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Mechanical performance of the working rat heart: Response to calcium
JOURNAL
OF SURGICAL
32, 57-64 (1982)
RESEARCH
Mechanical
Performance Response
of the Working to Calcium’
T. KUGIMIYA, M.D.,2 L. J. DROP,M.D., Anesthesia
PH.D.,~
AND
Rat Heart:
P. EWALENKO,
Services of the Massachusetts General Hospital and rhe Department Harvard Medical School, Boston, Massachusetts 02I 14 Submitted
for publication
M.D.4
of Anaesthesia,
December 26, 1980
Although calcium is generally considered a substance that can enhance cardiac pump performance, few data exist to demonstrate such effect when plasma ionized calcium is varied in the clinical concentration range. We have studied the relationship between perfusate ionized calcium concentration ([Ca”]) and myocardial mechanical performance in the isolated, blood perfused, ejecting rat heart. With pre- and afterload near constant, observations were made before and after steadystate increases in [Ca’+] from low (mean ? SEM: 0.74 * 0.01 mM) to normal (1.01 rt 0.04 mM); from low (1.01 f 0.02 mM) to higher than normal (1.45 ?I 0.04 mM, i.e., within the clinical hypercalcemia range); and to an excessive value (1.94 -t 0.03 mM). When [Ca*+] was increased from low to normal levels, aortic blood flow and stroke volume increased by approximately 25% of control (P < 0.025); heart rate did not change significantly. In contrast, when [Ca”] was raised from 1.03 mM (normal value) to 1.45 mM (within the clinical hypercalcemia range) or to 1.94 mM (above the clinical hypercalcemia range), the changes in aortic flow and stroke volume were less (P-c 0.05) as compared to those observed when ionized calcium was adjusted from a level below normal. We conclude that the isolated, blood-perfused heart with pre- and afterload near constant, responds to sustained increases in perfusate ionized calcium with increased aortic flow and stroke volume, but that these changes are disproportionally small when ionized calcium is elevated to above normal from a normal baseline value.
INTRODUCTION
The importance of the calcium ion for the myocardial contractile process has been recognized for almost a century. Although calcium is considered as a substance that can enhance myocardial performance [ 6, 12, 141, there are only few quantitative data on such effects when ionized calcium is varied in the clinical concentration range. Clinically, periods of hypercalcemia can occur following calcium infusion, initiated in anticipation of improved hemodynamic ’ Supported in part by United States Public Health Service (USPHS) Biomedical Research Supporting Grant RR-05486- 14 and USPHS Anesthesiology Training Grant S-T-01-GM 01273-13. ’ Present address: Department of Anesthesia, Faculty of Medicine, University of Tokyo, Hongo, Tokyo, Japan. ’ Address for reprints: L. J. Drop, Department of Anesthesia, Massachusetts General Hospital, Boston, Massachusetts 02114. ‘Present address: Institut Jules Bordet, lOOO-Bruxelles, Belgium.
performance. A consistent observation is the elevation of arterial blood pressure following calcium infusion[ 131. Since arterial blood pressure is a major determinant of ventricular function, alterations in blood pressure render interpretation of calcium-induced changes in performance of the heart difficult. The classical isolated frog heart study of McLean and Hastings [lo] demonstrated that it is the ionized moiety of plasma calcium that is physiologically active. Their data suggest that, whereas in the ionized calcium range from below normal to normal, elevation of ionized calcium was associated with enhanced amplitude of ventricular contraction, that variable was not consistently enhanced with calcium ion concentrations above normal. In these studies, the frog heart was perfused with red cell free electrolyte solutions. Using a calcium-selective electrode system for ionized calcium measurements, we
51
0022-4804/82/010057-08$01.00 Copyright 0 1982 by Academic Press, Inc. ALI rights of rcprodumon in any form rewrvsd.
58
JOURNAL OF SURGICAL
RESEARCH: VOL. 32, NO. 1, JANUARY
wanted to quantitate the response of the isolated ejecting rat heart preparation that was perfused with erythrocyte perfusate, to acute increases in ionized calcium, initiated from ionized calcium levels corresponding to either hypocalcemia and normocalcemia. In our experiments, pre- and afterload were held near constant. METHODS Blood-perfused
rat
heart
preparation.
The technique for use of the blood-perfused, isolated working rat heart has been described previously by Duvelleroy et al. [5] Briefly, rats of either sex (body weight ranging from 200 to 400 g) were anesthetized by inhalation of ether in ambient air. Following thoracotomy and systemic heparinization (2.5 mg), the heart was excised quickly with segments of the aorta, pulmonary artery, and vein attached, and immediately immersed in an ice-cold heparinized 0.9% sodium chloride solution. The aortic segment (approximately 5 mm in length) was fitted over and tied to the outflow cannula and initial retrograde perfusion of the heart with blood was achieved according to the Langendorff technique [9]. The inflow cannula into the heart was attached to the left atrium, and a catheter was pIaced into the right ventricle to collect coronary sinus blood. An occlusive roller pump (Holter Model RE 161) delivered blood perfusate (vide infra) from a thermostated (37°C) reservoir (250 ml) through a capillary membrane oxygenator (Bio-Fiber 5, Beaker BioRad Laboratories) to a second reservoir (also maintained at 37”C), from which the perfusate flowed by gravity via silastic rubber tubing (3.1 mm i.d.) into the left atrium. Positioning of the second reservoir at a level above the heart permitted the inflow pressure to be held constant at approximately 10 mm Hg. Inflow pressure was recorded via an appropriate transducer (Sanborn 267 BC) connected to the inflow tubing through a stiff-walled side arm. Appropriate blood filters (Ultipor blood filter, Pall Medical,
1982
Inc.) were placed before the oxygenator and the inflow cannula. The aortic cannula which returned the blood to the first reservoir was positioned vertically above the left ventricle to produce a blood column, the height of which was adjustable. The mean outflow pressure was maintained at approximately 80 mm Hg. Values of PO, were above 350 mm Hg and arterial pH and Pc02 were held within narrow limits (pH 7.38-7.41; Pc02, 32-35 mm Hg) by varying the flow rates of 97% oxygen/3% carbon dioxide or 95% oxygen/5% carbon dioxide supplied to the oxygenator. To determine the normal [Ca*+] value in the rat, heparinized blood samples were withdrawn from the great vessels prior to excision of the heart in seven animals. Perfusate. Whole blood was collected from a donor cow and heparinized (2.5 IU/ ml). The red cells were separated, washed, and stored at 4°C until the next day. Immediately prior to the experiment, the red cells were suspended in the Krebs-Hensleit buffer as described previously by Duvelleroy et al. [5]. In all experiments, the final hematocrit was adjusted to 30 f 1%. ANALYSES
AND PROTOCOL
All pressures in the perfusion circuit were recorded continuously on a multichannel direct writing polygraph (Hewlett-Packard) via appropriate transducers (Sanborn 267 BC), which were calibrated at frequent intervals with a mercury manometer. Mean pressure was determined by electronic integration. At each measurement period, recordings were made at a paper speed of 10 mm/set. Heart rate (HR) was calculated from the tracing. Coronary (CBF) and aortic (AF) blood flows (ml/min) were determined by timed volumetric collection. Stroke volume (SI/, ml/beat) was calculated by dividing blood flow per minute (AF + CBF) by heart rate. Blood oxygen concentration was determined in arterial and coronary sinus blood by use of the Lex-O-Con device. Recent data
KUGIMIYA,
DROP, AND
EWALENKO:
[Ca”]
AND
by Chitwood et al. [2] have demonstrated a close correlation between results obtained by this technique and those by manometric technique of Van Slyke and Neill, over the physiologic range. Blood gases and pH were measured by standard electrodes at 37°C. Plasma ionized calcium concentration was measured in a specimen of whole blood using a calcium-selective electrode system [ 31. Hematocrit, serum electrolytes, and osmolality were measured using standard analytical techniques. Myocardial oxygen consumption (&02, ml/min) was calculated as the product of coronary blood flow and arterial-to-coronary sinus blood oxygen content difference and expressed per gram LV weight. The contribution of the empty, nonworking right ventricle and atria to changes in MJ&, was ignored. At the conclusion of the experiment, the atria and right ventricle were cut away, the heart was blotted and weighed. Protocol. In each experiment, the reservoir was filled with reconstituted cow blood (pH adjusted to 7.40 + 0.01 by addition of sodium bicarbonate) and calcium chloride was added to a final [Ca”] value of 1.03 +- 0.02 mM. Following assembly of the circuit, hemodynamic and biochemical measurements were made at 10, 12, and 15 min to ascertain a steady state. Three groups of experiments were performed. In two groups, a new [Ca’+] plateau was established by addition of CaCl, to the perfusate in sufficient amounts to adjust [Ca”] to approximately 1.45 (mean value, Group B) or 1.95 mM (mean value, Group C). In the other group (Group A), the initial normocalcemic perfusate was first exchanged for blood perfusate in which [Ca”‘] had been adjusted to approximately 0.7 mit4. Hemodynamic and biochemical measurements were made at 12 and 15 min after this [Ca2+] plateau (0.7 mM) was established, following which [ Ca2+] was adjusted to approximately 1.05 (Group A). Measurements were then made again at the same time intervals. Of particular value was the fact that [Ca2+] values were known
CARDIAC
MECHANICAL
FUNCTION
59
within 3 min following blood sampling, permitting appropriate adjustment of [Ca’+] in the perfusate. Previous data [7] have suggested that the inotropic response of the rat heart to the addition of calcium is comparable to that of other species only when extracellular calcium levels are low (0.2-0.4 mA4). In view of these results, we wanted to define the performance of the ejecting rat heart at those [ Ca2+] levels. To achieve the low [ Ca2+] values, different quantities of a buffered (pH 7.40) citrate solution (acid-citrate-dextrose, Formula A, USP) were added to the perfusate in two experiments of each group and observations recorded. Citrate solution was titrated to pH 7.40 by addition of THAM [ tris(hydroxemethyl)aminomethane]; thus, a possible effect of a low pH could be excluded. Since the data on different [Ca”] levels were obtained in different animal groups, it was desirable to also study different [Ca”] levels in the same rat heart preparation. Thus, three additional animals were studied. In these studies, initial data were collected after a 15-min stabilization period (perfusate [Ca’+] = 1.03 mM). Three different [Ca”] were achieved, first by exchange of the normocalcemic perfusate with a blood perfusate in which [Ca’+] had been adjusted to 0.70 mM, followed by addition of CaC12, resulting in step increases in [Ca2+]. Each of these [Ca’+] plateaus was maintained for 15 min. The first experimental [Ca’+] level (0.70 mM) was then restored by use of an aliquot of corresponding perfusate. In view of the small changes in mechanical performance of the rat heart to calcium elevation from a normal baseline value, we wanted to ascertain its ability (under the conditions of the study) to substantially increase performance when [Ca”] was known to be normal. Thus, in five additional experiments, hemodynamic variables were recorded prior to and following additions of isoproterenol (final concentration, 4.5 pg/liter) to the perfusate in which [Ca”] was normal ([Ca2’] = 1.03 +- 0.02 mM). This
60
JOURNAL OF SURGICAL RESEARCH: VOL. 32, NO. 1, JANUARY
isoproterenol concentration had been selected to be in the range which has been previously demonstrated [ 81 to provide a significant increase in maximal velocity of contraction of the rat heart. In all experiments the changes in hemodynamic variables, expressed per gram of LV weight, were normalized (changes in percentage of control). Data of different groups were compared using Student’s t test for comparison of group means; within-group means, data were compared using Student’s t test for paired data. Values are expressed as mean -t SEM. RESULTS
The results on changes observed in the different experimental groups are presented in Fig. 1. Hypo-
to normocalcemia
(Group
A, n
= 5). Following an increase in mean [Ca”] (from 0.74 + 0.01 to 1.01 + 0.04 mM), AF increased (25.2 f 4.51%, P < 0.025), Why 25.3 + 7.7% (P < 0.05) and Mvo, by 13.7 f 5.4% (P < 0.05). There were no significant changes in HR or CBF.
1982
Several [Ca”] levels in the same rat heart. Increasing [Ca”] (from 0.70 f 0.01
to 1.03 f 0.02 mM) was associated with increased aortic flow (from 69 -t 1 to 89 f 1 ml/min/g); further increase in [Ca”] (to 1.50 -C 0.05 mM) produced a lesser increase (from 89 + 1 to 93 + 1 ml/min/g). Myocardial oxygen consumption increased from 0.219 -+ 0.008 to 0.251 + 0.003 ml/ min/g when [Ca”] increased from 0.70 to 1.03 mM, and the increase in Mvo, was from 0.251 f 0.003 to 0.293 + 0.007 ml/min/g when [Ca”] was increased further, to 1.50 mM. Following these step increasesin [ Ca*+], the initial experimental [Ca”] (0.70 mM) was restored and all measured variables returned toward their respective baseline values. Isoproterenol (n = 5). Addition of isoproterenol to the perfusate caused substantial rises in AF (P < 0.025) HR (P < 0.025, and SV (P < 0.025) (Table 1). Mr’o, increased by 100% and CBF nearly tripled (P < 0.001). Blood gases and biochemical
variables.
In all experiments, Pao, was above 350 mm Hg, Pace, ranged between 32 and 36 mm Hg, and pH between 7.38 and 7.41. Na ranged from 153 to 156 mM and K, from to moderate hypercalcemia Normo4.9 to 5.0 mM. Osmolality values ranged (Group B, n = 6). An increase in [Ca”] from 1.01 + 0.02 to 1.45 + 0.04 mM was asso- from 310 to 312 mOsm. These values reciated with increased AF (by 13.2 -C 2.99%, mained essentially unchanged during each experiment. P < 0.025). This change was less (P < 0.05) Normal [Ca”] values. The mean [Ca*‘] than that recorded in Group A. Mr’o, rose value (n = 7) in the rat during ether in room by 10.4 + 3.8% (P < 0.05). CBF increased air anesthesia immediately following openby 11.7 +_ 3.2% (P < 0.05). Changes in HR ing of the chest was 1.03 + 0.03 mM. and SV did not reach significance. Normo- to pronounced hypercalcemia (Group C, n = 6). An increase in [Ca”]
(from 0.95 f 0.03 to 1.94 * 0.05 mM) was associated with alterations in AF, CBF, and MV02 that were similar to those recorded in Group B. Stroke volume increased by 12.8 f 3% (P < 0.025). Heart rate remained unchanged. Citrate-induced hypocalcemia. In all experiments, stroke volume decreased to near zero when [Ca”] fell in the range of 0.48 to 0.53 mM (Fig. 2).
DISCUSSION
The most important finding of this study is that the response of the isolated, bloodperfused rat heart to acute increases in ionized calcium was dependent on the initial ionized calcium level. The rise in aortic flow and stroke volume was greater when a higher ionized calcium level was instituted from an initially low level as compared to when the initial ionized calcium level was normal.
KUGIMIYA,
DROP, AND
EWALENKO:
[Ca’+]
AND
CARDIAC
MECHANICAL
FUNCTION
61
Although the possibility of interspecies differences is recognized, these findings are consistent with previous data obtained in other animal species. For example, in their classical studies, McLean and Hastings [lo] utilized the frog heart. They found that, while the amplitude of ventricular contraction was proportional to the aqueous perfusate ionized calcium values (ranging from approximately 0.5 to 1.3 mM; their normal value was 1.22 mM), an elevation of ionized calcium above that value did not consistently further augment function. The frog heart is more dependent on extracellular calcium levels because of a poorly developed sarcoplasmic reticulum, an important regulator of intracellular calcium. In the dog, Mehmel and associates [ 111 found that, following calcium chloride infusion in two incremental doses, the second dose resulted in a much smaller change in myocardial dynamics than the first. Assuming that ionized calcium comprised 48% of the total calcium concentration, calcium infusion in these experiments may have resulted in increased ionized calcium from approximately 0.83 to 1.40 and to 1.90 mM, respectively. Our observations, together with these pre109 0 Control vious data, suggest that a calcium-induced l Excwimentol enhancement of cardiac function is more prominent whenever initial ionized calcium levels are below normal. A possible explanation is the impairment of ventricular funco 5 1” NormalRange’ tion that is characteristically associated with hypocalcemia. Whether impaired ventricuA B C n=6 n=5 n=5 lar function from other causes is also a conFIG. 1. Effects of changes in [Ca’+] on myocardial dition for calcium-induced enhancement remechanical and metabolic function of the blood-permains to be determined. fused, isolated rat heart. In Group A, [Ca”] was raised Our present findings of small changes in from 0.70 + 0.01 (open symbol) to 1.01 f 0.04 mechanical cardiac performance with insti- mM(solid symbol). Aortic flow (AF) and stroke volume tution of ionized hypercalcemia ([ Ca2+] (Sv) increased on the average by 25%. In contrast, when raised to 150% of normal and held at that [Ca’+] was raised from 1.01 + 0.02 mM to 1.45 f 0.04 level for 15 min) led us to perform the ex- (Group B) or 1.94 k 0.05 mM (Group C), changes in AF and SV were smaller as compared to when [Ca”] periments with excessive [ Ca2+] (ionized cal- was adjusted from below normal to normal. cium raised approximately 90% above normal and held at that level for 15 min). Since still below those obtained with initial hythe changes in aortic flow and stroke volume pocalcemia, we performed the isoproterenol observed at such high [Ca2+] values were experiments to ascertain that the rat heart
62
JOURNAL
OF SURGICAL
RESEARCH: VOL. 32, NO. 1, JANUARY
I Oni
I
I
0.2
0.3
1982
STROKE VOLUME (ml/g/beat) FIG. 2. Effect of citrate on the relationship between [ Ca’+] and stroke volume. The solid data points represent those obtained prior to citrate infusion, the open symbols those following citrate. The arrows indicate the direction in which the changes occurred. There was cessation of mechanical activity of the heart at a [Ca”] value of approximately 0.5 mM.
as used in this study was capable of a substantial increase in mechanical performance at normal [ Ca’+], given another potent stimulus. In view of the substantial alterations in mechanical function seen with isoproterenol, it seems that the relatively small changes with increased [Ca2’] from a normal baseline value is characteristic for the chances in extracellular calcium. This difference in response is striking because the action of catecholamines is thought to be also mediated through calcium flux into the cell. In our experiments, cessation of aortic
how occurred at a [Ca”] value ranging between 0.46 and 0.53 mM. These findings of a profound depression of function are of interest for two reasons. First, McLean and Hastings [ lo] also defined this hypocalcemia range as being incompatible with sustained cardiac action of the frog heart, thus suggesting that the response to severe hypocalcemia in two species is similar. With intact circulation, however, it is possible that mechanical performance of the heart, although impaired, may be sustained despite these low [Ca2+] levels, because of increased sympathetic activity. Second, Forester and Main-
KUGIMIYA,
DROP, AND EWALENKO:
[Ca’+] AND CARDIAC
MECHANICAL
63
FUNCTION
TABLE 1 COMPARATIVE EFFECTS OF IONIZED HYPERCALCAEMIA (WITHIN THE CLINICAL RANGE) AND ISOPROTERENOL ON THE BLOOD-PERFUSED RAT HEARTY
Perfusate [ Ca’+] (rnM) Normocalcemia 0.99 f 0.02 Hypercalcemia 1.50 + 0.05 Normocalcemia 1.03 no isoproterenol Normocalcemia 1.04 isoproterenol (4.5 pg/l)
n
AF (ml/ min/d
SV (ml/beat/g)
HR (bpm)
&oio, (ml/min/ 9)
CBF
(ml/min/s)
6
64?6
0.253 -r- 0.005
286 + 17
0.212 4 0.014
4.5 * 0.4
6
74 + 8***
0.265 f 0.008***
292 f 19
0.236 2 O.Oll***
5.0 2 0.4**
5
50?9
0.19 f 0.003
263 f 10
0.206 2 0.018
4.3 f 0.4
5
67 + 8***
0.224 + 0.007***
298 f i4***
0.446 f 0.031***
11.1 f 0.7*
Note. Comparative effects of ionized hypercalcemia (clinical hypercalcemia range) and isoproterenol on the blood-perfused rat heart. To ascertain the ability of the rat heart preparation to substantially augment function at an initially normal [ Cazf] level, hemodynamic variables during an hypercalcemic level (Group B) were compared with those at an isoproterenol, concentration of 4.5 pglliter. With isoproterenol, changes in aortic flow (AF), stroke volume (SF), heart rate (HR), myocardial oxygen consumption (MfoioJ, and coronary blood flow (CBF) were more pronounced than those observed with the ionized hypercalcemia levels. a In both instances, initial [Ca”] was normal. * P < 0.001. ** P < 0.05. *** P < 0.025.
wood [ 71 suggested that the rat myocardial response to [ Ca*+] alterations is comparable to those of other species only in the [Ca”] range of 0.2 to 0.4 mM, but derived these conclusions from data obtained in the stimulated rat heart papillary muscle. Although the limitations of comparison between isolated papillary muscle experiments and the whole heart is recognized, our results might not have been predicted from these previous data. The normal [Ca*+] value is in good agreement with those obtained previously [I] and close to values in the dog [ 41 and man [ 31. However, Forester and Mainwood [7] reported normal values in the rat which were above ours. The reason for this difference remains unclear. Finally, caution is appropriate when attempting to extrapolate our findings to the clinical situation. First, our results were obtained in the isolated, working rat heart when [Ca”‘] values were known and main-
tained in a steady-state. These conditions are different from those present following calcium bolus infusion (as practiced clinically), which is typically associated with an unsteady state. Second, the possibility of species differences may be considered. REFERENCES Bernstein, D. S., Aliapoulios, M. A., Hattner, R. S., Wachman, A., and Rose, B. Serum calcium ion activity: Effects of thyrocalcitonin and parathyroid extract in the rat. Endocrinology 85: 589, 1969. Chitwood, W. R., Sink, J. D., Hill, R. C., Wechsler, A. S., and Sabiston, D. C. The effect of hypothermia on myocardial oxygen consumption and transmural coronary blood Row in the potassium arrested heart. Ann. Surg. 190: 106, 1979. 3. Drop, L. J., Fuchs, C., and Stulz, P. M. Determination of blood ionized calcium in a large segment of the normal adult population. Clin. Chim. Acta 89: 503, 1978. 4. Drop, L. J., Geffin, G. A., O’Keefe, D. D., Chaffip, J. S., and Daggett, W. M. Left ventricular perfor-
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JOURNAL
OF SURGICAL
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mance during sustained hypo- and hypercalcemia. Surg. Forum 29: 259, 1978. Duvelleroy, M. A., Duruble, M., Martin, J. L., Tesseire, B., Droulez, J., and Cain, M. Blood-perfused working isolated rat heart. J. Appl. Physiol. 41: 603, 1976.
Feinberg, H. E., Boyd, E., and Katz, L. N. Calcium effect on performance of the heart. Amer. J, Physiol. 202: 643, 1962. Forester, G. V., and Mainwood, G. W. Interval dependent inotropic effects in the rat myocardium and the effect of calcium. Pjluegers Arch. 352: 189, 1914. Grassi, A. O.., Perez-Alzueta, A. D.. and Cingolani, H. E. Effect of isoproterenol on relation between maximal rate of contraction and maximal rate of relaxation. Amer. J. Physiol. 233: H404, 1977. Langendorff, 0. Untersuchungen am Uberlebenden Saugethierherzen. Arch. Ges. Physiol. 61: 291, 1895.
1982
10. McLean, F. C., and Hastings, A. B. A biological method for the estimation of calcium ion concentration. J. Biol. Chem. 107: 331, 1934. 11. Mehmel, H., Krayenbiihl, H. P., and Rutishauser, W. Peak measured velocity of shortening in the canine left ventricle. J. Appl. Physiol. 29: 637, 1970. 12. Sarnoff, S. J., Gilmore, J. P., McDonald, R. H., Daggett, W. M., Weisfeldt, M. L., and Mansfield, P. B. Relationship between myocardial K+ balance, O2 consumption and contractility. Amer. J. Physiof. 211: 361, 1966.
13. Scheidegger, D., Drop, L. J., and Schellenberg, J. C. Role of the systemic vasculature in the hemodynamic response to changes in plasma ionized calcium. Arch. Surg. 115: 206, 1980. 14. Siegel, J. H., Sonnenblick, E. H., Judge, R. D., and Wilson, R. D. The quantification of myocardial contractility in dog and man. Cardiologia 45: 189, 1964.
OF SURGICAL
32, 57-64 (1982)
RESEARCH
Mechanical
Performance Response
of the Working to Calcium’
T. KUGIMIYA, M.D.,2 L. J. DROP,M.D., Anesthesia
PH.D.,~
AND
Rat Heart:
P. EWALENKO,
Services of the Massachusetts General Hospital and rhe Department Harvard Medical School, Boston, Massachusetts 02I 14 Submitted
for publication
M.D.4
of Anaesthesia,
December 26, 1980
Although calcium is generally considered a substance that can enhance cardiac pump performance, few data exist to demonstrate such effect when plasma ionized calcium is varied in the clinical concentration range. We have studied the relationship between perfusate ionized calcium concentration ([Ca”]) and myocardial mechanical performance in the isolated, blood perfused, ejecting rat heart. With pre- and afterload near constant, observations were made before and after steadystate increases in [Ca’+] from low (mean ? SEM: 0.74 * 0.01 mM) to normal (1.01 rt 0.04 mM); from low (1.01 f 0.02 mM) to higher than normal (1.45 ?I 0.04 mM, i.e., within the clinical hypercalcemia range); and to an excessive value (1.94 -t 0.03 mM). When [Ca*+] was increased from low to normal levels, aortic blood flow and stroke volume increased by approximately 25% of control (P < 0.025); heart rate did not change significantly. In contrast, when [Ca”] was raised from 1.03 mM (normal value) to 1.45 mM (within the clinical hypercalcemia range) or to 1.94 mM (above the clinical hypercalcemia range), the changes in aortic flow and stroke volume were less (P-c 0.05) as compared to those observed when ionized calcium was adjusted from a level below normal. We conclude that the isolated, blood-perfused heart with pre- and afterload near constant, responds to sustained increases in perfusate ionized calcium with increased aortic flow and stroke volume, but that these changes are disproportionally small when ionized calcium is elevated to above normal from a normal baseline value.
INTRODUCTION
The importance of the calcium ion for the myocardial contractile process has been recognized for almost a century. Although calcium is considered as a substance that can enhance myocardial performance [ 6, 12, 141, there are only few quantitative data on such effects when ionized calcium is varied in the clinical concentration range. Clinically, periods of hypercalcemia can occur following calcium infusion, initiated in anticipation of improved hemodynamic ’ Supported in part by United States Public Health Service (USPHS) Biomedical Research Supporting Grant RR-05486- 14 and USPHS Anesthesiology Training Grant S-T-01-GM 01273-13. ’ Present address: Department of Anesthesia, Faculty of Medicine, University of Tokyo, Hongo, Tokyo, Japan. ’ Address for reprints: L. J. Drop, Department of Anesthesia, Massachusetts General Hospital, Boston, Massachusetts 02114. ‘Present address: Institut Jules Bordet, lOOO-Bruxelles, Belgium.
performance. A consistent observation is the elevation of arterial blood pressure following calcium infusion[ 131. Since arterial blood pressure is a major determinant of ventricular function, alterations in blood pressure render interpretation of calcium-induced changes in performance of the heart difficult. The classical isolated frog heart study of McLean and Hastings [lo] demonstrated that it is the ionized moiety of plasma calcium that is physiologically active. Their data suggest that, whereas in the ionized calcium range from below normal to normal, elevation of ionized calcium was associated with enhanced amplitude of ventricular contraction, that variable was not consistently enhanced with calcium ion concentrations above normal. In these studies, the frog heart was perfused with red cell free electrolyte solutions. Using a calcium-selective electrode system for ionized calcium measurements, we
51
0022-4804/82/010057-08$01.00 Copyright 0 1982 by Academic Press, Inc. ALI rights of rcprodumon in any form rewrvsd.
58
JOURNAL OF SURGICAL
RESEARCH: VOL. 32, NO. 1, JANUARY
wanted to quantitate the response of the isolated ejecting rat heart preparation that was perfused with erythrocyte perfusate, to acute increases in ionized calcium, initiated from ionized calcium levels corresponding to either hypocalcemia and normocalcemia. In our experiments, pre- and afterload were held near constant. METHODS Blood-perfused
rat
heart
preparation.
The technique for use of the blood-perfused, isolated working rat heart has been described previously by Duvelleroy et al. [5] Briefly, rats of either sex (body weight ranging from 200 to 400 g) were anesthetized by inhalation of ether in ambient air. Following thoracotomy and systemic heparinization (2.5 mg), the heart was excised quickly with segments of the aorta, pulmonary artery, and vein attached, and immediately immersed in an ice-cold heparinized 0.9% sodium chloride solution. The aortic segment (approximately 5 mm in length) was fitted over and tied to the outflow cannula and initial retrograde perfusion of the heart with blood was achieved according to the Langendorff technique [9]. The inflow cannula into the heart was attached to the left atrium, and a catheter was pIaced into the right ventricle to collect coronary sinus blood. An occlusive roller pump (Holter Model RE 161) delivered blood perfusate (vide infra) from a thermostated (37°C) reservoir (250 ml) through a capillary membrane oxygenator (Bio-Fiber 5, Beaker BioRad Laboratories) to a second reservoir (also maintained at 37”C), from which the perfusate flowed by gravity via silastic rubber tubing (3.1 mm i.d.) into the left atrium. Positioning of the second reservoir at a level above the heart permitted the inflow pressure to be held constant at approximately 10 mm Hg. Inflow pressure was recorded via an appropriate transducer (Sanborn 267 BC) connected to the inflow tubing through a stiff-walled side arm. Appropriate blood filters (Ultipor blood filter, Pall Medical,
1982
Inc.) were placed before the oxygenator and the inflow cannula. The aortic cannula which returned the blood to the first reservoir was positioned vertically above the left ventricle to produce a blood column, the height of which was adjustable. The mean outflow pressure was maintained at approximately 80 mm Hg. Values of PO, were above 350 mm Hg and arterial pH and Pc02 were held within narrow limits (pH 7.38-7.41; Pc02, 32-35 mm Hg) by varying the flow rates of 97% oxygen/3% carbon dioxide or 95% oxygen/5% carbon dioxide supplied to the oxygenator. To determine the normal [Ca*+] value in the rat, heparinized blood samples were withdrawn from the great vessels prior to excision of the heart in seven animals. Perfusate. Whole blood was collected from a donor cow and heparinized (2.5 IU/ ml). The red cells were separated, washed, and stored at 4°C until the next day. Immediately prior to the experiment, the red cells were suspended in the Krebs-Hensleit buffer as described previously by Duvelleroy et al. [5]. In all experiments, the final hematocrit was adjusted to 30 f 1%. ANALYSES
AND PROTOCOL
All pressures in the perfusion circuit were recorded continuously on a multichannel direct writing polygraph (Hewlett-Packard) via appropriate transducers (Sanborn 267 BC), which were calibrated at frequent intervals with a mercury manometer. Mean pressure was determined by electronic integration. At each measurement period, recordings were made at a paper speed of 10 mm/set. Heart rate (HR) was calculated from the tracing. Coronary (CBF) and aortic (AF) blood flows (ml/min) were determined by timed volumetric collection. Stroke volume (SI/, ml/beat) was calculated by dividing blood flow per minute (AF + CBF) by heart rate. Blood oxygen concentration was determined in arterial and coronary sinus blood by use of the Lex-O-Con device. Recent data
KUGIMIYA,
DROP, AND
EWALENKO:
[Ca”]
AND
by Chitwood et al. [2] have demonstrated a close correlation between results obtained by this technique and those by manometric technique of Van Slyke and Neill, over the physiologic range. Blood gases and pH were measured by standard electrodes at 37°C. Plasma ionized calcium concentration was measured in a specimen of whole blood using a calcium-selective electrode system [ 31. Hematocrit, serum electrolytes, and osmolality were measured using standard analytical techniques. Myocardial oxygen consumption (&02, ml/min) was calculated as the product of coronary blood flow and arterial-to-coronary sinus blood oxygen content difference and expressed per gram LV weight. The contribution of the empty, nonworking right ventricle and atria to changes in MJ&, was ignored. At the conclusion of the experiment, the atria and right ventricle were cut away, the heart was blotted and weighed. Protocol. In each experiment, the reservoir was filled with reconstituted cow blood (pH adjusted to 7.40 + 0.01 by addition of sodium bicarbonate) and calcium chloride was added to a final [Ca”] value of 1.03 +- 0.02 mM. Following assembly of the circuit, hemodynamic and biochemical measurements were made at 10, 12, and 15 min to ascertain a steady state. Three groups of experiments were performed. In two groups, a new [Ca’+] plateau was established by addition of CaCl, to the perfusate in sufficient amounts to adjust [Ca”] to approximately 1.45 (mean value, Group B) or 1.95 mM (mean value, Group C). In the other group (Group A), the initial normocalcemic perfusate was first exchanged for blood perfusate in which [Ca”‘] had been adjusted to approximately 0.7 mit4. Hemodynamic and biochemical measurements were made at 12 and 15 min after this [Ca2+] plateau (0.7 mM) was established, following which [ Ca2+] was adjusted to approximately 1.05 (Group A). Measurements were then made again at the same time intervals. Of particular value was the fact that [Ca2+] values were known
CARDIAC
MECHANICAL
FUNCTION
59
within 3 min following blood sampling, permitting appropriate adjustment of [Ca’+] in the perfusate. Previous data [7] have suggested that the inotropic response of the rat heart to the addition of calcium is comparable to that of other species only when extracellular calcium levels are low (0.2-0.4 mA4). In view of these results, we wanted to define the performance of the ejecting rat heart at those [ Ca2+] levels. To achieve the low [ Ca2+] values, different quantities of a buffered (pH 7.40) citrate solution (acid-citrate-dextrose, Formula A, USP) were added to the perfusate in two experiments of each group and observations recorded. Citrate solution was titrated to pH 7.40 by addition of THAM [ tris(hydroxemethyl)aminomethane]; thus, a possible effect of a low pH could be excluded. Since the data on different [Ca”] levels were obtained in different animal groups, it was desirable to also study different [Ca”] levels in the same rat heart preparation. Thus, three additional animals were studied. In these studies, initial data were collected after a 15-min stabilization period (perfusate [Ca’+] = 1.03 mM). Three different [Ca”] were achieved, first by exchange of the normocalcemic perfusate with a blood perfusate in which [Ca’+] had been adjusted to 0.70 mM, followed by addition of CaC12, resulting in step increases in [Ca2+]. Each of these [Ca’+] plateaus was maintained for 15 min. The first experimental [Ca’+] level (0.70 mM) was then restored by use of an aliquot of corresponding perfusate. In view of the small changes in mechanical performance of the rat heart to calcium elevation from a normal baseline value, we wanted to ascertain its ability (under the conditions of the study) to substantially increase performance when [Ca”] was known to be normal. Thus, in five additional experiments, hemodynamic variables were recorded prior to and following additions of isoproterenol (final concentration, 4.5 pg/liter) to the perfusate in which [Ca”] was normal ([Ca2’] = 1.03 +- 0.02 mM). This
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isoproterenol concentration had been selected to be in the range which has been previously demonstrated [ 81 to provide a significant increase in maximal velocity of contraction of the rat heart. In all experiments the changes in hemodynamic variables, expressed per gram of LV weight, were normalized (changes in percentage of control). Data of different groups were compared using Student’s t test for comparison of group means; within-group means, data were compared using Student’s t test for paired data. Values are expressed as mean -t SEM. RESULTS
The results on changes observed in the different experimental groups are presented in Fig. 1. Hypo-
to normocalcemia
(Group
A, n
= 5). Following an increase in mean [Ca”] (from 0.74 + 0.01 to 1.01 + 0.04 mM), AF increased (25.2 f 4.51%, P < 0.025), Why 25.3 + 7.7% (P < 0.05) and Mvo, by 13.7 f 5.4% (P < 0.05). There were no significant changes in HR or CBF.
1982
Several [Ca”] levels in the same rat heart. Increasing [Ca”] (from 0.70 f 0.01
to 1.03 f 0.02 mM) was associated with increased aortic flow (from 69 -t 1 to 89 f 1 ml/min/g); further increase in [Ca”] (to 1.50 -C 0.05 mM) produced a lesser increase (from 89 + 1 to 93 + 1 ml/min/g). Myocardial oxygen consumption increased from 0.219 -+ 0.008 to 0.251 + 0.003 ml/ min/g when [Ca”] increased from 0.70 to 1.03 mM, and the increase in Mvo, was from 0.251 f 0.003 to 0.293 + 0.007 ml/min/g when [Ca”] was increased further, to 1.50 mM. Following these step increasesin [ Ca*+], the initial experimental [Ca”] (0.70 mM) was restored and all measured variables returned toward their respective baseline values. Isoproterenol (n = 5). Addition of isoproterenol to the perfusate caused substantial rises in AF (P < 0.025) HR (P < 0.025, and SV (P < 0.025) (Table 1). Mr’o, increased by 100% and CBF nearly tripled (P < 0.001). Blood gases and biochemical
variables.
In all experiments, Pao, was above 350 mm Hg, Pace, ranged between 32 and 36 mm Hg, and pH between 7.38 and 7.41. Na ranged from 153 to 156 mM and K, from to moderate hypercalcemia Normo4.9 to 5.0 mM. Osmolality values ranged (Group B, n = 6). An increase in [Ca”] from 1.01 + 0.02 to 1.45 + 0.04 mM was asso- from 310 to 312 mOsm. These values reciated with increased AF (by 13.2 -C 2.99%, mained essentially unchanged during each experiment. P < 0.025). This change was less (P < 0.05) Normal [Ca”] values. The mean [Ca*‘] than that recorded in Group A. Mr’o, rose value (n = 7) in the rat during ether in room by 10.4 + 3.8% (P < 0.05). CBF increased air anesthesia immediately following openby 11.7 +_ 3.2% (P < 0.05). Changes in HR ing of the chest was 1.03 + 0.03 mM. and SV did not reach significance. Normo- to pronounced hypercalcemia (Group C, n = 6). An increase in [Ca”]
(from 0.95 f 0.03 to 1.94 * 0.05 mM) was associated with alterations in AF, CBF, and MV02 that were similar to those recorded in Group B. Stroke volume increased by 12.8 f 3% (P < 0.025). Heart rate remained unchanged. Citrate-induced hypocalcemia. In all experiments, stroke volume decreased to near zero when [Ca”] fell in the range of 0.48 to 0.53 mM (Fig. 2).
DISCUSSION
The most important finding of this study is that the response of the isolated, bloodperfused rat heart to acute increases in ionized calcium was dependent on the initial ionized calcium level. The rise in aortic flow and stroke volume was greater when a higher ionized calcium level was instituted from an initially low level as compared to when the initial ionized calcium level was normal.
KUGIMIYA,
DROP, AND
EWALENKO:
[Ca’+]
AND
CARDIAC
MECHANICAL
FUNCTION
61
Although the possibility of interspecies differences is recognized, these findings are consistent with previous data obtained in other animal species. For example, in their classical studies, McLean and Hastings [lo] utilized the frog heart. They found that, while the amplitude of ventricular contraction was proportional to the aqueous perfusate ionized calcium values (ranging from approximately 0.5 to 1.3 mM; their normal value was 1.22 mM), an elevation of ionized calcium above that value did not consistently further augment function. The frog heart is more dependent on extracellular calcium levels because of a poorly developed sarcoplasmic reticulum, an important regulator of intracellular calcium. In the dog, Mehmel and associates [ 111 found that, following calcium chloride infusion in two incremental doses, the second dose resulted in a much smaller change in myocardial dynamics than the first. Assuming that ionized calcium comprised 48% of the total calcium concentration, calcium infusion in these experiments may have resulted in increased ionized calcium from approximately 0.83 to 1.40 and to 1.90 mM, respectively. Our observations, together with these pre109 0 Control vious data, suggest that a calcium-induced l Excwimentol enhancement of cardiac function is more prominent whenever initial ionized calcium levels are below normal. A possible explanation is the impairment of ventricular funco 5 1” NormalRange’ tion that is characteristically associated with hypocalcemia. Whether impaired ventricuA B C n=6 n=5 n=5 lar function from other causes is also a conFIG. 1. Effects of changes in [Ca’+] on myocardial dition for calcium-induced enhancement remechanical and metabolic function of the blood-permains to be determined. fused, isolated rat heart. In Group A, [Ca”] was raised Our present findings of small changes in from 0.70 + 0.01 (open symbol) to 1.01 f 0.04 mechanical cardiac performance with insti- mM(solid symbol). Aortic flow (AF) and stroke volume tution of ionized hypercalcemia ([ Ca2+] (Sv) increased on the average by 25%. In contrast, when raised to 150% of normal and held at that [Ca’+] was raised from 1.01 + 0.02 mM to 1.45 f 0.04 level for 15 min) led us to perform the ex- (Group B) or 1.94 k 0.05 mM (Group C), changes in AF and SV were smaller as compared to when [Ca”] periments with excessive [ Ca2+] (ionized cal- was adjusted from below normal to normal. cium raised approximately 90% above normal and held at that level for 15 min). Since still below those obtained with initial hythe changes in aortic flow and stroke volume pocalcemia, we performed the isoproterenol observed at such high [Ca2+] values were experiments to ascertain that the rat heart
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I Oni
I
I
0.2
0.3
1982
STROKE VOLUME (ml/g/beat) FIG. 2. Effect of citrate on the relationship between [ Ca’+] and stroke volume. The solid data points represent those obtained prior to citrate infusion, the open symbols those following citrate. The arrows indicate the direction in which the changes occurred. There was cessation of mechanical activity of the heart at a [Ca”] value of approximately 0.5 mM.
as used in this study was capable of a substantial increase in mechanical performance at normal [ Ca’+], given another potent stimulus. In view of the substantial alterations in mechanical function seen with isoproterenol, it seems that the relatively small changes with increased [Ca2’] from a normal baseline value is characteristic for the chances in extracellular calcium. This difference in response is striking because the action of catecholamines is thought to be also mediated through calcium flux into the cell. In our experiments, cessation of aortic
how occurred at a [Ca”] value ranging between 0.46 and 0.53 mM. These findings of a profound depression of function are of interest for two reasons. First, McLean and Hastings [ lo] also defined this hypocalcemia range as being incompatible with sustained cardiac action of the frog heart, thus suggesting that the response to severe hypocalcemia in two species is similar. With intact circulation, however, it is possible that mechanical performance of the heart, although impaired, may be sustained despite these low [Ca2+] levels, because of increased sympathetic activity. Second, Forester and Main-
KUGIMIYA,
DROP, AND EWALENKO:
[Ca’+] AND CARDIAC
MECHANICAL
63
FUNCTION
TABLE 1 COMPARATIVE EFFECTS OF IONIZED HYPERCALCAEMIA (WITHIN THE CLINICAL RANGE) AND ISOPROTERENOL ON THE BLOOD-PERFUSED RAT HEARTY
Perfusate [ Ca’+] (rnM) Normocalcemia 0.99 f 0.02 Hypercalcemia 1.50 + 0.05 Normocalcemia 1.03 no isoproterenol Normocalcemia 1.04 isoproterenol (4.5 pg/l)
n
AF (ml/ min/d
SV (ml/beat/g)
HR (bpm)
&oio, (ml/min/ 9)
CBF
(ml/min/s)
6
64?6
0.253 -r- 0.005
286 + 17
0.212 4 0.014
4.5 * 0.4
6
74 + 8***
0.265 f 0.008***
292 f 19
0.236 2 O.Oll***
5.0 2 0.4**
5
50?9
0.19 f 0.003
263 f 10
0.206 2 0.018
4.3 f 0.4
5
67 + 8***
0.224 + 0.007***
298 f i4***
0.446 f 0.031***
11.1 f 0.7*
Note. Comparative effects of ionized hypercalcemia (clinical hypercalcemia range) and isoproterenol on the blood-perfused rat heart. To ascertain the ability of the rat heart preparation to substantially augment function at an initially normal [ Cazf] level, hemodynamic variables during an hypercalcemic level (Group B) were compared with those at an isoproterenol, concentration of 4.5 pglliter. With isoproterenol, changes in aortic flow (AF), stroke volume (SF), heart rate (HR), myocardial oxygen consumption (MfoioJ, and coronary blood flow (CBF) were more pronounced than those observed with the ionized hypercalcemia levels. a In both instances, initial [Ca”] was normal. * P < 0.001. ** P < 0.05. *** P < 0.025.
wood [ 71 suggested that the rat myocardial response to [ Ca*+] alterations is comparable to those of other species only in the [Ca”] range of 0.2 to 0.4 mM, but derived these conclusions from data obtained in the stimulated rat heart papillary muscle. Although the limitations of comparison between isolated papillary muscle experiments and the whole heart is recognized, our results might not have been predicted from these previous data. The normal [Ca*+] value is in good agreement with those obtained previously [I] and close to values in the dog [ 41 and man [ 31. However, Forester and Mainwood [7] reported normal values in the rat which were above ours. The reason for this difference remains unclear. Finally, caution is appropriate when attempting to extrapolate our findings to the clinical situation. First, our results were obtained in the isolated, working rat heart when [Ca”‘] values were known and main-
tained in a steady-state. These conditions are different from those present following calcium bolus infusion (as practiced clinically), which is typically associated with an unsteady state. Second, the possibility of species differences may be considered. REFERENCES Bernstein, D. S., Aliapoulios, M. A., Hattner, R. S., Wachman, A., and Rose, B. Serum calcium ion activity: Effects of thyrocalcitonin and parathyroid extract in the rat. Endocrinology 85: 589, 1969. Chitwood, W. R., Sink, J. D., Hill, R. C., Wechsler, A. S., and Sabiston, D. C. The effect of hypothermia on myocardial oxygen consumption and transmural coronary blood Row in the potassium arrested heart. Ann. Surg. 190: 106, 1979. 3. Drop, L. J., Fuchs, C., and Stulz, P. M. Determination of blood ionized calcium in a large segment of the normal adult population. Clin. Chim. Acta 89: 503, 1978. 4. Drop, L. J., Geffin, G. A., O’Keefe, D. D., Chaffip, J. S., and Daggett, W. M. Left ventricular perfor-
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mance during sustained hypo- and hypercalcemia. Surg. Forum 29: 259, 1978. Duvelleroy, M. A., Duruble, M., Martin, J. L., Tesseire, B., Droulez, J., and Cain, M. Blood-perfused working isolated rat heart. J. Appl. Physiol. 41: 603, 1976.
Feinberg, H. E., Boyd, E., and Katz, L. N. Calcium effect on performance of the heart. Amer. J, Physiol. 202: 643, 1962. Forester, G. V., and Mainwood, G. W. Interval dependent inotropic effects in the rat myocardium and the effect of calcium. Pjluegers Arch. 352: 189, 1914. Grassi, A. O.., Perez-Alzueta, A. D.. and Cingolani, H. E. Effect of isoproterenol on relation between maximal rate of contraction and maximal rate of relaxation. Amer. J. Physiol. 233: H404, 1977. Langendorff, 0. Untersuchungen am Uberlebenden Saugethierherzen. Arch. Ges. Physiol. 61: 291, 1895.
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10. McLean, F. C., and Hastings, A. B. A biological method for the estimation of calcium ion concentration. J. Biol. Chem. 107: 331, 1934. 11. Mehmel, H., Krayenbiihl, H. P., and Rutishauser, W. Peak measured velocity of shortening in the canine left ventricle. J. Appl. Physiol. 29: 637, 1970. 12. Sarnoff, S. J., Gilmore, J. P., McDonald, R. H., Daggett, W. M., Weisfeldt, M. L., and Mansfield, P. B. Relationship between myocardial K+ balance, O2 consumption and contractility. Amer. J. Physiof. 211: 361, 1966.
13. Scheidegger, D., Drop, L. J., and Schellenberg, J. C. Role of the systemic vasculature in the hemodynamic response to changes in plasma ionized calcium. Arch. Surg. 115: 206, 1980. 14. Siegel, J. H., Sonnenblick, E. H., Judge, R. D., and Wilson, R. D. The quantification of myocardial contractility in dog and man. Cardiologia 45: 189, 1964.