, and have been shown to reduce mortality (Diaz et al., 1998; Stanley et al., 1997). Energy substrate metabolism is complex and requires simultaneous
measurement of a variety of processes in order to obtain a thorough understanding of the biochemical mechanisms underlying any functional response. This review will present various in vivo techniques that are useful for evaluating the efficacy and metabolic effects of potential drugs for the treatment of myocardial ischemia. 3. Assessment of myocardial metabolism in vivo The general goal of in vivo studies on a potential antiischemic drug is to determine if the compound is efficacious and to learn about the pharmacodynamics and pharmacokinetics of the agent. Metabolic studies usually require assessment of the cardiovascular effects of the compound of interest, thus, one must make simultaneous measurements of the standard indices of cardiovascular function, such as aortic and left ventricular pressure, regional ventricular wall motion, and coronary or regional myocardial blood flow. 3.1. Use of small animals to study myocardial metabolism in vivo Small animals (e.g. mouse, rat, guinea pig, and rabbit) are generally not very useful in the study of cardiac metabolism in vivo because it is not possible to sample the coronary venous drainage and measure the rate of substrate uptake or metabolite efflux. It is possible to assess glucose uptake by the heart in the intact rat with radiolabeled deoxyglucose (Kraegen et al., 1993), however, the fate of the glucose taken up by the heart cannot be traced. These limitations lead investigators to study the in vitro isolated buffer-perfused rat heart. In this preparation, the venous effluent can be collected, and, thus, the exchange of substances across the heart can be measured.
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With the advent of transgenic mice, there is great interest in measuring metabolism in the mouse heart. Methods have recently been established for measuring metabolism in isolated mouse cardiomyocytes (Schaap, Binas, Danneberg, van der Vusse, & Glatz, 1999) and in the isolated working mouse heart (Belke, Larsen, Lopaschuk, & Severson, 1999). Analysis of substrate concentrations and enzyme activities can be made in rapidly harvested mouse hearts, however, the assessment of substrate flux in the transgenic rodent heart is limited to ex vivo conditions. 3.2. Justification for using swine and dog model In light of the widespread use of the isolated perfused rodent heart in the study of myocardial metabolism, it is important to justify the use of large animals. The main justification is that the in vivo dog or pig heart (Fig. 3) is blood perfused, and has very human-like myocardial blood flow, ventricular mechanics, and rates of metabolic fluxes. Recent quantitative studies in isolated buffer-perfused rodent hearts have demonstrated that there is a high rate of anaerobic glycolysis and low rates of glucose oxidation under well-perfused conditions (Gamble & Lopaschuk, 1994; Jeffrey, Diczku, Sherry, & Malloy, 1995; Lopaschuk et al., 1993). Poizat, Keriel, and Cuchet (1994) noted that the rate of lactate production by the isolated buffer-perfused rat heart produces lactate under condition of moderate contractile load, but not at low workloads, suggesting that the oxygen supply is insufficient in this model even when the PO2 is high. As noted above, the healthy human myocardium is a net lactate consumer (Gertz, Wisneski, Stanley, & Neese, 1988; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski, Gertz, Neese, Gruenke, Morris, & Craig, 1985; Wisneski, Gertz, Neese, & Mayr, 1987; Wisneski, Stanley, Gertz, & Neese, 1990), as are dogs (Mazer et al., 1990) and open-chest swine (Cason et al., 1992; Guth et al., 1990; Mazer et al., 1994; Stanley, Hall, Stone, & Hacker, 1992).
Fig. 3. Diagram of the coronary by-pass circuit in the pig.
The isolated buffer-perfused rodent heart must have extremely high rates of buffer flow in order to maintain sufficient oxygen delivery to support normal oxygen consumption and ventricular power. Due to the absence of hemoglobin, the oxygen content of Krebs buffer equilibrated with 95% oxygen is approximately 2 ml/dl, compared to 18 ml/dl in a healthy human. Myocardial blood flow in vivo is approximately 1 ml g tissue ÿ 1 min ÿ 1, but is roughly nine times this value when the heart is perfused with Krebs buffer in vitro. Thus, oxygen delivery is maintained by supra-physiologic flows. This becomes a factor during ischemia because there is a much higher rate of substrate delivery and a greater washout of noxious end products (lactate and H + ). In addition, openchest swine or dog models allow the simultaneous serial measurement of myocardial substrate utilization and the activities and contents of key enzymes and metabolites (Arai, Pantely, Anselone, Bristow, & Bristow, 1991; Hall, Henderson, Hernandez, & Stanley, 1996). The swine model is particularly good because it has a very humanlike coronary anatomy (i.e. no innate collateral coronary arteries as occurs in the dog), which allows one to induce regional myocardial ischemia in the left anterior descending coronary artery (LAD) and sample the venous effluent from the anterior interventricular vein (Renstrom, Nellis, & Liedtke, 1989). For the purpose of this review, we will focus on the use of large animal models for the assessment of myocardial metabolism in vivo. Metabolic studies in large animals have been performed mainly in anesthetized open-chest dogs and pigs. 3.3. Measurement of substrate uptake and output Assessment of myocardial metabolism generally involves measurement of the uptake by the myocardium of energy substrates (oxygen, free fatty acids, lactate, and glucose), and the release of end products (lactate and carbon dioxide). Often, one is also interested in measuring the release of other compounds by the heart, such as adenosine, norepinephrine, or nitrate and nitrite formed from nitric oxide. Additionally, one can measure the uptake by the heart of the drug candidate itself, though this requires a plasma or blood assay of acceptable precision. The uptake is calculated using the Fick principle (uptake = myocardial blood flow (arterial concentration ÿ venous concentration)). The rate of uptake is usually expressed in mmol g tissue ÿ 1 min ÿ 1. A negative arterial ± venous concentration difference and uptake means that the myocardium is producing the substrate. Sometimes, for technical or financial reasons, it is not possible to measure the rate of myocardial blood flow, so in this case one reports only the arterial ± venous concentration differences (usually expressed as mM). The lack of measurement of myocardial blood flow can severely limit the usefulness of the results, particularly if the compound effects coronary blood flow or the trans-
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mural distribution of flow, or if the degree of ischemia needs to be accurately measured. Arterial blood samples can be drawn from any artery, however, the venous sample must come from a vein that drains the region of interest. This can become an issue when studying myocardial ischemia. In swine, ischemia is usually induced by a reduction in flow to the LAD. The venous blood from the LAD perfusion territory is collected from the anterior interventricular vein. Not all of the blood flowing out of the interventricular vein, however, originates from the LAD. Renstrom et al. (1989) infused indocyanine green dye into the LAD via and extracorporeal perfusion circuit and found that the concentration of green dye in the interventricular vein was 91% of the concentration in the LAD. When the LAD flow was reduce by 60% this value fell to 78%. Thus, there is a modest but significant dilution of the venous effluent from the LAD territory. The main site of error in measuring the uptake of a drug or substrate by the myocardium is usually the measurement error in the arterial ±venous concentration difference. For example, the arterial ± venous difference for glucose is typically 0.10 mM, and the arterial glucose concentration is 5.00 mM, then the percent extraction of arterial glucose is 2%. If the coefficient of variation on the glucose assay (the standard error divided by the mean) is 1%, than the coefficient of variation on the measurement of the arterial ±venous glucose difference will be about 50%. The precision of the assay is less of an issue for fatty acids and lactate because of a greater fractional extraction by the myocardium, and a relatively greater arterial ± venous difference (Gertz et al., 1988; Guth et al., 1990; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985). In any case, it is particularly important to precisely measure the concentration of the substrate in the blood. 3.4. Use of isotopic tracers in metabolic studies In addition to measuring the rate of uptake of a substrate by the myocardium, it is also possible to study the conversion of a substrate to a product using isotopic tracers, usually 14C or 3H (Gertz et al., 1988; Hall, Henderson, et al., 1996; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985; Wisneski et al., 1987, 1990). With this method, the tracer is infused either systemically (Gertz et al., 1988; Hall, Henderson et al., 1996; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski et al., 1987, 1990) or directly into the coronary arteries via an extracorporeal perfusion circuit (Liedtke & Nellis, 1978; Renstrom et al., 1989; Stanley et al., 1992). The rate of substrate oxidation (e.g. glucose, lactate or fatty acids) are expressed as mmol g ÿ 1 min ÿ 1. The general formula for
calculating substrate oxidation for a strate is:
137 14
C-labeled sub-
Substrate oxidation myocardial blood flow
14 CO2 vein ÿ 14 CO2 artery arterial specific radioactivity
where the myocardial blood flow is expressed as ml g ÿ 1 min ÿ 1, the 14CO2 in the blood as dpm/ml, and the arterial specific radioactivity (dpm/mmol) is the ratio of the concentration of radioactive substrate in the blood divided by the total concentration of the substrate in the blood. Tritium can be used instead of 14C to measure oxidation of fatty acids (i.e. oleate or palmitate), and the same equation is used substituting the venous ±arterial difference of 3H2O for 14CO2. This approach can also be used to measure the uptake of the substrate and its conversion to an intermediate, such as the uptake of 14C-glucose and its conversion to 14C-lactate (Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985). Isotopic tracers are particularly useful for assessing the metabolism of lactate by the heart. The healthy in vivo heart is a net consumer of lactate, however, studies with 13 C-lactate or 14C-lactate tracers demonstrate that the nonischemic heart simultaneously consumes and produces lactate. Studies in humans (Gertz et al., 1981), dogs (Mazer et al., 1990), and swine (Guth et al., 1990) showed that the fractional extraction of 14C-lactate exceeded the net lactate extraction in the healthy human heart. The difference between the net extraction of lactate and the 14C-lactate tracer extraction is the release of lactate from the tissue. This method is particularly sensitive for measuring myocardial lactate production in CAD patient (Gertz et al., 1981; Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985). It is possible to substitute nonradioactive 13C-lactate tracer for 14C-lactate tracer (Hall, Henderson, et al., 1996; Neese, Gertz, Wisneski, Gruenke, & Craig, 1983). It is also possible to combine 13 C-lactate tracer with 14C-glucose so that glucose oxidation, lactate uptake, and lactate production can be measured simultaneously (Gertz et al., 1988; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985; Wisneski et al., 1990). 3.5. Tissue measurements of metabolic substrates, intermediates, and products Often, it is important to measure the concentration of metabolic intermediates (e.g. lactate, long- and short-chain CoA esters, glycogen, etc), high-energy phosphates (ATP and CP), or the activation or inhibition of an enzyme before and after drug treatment, or at various time points during ischemia and reperfusion (Bùtker, Helligsù, Kimose, Thomassen, & Nielsen, 1994). The traditional method for assaying tissue is to take a transmural biopsy from the region of interest, quickly freeze it (within 0.5± 3
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s), and perform ``wet'' biochemical analysis at a later date. This technique requires a large heart, and is generally limited to large animals (dogs and pigs). Biopsies can be taken with a pneumatic cardiac biopsy drill (Allard, Conhaim, Vlahakes, O'Neill, & Hoffman, 1981), or a biopsy needle (Hall et al., 1995; Hall, Henderson, et al., 1996). A cardiac biopsy drill has been developed with an air turbine (10,000 rpm) to turn a stainless-steel bore (2.0 ±5 mm diameter) (Allard et al., 1981). With the drill method, it is necessary to tie a ``purse string'' suture circumventing the incision point of the bore. It is important to punch the bore through the wall and out very quickly, and at an angle so that more tissue is collected. The bore is attached to an insulated vacuum canister containing liquid nitrogen. The biopsy is very rapidly drawn into the liquid nitrogen and frozen ( 0.3± 1.5 s, depending on the size). Immediately after the bit is removed, the suture is pulled tight to prevent bleeding out from the ventricle. The advantage of the drill method is that large samples can be obtained ( 30 ± 150 mg, depending on the bore size, the angle of insertion, and the wall thickness). A potential disadvantage to this method, however, is that severe ventricular bleeding can occur, causing impaired ventricular function and damage to the area of myocardium under study. The drill described by Allard et al. is, to the author's knowledge, no longer commercially available. The needle biopsy method is less traumatic, but gives a smaller biopsy (10 ± 20 mg) using a 14-gauge breast biopsy needle. If the needle is inserted at a relatively flat angle, it is often not necessary to close the point of insertion with a purse string suture, even if the cut is transmural. To obtain large biopsies, it is important to apply upward pressure on the needle as the cutting trocar is advanced forward. In general, it is possible with either the drill or the needle technique to take four to six biopsies over the course of an experiment without severely disrupting ventricular function. It is also possible to separate the subendocardial region from the subepicardial region by staining the epicardium with a spot of dye before taking the biopsy. The frozen biopsy can be divided into two or three layers using a scalpel and forceps precooled in liquid nitrogen. For many metabolism-related assays large amounts of tissue are frequently required for biochemical assay (>200 mg). This amount can be taken using the terminal ``punch biopsy'' technique (Liedtke, Nellis, & Mjos, 1984). With this method, the vena cava is severed and immediately the heart is lifted up and a sharp cylindrical brass or steel cork borer is pressed through the anterior and posterior free walls of the ventricle. The tissue is rapidly pulled out of the borer onto aluminum blocks precooled in liquid nitrogen and immediately freeze clamped. High-energy phosphates and pH can be measured serially in open-chest large animals using 31P-nuclear magnetic resonance (NMR) (Schaefer et al., 1992). With this technique, NMR spectra are collected acquired continuously and averaged every few minutes. The spectra are analyzed
offline for the relative concentrations of ATP, CP, ADP and Pi, and the pH. This is a technically difficult method that requires access to expensive equipment, however, it has advantages over the traditional biopsy method in that it does not damage the heart, and it allows one to measure concentrations of free ADP and Pi rather than total tissue concentrations. On the other hand, NMR does not measure the absolute concentration of high-energy phosphates, but only the relative concentrations. 3.6. Interstitial measurements with microdialysis Sometimes, it is important to assess the interstitial concentration of metabolites. Van Wylen et al. (1990) introduced the myocardial microdialysis technique for measuring small molecules in the interstitium in the heart. They were able to show that ischemia caused enormous increases in interstitial adenosine concentration, which was much greater than the rise in tissue adenosine content. Several groups have made their own microdialysis probes (Van Wylen et al., 1990), however, commercial probes are available (Hall, Hernandez, Henderson, Kellerman, & Stanley, 1994; Hall et al., 1995). With this method, the probes are inserted into the myocardium at the desired sights and depth, and buffer is pumped through the probe until the effluent concentration of the compound of interest is constant. Effluent fractions are collected over the course of the study protocol. Several probes can be inserted into a large animal heart at the same time, and it is possible to position the probes in both the subendocardium and subepicardium, and assess transmural differences during myocardial ischemia and reperfusion (Hall et al., 1994, 1995). This powerful method can be used to measure changes in the interstitial concentration of a variety of compounds, including drugs, metabolites (adenosine, glucose, lactate, etc.), or neurotransmitters (norepinephrine, dopamine, and neuropeptide Y) (Mertes et al., 1996). It is particularly useful for measuring adenosine concentration, as adenosine exerts its effects on cardiomyocytes and vascular smooth muscle from the interstitial space, but is rapidly cleared from the vascular and intracellular compartments. Myocardial ischemia results in a modest increase in whole tissue and coronary venous adenosine concentration, but approximately 5- to 10-fold greater increases in interstitial levels as measured by microdialysis (Van Wylen et al., 1990). Studies using microdialysis in open-chest anesthetized swine have shown that the adenosine transport blocker R-75231 causes a significant increase in microdialysate adenosine concentration during ischemia (Martin, Lasley, & Mentzer, 1997). 4. Extracorporeal perfused swine heart It is frequently desirable to perform acute large animal studies with regional myocardial ischemia. In addition, it is
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sometimes necessary to deliver the drug of interest directly down the coronary artery. The extracorporeal perfused swine heart works very well for these purposes. With this method, blood is pumped out of the femoral artery into one or several coronary arteries. Liedtke et al. (Liedtke & Nellis, 1978; Renstrom et al., 1989; Stanley et al., 1992) perfected this model with a triple vessel perfusion (right main coronary artery, left main, and LAD). Many groups prefer the more simple preparation with perfusion of only the LAD, with blood sampled from the anterior interventricular vein (Cason et al., 1992; Guth et al., 1990; Hall et al., 1994, 1995; Ito, 1995; Schultz, Guth, Pieper, Martin, & Heusch, 1992). The procedure is usually done using domestic swine (30 ± 50 kg). The heart is exposed by a midline sternotomy with partial left side rib resection, and suspended in a pericardial sling. The pig is heparinized (500 U/kg iv bolus, followed by 250 U/kg/h). The femoral artery is cannulated, and blood is pumped through medical grade tygon tubing using a roller pump. Some groups have included a 40-mm blood transfusion filter in the perfusion line distal to the roller pump (Liedtke & Nellis, 1978; Liedtke et al., 1984), however, it does not appear to be necessary (Guth et al., 1990; Ito, 1995; Schultz et al., 1992). The end of the perfusion line is outfitted with a tapered polyethylene cannula with a 70° bend approximately 1.5± 2.0 cm from the tip. The tip is fluted so that it will remain in place when tied into the coronary artery. After the perfusion line is primed, the LAD is ligated above the first diagonal branch, cannulated, and the flow restored. Usually, the total time of ischemia is 30± 50 s from the time of ligation to restoration of flow. The flow setting on the pump is adjusted so that the mean pressure in the LAD matches the mean aortic pressure, and the interventricular venous hemoglobin saturation is between, in the normal in vivo range, 35% and 40% saturation. In this preparation, the myocardial blood flow can be measured by calibrating the perfusion pump before the study and measuring the mass of the LAD perfusion bed at the end of the study. If one wants to measure regional myocardial flow (i.e. subendocardial and subepicardial blood flow) either radioactive (Cason et al., 1992; Guth et al., 1990; Stanley et al., 1992) or nonradioactive (Hall, Henderson, et al., 1996; Hall et al., 1994; Hall, Stanley, et al., 1996; Hall, et al., 1995; Stanley et al., 1996) microspheres can be used. With this method, 15-mm latex spheres containing either gamma-emitting isotopes or fluorescent dye are injected arterially, and the concentration of sphere is measured in the tissue ex vivo (see Berman, Lister, Pitt, & Hoffman, 1988; Glenny, Bernard, & Brinkley, 1993 for a detailed description of the method). The microspheres can be injected either in the left atrium (Hall, Henderson, et al., 1996; Hall et al., 1994; Hall, Stanley, et al., 1996; Hall, et al. 1995),or directly into the perfusion circuit (Cason et al., 1992; Guth et al., 1990; Stanley et al., 1992).
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5. Summary Evaluating the efficacy of novel drug candidates takes a variety of experimental tools. In this age of cell culture and transgenic mice, it is important to remember the role of in vivo evaluation of novel pharmacotherapies and drug candidates in large animal models. Clearly, large animal in vivo models of myocardial ischemia are necessary as predictors of clinical utility prior to initiating patient trials. Moreover, properly designed large animal cardiac metabolism studies provide critical information on the biochemical mechanisms and effects of experimental drugs Ð information that cannot usually be obtained from studies on isolated hearts or in the clinic setting. References Allard, J. R., Conhaim, R. L., Vlahakes, G. J., O'Neill, M. J., & Hoffman, J. I. (1981). Rapid-freezing transmuralcardiac biopsy drill. American Journal of Physiology, 240, H126 ± H132. Arai, A. E., Pantely, G. A., Anselone, C. G., Bristow, J., & Bristow, J. D. (1991). Active down-regulation of myocardial energy requirements during prolonged moderate ischemia in swine. Circulation Research, 69, 1458 ± 1469. Belke, D. D., Larsen, T. S., Lopaschuk, G. D., & Severson, D. L. (1999). Glucose and fatty acid metabolism in the isolated working mouse heart. American Journal of Physiology, 277, R1210 ± R1217. Berman, W. Jr., Lister, G. Jr., Pitt, B. R., & Hoffman, J. I. (1988). Measurements of blood flow. Advances in Pediatrics, 35, 427 ± 481. Bùtker, H. E., Helligsù, P., Kimose, H. H., Thomassen, A. R., & Nielsen, T. T. (1994). Determination of high-energy phosphates and glycogen in cardiac and skeletal muscle biopsies with special reference to influence of biopsy technique and delayed freezing. Cardiovascular Research, 24, 524 ± 527. Cason, B. A., Wisneski, J. A., Neese, R. A., Stanley, W. C., Hickey, R. F., Shnier, C. B., & Gertz, E. W. (1992). Effects of high arterial oxygen tension on function, blood flow distribution and metabolism in ischemic myocardium. Circulation, 85, 828 ± 838. Diaz, R., Paolasso, E. A., Piegas, L. S., Tajer, C. D., Moreno, M. G., Corvalan, R., Isea, J. E., & Romero, G. (1998). Metabolic modulation of acute myocardial infarction. The ECLA (Estudios Cardiologicos Latinoamerica) collaborative group. Circulation, 98, 2227 ± 2234. Gamble, J., & Lopaschuk, G. D. (1994). Glycolysis and glucose oxidation during reperfusion of ischemic hearts from diabetic rats. Biochimica et Biophysica Acta, 1225, 191 ± 199. Gertz, E. W., Wisneski, J. A., Neese, R. A., Bristow, J. D., Searle, G. L., & Hanlon, J. T. (1981). Myocardial lactate metabolism: evidence of lactate release during net chemical extraction in man. Circulation, 63, 1273 ± 1279. Gertz, E. W., Wisneski, J. A., Stanley, W. C., & Neese, R. A. (1988). Myocardial substrate utilization during exercise in humans: dual carbon-labeled carbohydrate isotope experiments. Journal of Clinical Investigation, 82, 2017 ± 2025. Glenny, R. W., Bernard, S., & Brinkley, M. (1993). Validation of fluorescent-labeled microspheres for measurement of regional organ perfusion. Journal of Applied Physiology, 74, 2585 ± 2597. Guth, B. D., Wisneski, J. A., Neese, R. A., White, F. C., Heusch, G., & Mazer, C. D. (1990). Myocardial lactate release during ischemia in swine. Relation to regional blood flow. Circulation, 81, 1948 ± 1958. Hall, J. L., Henderson, J., Hernandez, L. A., & Stanley, W. C. (1996). Hyperglycemia causes an increase in myocardial interstitial glucose and glucose uptake during ischemia in swine. Metabolism, 45, 542 ± 549.
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Hall, J. L., Hernandez, L. A., Henderson, J., Kellerman, L., & Stanley, W. C. (1994). Effects of ischemia and reperfusion on microdialysate glucose and lactate levels in swine. Basic Research in Cardiology, 89, 468 ± 486. Hall, J. L., Lopaschuk, G. D., Pizzurro, R. D., Bringas, J., & Stanley, W. C. (1996). Increased cardiac fatty acid uptake with dobutamine infusion in swine is accompanied by a decrease in malonyl CoA levels. Cardiovascular Research, 32, 879 ± 885. Hall, J. L., Stanley, W. C., Lopaschuk, G. D., Wisneski, J. A., Pizzurro, R. D., Hamilton, C. D., & McCormack, J. G. (1996). Impaired pyruvate oxidation but not glucose uptake in diabetic pig heart during dobutamine stress. American Journal of Physiology, 271, H2320 ± H2329. Hall, J. L., Van Wylen, D. G. L., Pizzurro, R. D., Hamilton, C. D., Reiling, C. M., & Stanley, W. C. (1995). Myocardial interstitial purine metabolites and lactate with increased work in swine. Cardiovascular Research, 30, 351 ± 356. Ito, B. R. (1995). Gradual onset of myocardial ischemia results in reduced myocardial infarction. Association with reduced contractile function and metabolic down-regulation. Circulation, 91, 2058 ± 2070. Jeffrey, F. M. H., Diczku, V., Sherry, A. D., & Malloy, C. R. (1995). Substrate selection in the isolated working rat heart: effects of reperfusion, afterload and concentration. Basic Research in Cardiology, 90, 388 ± 396. Kantor, P. F., Lucien, A., Kozak, R., & Lopaschuk, G. D. (2000). The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase. Circulation Research, 86, 580 ± 588. Kraegen, E. W., Sowden, J. A., Halstead, M. B., Clark, P. W., Rodnick, K. J., Chisholm, D. J., & James, D. E. (1993). Glucose transporters and in vivo glucose uptake in skeletal and cardiac muscle: fasting, insulin stimulation and immunoisolation studies of GLUT1 and GLUT4. Biochemical Journal, 295, 287 ± 293. Lewandowski, E. D. (2000). Metabolic mechanisms associated with antianginal therapy. Circulation Research, 86, 487 ± 489. Liedtke, A. J. (1981). Alterations in carbohydrate and lipid metabolism in the acutely ischemic heart. Progress in Cardiovascular Diseases, 23, 321 ± 326. Liedtke, A. J., Hughes, H. C., & Neely, J. R. (1975). Metabolic responses to varying restrictions of coronary blood flow in swine. American Journal of Physiology, 228, 655 ± 662. Liedtke, A. J., & Nellis, S. H. (1978). Effects of buffered pyruvate on regional cardiac function in moderate, short-term ischemia. Circulation Research, 43, 189 ± 199. Liedtke, A. J., Nellis, S. H., & Mjos, O. D. (1984). Effects of reducing fatty acid metabolism on mechanical function in regionally ischemic hearts. American Journal of Physiology, 247, H387 ± H394. Lopaschuk, G. D., Wambolt, R. B., & Barr, R. L. (1993). An imbalance between glycolysis and glucose oxidation is a possible explanation for the detrimental effects of high levels of fatty acids during aerobic reperfusion of ischemic hearts. Journal of Pharmacology and Experimental Therapeutics, 264, 135 ± 144. Martin, B. J., Lasley, R. D., & Mentzer, R. M. (1997). Infarct size reduction with the nucleoside transport inhibitor R-75231 in swine. American Journal of Physiology, 272, H1857 ± H1865. Mazer, C. D., Cason, B. A., Stanley, W. C., Shnier, C. B., Wisneski, J. A., & Hickey, R. F. (1994). Dichloracetate stimulates carbohydrate metabolism but does not improve systolic function in ischemic pig heart. American Journal of Physiology, 268, H879 ± H885. Mazer, C. D., Stanley, W. C., Hickey, R. F., Neese, R. A., Cason, B. A., Demas, K. A., Wisneski, J. A., & Gertz, E. W. (1990). Myocardial metabolism during hypoxia: maintained lactate oxidation during increased glycolysis. Metabolism, 39, 913 ± 918.
McCormack, J. G., Stanley, W. C., & Wolff, A. A. (1998). Pharmacology of ranolazine: a novel metabolic modulator for the treatment of angina. General Pharmacology, 30, 639 ± 645. McNulty, P. H., Sinusas, A. J., Shi, C. Q., Dione, D., Young, L. H., Cline, G. C., & Shulman, G. I. (1996). Glucose metabolism distal to a critical coronary stenosis in a canine model of low-flow myocardial ischemia. Journal of Clinical Investigation, 98, 62 ± 69. Mertes, P. M., el-Abbassi, K., Jaboin, Y., Michel, C., Beck, B., Pinelli, G., Carteaux, J. P., Villemot, J. P., & Burlet, C. (1996). Consequences of coronary occlusion on changes in regional interstitial myocardial neuropeptide Y and norepinephrine concentrations. Journal of Molecular and Cellular Cardiology, 28, 1995 ± 2004. Neese, R. A., Gertz, E. W., Wisneski, J. A., Gruenke, L. D., & Craig, L. C. (1983). A stable isotope technique for investigating lactate metabolism in humans. Biomedical Mass Spectroscopy, 10, 458 ± 462. Poizat, C., Keriel, C., & Cuchet, P. (1994). Is oxygen supply sufficient to induce normoxic conditions in isolated rat heart? Basic Research in Cardiology, 89, 535 ± 544. Renstrom, B., Nellis, S. H., & Liedtke, A. J. (1989). Metabolic oxidation of glucose during early myocardial reperfusion. Circulation Research, 65, 1094 ± 1101. Schaap, F. G., Binas, B., Danneberg, H., van der Vusse, G. J., & Glatz, J. F. (1999). Impaired long-chain fatty acid utilization by cardiac myocytes isolated from mice lacking the heart-type fatty acid binding protein gene. Circulation Research, 85, 329 ± 337. Schaefer, S., Schwartz, G., Wisneski, J. A., Trocha, S. D., Christoph, I., Steinman, S. K., Garcia, J., Massie, B. M., & Weiner, M. (1992). Response of high-energy phosphates and lactate release during prolonged regional ischemia in vivo. Circulation, 85, 342 ± 349. Schultz, R., Guth, B. D., Pieper, K., Martin, C., & Heusch, G. (1992). Recruitment of an inotropic reserve in moderately ischemic myocardium at the expense of metabolic recovery. Circulation Research, 70, 1282 ± 1295. Stanley, W. C., Hall, J. L., Stone, C. K., & Hacker, T. A. (1992). Acute myocardial ischemia causes a transmural gradient in glucose extraction but not glucose uptake. American Journal of Physiology, 262, H91 ± H96. Stanley, W. C., Hernandez, L. A., Spires, D. A., Bringas, J., Wallace, S., & McCormack, J. G. (1996). Pyruvate dehydrogenase activity and malonyl CoA levels in normal and ischemic swine myocardium: effects of dichloroacetate. Journal of Molecular and Cellular Cardiology, 29, 905 ± 914. Stanley, W. C., Lopaschuk, G. D., Hall, J. L., & McCormack, J. G. (1997). Regulation of myocardial carbohydrate metabolism under normal and ischemic conditions: potential for pharmacological interventions. Cardiovascular Research, 33, 243 ± 257. Van Wylen, D. G. L., Willis, J., Sodhi, J., Weiss, R. J., Lasly, R. D., & Mentzer, R. M. Jr. (1990). Cardiac microdialysis to estimate interstitial adenosine and coronary blood flow. American Journal of Physiology, 258, H1642 ± H1649. Wisneski, J. A., Gertz, E. W., Neese, R. A., Gruenke, L. D., & Craig, J. C. (1985). Dual carbon-labeled isotope experiments using D-[6-14C] glucose and L-[1,2,3-13C3] lactate: a new approach for investigating human myocardial metabolism during ischemia. Journal of the American College of Cardiology, 5, 1138 ± 1146. Wisneski, J. A., Gertz, E. W., Neese, R. A., Gruenke, L. D., Morris, D. L., & Craig, J. C. (1985). Metabolic fate of extracted glucose in normal human myocardium. Journal of Clinical Investigation, 76, 1819 ± 1827. Wisneski, J. A., Gertz, E. W., Neese, R. A., & Mayr, M. (1987). Myocardial metabolism of free fatty acids: studies with 14C-labeled substrates in humans. Journal of Clinical Investigation, 79, 359 ± 366. Wisneski, J. A., Stanley, W. C., Gertz, E. W., & Neese, R. A. (1990). Effects of hyperglycemia on myocardial glycolytic activity in normal humans. Journal of Clinical Investigation, 85, 1648 ± 1656.
In vivo models of myocardial metabolism during ischemia Application to drug discovery and evaluation William C. Stanley* Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4970, USA Received 1 June 2000; accepted 1 July 2000
Abstract This review examines the in vivo techniques that are available for evaluation of the metabolic effects of agents intended for the treatment of myocardial ischemia. Energy substrate metabolism is complex, and requires simultaneous measurement of a variety of processes in order to obtain a thorough understanding of the biochemical mechanisms underlying any functional response. Small animals (from the mouse to the rabbit) are generally not very useful in the study of cardiac metabolism in vivo because it is not possible to sample the coronary venous drainage and measure the rate of substrate uptake or metabolic efflux. Anesthetized open-chest swine or dog models allow simultaneous serial measurement of myocardial substrate use, and repeated tissue sampling for the activities and contents of key enzymes and metabolites. The swine model is particularly good because pigs, like humans, lack innate collateral vessels, thus one can induce regional myocardial ischemia in the left anterior descending coronary artery and sample the venous effluent from the anterior interventricular vein. In this review the biochemical and physiological methods that can be used in conjunction with this preparation are described. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Angina; Animal models; Cardiac; Coronary artery disease; Heart; Metabolism; Microdialysis; Swine
1. Introduction Coronary artery disease (CAD) induces myocardial ischemia. The definition of myocardial ischemia is somewhat elusive, but can be broadly given as an insufficient blood flow to meet the normal demand for oxygen for the given mechanical power output of the myocardium. Clinically, myocardial ischemia initially presents a failure to meet the demand for myocardial blood flow and oxygen delivery in response to an increase in the demand for contractile work. When the arterial blockage is more severe, ischemia occurs even under resting conditions. Traditional pharmacological treatments for chronic stable myocardial ischemia (clinically referred to as chronic stable angina) are aimed at re-establishing the balance between oxygen delivery to the myocardium and the demand for oxygen by the tissue by (1) increasing the blood flow to the myocardium (e.g. via coronary vasodilation), or (2) decreasing the mechanical power output of the tissue through a reduction in heart rate,
* Tel.: +1-216-368-5585; fax: +1-216-368-3952. E-mail address: [email protected] (W.C. Stanley).
blood pressure, and contractility. Drugs designed for the treatment of stable angina are evaluated and approved by regulatory agencies based on their ability to improve the patient's exercise time without chest pain or classic S-T segment depression on the electrocardiogram during an incremental exercise test. More severe ischemia is caused by an acute myocardial infarction induced by formation of an intra-arterial thrombus, which is treated by rapidly restoring blood flow using thrombolytic therapy or coronary angioplasty. Thus, traditional therapies for CAD and AMI are aimed at better matching coronary flow to the demand for myocardial mechanical power. These hemodynamic and ``plumbing'' approaches to the treatment of ischemic heart disease have proven relatively effective, especially for the treatment of acute myocardial infarction. There are, however, CAD patients with severe symptoms of myocardial ischemia who have undergone revascularization procedures (i.e. angioplasty or coronary by-pass surgery) and are on maximally tolerated medications for stable angina. Additionally, there are AMI patients who die or develop infarcts despite optimal thrombolytic therapy. An adjunctive treatment for ischemia is to manipulate energy metabolism in the ischemic zone in a way that
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optimizes the metabolism of the ischemic cardiac muscle (Lewandowski, 2000; Stanley, Lopaschuk, Hall, & McCormack, 1997). This approach, termed ``metabolic modulation,'' reduces the tissue content and release of anginaproducing noxious stimuli (e.g. K + , H + , and adenosine), and could potentially increase the rate of mechanical power generated for a given rate of myocardial oxygen consumption (Stanley et al., 1997). 2. Rationale for metabolic therapies for CAD Cardiac muscle requires a constant high rate of carbon substrate oxidation to generate the reducing equivalents for the electron transport chain and drive ATP formation by oxidative phosphorylation (Liedtke, 1981; Stanley et al., 1997). ATP synthesis is matched by ATP hydrolysis, which provides the energy to fuel contractile work and Ca2 + reuptake into the sarcoplasmic reticulum for diastolic relaxation (Fig. 1). The oxidation of fatty acids in the mitochondria normally supplies approximately two-thirds of the energy for oxidative phosphorylation, and the remaining one-third is provided by the oxidation of glucose and lactate. Myocardial ischemia results in a profound disruption in myocardial energy metabolism. Reduction in coronary flow ( > 30%) results in an immediate burst of glycolysis, glycogen breakdown, a switch from lactate uptake to lactate production, lactate accumulation in the tissue, a fall in ATP concentration and a decrease in intracellular pH (Stanley et al., 1997). This coincides with severe or complete contractile dysfunction and, if the ischemia is of sufficient severity and duration, myocardial necrosis and infarction. It is important to note that during
myocardial ischemia, there is a reduction, not elimination, of blood flow, and, thus, there is continued oxidative phosphorylation and oxidation of carbon substrates, although at a reduced rate. Paradoxically, studies in pigs (Liedtke, 1981; Liedtke, Hughes, & Neely, 1975) and dogs (McNulty et al., 1996) demonstrate that the primary source of carbon substrate is fatty acids during partial myocardial ischemia (30 ± 60% reduction in coronary flow). Thus, there is lactate production and a fall in pH during ischemia despite a continued oxidation of fatty acids in the mitochondria. An alternative approach for the treatment of myocardial ischemia and post-ischemic reperfusion injury is to prevent or reduce the disruption to cardiomyocyte energy metabolism. Specifically, much effort has been placed on increasing the rate of pyruvate oxidation and thus reducing the accumulation of lactate in the cell and the associated fall in pH. Myocardial ischemia results in profound derangements in myocardial substrate utilization, particularly impaired pyruvate oxidation and an increase in anaerobic glycolysis (Stanley et al., 1997). Post-ischemic reperfused myocardium rapidly returns to a normal rate of oxygen consumption, but has an accelerated rate of anaerobic glycolysis, impaired pyruvate oxidation, and a disproportionately high rate of fatty acid oxidation, which all correspond with a decrease in contractile work (Lopaschuk, Wambolt, & Barr, 1993; Stanley et al., 1997). Pyruvate is oxidized in the mitochondrial matrix by the pyruvate dehydrogenase complex (PDH), which is strongly inhibited by its products, acetyl-CoA and NADH (Fig. 2). PDH is phosphorylated and inhibited by PDH kinase, and is dephosphorylated and activated by PDH phosphatase. PDH kinase is activated by increases in the NADH/NAD + and acetyl-CoA/CoA ratios, and inhibited by pyruvate and the
Fig. 1. Schematic depiction of myocardial metabolism. Abbreviations: GLUT, glucose transporters; G 6-P, glucose 6-phosphate; PDH, pyruvate dehydrogenase; CAC, citric acid cycle; CPT-I, carnitine palmitoyl transferase I.
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Fig. 2. Regulation of pyruvate dehydrogenase (PDH) by phosphorylation and product inhibition.
pyruvate analogue dichloroacetate (DCA) (Fig. 2). Acetyl CoA and NADH are also the products of b-oxidation of fatty acids, thus fatty acid oxidation inhibits pyruvate oxidation (Figs. 1 and 2). Pyruvate oxidation can be increased by partially inhibiting fatty acid oxidation, which removes product inhibition on PDH (Stanley et al., 1997). The rate of fatty acid oxidation in cardiomyocytes is primarily controlled by the activity of carnitine palmitoyl transferase I (CPT I). Pharmacological stimulation of PDH with the PDH kinase inhibitor DCA, or by inhibition of CPT-I (e.g. with oxfenicine or etomoxir) or fatty acid beta-oxidation results in a significant improvement in left ventricular function following ischemia without an increase in myocardial oxygen consumption (Stanley et al., 1997). The enhanced pyruvate oxidation lowers lactate accumulation and efflux from the cell, and helps maintain myocyte pH and ion homeostasis. This would allow more efficient use of ATP for contractile work by myosin ATPases and for relaxation by the sarcoplasmic Ca2 + pump, and result in higher ATP levels. At present, there is interest in developing drugs that will optimize myocardial energy metabolism during and after ischemia (Lewandowski, 2000). The partial fatty acid oxidation inhibitors trimetazidine (Kantor, Lucien, Kozak, & Lopaschuk, 2000) and ranolazine (McCormack, Stanley, & Wolff, 1998) have been used successfully for the treatment of stable angina (Stanley et al., 1997), without eliciting any of the classic anti-ischemic effects of traditional therapies (e.g. decrease heart rate, coronary vasodilation and decreased arterial blood pressure). Adjunctive treatment with infusions of glucose and insulin during an AMI reduce plasma free fatty acid concentration and presumably stimulate pyruvate oxidation, and have been shown to reduce mortality (Diaz et al., 1998; Stanley et al., 1997). Energy substrate metabolism is complex and requires simultaneous
measurement of a variety of processes in order to obtain a thorough understanding of the biochemical mechanisms underlying any functional response. This review will present various in vivo techniques that are useful for evaluating the efficacy and metabolic effects of potential drugs for the treatment of myocardial ischemia. 3. Assessment of myocardial metabolism in vivo The general goal of in vivo studies on a potential antiischemic drug is to determine if the compound is efficacious and to learn about the pharmacodynamics and pharmacokinetics of the agent. Metabolic studies usually require assessment of the cardiovascular effects of the compound of interest, thus, one must make simultaneous measurements of the standard indices of cardiovascular function, such as aortic and left ventricular pressure, regional ventricular wall motion, and coronary or regional myocardial blood flow. 3.1. Use of small animals to study myocardial metabolism in vivo Small animals (e.g. mouse, rat, guinea pig, and rabbit) are generally not very useful in the study of cardiac metabolism in vivo because it is not possible to sample the coronary venous drainage and measure the rate of substrate uptake or metabolite efflux. It is possible to assess glucose uptake by the heart in the intact rat with radiolabeled deoxyglucose (Kraegen et al., 1993), however, the fate of the glucose taken up by the heart cannot be traced. These limitations lead investigators to study the in vitro isolated buffer-perfused rat heart. In this preparation, the venous effluent can be collected, and, thus, the exchange of substances across the heart can be measured.
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With the advent of transgenic mice, there is great interest in measuring metabolism in the mouse heart. Methods have recently been established for measuring metabolism in isolated mouse cardiomyocytes (Schaap, Binas, Danneberg, van der Vusse, & Glatz, 1999) and in the isolated working mouse heart (Belke, Larsen, Lopaschuk, & Severson, 1999). Analysis of substrate concentrations and enzyme activities can be made in rapidly harvested mouse hearts, however, the assessment of substrate flux in the transgenic rodent heart is limited to ex vivo conditions. 3.2. Justification for using swine and dog model In light of the widespread use of the isolated perfused rodent heart in the study of myocardial metabolism, it is important to justify the use of large animals. The main justification is that the in vivo dog or pig heart (Fig. 3) is blood perfused, and has very human-like myocardial blood flow, ventricular mechanics, and rates of metabolic fluxes. Recent quantitative studies in isolated buffer-perfused rodent hearts have demonstrated that there is a high rate of anaerobic glycolysis and low rates of glucose oxidation under well-perfused conditions (Gamble & Lopaschuk, 1994; Jeffrey, Diczku, Sherry, & Malloy, 1995; Lopaschuk et al., 1993). Poizat, Keriel, and Cuchet (1994) noted that the rate of lactate production by the isolated buffer-perfused rat heart produces lactate under condition of moderate contractile load, but not at low workloads, suggesting that the oxygen supply is insufficient in this model even when the PO2 is high. As noted above, the healthy human myocardium is a net lactate consumer (Gertz, Wisneski, Stanley, & Neese, 1988; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski, Gertz, Neese, Gruenke, Morris, & Craig, 1985; Wisneski, Gertz, Neese, & Mayr, 1987; Wisneski, Stanley, Gertz, & Neese, 1990), as are dogs (Mazer et al., 1990) and open-chest swine (Cason et al., 1992; Guth et al., 1990; Mazer et al., 1994; Stanley, Hall, Stone, & Hacker, 1992).
Fig. 3. Diagram of the coronary by-pass circuit in the pig.
The isolated buffer-perfused rodent heart must have extremely high rates of buffer flow in order to maintain sufficient oxygen delivery to support normal oxygen consumption and ventricular power. Due to the absence of hemoglobin, the oxygen content of Krebs buffer equilibrated with 95% oxygen is approximately 2 ml/dl, compared to 18 ml/dl in a healthy human. Myocardial blood flow in vivo is approximately 1 ml g tissue ÿ 1 min ÿ 1, but is roughly nine times this value when the heart is perfused with Krebs buffer in vitro. Thus, oxygen delivery is maintained by supra-physiologic flows. This becomes a factor during ischemia because there is a much higher rate of substrate delivery and a greater washout of noxious end products (lactate and H + ). In addition, openchest swine or dog models allow the simultaneous serial measurement of myocardial substrate utilization and the activities and contents of key enzymes and metabolites (Arai, Pantely, Anselone, Bristow, & Bristow, 1991; Hall, Henderson, Hernandez, & Stanley, 1996). The swine model is particularly good because it has a very humanlike coronary anatomy (i.e. no innate collateral coronary arteries as occurs in the dog), which allows one to induce regional myocardial ischemia in the left anterior descending coronary artery (LAD) and sample the venous effluent from the anterior interventricular vein (Renstrom, Nellis, & Liedtke, 1989). For the purpose of this review, we will focus on the use of large animal models for the assessment of myocardial metabolism in vivo. Metabolic studies in large animals have been performed mainly in anesthetized open-chest dogs and pigs. 3.3. Measurement of substrate uptake and output Assessment of myocardial metabolism generally involves measurement of the uptake by the myocardium of energy substrates (oxygen, free fatty acids, lactate, and glucose), and the release of end products (lactate and carbon dioxide). Often, one is also interested in measuring the release of other compounds by the heart, such as adenosine, norepinephrine, or nitrate and nitrite formed from nitric oxide. Additionally, one can measure the uptake by the heart of the drug candidate itself, though this requires a plasma or blood assay of acceptable precision. The uptake is calculated using the Fick principle (uptake = myocardial blood flow (arterial concentration ÿ venous concentration)). The rate of uptake is usually expressed in mmol g tissue ÿ 1 min ÿ 1. A negative arterial ± venous concentration difference and uptake means that the myocardium is producing the substrate. Sometimes, for technical or financial reasons, it is not possible to measure the rate of myocardial blood flow, so in this case one reports only the arterial ± venous concentration differences (usually expressed as mM). The lack of measurement of myocardial blood flow can severely limit the usefulness of the results, particularly if the compound effects coronary blood flow or the trans-
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mural distribution of flow, or if the degree of ischemia needs to be accurately measured. Arterial blood samples can be drawn from any artery, however, the venous sample must come from a vein that drains the region of interest. This can become an issue when studying myocardial ischemia. In swine, ischemia is usually induced by a reduction in flow to the LAD. The venous blood from the LAD perfusion territory is collected from the anterior interventricular vein. Not all of the blood flowing out of the interventricular vein, however, originates from the LAD. Renstrom et al. (1989) infused indocyanine green dye into the LAD via and extracorporeal perfusion circuit and found that the concentration of green dye in the interventricular vein was 91% of the concentration in the LAD. When the LAD flow was reduce by 60% this value fell to 78%. Thus, there is a modest but significant dilution of the venous effluent from the LAD territory. The main site of error in measuring the uptake of a drug or substrate by the myocardium is usually the measurement error in the arterial ±venous concentration difference. For example, the arterial ± venous difference for glucose is typically 0.10 mM, and the arterial glucose concentration is 5.00 mM, then the percent extraction of arterial glucose is 2%. If the coefficient of variation on the glucose assay (the standard error divided by the mean) is 1%, than the coefficient of variation on the measurement of the arterial ±venous glucose difference will be about 50%. The precision of the assay is less of an issue for fatty acids and lactate because of a greater fractional extraction by the myocardium, and a relatively greater arterial ± venous difference (Gertz et al., 1988; Guth et al., 1990; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985). In any case, it is particularly important to precisely measure the concentration of the substrate in the blood. 3.4. Use of isotopic tracers in metabolic studies In addition to measuring the rate of uptake of a substrate by the myocardium, it is also possible to study the conversion of a substrate to a product using isotopic tracers, usually 14C or 3H (Gertz et al., 1988; Hall, Henderson, et al., 1996; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985; Wisneski et al., 1987, 1990). With this method, the tracer is infused either systemically (Gertz et al., 1988; Hall, Henderson et al., 1996; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski et al., 1987, 1990) or directly into the coronary arteries via an extracorporeal perfusion circuit (Liedtke & Nellis, 1978; Renstrom et al., 1989; Stanley et al., 1992). The rate of substrate oxidation (e.g. glucose, lactate or fatty acids) are expressed as mmol g ÿ 1 min ÿ 1. The general formula for
calculating substrate oxidation for a strate is:
137 14
C-labeled sub-
Substrate oxidation myocardial blood flow
14 CO2 vein ÿ 14 CO2 artery arterial specific radioactivity
where the myocardial blood flow is expressed as ml g ÿ 1 min ÿ 1, the 14CO2 in the blood as dpm/ml, and the arterial specific radioactivity (dpm/mmol) is the ratio of the concentration of radioactive substrate in the blood divided by the total concentration of the substrate in the blood. Tritium can be used instead of 14C to measure oxidation of fatty acids (i.e. oleate or palmitate), and the same equation is used substituting the venous ±arterial difference of 3H2O for 14CO2. This approach can also be used to measure the uptake of the substrate and its conversion to an intermediate, such as the uptake of 14C-glucose and its conversion to 14C-lactate (Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985). Isotopic tracers are particularly useful for assessing the metabolism of lactate by the heart. The healthy in vivo heart is a net consumer of lactate, however, studies with 13 C-lactate or 14C-lactate tracers demonstrate that the nonischemic heart simultaneously consumes and produces lactate. Studies in humans (Gertz et al., 1981), dogs (Mazer et al., 1990), and swine (Guth et al., 1990) showed that the fractional extraction of 14C-lactate exceeded the net lactate extraction in the healthy human heart. The difference between the net extraction of lactate and the 14C-lactate tracer extraction is the release of lactate from the tissue. This method is particularly sensitive for measuring myocardial lactate production in CAD patient (Gertz et al., 1981; Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985). It is possible to substitute nonradioactive 13C-lactate tracer for 14C-lactate tracer (Hall, Henderson, et al., 1996; Neese, Gertz, Wisneski, Gruenke, & Craig, 1983). It is also possible to combine 13 C-lactate tracer with 14C-glucose so that glucose oxidation, lactate uptake, and lactate production can be measured simultaneously (Gertz et al., 1988; Wisneski, Gertz, Neese, Gruenke, & Craig, 1985; Wisneski, Gertz, Neese, Gruenke, Morris, et al., 1985; Wisneski et al., 1990). 3.5. Tissue measurements of metabolic substrates, intermediates, and products Often, it is important to measure the concentration of metabolic intermediates (e.g. lactate, long- and short-chain CoA esters, glycogen, etc), high-energy phosphates (ATP and CP), or the activation or inhibition of an enzyme before and after drug treatment, or at various time points during ischemia and reperfusion (Bùtker, Helligsù, Kimose, Thomassen, & Nielsen, 1994). The traditional method for assaying tissue is to take a transmural biopsy from the region of interest, quickly freeze it (within 0.5± 3
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s), and perform ``wet'' biochemical analysis at a later date. This technique requires a large heart, and is generally limited to large animals (dogs and pigs). Biopsies can be taken with a pneumatic cardiac biopsy drill (Allard, Conhaim, Vlahakes, O'Neill, & Hoffman, 1981), or a biopsy needle (Hall et al., 1995; Hall, Henderson, et al., 1996). A cardiac biopsy drill has been developed with an air turbine (10,000 rpm) to turn a stainless-steel bore (2.0 ±5 mm diameter) (Allard et al., 1981). With the drill method, it is necessary to tie a ``purse string'' suture circumventing the incision point of the bore. It is important to punch the bore through the wall and out very quickly, and at an angle so that more tissue is collected. The bore is attached to an insulated vacuum canister containing liquid nitrogen. The biopsy is very rapidly drawn into the liquid nitrogen and frozen ( 0.3± 1.5 s, depending on the size). Immediately after the bit is removed, the suture is pulled tight to prevent bleeding out from the ventricle. The advantage of the drill method is that large samples can be obtained ( 30 ± 150 mg, depending on the bore size, the angle of insertion, and the wall thickness). A potential disadvantage to this method, however, is that severe ventricular bleeding can occur, causing impaired ventricular function and damage to the area of myocardium under study. The drill described by Allard et al. is, to the author's knowledge, no longer commercially available. The needle biopsy method is less traumatic, but gives a smaller biopsy (10 ± 20 mg) using a 14-gauge breast biopsy needle. If the needle is inserted at a relatively flat angle, it is often not necessary to close the point of insertion with a purse string suture, even if the cut is transmural. To obtain large biopsies, it is important to apply upward pressure on the needle as the cutting trocar is advanced forward. In general, it is possible with either the drill or the needle technique to take four to six biopsies over the course of an experiment without severely disrupting ventricular function. It is also possible to separate the subendocardial region from the subepicardial region by staining the epicardium with a spot of dye before taking the biopsy. The frozen biopsy can be divided into two or three layers using a scalpel and forceps precooled in liquid nitrogen. For many metabolism-related assays large amounts of tissue are frequently required for biochemical assay (>200 mg). This amount can be taken using the terminal ``punch biopsy'' technique (Liedtke, Nellis, & Mjos, 1984). With this method, the vena cava is severed and immediately the heart is lifted up and a sharp cylindrical brass or steel cork borer is pressed through the anterior and posterior free walls of the ventricle. The tissue is rapidly pulled out of the borer onto aluminum blocks precooled in liquid nitrogen and immediately freeze clamped. High-energy phosphates and pH can be measured serially in open-chest large animals using 31P-nuclear magnetic resonance (NMR) (Schaefer et al., 1992). With this technique, NMR spectra are collected acquired continuously and averaged every few minutes. The spectra are analyzed
offline for the relative concentrations of ATP, CP, ADP and Pi, and the pH. This is a technically difficult method that requires access to expensive equipment, however, it has advantages over the traditional biopsy method in that it does not damage the heart, and it allows one to measure concentrations of free ADP and Pi rather than total tissue concentrations. On the other hand, NMR does not measure the absolute concentration of high-energy phosphates, but only the relative concentrations. 3.6. Interstitial measurements with microdialysis Sometimes, it is important to assess the interstitial concentration of metabolites. Van Wylen et al. (1990) introduced the myocardial microdialysis technique for measuring small molecules in the interstitium in the heart. They were able to show that ischemia caused enormous increases in interstitial adenosine concentration, which was much greater than the rise in tissue adenosine content. Several groups have made their own microdialysis probes (Van Wylen et al., 1990), however, commercial probes are available (Hall, Hernandez, Henderson, Kellerman, & Stanley, 1994; Hall et al., 1995). With this method, the probes are inserted into the myocardium at the desired sights and depth, and buffer is pumped through the probe until the effluent concentration of the compound of interest is constant. Effluent fractions are collected over the course of the study protocol. Several probes can be inserted into a large animal heart at the same time, and it is possible to position the probes in both the subendocardium and subepicardium, and assess transmural differences during myocardial ischemia and reperfusion (Hall et al., 1994, 1995). This powerful method can be used to measure changes in the interstitial concentration of a variety of compounds, including drugs, metabolites (adenosine, glucose, lactate, etc.), or neurotransmitters (norepinephrine, dopamine, and neuropeptide Y) (Mertes et al., 1996). It is particularly useful for measuring adenosine concentration, as adenosine exerts its effects on cardiomyocytes and vascular smooth muscle from the interstitial space, but is rapidly cleared from the vascular and intracellular compartments. Myocardial ischemia results in a modest increase in whole tissue and coronary venous adenosine concentration, but approximately 5- to 10-fold greater increases in interstitial levels as measured by microdialysis (Van Wylen et al., 1990). Studies using microdialysis in open-chest anesthetized swine have shown that the adenosine transport blocker R-75231 causes a significant increase in microdialysate adenosine concentration during ischemia (Martin, Lasley, & Mentzer, 1997). 4. Extracorporeal perfused swine heart It is frequently desirable to perform acute large animal studies with regional myocardial ischemia. In addition, it is
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sometimes necessary to deliver the drug of interest directly down the coronary artery. The extracorporeal perfused swine heart works very well for these purposes. With this method, blood is pumped out of the femoral artery into one or several coronary arteries. Liedtke et al. (Liedtke & Nellis, 1978; Renstrom et al., 1989; Stanley et al., 1992) perfected this model with a triple vessel perfusion (right main coronary artery, left main, and LAD). Many groups prefer the more simple preparation with perfusion of only the LAD, with blood sampled from the anterior interventricular vein (Cason et al., 1992; Guth et al., 1990; Hall et al., 1994, 1995; Ito, 1995; Schultz, Guth, Pieper, Martin, & Heusch, 1992). The procedure is usually done using domestic swine (30 ± 50 kg). The heart is exposed by a midline sternotomy with partial left side rib resection, and suspended in a pericardial sling. The pig is heparinized (500 U/kg iv bolus, followed by 250 U/kg/h). The femoral artery is cannulated, and blood is pumped through medical grade tygon tubing using a roller pump. Some groups have included a 40-mm blood transfusion filter in the perfusion line distal to the roller pump (Liedtke & Nellis, 1978; Liedtke et al., 1984), however, it does not appear to be necessary (Guth et al., 1990; Ito, 1995; Schultz et al., 1992). The end of the perfusion line is outfitted with a tapered polyethylene cannula with a 70° bend approximately 1.5± 2.0 cm from the tip. The tip is fluted so that it will remain in place when tied into the coronary artery. After the perfusion line is primed, the LAD is ligated above the first diagonal branch, cannulated, and the flow restored. Usually, the total time of ischemia is 30± 50 s from the time of ligation to restoration of flow. The flow setting on the pump is adjusted so that the mean pressure in the LAD matches the mean aortic pressure, and the interventricular venous hemoglobin saturation is between, in the normal in vivo range, 35% and 40% saturation. In this preparation, the myocardial blood flow can be measured by calibrating the perfusion pump before the study and measuring the mass of the LAD perfusion bed at the end of the study. If one wants to measure regional myocardial flow (i.e. subendocardial and subepicardial blood flow) either radioactive (Cason et al., 1992; Guth et al., 1990; Stanley et al., 1992) or nonradioactive (Hall, Henderson, et al., 1996; Hall et al., 1994; Hall, Stanley, et al., 1996; Hall, et al., 1995; Stanley et al., 1996) microspheres can be used. With this method, 15-mm latex spheres containing either gamma-emitting isotopes or fluorescent dye are injected arterially, and the concentration of sphere is measured in the tissue ex vivo (see Berman, Lister, Pitt, & Hoffman, 1988; Glenny, Bernard, & Brinkley, 1993 for a detailed description of the method). The microspheres can be injected either in the left atrium (Hall, Henderson, et al., 1996; Hall et al., 1994; Hall, Stanley, et al., 1996; Hall, et al. 1995),or directly into the perfusion circuit (Cason et al., 1992; Guth et al., 1990; Stanley et al., 1992).
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5. Summary Evaluating the efficacy of novel drug candidates takes a variety of experimental tools. In this age of cell culture and transgenic mice, it is important to remember the role of in vivo evaluation of novel pharmacotherapies and drug candidates in large animal models. Clearly, large animal in vivo models of myocardial ischemia are necessary as predictors of clinical utility prior to initiating patient trials. Moreover, properly designed large animal cardiac metabolism studies provide critical information on the biochemical mechanisms and effects of experimental drugs Ð information that cannot usually be obtained from studies on isolated hearts or in the clinic setting. References Allard, J. R., Conhaim, R. L., Vlahakes, G. J., O'Neill, M. J., & Hoffman, J. I. (1981). Rapid-freezing transmuralcardiac biopsy drill. American Journal of Physiology, 240, H126 ± H132. Arai, A. E., Pantely, G. A., Anselone, C. G., Bristow, J., & Bristow, J. D. (1991). Active down-regulation of myocardial energy requirements during prolonged moderate ischemia in swine. Circulation Research, 69, 1458 ± 1469. Belke, D. D., Larsen, T. S., Lopaschuk, G. D., & Severson, D. L. (1999). Glucose and fatty acid metabolism in the isolated working mouse heart. American Journal of Physiology, 277, R1210 ± R1217. Berman, W. Jr., Lister, G. Jr., Pitt, B. R., & Hoffman, J. I. (1988). Measurements of blood flow. Advances in Pediatrics, 35, 427 ± 481. Bùtker, H. E., Helligsù, P., Kimose, H. H., Thomassen, A. R., & Nielsen, T. T. (1994). Determination of high-energy phosphates and glycogen in cardiac and skeletal muscle biopsies with special reference to influence of biopsy technique and delayed freezing. Cardiovascular Research, 24, 524 ± 527. Cason, B. A., Wisneski, J. A., Neese, R. A., Stanley, W. C., Hickey, R. F., Shnier, C. B., & Gertz, E. W. (1992). Effects of high arterial oxygen tension on function, blood flow distribution and metabolism in ischemic myocardium. Circulation, 85, 828 ± 838. Diaz, R., Paolasso, E. A., Piegas, L. S., Tajer, C. D., Moreno, M. G., Corvalan, R., Isea, J. E., & Romero, G. (1998). Metabolic modulation of acute myocardial infarction. The ECLA (Estudios Cardiologicos Latinoamerica) collaborative group. Circulation, 98, 2227 ± 2234. Gamble, J., & Lopaschuk, G. D. (1994). Glycolysis and glucose oxidation during reperfusion of ischemic hearts from diabetic rats. Biochimica et Biophysica Acta, 1225, 191 ± 199. Gertz, E. W., Wisneski, J. A., Neese, R. A., Bristow, J. D., Searle, G. L., & Hanlon, J. T. (1981). Myocardial lactate metabolism: evidence of lactate release during net chemical extraction in man. Circulation, 63, 1273 ± 1279. Gertz, E. W., Wisneski, J. A., Stanley, W. C., & Neese, R. A. (1988). Myocardial substrate utilization during exercise in humans: dual carbon-labeled carbohydrate isotope experiments. Journal of Clinical Investigation, 82, 2017 ± 2025. Glenny, R. W., Bernard, S., & Brinkley, M. (1993). Validation of fluorescent-labeled microspheres for measurement of regional organ perfusion. Journal of Applied Physiology, 74, 2585 ± 2597. Guth, B. D., Wisneski, J. A., Neese, R. A., White, F. C., Heusch, G., & Mazer, C. D. (1990). Myocardial lactate release during ischemia in swine. Relation to regional blood flow. Circulation, 81, 1948 ± 1958. Hall, J. L., Henderson, J., Hernandez, L. A., & Stanley, W. C. (1996). Hyperglycemia causes an increase in myocardial interstitial glucose and glucose uptake during ischemia in swine. Metabolism, 45, 542 ± 549.
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