J
Mol Cell Cardiol22,697-705
(1990)
Effects of Hypoxia, on Repriming
Motoyoshi
Shim&u,
Acidosis, and Simulated of Caffeine Contracture Myocardium* Shinichi and Arthur
Kimura, Robert L. Bassett
Ischemia in Rat
J. Myerburg
Departments of Pharmacology and Medicine (Division of Cardiology), University of Miami Schpol of Medicine, Miami, FL 33101, USA (Received 28 April 1989, accepted in revisedform
19 January 1990)
M. SHIMIZU, S. KIMURA, R. J. MYERBURG, AND A. L. BASSETT. Effects of Hypoxia, Acidosis, and Simulated Ischemia on Repriming of Caffeine Contracture in Rat Myocardium. Journal of Molecular and Cellular Cardiology (1990) 22,697-705. This study was designed to examine the effects ofhypoxia, acidosis, glucose-free medium and their combination on contraction and sarcoplasmic reticulum (SR) function in rat ventricular trabeculae. The isometric twitch tension was measured during superfusion with hypoxic (PO2 < 30 mmHg), acidic (pH 6.80), glucose-free, or their combined (“ischemic”) Tyrode’s solution at 20°C. The time needed to fully recover the contraction induced by 10 mn caffeine (repriming time) was measured to indirectly estimate the Ca2+ uptake of the SR. In “ischemia” and acidosis, the peak developed tension decreased progressively for the first 30 min (37.6 _+ 9.2% and 56.6 + 8.4% ofcontrol at 30 min, respectively), and then became steady. In hypoxic solution, the peak developed tension decreased moderately for the first 30 min (86.8 + 4.8% of control at 30 min), and thereafter remained steady. Developed tension did not change significantly during 60 min of superfusion with glucose-free solution. The repriming time of caffeine contraction was significantly delayed in both “ischemic” and hypoxic solutions, but was unchanged in acidic and glucose-free solutions. These results lead us to suggest to the decline in tension in ischemia and that depressed SR function to accumulate Ca’ + may contribute hypoxia, but that other mechanisms are important in the tension decline induced by acidosis. KEY WORDS: Contractile
function;
Sarcoplasmic
reticulum;
Introduction Myocardial &hernia and hypoxia are initially characterized by a decrease in cardiac developed tension without change in resting tension. Subsequently, developed tension continues to decrease and resting tension starts to increase, and then contracture may develop with a significant increase in resting tension without the capability to generate active tension [28]. The late phase with an increase in resting tension is probably due to rigor resulted from ATP depletion [.?3]. The initial phase, characterized by a decrease in developed tension without change in resting tension, has been postulated to be due to a
Caffeine;
Hypoxia;
Acidosis.
breakdown in the excitation-contraction coupling system [I, 181, but the mechanisms underlying the mechanical properties in the initial phase of ischemia and hypoxia are not well understood. The sarcoplasmic reticulum (SR) plays a. prominent role in the activation and relax-, ation of the contractile apparatus [&YJ, and changes in its functional properties could result in the loss of regulation of [Ca2+li and a decrease in tension development. Caffeine at high concentrations induces transient contracture by releasing Ca2 + from the SR [2, II, 241. Caffeine cannot induce contracture in rapid succession and time is
*This work was supported by National Heart, Lung, and Blood Institute grants HL-19044 and HL-21735, and Grants-in-Aid from the American Heart Association, Greater Miami Chapter and Florida Affiliate, and a program grant from the Florida Affiliate. Please address all correspondence to: Arthur L. Bassett, Department of Pharmacology, University of Miami School of Medicine, Miami, FL 33101, USA. 0022-2828/90/060697
+ 09 $03.00/O
0 1990 Academic
Press Limited1
698
M. Shimizu
necessary to reprime the SR with Ca’+ [II]. The repriming (recovery time) of caffeine contracture reflects in an indirect fashion the rate of Ca2+ uptake by the SR [Z, II]. In the present study, we examined the effects of hypoxia, acidosis, glucose-free medium and their combination (simulated ischemia) on the repriming of caffeine contracture in a limited experimental circumstance to gain further insight into the mechanisms of contractile dysfunction during the early phase of &hernia, hypoxia, and acidosis.
Methods Preparations
and solutions
Male Sprague-Dawley rats (280-400 g) were anesthetized with sodium pentobarbital (35 mg/kg, i.p.), and treated with heparin (200 IU, i.p.). After the thorax was opened, the heart was rapidly removed, and placed in a modified Tyrode’s solution gassed with 0s at 20°C. Small free-running trabeculae, 100-300 urn in diameter and 1-5 mm long, were isolated from the left ventricle under a dissecting microscope. The preparation was mounted in a tissue bath designed for measurement of isometric tension and rapid change of the and with superfusate, was superfused oxygenated Tyrode’s solution at a rate of 20 ml/min. The preparation was stimulated by a Grass Model S88 stimulator delivering 5 ms square wave pulses through a pair of platinum plate electrodes (field stimulation) at a rate of 12/min. Stimulation voltages were set at twice threshold. After a 20-30 min equilibration period, muscle length was adjusted in steps by a micromanipulator to obtain maximum tension development. The tension was recorded on a polygraph (Grass, model 79). Preparations that developed oscillations in the resting tension or spontaneous activity were discarded. The normal modified Tyrode’s solution had the following composition (in mM): NaCl 140.0, KC1 4.0, CaClz 1.8, MgClz 1.0, NaH2P04 1.8, glucose 5.5, HEPES 5.0 (pH 7.4 with NaOH), and was continuously gassed with lOOo/o 0s (Paz > 500 mmHg). The acidic Tyrode’s solution was gassed with 100% 02, and its pH was adjusted to 6.8 with HCl. Glucose-free Tyrode’s solution was
et al.
identical to normal Tyrode’s solution except that it contained no glucose. The hypoxic Tyrode’s solution had the identical composition as the normal Tyrode’s solution, but was gassed with lOOr$‘o Nz(Po2 < 30 mmHg). The “ischemic” solution contained no glucose; its pH was adjusted to 6.8 and it was gassed with 100% Ns. Each preparation was exposed to only one test solution. Experimental
protocol
Protocol 1. In the first series of experiments, we monitored peak developed tension during super-fusion with hypoxic, acidic, glucose-free, and “ischemic” solutions. The preparations were electrically stimulated at 0.2 Hz. After a 30 min equilibration period in normal Tyrode’s solution, each preparation was superfused with a test solution at a rate of 20 ml/min for 60 min, followed by 30 min washout with normal Tyrode’s solution; twitch tension was monitored continuously. Protocol 2. The contracture evoked by the sudden application of caffeine to isolated cardiac muscle is transient, and it relaxes spontaneously even in the presence of caffeine [2, II]. When rat ventricular muscle is returned to caffeine-free solution, a second application of caffeine, made after a minute, induces a much smaller response. The full recovery, or repriming, of the subsequent caffeine contracture on rat ventricle is generally complete after 3-7 min in caffeine-free solution at 20°C after a prior caffeine contracture [2, II]. This repriming time reflects indirectly the rate of Ca’ + uptake by the SR [2, U]. We measured the repriming time of caffeine contracture to estimate the ability of the SR to accumulate Ca2+. The time course of the repriming of caffeine contracture was determined by varying the time between caffeine application [2]. As schematically shown in Figure 1 panel (A) each preparation was exposed to 10 mM caffeine at time 0 and then at intervals of 1, 3, 5, 7, 10 min; each caffeine contracture was followed by washout with caffeine-free solution (continuous super-fusion at a rate of 20 ml/min). The time interval (1, 3, 5, 7, and 10 min) was measured from the time of the prior
Caffeine
IHHl---if--dl--‘+-ll I min 3 min 5 min o
b
c
7 min d
30 min Superfused
with normal
e
Response
IO min f
30min Tyrode’s
solution
c.e Normal
Tyrode’s
solution
Test solution
Normal
Tyrode’s
solvt~on
FIGURE 1. Schematic representation of the experimental protocols. Panel A shows the protocol for the repriming of the caffeine contracture. Upward arrows (7): application of ICI rn~ caffeine solution. Downward arrows (1): washout with caffeine-free solution. a: initial caffeineinduced contracture; b: contracture induced 1 min after initial (a) caffeine contracture; c: contracture induced 3 min after prior (b) caffeine contracture; d: contracture induced 5 min after prior (c) caffeine contracture; e: contracture induced 7 min after prior (d) caffeine contracture; f: contracture induced 10 min after prior (e) caffeine contracture. Contractures e and fwere induced only when full recovery was not observed at d, contracture induced 5 min after the prior application of caffeine; recovery of full (control) magnitude of caffeine contracture was usually complete at 5 min. Panel B shows the protocol used in testing reproducibility of repriming of caffeine contracture. Normal Tyrode’s solution was used to superfuse each preparation throughout this protocol. The tests of repriming shown in panel A were repeated at intervals of 30 mins. “At ” , ‘ 0.93. We calculated the slope of the exponential regression line for each intervention.
Tension development during intervention Figure 2 shows the time course of the changes in tension development during superfusion with various test solutions. In “ischemic” solution, the peak developed tension was progressively reduced for the first 30 min (37.6 f 9.2% of control at 30 min), and then became constant (35.2 f 9.3% of control at 60 min). The tension returned to normal within 30 min of washout with normal Tyrode’s solution. Hypoxic solution increased the peak developed tension slightly for the first 5 min (104.8 + 3.1 o/o of control at 5 min). Then, tension declined to 86.8 k 4.8% of control at 30 min. Developed tension remained constant during the final 30 min of superfusion with hypoxic solution (81.2 f 5.5% of control at 60 min). During exposure to acidic solution, the peak developed tension decreased progressively for the first 30 min, and then became constant (56.6 f 8.4% and 52.6 + 7.3% of control at 30 min and 60 min, respectively). The tension exceeded control values immediately after reexposure to normal Tyrode’s solution but returned to normal within 10 min of initiating washout.
IIII 03510
I 20
I 30
1 40
The peak developed tension did not change significantly during the entire 60 min period of superfusion with glucose-free solution or 30 min washout (data not shown). The repriming
Results
‘: a!
et al.
I 50
wostiout I,, 60510
, 20
I 30
Time (mid FIGURE 2. The time course of the changes in tension development of isolated trabecular preparations from rat left ventricles during 60 min of superfusion with “ischemic” (combined hypoxia, acidosis, and glucosefree), hypoxic, and acidic solutions, and 30 min of “washout” during exposure to normal Tyrode’s solution. Values are expressed as a o/0 of pre-intervention control, and standard errors are indicated by bars.
of caffeine contractwe during superfusion
Figure 3 shows a representative experiment establishing the reproducibility of the rate of repriming in one preparation during exposure to normal Tyrode’s solution. Full recovery of the magnitude of the caffeine contracture (“Al”). When the repriming procedures were repeated at an interval of 30 mins (“AZ” and “As”), the rate of repriming of caffeine contracture was very similar, and full recovery was again observed at 5 min. Such reproducibility of response enabled us to compare the rate of repriming of caffeine contracture obtained before and during exposure to a test solution in each preparation. As shown in Figure 2, the peak developed tension became steady after 30 min of superfusion with any of the test solutions. Thus, the rate of repriming of caffeine contracture could be examined without the influence or association of declining tension. Figure 4 shows representative experiments showing the effects of the test solutions on the repriming of caffeine contracture. Full recovery of caffeine contracture was observed at 5 min in normal Tyrode’s solution (left panels in Fig. 5). However, the caffeine response was not completely recovered at 5 min in “ischemic” (right panel a) and hypoxic (right panel b) solutions. On the other hand, the caffeine response was completely recovered at 5 min in acidic (right panel c) and glucose-free (not shown in Fig. 4) solutions. In Figure 5, the relative caffeine responses are plotted as a function of time between caffeine application (n = 6 each group). In “ischemic” solution, the time to maximum repriming of caffeine contracture was significantly longer than that monitored in control solution [Fig. 6(a)]. Even after washout with normal Tyrode’s solution, the repriming of caffeine contracture was delayed. In hypoxic solution [Fig. 6(b)] the repriming of caffeine contraction was slightly but significantly delayed, and returned to the control after washout with normal Tyrode’s solution. In contrast, acidic solution [Fig. S(c)] or
Caffeine
Response
in Ischemia
701
t
t
Initial
5min
FIGURE 3. Reproducibility of the rate of the repriming of caffeine contracture. The schematic protocol for this experiment is shown in Figure 1 panel B. The preparation was superfused with normal Tyrode’s solution, When thle repriming procedures were repeated at interval of 30 minutes, “Ar”, “As” and “As”, full recovery of caffeine contracture was always recorded at 5 min. Upward arrows (T): application of 10 nm caffeine. Caffeine contractures at 1 and 3 minutes for “Al”, “AZ” and “AJ” series are not shown.
Normal
Tyrode’s Control
Test
(A,)
(A,)
solution Ischemia
Hypowo
(b)
Acidosis
(c 1
J
IO mg
IO s Initial Caffeine
5 min
responses
FIGURE 4. Representative experiments showing the effects of the test solutions on the repriming of caffeine contracture. The protocol for this experiment is shown in Figure 1 panel (C). Full recovery of the subsequent caffeine contracture was observed at 5 min after the prior application of caffeine in each control experiment in normal Tyrode’s and hypoxic test solutions, full recovery was not observed at 5 solution. However during super-fusion with “ischemic” min after the prior application of caffeine. In acidic test solution, full recovery was observed at 5 min after the prior application of caffeine. Test of caffeine repriming during washout is not shown.
M. Shimizu
702
Time
between
et al.
contractlon
(mid
FIGURE 5. Relative caffeine-induced responses are plotted as a function of time between caffeine contractme; protocol is shown in Figure 1 panel C. Control test of repriming in normal Tyrode’s solution (Ai, 0 - 0); intervention (panel a), hypoxic (panel repriming (As, 0 - 0 ); washout repriming (As, A - A). Solutions used were “ischemic” and hypoxic solutions the repriming of caffeine b), acidic (panel c), and glucose-free (panel d) solutions. In “ischemic” contracture required significantly longer time than that noted during control (normal) solution. Acidic and glucose-free solutions did not affect the repriming of caffeine contracture. n = 6 for all groups; *P < 0.05 vs control.
glucose-free solution [Fig. 6(d)] did not affect the repriming time of caffeine contracture. Data on the repriming of caffeine contracture were fitted to a single exponential function, and thus we calculated the slope of the exponential regression line for each intervention (Table 1). The slope was significantly reduced by “ischemic” and hypoxic solutions, but was not changed by acidic nor glucosefree solution. Discussion The present study demonstrates that acidic (pH = 6.8) depresses phasic solution TABLE
1.
Slopes
of the regression
lines
Control “ischemia” hypoxia acidosis glucose-free
0.45 0.35 0.44 0.54
&+ + +
electrically-induced contractions without affecting the repriming of caffeine contracture, while hypoxic and “ischemic” (combined hypoxia, acidosis, and glucose-free) solutions depress both phasic contraction and the repriming of caffeine contracture. “Ischemic” solution is more depressant on the repriming of caffeine contraction than the hypoxic solution alone. Caffeine contracture is thought to reflect Ca2+ release from intracellular (SR) Ca2+ storage sites [Z, II]. Caffeine cannot induce contracture in rapid succession and time is evidently necessary to reprime the SR with Cazf for each successive contracture. Previ-
of caffeine
responses Intervention
0.07 0.02 0.04 0.04
0.23 0.25 0.51 0.49
+ f f +
Washout 0.04* 0.04* 0.05 0.09
0.28 0.42 0.53 0.48
* + f f
0.03 0.02 0.02 0.06
control; The slope (B) was Data are expressed as mean + s.E.; *P < 0.05 versus calculated by using the equation: Y = Ae-s’, where Y : 100 - relative caffeine response (%), A: intercept, t: time. Data on the repriming of caffeine response were fitted to single exponential regression lines. The correlation coefficient of the exponential regression lines was 20.93.
Caffeine
Response
ous work by Chapman and Leoty showed that the repriming of caffeine contracture had a sigmoidal time course, while our data revealed a single exponential function. This discrepancy is likely due to the fact that the repriming of caffeine contracture during the earlier phase (within 1 min) were not examined in the present study. Because of the limitation of time for caffeine tests during superfusion with test solutions (30 min), we could not examine the initial stages of repriming of caffeine contracture. The repriming time may be influenced by various factors including changes in Ca” entry through slow channels and Na+-Ca2+ exchange, CaZf uptake by the SR, and Ca2+ store (or binding) in other sites. Inward Ca2+ currents may be reduced by ischemia and hypoxia [12, 221. However, in our study the preparations were quiescent (not stimulated) during the caffeine test, and, thus, it is unlikely that changes in inward Ca2+ currents are involved in the delayed repriming of caffeine contracture in “ischemic” and hypoxic solutions. The contribution of the Na+-Ca2+ exchange to Ca2 + influx and efflux during ischemia and hypoxia remains uncertain. However, the intracellular Na+ concentration does not increase during the early phase of ischemia and hypoxia [lo, 17, 231. Even if the intracellular Na+ concentration increases, Naf-Ca2+ exchange would result in an increase in the intracellular Cazf concentration. One might predict that repriming time of caffeine contracture is shortened in such a situation. Thus, we suggest that the most probable explanation for the prolongation of the repriming time of caffeine contracture in “ischemic” and hypoxic solutions is that the SR accumulation of Ca2+ is depressed, although we cannot exclude the possibility that changes in intracellular Ca2+ binding sites may be involved in the delayed repriming. The mechanisms responsible for the decline of tension during the early phase of ischemia and hypoxia without concomitant changes in resting tension are not well understood. However, it is quite possible that failure of Ca2+ delivery to the contractile proteins is one of the causes of the decline of tension in ischemia and hypoxia. The sarcoplasmic reticulum serves as the source and sink for coupling Ca2+ in the
in Ischemia
703
excitation-contraction coupling system [4, ,5, f?J. Depression of SR function to release CaZ+ and/or to accumulate CaZf potentially available for release would contribute to the decline of tension. Our data suggest that the ra’te of Ca2+ uptake by the SR was prolonged in “ischemic” and hypoxic solutions. Krause and Hess [12] also have shown biochemically that Ca2+ transport is depressed during the early phase of ischemia in the whole heart homogenate and the isolated SR in dogs. These findings suggest that depressed SR function also may be a causal factor for the decline of tension during early phase of ischemia and hypoxia, although it should be noted that relative importance of the SR in the excitation-contraction coupling system may differ in different species. Depletion of high-energy phosphates ma.y affect the SR function. Kammermeier et al. [.!?I have shown that the free energy change required for maintenance of SR Ca2+ pumping is the highest among all the ion pumping systems. Thus, it is likely that changes in high energy phosphates in ‘5schemic” and hypoxic solution slowed the repriming of caffeine contracture. Although [ATP]i has been shown to fall by only a small amount during the ear1.y phase of ischemia, there may be compartmentation of ATP within the myoplasm, with [ATP] falling to much lower levels in some critical region of the cell [8, IS, 201. It is interesting that acidic solution was more depressant on tension than hypoxic solution, but did not affect the repriming of caffeine contracture. Fabiato and Fabiato [.!i] have shown that SR ability to accumulate Ca2+ is markedly depressed by lowering pH to 6.2 [4] Fabiato [fl also showed that acidosis may modify the Ca 2’ dependency of the Ca’+-induced Ca2+ release. However, oxygenated Tyrode’s solution with pH of 6..8 was used in the present study, which may not have changed intracellular pH significantly enough to alter Ca2+ uptake by the SR.. Deitmer and Ellis reported that the intracellular pH changed linearly with the extralcellular pH by 0.23 pH units/extracellular pH unit change in sheep Purkinje fibers. Assumeing that the intracellular pH is 7.0 at the extracellular pH of 7.4, reduction of the extracellular pH to 6.8 would result in a moderate change in intracellular pH to 6.86. Also, a
704
M. Shim&u
recent study shows that intracellular pH does not fall significantly during superfusion with solution containing Paz 30 mmHg, KC1 9 mM, cyanide 1 mM, and pH 6.5 [15]. Despite the assumed modest fall in intracellular pH, developed tension was markedly depressed by extracellular acidosis (pH 6.8) in the present study. This decline of tension may be explained by pH effects on various sarcolemmal functional properties including Na+-Ca2+ exchange [19], and transmembrane Na+ [251 and Ca2+ currents [21]. More recently, sar-
et al. colemmal Ca2+ binding has also been shown to be affected by the extracellular pH [la]. In conclusion, the results of the present study lead us to suggest that the dysfunction of the SR may contribute to the decline in developed tension during the early phase of ischemia and hypoxia, but that other mechanisms, in addition to the intracellular pH effects on the contractile proteins and SR function, play important roles in the tension decline induced by acidosis.
References 1 2 3 4 5 6 7 a 9 10 11 12 13 14 15 16 17 ia 19 20 21 22
D. G., ORCHARD, C. H. Myocardial Contractile Function During Ischemia and Hypoxia. Circ Res 60, 153-168 (1987). CHAPMAN, R. A., LEOTY, C. The time-dependent and dose-dependent effects of caffeine on the contraction of the ferret heart. J Physio1256, 287-314 (1976). DEITMER, J. W., ELLIS, D. Interactions between the regulation of the intracellular pH and sodium activity of sheep cardiac Purkinje fibers. J Physiol304,471-488 (1980). FABIATO, A., FABIATO, F. Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells. J Physiol249, 469495 (1975). FAEXATO, A., FABIATO, F. Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. J Physio1276, 233-255 (1978). FABIATO, A., FABIATO, F. Calcium-induced release of calcium ions from the cardiac sarcoplasmic reticulum. Am J Physio1245, Cl-Cl4 (1983). FABIATO, A. Use of aequorin for the appraisal of the hypothesis of the release of calcium from the sarcoplasmic reticulum induced by a change of pH in skinned cardiac cells. Cell Calcium 6,95-108 (1985). GEISBUHLER, T., ALTSCHULD, R. A., TREWYN, R. W., ANSEL, A. Z., LAMKA, K., BRIERLEY, G. P. Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells. Circ Res 51: 536-546 (1984). KAMMERMEIER, H., SCHMIDT, P., JUNGLING, E. Free energy change of ATP-hydrolysis: a causal factor of early hypoxic failure of the myocardium? J Mol Cell CardiolI4,267-277 (1982). KLEBER, A. G. Resting membrane potential, extracellular potassium activity, and intracellular sodium activity during acute global &hernia in isolated perfused guinea pig hearts. Circ Res 52,4424150 (1983). KONISHI, M., KURIHARA, S., SAKAI, T. The effects of caffeine on tension development and intracellular calcium transients in rat ventricular muscle. J Physio1355, 605-618 (1984). KOULHARDT, M., KUBLER, M. The influence of metabolic inhibitors upon the transmembrane slow inward current in the mammalian ventricular myocardium. Naunyn-Schmiedebergs Arch Pharmacol290,265-274 (1975). KRAUSE, S. M., HESS, M. L. Characterization of cardiac sarcoplasmic reticulum dysfunction during short-term, normothermic, global ischemia. Circ Res 55, 176-184 (1984). LANGER, G. A. The effect of pH on cellular and membrane calcium binding and contraction of myocardium. Circ R~s 57,374-382 ( 1985). MCCARTHY, J. J., MURPHY, E., STEENBERGEN, C., GETTES, L. S. Investigation ofsimulated ischemia with cyanide in guinea pig myocardium. Circulation 80 [Suppl II] 193 (1989). MEYER, R. A., SWEENEY, H. L., KUSHMERICK, M. J. A simple analysis of the “phosphocreatine shuttle”. Am J Physio1246, C365-C377 (1984). NAKAYA, H., KIMURA, S., KANNO, M. Intracellular K+ and Na+ activities under hypoxia, acidosis, and no glucose in dog hearts. Am J Physiol249, H107&H1085 (1985). NAYLER, W. G., POOLE-WILSON, P. A., WILLIAMS, A. Hypoxia and Calcium. J Mol Cell Cardiol 11, 683-706 (1979). PHILIPSON, K. D., BERSOHN, M. M., NISHIMOTO, A. Y. Effects of pH on Na+-Cazf exchange in canine cardiac sarcolemma vesicles. Circ Res 50, 287-293 (1982). PIERCE, G. N., PHILIPSON, K. D. Binding of glycolytic enzymes to cardiac sarcolemmal and sarcoplasmic reticular membranes. J Biol Chem 260,6862-6878 (1985). SATO, R., NOMA, A., KURACHI, Y., IRISAWA, H. Effects of intracellular acidification on membrane currents in ventricular cells of the guinea pig. Circ Res 57, 553-561 (1985). TANIGUCHI, J., NOMA, A., IRISAWA, H. Modification of the cardiac action potential by intracellular injection of adenosine triphosphate and related substances in guinea-pig single ventricular cells. Circ Res 53, 131-139 (1983). ALLEN,
CaRehe 23 24 25
Response
in Ischemis
705
WALSH, L. G., TOFXEY, J. M. Subcellular electrolyte shifts during in vitro myocardial ischemia and reperkon. Am J Physiol255, H917-H928 (1988). WEBER, A., HERZ, R. The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J Gen Physiol52, 750-759 ( 1968). YATANI, A., BROWN, A. M., AKAIKE, N. Effect of extracellular pH on sodium current in isolated, single rat ventricular cells. J Membr Biol78, 163-168 (1984).
Mol Cell Cardiol22,697-705
(1990)
Effects of Hypoxia, on Repriming
Motoyoshi
Shim&u,
Acidosis, and Simulated of Caffeine Contracture Myocardium* Shinichi and Arthur
Kimura, Robert L. Bassett
Ischemia in Rat
J. Myerburg
Departments of Pharmacology and Medicine (Division of Cardiology), University of Miami Schpol of Medicine, Miami, FL 33101, USA (Received 28 April 1989, accepted in revisedform
19 January 1990)
M. SHIMIZU, S. KIMURA, R. J. MYERBURG, AND A. L. BASSETT. Effects of Hypoxia, Acidosis, and Simulated Ischemia on Repriming of Caffeine Contracture in Rat Myocardium. Journal of Molecular and Cellular Cardiology (1990) 22,697-705. This study was designed to examine the effects ofhypoxia, acidosis, glucose-free medium and their combination on contraction and sarcoplasmic reticulum (SR) function in rat ventricular trabeculae. The isometric twitch tension was measured during superfusion with hypoxic (PO2 < 30 mmHg), acidic (pH 6.80), glucose-free, or their combined (“ischemic”) Tyrode’s solution at 20°C. The time needed to fully recover the contraction induced by 10 mn caffeine (repriming time) was measured to indirectly estimate the Ca2+ uptake of the SR. In “ischemia” and acidosis, the peak developed tension decreased progressively for the first 30 min (37.6 _+ 9.2% and 56.6 + 8.4% ofcontrol at 30 min, respectively), and then became steady. In hypoxic solution, the peak developed tension decreased moderately for the first 30 min (86.8 + 4.8% of control at 30 min), and thereafter remained steady. Developed tension did not change significantly during 60 min of superfusion with glucose-free solution. The repriming time of caffeine contraction was significantly delayed in both “ischemic” and hypoxic solutions, but was unchanged in acidic and glucose-free solutions. These results lead us to suggest to the decline in tension in ischemia and that depressed SR function to accumulate Ca’ + may contribute hypoxia, but that other mechanisms are important in the tension decline induced by acidosis. KEY WORDS: Contractile
function;
Sarcoplasmic
reticulum;
Introduction Myocardial &hernia and hypoxia are initially characterized by a decrease in cardiac developed tension without change in resting tension. Subsequently, developed tension continues to decrease and resting tension starts to increase, and then contracture may develop with a significant increase in resting tension without the capability to generate active tension [28]. The late phase with an increase in resting tension is probably due to rigor resulted from ATP depletion [.?3]. The initial phase, characterized by a decrease in developed tension without change in resting tension, has been postulated to be due to a
Caffeine;
Hypoxia;
Acidosis.
breakdown in the excitation-contraction coupling system [I, 181, but the mechanisms underlying the mechanical properties in the initial phase of ischemia and hypoxia are not well understood. The sarcoplasmic reticulum (SR) plays a. prominent role in the activation and relax-, ation of the contractile apparatus [&YJ, and changes in its functional properties could result in the loss of regulation of [Ca2+li and a decrease in tension development. Caffeine at high concentrations induces transient contracture by releasing Ca2 + from the SR [2, II, 241. Caffeine cannot induce contracture in rapid succession and time is
*This work was supported by National Heart, Lung, and Blood Institute grants HL-19044 and HL-21735, and Grants-in-Aid from the American Heart Association, Greater Miami Chapter and Florida Affiliate, and a program grant from the Florida Affiliate. Please address all correspondence to: Arthur L. Bassett, Department of Pharmacology, University of Miami School of Medicine, Miami, FL 33101, USA. 0022-2828/90/060697
+ 09 $03.00/O
0 1990 Academic
Press Limited1
698
M. Shimizu
necessary to reprime the SR with Ca’+ [II]. The repriming (recovery time) of caffeine contracture reflects in an indirect fashion the rate of Ca2+ uptake by the SR [Z, II]. In the present study, we examined the effects of hypoxia, acidosis, glucose-free medium and their combination (simulated ischemia) on the repriming of caffeine contracture in a limited experimental circumstance to gain further insight into the mechanisms of contractile dysfunction during the early phase of &hernia, hypoxia, and acidosis.
Methods Preparations
and solutions
Male Sprague-Dawley rats (280-400 g) were anesthetized with sodium pentobarbital (35 mg/kg, i.p.), and treated with heparin (200 IU, i.p.). After the thorax was opened, the heart was rapidly removed, and placed in a modified Tyrode’s solution gassed with 0s at 20°C. Small free-running trabeculae, 100-300 urn in diameter and 1-5 mm long, were isolated from the left ventricle under a dissecting microscope. The preparation was mounted in a tissue bath designed for measurement of isometric tension and rapid change of the and with superfusate, was superfused oxygenated Tyrode’s solution at a rate of 20 ml/min. The preparation was stimulated by a Grass Model S88 stimulator delivering 5 ms square wave pulses through a pair of platinum plate electrodes (field stimulation) at a rate of 12/min. Stimulation voltages were set at twice threshold. After a 20-30 min equilibration period, muscle length was adjusted in steps by a micromanipulator to obtain maximum tension development. The tension was recorded on a polygraph (Grass, model 79). Preparations that developed oscillations in the resting tension or spontaneous activity were discarded. The normal modified Tyrode’s solution had the following composition (in mM): NaCl 140.0, KC1 4.0, CaClz 1.8, MgClz 1.0, NaH2P04 1.8, glucose 5.5, HEPES 5.0 (pH 7.4 with NaOH), and was continuously gassed with lOOo/o 0s (Paz > 500 mmHg). The acidic Tyrode’s solution was gassed with 100% 02, and its pH was adjusted to 6.8 with HCl. Glucose-free Tyrode’s solution was
et al.
identical to normal Tyrode’s solution except that it contained no glucose. The hypoxic Tyrode’s solution had the identical composition as the normal Tyrode’s solution, but was gassed with lOOr$‘o Nz(Po2 < 30 mmHg). The “ischemic” solution contained no glucose; its pH was adjusted to 6.8 and it was gassed with 100% Ns. Each preparation was exposed to only one test solution. Experimental
protocol
Protocol 1. In the first series of experiments, we monitored peak developed tension during super-fusion with hypoxic, acidic, glucose-free, and “ischemic” solutions. The preparations were electrically stimulated at 0.2 Hz. After a 30 min equilibration period in normal Tyrode’s solution, each preparation was superfused with a test solution at a rate of 20 ml/min for 60 min, followed by 30 min washout with normal Tyrode’s solution; twitch tension was monitored continuously. Protocol 2. The contracture evoked by the sudden application of caffeine to isolated cardiac muscle is transient, and it relaxes spontaneously even in the presence of caffeine [2, II]. When rat ventricular muscle is returned to caffeine-free solution, a second application of caffeine, made after a minute, induces a much smaller response. The full recovery, or repriming, of the subsequent caffeine contracture on rat ventricle is generally complete after 3-7 min in caffeine-free solution at 20°C after a prior caffeine contracture [2, II]. This repriming time reflects indirectly the rate of Ca’ + uptake by the SR [2, U]. We measured the repriming time of caffeine contracture to estimate the ability of the SR to accumulate Ca2+. The time course of the repriming of caffeine contracture was determined by varying the time between caffeine application [2]. As schematically shown in Figure 1 panel (A) each preparation was exposed to 10 mM caffeine at time 0 and then at intervals of 1, 3, 5, 7, 10 min; each caffeine contracture was followed by washout with caffeine-free solution (continuous super-fusion at a rate of 20 ml/min). The time interval (1, 3, 5, 7, and 10 min) was measured from the time of the prior
Caffeine
IHHl---if--dl--‘+-ll I min 3 min 5 min o
b
c
7 min d
30 min Superfused
with normal
e
Response
IO min f
30min Tyrode’s
solution
c.e Normal
Tyrode’s
solution
Test solution
Normal
Tyrode’s
solvt~on
FIGURE 1. Schematic representation of the experimental protocols. Panel A shows the protocol for the repriming of the caffeine contracture. Upward arrows (7): application of ICI rn~ caffeine solution. Downward arrows (1): washout with caffeine-free solution. a: initial caffeineinduced contracture; b: contracture induced 1 min after initial (a) caffeine contracture; c: contracture induced 3 min after prior (b) caffeine contracture; d: contracture induced 5 min after prior (c) caffeine contracture; e: contracture induced 7 min after prior (d) caffeine contracture; f: contracture induced 10 min after prior (e) caffeine contracture. Contractures e and fwere induced only when full recovery was not observed at d, contracture induced 5 min after the prior application of caffeine; recovery of full (control) magnitude of caffeine contracture was usually complete at 5 min. Panel B shows the protocol used in testing reproducibility of repriming of caffeine contracture. Normal Tyrode’s solution was used to superfuse each preparation throughout this protocol. The tests of repriming shown in panel A were repeated at intervals of 30 mins. “At ” , ‘ 0.93. We calculated the slope of the exponential regression line for each intervention.
Tension development during intervention Figure 2 shows the time course of the changes in tension development during superfusion with various test solutions. In “ischemic” solution, the peak developed tension was progressively reduced for the first 30 min (37.6 f 9.2% of control at 30 min), and then became constant (35.2 f 9.3% of control at 60 min). The tension returned to normal within 30 min of washout with normal Tyrode’s solution. Hypoxic solution increased the peak developed tension slightly for the first 5 min (104.8 + 3.1 o/o of control at 5 min). Then, tension declined to 86.8 k 4.8% of control at 30 min. Developed tension remained constant during the final 30 min of superfusion with hypoxic solution (81.2 f 5.5% of control at 60 min). During exposure to acidic solution, the peak developed tension decreased progressively for the first 30 min, and then became constant (56.6 f 8.4% and 52.6 + 7.3% of control at 30 min and 60 min, respectively). The tension exceeded control values immediately after reexposure to normal Tyrode’s solution but returned to normal within 10 min of initiating washout.
IIII 03510
I 20
I 30
1 40
The peak developed tension did not change significantly during the entire 60 min period of superfusion with glucose-free solution or 30 min washout (data not shown). The repriming
Results
‘: a!
et al.
I 50
wostiout I,, 60510
, 20
I 30
Time (mid FIGURE 2. The time course of the changes in tension development of isolated trabecular preparations from rat left ventricles during 60 min of superfusion with “ischemic” (combined hypoxia, acidosis, and glucosefree), hypoxic, and acidic solutions, and 30 min of “washout” during exposure to normal Tyrode’s solution. Values are expressed as a o/0 of pre-intervention control, and standard errors are indicated by bars.
of caffeine contractwe during superfusion
Figure 3 shows a representative experiment establishing the reproducibility of the rate of repriming in one preparation during exposure to normal Tyrode’s solution. Full recovery of the magnitude of the caffeine contracture (“Al”). When the repriming procedures were repeated at an interval of 30 mins (“AZ” and “As”), the rate of repriming of caffeine contracture was very similar, and full recovery was again observed at 5 min. Such reproducibility of response enabled us to compare the rate of repriming of caffeine contracture obtained before and during exposure to a test solution in each preparation. As shown in Figure 2, the peak developed tension became steady after 30 min of superfusion with any of the test solutions. Thus, the rate of repriming of caffeine contracture could be examined without the influence or association of declining tension. Figure 4 shows representative experiments showing the effects of the test solutions on the repriming of caffeine contracture. Full recovery of caffeine contracture was observed at 5 min in normal Tyrode’s solution (left panels in Fig. 5). However, the caffeine response was not completely recovered at 5 min in “ischemic” (right panel a) and hypoxic (right panel b) solutions. On the other hand, the caffeine response was completely recovered at 5 min in acidic (right panel c) and glucose-free (not shown in Fig. 4) solutions. In Figure 5, the relative caffeine responses are plotted as a function of time between caffeine application (n = 6 each group). In “ischemic” solution, the time to maximum repriming of caffeine contracture was significantly longer than that monitored in control solution [Fig. 6(a)]. Even after washout with normal Tyrode’s solution, the repriming of caffeine contracture was delayed. In hypoxic solution [Fig. 6(b)] the repriming of caffeine contraction was slightly but significantly delayed, and returned to the control after washout with normal Tyrode’s solution. In contrast, acidic solution [Fig. S(c)] or
Caffeine
Response
in Ischemia
701
t
t
Initial
5min
FIGURE 3. Reproducibility of the rate of the repriming of caffeine contracture. The schematic protocol for this experiment is shown in Figure 1 panel B. The preparation was superfused with normal Tyrode’s solution, When thle repriming procedures were repeated at interval of 30 minutes, “Ar”, “As” and “As”, full recovery of caffeine contracture was always recorded at 5 min. Upward arrows (T): application of 10 nm caffeine. Caffeine contractures at 1 and 3 minutes for “Al”, “AZ” and “AJ” series are not shown.
Normal
Tyrode’s Control
Test
(A,)
(A,)
solution Ischemia
Hypowo
(b)
Acidosis
(c 1
J
IO mg
IO s Initial Caffeine
5 min
responses
FIGURE 4. Representative experiments showing the effects of the test solutions on the repriming of caffeine contracture. The protocol for this experiment is shown in Figure 1 panel (C). Full recovery of the subsequent caffeine contracture was observed at 5 min after the prior application of caffeine in each control experiment in normal Tyrode’s and hypoxic test solutions, full recovery was not observed at 5 solution. However during super-fusion with “ischemic” min after the prior application of caffeine. In acidic test solution, full recovery was observed at 5 min after the prior application of caffeine. Test of caffeine repriming during washout is not shown.
M. Shimizu
702
Time
between
et al.
contractlon
(mid
FIGURE 5. Relative caffeine-induced responses are plotted as a function of time between caffeine contractme; protocol is shown in Figure 1 panel C. Control test of repriming in normal Tyrode’s solution (Ai, 0 - 0); intervention (panel a), hypoxic (panel repriming (As, 0 - 0 ); washout repriming (As, A - A). Solutions used were “ischemic” and hypoxic solutions the repriming of caffeine b), acidic (panel c), and glucose-free (panel d) solutions. In “ischemic” contracture required significantly longer time than that noted during control (normal) solution. Acidic and glucose-free solutions did not affect the repriming of caffeine contracture. n = 6 for all groups; *P < 0.05 vs control.
glucose-free solution [Fig. 6(d)] did not affect the repriming time of caffeine contracture. Data on the repriming of caffeine contracture were fitted to a single exponential function, and thus we calculated the slope of the exponential regression line for each intervention (Table 1). The slope was significantly reduced by “ischemic” and hypoxic solutions, but was not changed by acidic nor glucosefree solution. Discussion The present study demonstrates that acidic (pH = 6.8) depresses phasic solution TABLE
1.
Slopes
of the regression
lines
Control “ischemia” hypoxia acidosis glucose-free
0.45 0.35 0.44 0.54
&+ + +
electrically-induced contractions without affecting the repriming of caffeine contracture, while hypoxic and “ischemic” (combined hypoxia, acidosis, and glucose-free) solutions depress both phasic contraction and the repriming of caffeine contracture. “Ischemic” solution is more depressant on the repriming of caffeine contraction than the hypoxic solution alone. Caffeine contracture is thought to reflect Ca2+ release from intracellular (SR) Ca2+ storage sites [Z, II]. Caffeine cannot induce contracture in rapid succession and time is evidently necessary to reprime the SR with Cazf for each successive contracture. Previ-
of caffeine
responses Intervention
0.07 0.02 0.04 0.04
0.23 0.25 0.51 0.49
+ f f +
Washout 0.04* 0.04* 0.05 0.09
0.28 0.42 0.53 0.48
* + f f
0.03 0.02 0.02 0.06
control; The slope (B) was Data are expressed as mean + s.E.; *P < 0.05 versus calculated by using the equation: Y = Ae-s’, where Y : 100 - relative caffeine response (%), A: intercept, t: time. Data on the repriming of caffeine response were fitted to single exponential regression lines. The correlation coefficient of the exponential regression lines was 20.93.
Caffeine
Response
ous work by Chapman and Leoty showed that the repriming of caffeine contracture had a sigmoidal time course, while our data revealed a single exponential function. This discrepancy is likely due to the fact that the repriming of caffeine contracture during the earlier phase (within 1 min) were not examined in the present study. Because of the limitation of time for caffeine tests during superfusion with test solutions (30 min), we could not examine the initial stages of repriming of caffeine contracture. The repriming time may be influenced by various factors including changes in Ca” entry through slow channels and Na+-Ca2+ exchange, CaZf uptake by the SR, and Ca2+ store (or binding) in other sites. Inward Ca2+ currents may be reduced by ischemia and hypoxia [12, 221. However, in our study the preparations were quiescent (not stimulated) during the caffeine test, and, thus, it is unlikely that changes in inward Ca2+ currents are involved in the delayed repriming of caffeine contracture in “ischemic” and hypoxic solutions. The contribution of the Na+-Ca2+ exchange to Ca2 + influx and efflux during ischemia and hypoxia remains uncertain. However, the intracellular Na+ concentration does not increase during the early phase of ischemia and hypoxia [lo, 17, 231. Even if the intracellular Na+ concentration increases, Naf-Ca2+ exchange would result in an increase in the intracellular Cazf concentration. One might predict that repriming time of caffeine contracture is shortened in such a situation. Thus, we suggest that the most probable explanation for the prolongation of the repriming time of caffeine contracture in “ischemic” and hypoxic solutions is that the SR accumulation of Ca2+ is depressed, although we cannot exclude the possibility that changes in intracellular Ca2+ binding sites may be involved in the delayed repriming. The mechanisms responsible for the decline of tension during the early phase of ischemia and hypoxia without concomitant changes in resting tension are not well understood. However, it is quite possible that failure of Ca2+ delivery to the contractile proteins is one of the causes of the decline of tension in ischemia and hypoxia. The sarcoplasmic reticulum serves as the source and sink for coupling Ca2+ in the
in Ischemia
703
excitation-contraction coupling system [4, ,5, f?J. Depression of SR function to release CaZ+ and/or to accumulate CaZf potentially available for release would contribute to the decline of tension. Our data suggest that the ra’te of Ca2+ uptake by the SR was prolonged in “ischemic” and hypoxic solutions. Krause and Hess [12] also have shown biochemically that Ca2+ transport is depressed during the early phase of ischemia in the whole heart homogenate and the isolated SR in dogs. These findings suggest that depressed SR function also may be a causal factor for the decline of tension during early phase of ischemia and hypoxia, although it should be noted that relative importance of the SR in the excitation-contraction coupling system may differ in different species. Depletion of high-energy phosphates ma.y affect the SR function. Kammermeier et al. [.!?I have shown that the free energy change required for maintenance of SR Ca2+ pumping is the highest among all the ion pumping systems. Thus, it is likely that changes in high energy phosphates in ‘5schemic” and hypoxic solution slowed the repriming of caffeine contracture. Although [ATP]i has been shown to fall by only a small amount during the ear1.y phase of ischemia, there may be compartmentation of ATP within the myoplasm, with [ATP] falling to much lower levels in some critical region of the cell [8, IS, 201. It is interesting that acidic solution was more depressant on tension than hypoxic solution, but did not affect the repriming of caffeine contracture. Fabiato and Fabiato [.!i] have shown that SR ability to accumulate Ca2+ is markedly depressed by lowering pH to 6.2 [4] Fabiato [fl also showed that acidosis may modify the Ca 2’ dependency of the Ca’+-induced Ca2+ release. However, oxygenated Tyrode’s solution with pH of 6..8 was used in the present study, which may not have changed intracellular pH significantly enough to alter Ca2+ uptake by the SR.. Deitmer and Ellis reported that the intracellular pH changed linearly with the extralcellular pH by 0.23 pH units/extracellular pH unit change in sheep Purkinje fibers. Assumeing that the intracellular pH is 7.0 at the extracellular pH of 7.4, reduction of the extracellular pH to 6.8 would result in a moderate change in intracellular pH to 6.86. Also, a
704
M. Shim&u
recent study shows that intracellular pH does not fall significantly during superfusion with solution containing Paz 30 mmHg, KC1 9 mM, cyanide 1 mM, and pH 6.5 [15]. Despite the assumed modest fall in intracellular pH, developed tension was markedly depressed by extracellular acidosis (pH 6.8) in the present study. This decline of tension may be explained by pH effects on various sarcolemmal functional properties including Na+-Ca2+ exchange [19], and transmembrane Na+ [251 and Ca2+ currents [21]. More recently, sar-
et al. colemmal Ca2+ binding has also been shown to be affected by the extracellular pH [la]. In conclusion, the results of the present study lead us to suggest that the dysfunction of the SR may contribute to the decline in developed tension during the early phase of ischemia and hypoxia, but that other mechanisms, in addition to the intracellular pH effects on the contractile proteins and SR function, play important roles in the tension decline induced by acidosis.
References 1 2 3 4 5 6 7 a 9 10 11 12 13 14 15 16 17 ia 19 20 21 22
D. G., ORCHARD, C. H. Myocardial Contractile Function During Ischemia and Hypoxia. Circ Res 60, 153-168 (1987). CHAPMAN, R. A., LEOTY, C. The time-dependent and dose-dependent effects of caffeine on the contraction of the ferret heart. J Physio1256, 287-314 (1976). DEITMER, J. W., ELLIS, D. Interactions between the regulation of the intracellular pH and sodium activity of sheep cardiac Purkinje fibers. J Physiol304,471-488 (1980). FABIATO, A., FABIATO, F. Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells. J Physiol249, 469495 (1975). FAEXATO, A., FABIATO, F. Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. J Physio1276, 233-255 (1978). FABIATO, A., FABIATO, F. Calcium-induced release of calcium ions from the cardiac sarcoplasmic reticulum. Am J Physio1245, Cl-Cl4 (1983). FABIATO, A. Use of aequorin for the appraisal of the hypothesis of the release of calcium from the sarcoplasmic reticulum induced by a change of pH in skinned cardiac cells. Cell Calcium 6,95-108 (1985). GEISBUHLER, T., ALTSCHULD, R. A., TREWYN, R. W., ANSEL, A. Z., LAMKA, K., BRIERLEY, G. P. Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells. Circ Res 51: 536-546 (1984). KAMMERMEIER, H., SCHMIDT, P., JUNGLING, E. Free energy change of ATP-hydrolysis: a causal factor of early hypoxic failure of the myocardium? J Mol Cell CardiolI4,267-277 (1982). KLEBER, A. G. Resting membrane potential, extracellular potassium activity, and intracellular sodium activity during acute global &hernia in isolated perfused guinea pig hearts. Circ Res 52,4424150 (1983). KONISHI, M., KURIHARA, S., SAKAI, T. The effects of caffeine on tension development and intracellular calcium transients in rat ventricular muscle. J Physio1355, 605-618 (1984). KOULHARDT, M., KUBLER, M. The influence of metabolic inhibitors upon the transmembrane slow inward current in the mammalian ventricular myocardium. Naunyn-Schmiedebergs Arch Pharmacol290,265-274 (1975). KRAUSE, S. M., HESS, M. L. Characterization of cardiac sarcoplasmic reticulum dysfunction during short-term, normothermic, global ischemia. Circ Res 55, 176-184 (1984). LANGER, G. A. The effect of pH on cellular and membrane calcium binding and contraction of myocardium. Circ R~s 57,374-382 ( 1985). MCCARTHY, J. J., MURPHY, E., STEENBERGEN, C., GETTES, L. S. Investigation ofsimulated ischemia with cyanide in guinea pig myocardium. Circulation 80 [Suppl II] 193 (1989). MEYER, R. A., SWEENEY, H. L., KUSHMERICK, M. J. A simple analysis of the “phosphocreatine shuttle”. Am J Physio1246, C365-C377 (1984). NAKAYA, H., KIMURA, S., KANNO, M. Intracellular K+ and Na+ activities under hypoxia, acidosis, and no glucose in dog hearts. Am J Physiol249, H107&H1085 (1985). NAYLER, W. G., POOLE-WILSON, P. A., WILLIAMS, A. Hypoxia and Calcium. J Mol Cell Cardiol 11, 683-706 (1979). PHILIPSON, K. D., BERSOHN, M. M., NISHIMOTO, A. Y. Effects of pH on Na+-Cazf exchange in canine cardiac sarcolemma vesicles. Circ Res 50, 287-293 (1982). PIERCE, G. N., PHILIPSON, K. D. Binding of glycolytic enzymes to cardiac sarcolemmal and sarcoplasmic reticular membranes. J Biol Chem 260,6862-6878 (1985). SATO, R., NOMA, A., KURACHI, Y., IRISAWA, H. Effects of intracellular acidification on membrane currents in ventricular cells of the guinea pig. Circ Res 57, 553-561 (1985). TANIGUCHI, J., NOMA, A., IRISAWA, H. Modification of the cardiac action potential by intracellular injection of adenosine triphosphate and related substances in guinea-pig single ventricular cells. Circ Res 53, 131-139 (1983). ALLEN,
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WALSH, L. G., TOFXEY, J. M. Subcellular electrolyte shifts during in vitro myocardial ischemia and reperkon. Am J Physiol255, H917-H928 (1988). WEBER, A., HERZ, R. The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J Gen Physiol52, 750-759 ( 1968). YATANI, A., BROWN, A. M., AKAIKE, N. Effect of extracellular pH on sodium current in isolated, single rat ventricular cells. J Membr Biol78, 163-168 (1984).
