The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae

The control of force by [Ca2+] was investigated in rat cardiac trabeculae loaded with fura-2 salt. At sarcomere lengths of 2.1-2.3 microns, the steady state force-[Ca2+]i relationship during tetanization in the presence of ryanodine was half maximally activated at a [Ca2+]i of 0.65 +/- 0.19 microM with a Hill coefficient of 5.2 +/- 1.2 (mean +/- SD, n = 9), and the maximal stress produced at saturating [Ca2+]i equalled 121 +/- 35 mN/mm2 (n = 9). The dependence of steady state force on [Ca2+]i was identical in muscles tetanized in the presence of the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). The force-[Ca2+]i relationship during the relaxation of twitches in the presence of CPA coincided exactly to that measured at steady state during tetani, suggesting that CPA slows the decay rate of [Ca2+]i sufficiently to allow the force to come into a steady state with the [Ca2+]i. In contrast, the relationship of force to [Ca2+]i during the relaxation phase of control twitches was shifted leftward relative to the steady state relationship, establishing that relaxation is limited by the contractile system itself, not by Ca2+ removal from the cytosol. Under control conditions the force-[Ca2+]i relationship, quantified at the time of peak twitch force (i.e., dF/dt = 0), coincided fairly well with steady state measurements in some trabeculae (i.e., three of seven). However, the force-[Ca2+]i relationship at peak force did not correspond to the steady state measurements after the application of 5 mM 2,3-butanedione monoxime (BDM) (to accelerate cross-bridge kinetics) or 100 microM CPA (to slow the relaxation of the [Ca2+]i transient). Therefore, we conclude that the relationship of force to [Ca2+]i during physiological twitch contractions cannot be used to predict the steady state relationship.     

Measurement of myofilament calcium sensitivity at physiological temperature in intact cardiac trabeculae

Cardiac contraction-relaxation coupling is determined by both the free intracellular calcium concentration ([Ca2+]i) and myofilament properties. We set out to develop a technique where we could assess these parameters (twitch and steady-state force [Ca2+]i) under near physiological conditions. Bis-fura-2 was iontophorically introduced into ultrathin rat trabeculae preparations to monitor the [Ca2+]i, and steady-state contractures were achieved by using a modified Krebs-Henseleit solution containing high K+. During K+ contractures, the very slow changes in [Ca2+]i and force development were in equilibrium and allowed for the construction of a steady-state, force-[Ca2+]i relationship. Twitch contractions before and after this myofilament calcium sensitivity assessment were unaltered, and this protocol could be repeated several times. For the first time, this novel protocol allows us to measure myofilament calcium sensitivity under physiological temperature. Not only do the data so obtained allow us to assess myofilament calcium sensitivity, the data also will allow us, in the same preparation under nearly identical conditions, to compare the dynamic to the steady-state, force-calcium relationship. To test whether the steady-state relationship between force and calcium in our novel protocol reproduces expected changes, we determined this relationship in the presence of isoproterenol and under acidosis and alkalosis. As expected, beta-adrenergic stimulation resulted in an increase of calcium amplitude and twitch force and a desensitization of the myofilaments as indicated by a rightward shift of the obtained steady-state, force-calcium relationship. An increase in pH shifted the curve leftward, whereas a decrease in pH resulted in the expected rightward shift.     

Relationship between force and intracellular [Ca2+] in tetanized mammalian heart muscle

To determine features of the steady state [Ca2+]-tension relationship in intact heart, we measured steady force and intracellular [Ca2+] ([Ca2+]i) in tetanized ferret papillary muscles. [Ca2+]i was estimated from the luminescence emitted by muscles that had been microinjected with aequorin, a Ca2+-sensitive, bioluminescent protein. We found that by raising extracellular [Ca2+] and/or by exposing muscles to the Ca2+ channel agonist Bay K 8644, tension development could be varied from rest to an apparently saturating level, at which increases in [Ca2+]i produced no further rise in force. 95% of maximal Ca2+-activated force was reached at a [Ca2+]i of 0.85 +/- 0.06 microM (mean +/- SEM; n = 7), which suggests that the sensitivity of the myofilaments to [Ca2+]i is far greater than anticipated from studies of skinned heart preparations (or from previous studies using Ca2+-sensitive microelectrodes in intact heart). Our finding that maximal force was reached by approximately 1 microM also allowed us to calculate that the steady state [Ca2+]i-tension relationship, as it might be observed in intact muscle, should be steep (Hill coefficient of greater than 4), which is consistent with the Hill coefficient estimated from the entire [Ca2+]i-tension relationship derived from families of variably activated tetani (6.08 +/- 0.68; n = 7). Finally, with regard to whether steady state measurements can be applied directly toward understanding physiological contractions, we found that the relation between steady force and [Ca2+]i obtained during tetani was steeper than that between peak force and peak [Ca2+]i observed during physiological twitches.     

Effect of transient stretch on intracellular Ca2+ during triggered propagated contractions in intact trabeculae

Transient stretch of cardiac muscle during a twitch contraction may dissociate Ca2+ from myofilaments into the cytosol at the moment of quick release of the muscle. We studied the effect of stretch and quick release of trabeculae on changes in intracellular Ca2+ ([Ca2+]i) during triggered propagated contractions (TPCs). Trabeculae were dissected from the right ventricle of 9 rat hearts. [Ca2+]i was measured using electrophoretically injected fura-2. Force was measured using a silicon strain gauge and sarcomere length was measured using laser diffraction techniques. Reproducible TPCs (n = 13) were induced by trains of electrical stimuli (378 +/- 19 ms interval) for 7.5 s at [Ca2+]o of 2.0 mM (27.9 +/- 0.2 degrees C). The latency of the TPC force and the underlying increase in [Ca2+]i was calculated from the time (TimeF) between the last stimulus and the peak of TPC force (PeakF), or the time (TimeCa) between the last stimulus and the peak of the increase in [Ca2+]i during the TPCs (PeakCa). As a result of a 10% increase in muscle length for 150-200 ms during the last stimulated twitches, TimeF and TimeCa decreased and PeakF and PeakCa increased significantly (n = 13). In addition, transient stretch sometimes induced a twitch contraction subsequent to the accelerated TPC and its underlying increase in [Ca2+]i. These results suggest that Ca2+ binding and dissociation from the myofilaments by the stretch and quick release of muscle may modulate the TPC force and the underlying increases in [Ca2+]i and play an important role in the induction of arrhythmias.     

Dissociation of changes in apparent myofibrillar Ca2+ sensitivity and twitch relaxation induced by adrenergic and cholinergic stimulation in isolated ferret cardiac muscle

In isolated, aequorin-injected ferret cardiac muscle we measured the apparent myofilament Ca2+ sensitivity and its relationship to twitch relaxation time in the presence of autonomic perturbations. The Ca2+-tension relation was determined from the peak aequorin luminescence and peak twitch tension measured in muscles across a broad range of bathing [Ca2+] in the presence and absence of acetylcholine (ACh) (1 microM) or isoproterenol (ISN) (1 microM), or both drugs. ACh shifted the relationship of peak tension to (peak) aequorin light leftward, which suggests an increase in myofilament Ca2+ sensitivity, but it did not alter relaxation, which was measured as the time for peak tension to decay by 50% (t 1/2 R). ISN produced its previously documented effects, i.e., a rightward shift of the relationship of peak tension to peak aequorin light and a decrease in t1/2R. ACh abolished the ISN effect on the peak tension-aequorin light relationship but did not reverse the effect of ISN to decrease t1/2R. The effects of ACh and ISN of modulating the apparent myofilament Ca2+ sensitivity in intact muscles, corroborate findings of previous studies in isolated myofibrillar preparations. However, these perturbations of myofilament Ca2+ sensitivity in the intact muscle do not relate to twitch relaxation, measured as t1/2R, since (a) ACh affects the former but not the later and (b) the effect of ISN on the Ca2+-tension relationship is abolished by ACh, while the relaxant effect persists.     

Thyroid status and beta-agonistic effects on cytosolic calcium concentrations in single rat cardiac myocytes activated by electrical stimulation or high-K+ depolarization.

The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.     

Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle

Intact cardiac cells from the adult rat or rabbit ventricle were isolated by enzymatic digestion with a progressive increase of the [free Ca2+] in the solution. These cells were electrically stimulated in the presence of 2.50 mM free Ca2+, and a twitch of maximum amplitude was elicited by the positive inotropic interventions that were found to be optimum. Then the cells were chemically skinned, and the maximum tension induced by a saturating [free Ca2+] was used as a reference to express the tension developed during the twitch of the intact cells. The myoplasmic [free Ca2+] reached during the twitch was inferred from the tension-pCa curve. In mechanically skinned cells of the same animal species, the myoplasmic [free Ca2+] reached during Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR) was inferred by two methods using (a) the tension-pCa curve and (b) a direct calibration of the transients of aequorin bioluminescence. The induction of a maximum Ca2+ release from the SR required a larger Ca2+ preload of the SR and a higher [free Ca2+] trigger in the rabbit than in the rat skinned cells. However, the results obtained with the two methods of inference of the myoplasmic [free Ca2+] suggest that in both animal species a maximum myoplasmic [free Ca2+] of pCa approximately 5.40 was reached during both the optimum Ca2+-induced release of Ca2+ from the SR of the skinned cells and the optimum twitch of the intact cells. This was much lower than the [free Ca2+] necessary for the full activation of the myofilaments (pCa approximately 4.90).     

Dexamethasone inhibits endotoxin-induced changes in calcium and contractility in rat isolated papillary muscle

This study investigates whether endotoxin-induced contractile dysfunction is associated with a defect in the modulation of calcium homeostasis and the potential mechanisms involved. Treatment of rats in vivo with endotoxin significantly decreased the magnitude of contractile transients in electrically stimulated left ventricular papillary muscle isolated after an equilibration period of 6 hours. Although no significant difference was found in the peak intracellular calcium concentration ([Ca2+]i) between the endotoxin-treated and control groups, resting [Ca2+]i) was significantly elevated in the endotoxin-treated group, producing a smaller Ca2+ transient (basal-peak difference) in this group. Pretreatment of rats with dexamethasone prevented the endotoxin-induced decrease in peak tension and inhibited the elevation in resting [Ca2+]i, with a resultant maintenance of Ca2+ transient magnitude. Similar observations were made during stimulation of the muscles by the beta-adrenoceptor agonist, isoprenaline. These results show that endotoxin-induced reduction of cardiac contractile performance is mediated, at least in part, by elevating resting [Ca2+]i, and a glucocorticoid protected from these negative effects. While endotoxin reduces the magnitude of the Ca2+ transient it does not alter peak [Ca2+]i availability. Further investigation is required to determine whether endotoxin decreases contractile performance by reducing the sensitivity of cardiac myofilaments to calcium.     

Genistein attenuates postischemic depressed myocardial function by increasing myofilament Ca2+ sensitivity in rat myocardium

The present study investigated whether genistein, a broad-spectrum tyrosine kinase inhibitor, could increase the myofilament Ca(2+) sensitivity and partially reverse postischemic depressed myocardial function. Left ventricular papillary muscles were isolated from adult Wistar rats and loaded with the Ca2+ indicator, aequorin. The use of fluorocarbon immersion with hypoxia simulated a model of ischemia. Myofilament responsiveness to Ca2+ was evaluated from force-[Ca2+]i relationship recorded during tetani in papillary muscles. Protein levels of troponin I (TnI) were measured in postischemic papillary muscles with the Western blot technique. Isometric contraction was depressed during the period of ischemia and remained low after 60 min of reoxygenation without a corresponding significant change of peak [Ca2+]i in the control group (n = 7). In contrast, the depression of isometric contraction was ameliorated during ischemia in muscle preparations in the presence of genistein (2 micro M; n = 8), and postischemic depressed myocardial contractility partially recovered after a 60-min reperfusion. The myofilament Ca2+ responsiveness was significantly increased in papillary muscles in the presence of genistein. Protein levels of TnI were reduced in postischemic papillary muscles, whereas genistein partially restored decreased protein levels of TnI. Our results reveal that genistein produces an effective attenuation of postischemic depressed myocardial function and improves myofibrillar Ca2+ responsiveness in rat myocardium.     

Sarcomere mechanics in uniform and non-uniform cardiac muscle: a link between pump function and arrhythmias

Starling's Law and the well-known end-systolic pressure-volume relationship (ESPVR) of the left ventricle reflect the effect of sarcomere length (SL) on stress (sigma) development and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL-[Ca2+]free relationships (sigma-SL-CaR) at submaximal [Ca2+] in intact and skinned trabeculae were similar, albeit that the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-CaR using a kinetic model where we assumed that the rates of Ca2+ binding by Troponin-C (Tn-C) and/or cross-bridge (XB) cycling are determined by SL, [Ca2+] or stress. We analyzed the correlation between the model results and steady state stress measurements at varied SL and [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that: (i) the dominant feedback mechanism is SL, stress or [Ca2+]-dependent; and (ii) the feedback mechanism regulates: Tn-C-Ca2+ affinity, XB kinetics or, unitary XB-force. The analysis strongly suggests that feedback of the number of strong XBs to cardiac Tn-C-Ca2+ affinity is the dominant mechanism that regulates XB recruitment. Application of this concept in a mathematical model of twitch-stress accurately reproduced the sigma-SL-CaR and the time course of twitch-stress as well as the time course of intracellular [Ca2+]i. Modeling of the response of the cardiac twitch to rapid stress changes using the above feedback model uniquely predicted the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. The above concept has important repercussions for the non-uniformly contracting heart in which arrhythmogenic Ca2+ waves arise from weakened areas in cardiac muscle. These Ca2+ waves can reversibly be induced in muscle with non-uniform excitation contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). These observations suggest the unifying hypothesis that force feedback to Ca2+ binding by Tn-C is responsible for Starling's Law and the ESPVR in uniform myocardium and leads in non-uniform myocardium to a surge of Ca2+ released by the myofilaments during relaxation, which initiates arrhythmogenic propagating Ca2+ release by the SR.     

Modulation of stimulation frequency responses and calcium dependency of functional parameters in hyperthyroid rat ventricular papillary muscles

The effects of stimulation frequency (0.2-1.5 Hz) and extracellular calcium concentration ([Ca2+]o) (0.6-15.0 mM) on the contractile function of thin papillary muscles of euthyroid and hyperthyroid rats were studied. Hyperthyroidism led to a decrease in developed tension (DT) and time to peak tension (TPT), but it exhibited no influence on the maximal rates of contraction (+dT/dt) and relaxation (-dT/dt). Also, the mean rates of contraction were similar in euthyroid and hyperthyroid muscle groups. The increase in stimulation frequency brought about a marked decrease in DT, +dT/dt, and -dT/dt of euthyroid papillary muscles at lower frequencies in comparison to papillary muscles in the hyperthyroid group. At stimulation frequencies above 1.0 Hz, the absolute and relative levels of DT and -dT/dt of hyperthyroid myocardium were elevated over euthyroid preparations. At the same time, TPT was unchanged in any of the muscle groups. Hyperthyroidism modulated the relationships between contractile parameters and [Ca2+]o. At a [Ca2+]o of 1.0-4.0 mM, the DT of hyperthyroid papillary muscles was lower than in euthyroid muscle. At 4.0 and 8.0 mM of [Ca2+]o, the equal values of maximal DT were registered for euthyroid and hyperthyroid papillary muscles, respectively. An increase in the [Ca2+]o in the range of 1.0-15.0 mM was accompanied by an increase in TPT of both muscle groups, but to a greater extent in hyperthyroid myocardium. In conclusion, the myocardium of hyperthyroid rat appeared to exhibit decreased sensitivity to calcium as well as to the negative inotropic effect of enhanced stimulation frequency. Alterations of the processes of transsarcolemmal movement and intracellular recycling of Ca2 may be implicated.     

Effect of thyroid state on cytosolic free calcium in resting and electrically stimulated cardiac myocytes

The effects of the thyroid state on the cytosolic free Ca2+ concentration, [Ca2+]i, of resting and K+-depolarized cardiomyocytes were studied using the fluorescent Ca2+ indicator fura2. The mean resting [Ca2+]i in euthyroid myocytes (89 +/- 8 nM) was not significantly different from that in hyperthyroid myocytes (100 +/- 14 nM). The resting O2-consumption rate was identical for both groups when expressed per mg protein, but a 35% higher value was observed in the hyperthyroid group when expressed per cell on account of the cellular hypertrophy induced by thyroid hormone. Potassium induced depolarization (50 mM [K+]0) raised the level of [Ca2+]i by 50% in both groups. When ATP-coupled respiration was blocked with oligomycin, the 50 mM K+-induced rise in [Ca2+]i was accompanied in both groups by a 40% rise in glycolytic activity as inferred from measurement of lactate production. Ca2+-fluorescence transients were recorded from electrically stimulated myocytes of euthyroid, hyperthyroid and hypothyroid rats. The time taken to reach peak fluorescence (TPL) and that to 50% decay of peak fluorescence (RL0.5) decreased in the direction hypothyroid----hyperthyroid, indicating an increase in Ca2+ fluxes in the same direction. Isoproterenol (1 microM) enhanced the peak Ca2+ fluorescence in electrically stimulated hypothyroid and euthyroid myocytes but not in hyperthyroid myocytes. Both the TPL and RL0.5 were decreased by isoproterenol in euthyroid, but more so in hypothyroid myocytes. None of these parameters were influenced by isoproterenol in the hyperthyroid group. We conclude that (1) thyroid hormone increases neither the O2-consumption rate nor the level of [Ca2+]i of resting cardiomyocytes and (2) the effects of the beta-receptor-agonist isoproterenol on Ca2+ transients of electrically stimulated myocytes, are inversely related to the documented changes in beta-receptor density in heart tissue occurring with alterations in the thyroid state.     

Steady-state [Ca2+]i-force relationship in intact twitching cardiac muscle: direct evidence for modulation by isoproterenol and EMD 53998.

We have developed a novel method for measuring steady-state force-[Ca2+]i relations in isolated, membrane-intact rat trabeculae that are microinjected with Fura-2 salt. Twitches are markedly slowed after inhibition of phasic Ca2+ release and uptake from the sarcoplasmic reticulum by addition of cyclopiazonic acid and ryanodine. During relaxation of slowed twitches, force and [Ca2+]i trace a common trajectory in plots of force versus [Ca2+]i, despite very different histories of contraction. The common trajectory thereby provides a high resolution determination of the steady-state relation between force and [Ca2+]i. Using this method, we show that 1 microM isoproterenol, a beta-adrenergic agonist, causes a rightward shift (Hill function K1/2 increased from 0.39 +/- 0.07 microM to 0.82 +/- 0.23 microM, p < 0.02, n = 6) and a decreased slope (nH decreased from 5.4 +/- 1.1 to 4.0 +/- 1.4, p < 0.02) of the steady-state force-[Ca2+]i curve, with no change in maximal force (Fmax = 99.2 +/- 2.2% of control). In contrast, 2 microM EMD 53998, a racemic thiadiazinone derivative, causes a leftward shift (K1/2 decreased from 0.42 +/- 0.02 microM to 0.30 +/- 0.06 microM, p < 0.02, n = 4) with no change in slope of the steady-state force-[Ca2+]i curve, accompanied by a modest increase in maximal force (Fmax = 107.1 +/- 4.6% of control, p < 0.02). To gain mechanistic insight into these modulatory events, we developed a simple model of cooperative thin filament activation that predicts steady-state force-[Ca2+]i relationships. Model analysis suggests that isoproterenol decreases cooperativity arising from nearest-neighbor interactions between regulatory units on the thin filament, without change in the equilibrium constant for Ca2+ binding. In contrast, the effects of EMD 53998 are consistent with an increase in the affinity of strong-binding cross-bridges, without change in either the affinity of troponin C for Ca2+ or cooperative interactions.     

Maximal Ca2+-activated force and myofilament Ca2+ sensitivity in intact mammalian hearts. Differential effects of inorganic phosphate and hydrogen ions

Myofilament Ca2+ sensitivity and maximal Ca2+-activated force are fundamental properties of the contractile proteins in the heart. Although these properties can be evaluated directly in skinned preparations, they have remained elusive in intact tissue. A novel approach is described that allows maximal Ca2+-activated force to be measured and myofilament Ca2+ sensitivity to be deduced from isovolumic pressure in intact perfused ferret hearts. Phosphorus nuclear magnetic resonance spectra are obtained sequentially to measure the intracellular inorganic phosphate (Pi) and hydrogen ion (H+) concentrations. After a period of perfusion with oxygenated, HEPES-buffered Tyrode solution, hypoxia is induced as a means of elevating [Pi]. The decline in twitch pressure can then be related to the measured increase in [Pi]. After recovery, hearts are perfused with ryanodine to enable tetanization and the measurement of maximal Ca2+-activated pressure. Hypoxia is induced once again, and maximal pressure is correlated with [Pi]. We then compare the relations between [Pi] and maximal pressure on the one hand, and [Pi] and twitch pressure on the other. If the two relations differ only by a constant scaling factor, then the decline in twitch pressure can be attributed solely to a decline in maximal pressure, with no change in myofilament sensitivity. We obtained such a result during hypoxia, which indicated that Pi accumulation decreases maximal force but does not change myofilament sensitivity. We compared these results with acidosis (induced by bubbling with 5% CO2). In contrast with Pi, the accumulation of H+ decreases twitch force primarily by shifting myofilament Ca2+ sensitivity. This approach in intact tissue has strengths and limitations complementary to those of skinned muscle experiments.     

Attenuation of extracellular ATP response in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Extracellular ATP is known to augment cardiac contractility by increasing intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes; however, the status of ATP-mediated Ca2+ mobilization in hearts undergoing ischemia-reperfusion (I/R) has not been examined previously. In this study, therefore, isolated rat hearts were subjected to 10-30 min of global ischemia and 30 min of reperfusion, and the effect of extracellular ATP on [Ca2+]i was measured in purified cardiomyocytes by fura-2 microfluorometry. Reperfusion for 30 min of 20-min ischemic hearts, unlike 10-min ischemic hearts, revealed a partial depression in cardiac function and ATP-induced increase in [Ca2+]i; no changes in basal [Ca2+]i were evident in 10- or 20-min I/R preparations. On the other hand, reperfusion of 30-min ischemic hearts for 5, 15, or 30 min showed a marked depression in both cardiac function and ATP-induced increase in [Ca2+]i and a dramatic increase in basal [Ca2+]i. The positive inotropic effect of extracellular ATP was attenuated, and the maximal binding characteristics of 35S-labeled adenosine 5'-[gamma-thio]triphosphate with crude membranes from hearts undergoing I/R was decreased. ATP-induced increase in [Ca2+]i in cardiomyocytes was depressed by verapamil and Cibacron Blue in both control and I/R hearts; however, this response in I/R hearts, unlike control hearts, was not affected by ryanodine. I/R-induced alterations in cardiac function and ATP-induced increase in [Ca2+]i were attenuated by treatment with an antioxidant mixture and by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2. The results suggest an impairment of extracellular ATP-induced Ca2+ mobilization in I/R hearts, and this defect appears to be mediated through oxidative stress.     

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

A change of pH did not modify the sensitivity of aequorin to Ca2+, but an increase of pH enhanced the Ca2+ sensitivity of the myofilaments of a skinned canine cardiac Purkinje cell. The tension-pCa curve did not present any hysteresis when a given [free Ca2+] was reached from a higher versus from a lower [free Ca2+] in the presence of pH 6.60, 7.10 or 7.40. A rapid variation of pH in either direction failed to induce Ca2+ release from the sarcoplasmic reticulum (SR). The proton ionophores CCCP and gramicidin also failed to induce Ca2+ release from the SR. Increase of pH from 7.10 to 7.40 enhanced Ca2+ accumulation into the SR and, thereby, augmented the Ca2+ content of the SR. Consequently, the amplitude of a subsequent Ca2+ release triggered by a rapid increase of [free Ca2+] at the outer surface of the SR was increased. Conversely, a decrease of pH from 7.10 to 6.60 diminished the Ca2+ accumulation into the SR, the Ca2+ content of the SR and the amplitude of a subsequent Ca2+-induced release of Ca2+ from the SR. In addition, the optimum [free Ca2+] for triggering Ca2+-induced release of Ca2+ was shifted to higher [free Ca2+] values by a decrease of pH from 7.40 to 7.10 or 7.10 to 6.60. This may help to explain the enhancement of the aequorin light transient during acidosis in the intact cardiac muscle inasmuch as acidosis may increase the [free Ca2+] trigger at the outer surface of the SR by inhibiting Na+-Ca2+ exchange across the sarcolemma.     

Effects of ryanodine in skinned cardiac cells

Ryanodine (1 X 10(-5) M) did not affect the Ca2+ sensitivity of the myofilaments of skinned (sarcolemma removed by microdissection) cardiac cells from the rat ventricle. Ryanodine (1 X 10(-5) M) inhibited three types of Ca2+ release from the sarcoplasmic reticulum (SR), which have different mechanisms: 1) Ca2+-induced release of Ca2+ triggered by a rapid and transient increase of [free Ca2+] at the outer surface of the SR; 2) caffeine-induced release of Ca2+; 3) spontaneous cyclic release of Ca2+ occurring in the continuous presence of a [free Ca2+] sufficient to overload the SR. These results suggest that the three types of Ca2+ release are through the same channel across the SR membrane, although the gating mechanisms are different for the three types. Ryanodine also diminished the rate of Ca2+ accumulation into the SR. Even in the presence of 1 X 10(-5) M ryanodine the SR accumulated Ca2+ that could be released when the SR was sufficiently overloaded with Ca2+. Thus, ryanodine pretreatment did not permit the direct activation of the myofilaments by externally applied Ca2+. The approximately 1000-fold difference in the effective concentrations of ryanodine in intact vs. skinned cardiac cells suggests that low concentrations of ryanodine act in the intact cardiac tissues through processes or on structures that are destroyed by the skinning procedure. No significant differences were observed in the effects of ryanodine in skinned cardiac cells from different adult mammalian species.     

Stretch and quick release of rat cardiac trabeculae accelerates Ca2+ waves and triggered propagated contractions

Rapid shortening of active cardiac muscle [quick release (QR)] dissociates Ca2+ from myofilaments. We studied, using muscle stretches and QR, whether Ca2+ dissociation affects triggered propagated contractions (TPCs) and Ca2+ waves. The intracellular Ca2+ concentration was measured by a SIT camera in right ventricular trabeculae dissected from rat hearts loaded with fura 2 salt, force was measured by a silicon strain gauge, and sarcomere length was measured by laser diffraction while a servomotor controlled muscle length. TPCs (n = 27) were induced at 28 degrees C by stimulus trains (7.5 s at 2.65 +/- 0.13 Hz) at an extracellular Ca2+ concentration ([Ca2+]o) = 2.0 mM or with 10 microM Gd3+ at [Ca2+]o = 5.2 +/- 0.73 mM. QR during twitch relaxation after a 10% stretch for 100-200 ms reduced both the time between the last stimulus and the peak TPC (PeakTPC) and the time between the last stimulus and peak Ca2+ wave (PeakCW) and increased PeakTPC and PeakCW (n = 13) as well as the propagation velocity (Vprop; n = 8). Active force during stretch also increased Vprop (r = 0.84, n = 12, P < 0.01), but Gd3+ had no effect (n = 5). These results suggest that Ca2+ dissociation by QR during relaxation accelerates the initiation and propagation of Ca2+ waves.     

Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Although ischemia-reperfusion (I/R) has been shown to affect subcellular organelles that regulate the intracellular Ca2+ concentration ([Ca2+]i), very little information regarding the Ca2+ handling ability of cardiomyocytes obtained from I/R hearts is available. To investigate changes in [Ca2+]i due to I/R, rat hearts in vitro were subjected to 10-30 min of ischemia followed by 5-30 min of reperfusion. Cardiomyocytes from these hearts were isolated and purified; [Ca2+]i was measured by employing fura-2 microfluorometry. Reperfusion for 30 min of the 20-min ischemic hearts showed attenuated cardiac performance, whereas basal [Ca2+]i as well as the KCl-induced increase in [Ca2+]i and isoproterenol (Iso)-induced increase in [Ca2+]i in cardiomyocytes remained unaltered. On the other hand, reperfusion of the 30-min ischemic hearts for different periods revealed marked changes in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i without any alterations in the KCl-induced increase in [Ca2+]i or S(-)-BAY K 8644-induced increase in [Ca2+]i. The I/R-induced alterations in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i in cardiomyocytes were attenuated by an antioxidant mixture containing superoxide dismutase and catalase as well as by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2 for 30 min. These results suggest that abnormalities in basal [Ca2+]i as well as mobilization of [Ca2+]i upon beta-adrenoceptor stimulation in cardiomyocytes are dependent on the duration of ischemic injury to the myocardium. Furthermore, Ca2+ handling defects in cardiomyocytes appear to be mediated through oxidative stress in I/R hearts.     

Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+- dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium

We hypothesized that the occurrence of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), in diastole, might be a mechanism for the saturation of twitch potentiation common to a variety of inotropic perturbations that increase the total cell Ca. We used a videomicroscopic technique in single cardiac myocytes to quantify the amplitude of electrically stimulated twitches and to monitor the occurrence of the mechanical manifestation of spontaneous SR Ca2+ release, i.e., the spontaneous contractile wave. In rat myocytes exposed to increasing bathing [Ca2+] (Cao) from 0.25 to 10 mM, the Cao at which the peak twitch amplitude occurred in a given cell was not unique but varied with the rate of stimulation or the presence of drugs: in cells stimulated at 0.2 Hz in the absence of drugs, the maximum twitch amplitude occurred in 2 mM Cao; a brief exposure to 50 nM ryanodine before stimulation at 0.2 Hz shifted the Cao of the maximum twitch amplitude to 7 mM. In cells stimulated at 1 Hz in the absence of drugs, the maximum twitch amplitude occurred in 4 mM Cao; 1 microM isoproterenol shifted the Cao of the maximum twitch amplitude to 3 mM. Regardless of the drug or the stimulation frequency, the Cao at which the twitch amplitude saturated varied linearly with the Cao at which spontaneous Ca2+ release first occurred, and this relationship conformed to a line of identity (r = 0.90, p = less than 0.001, n = 25). The average peak twitch amplitude did not differ among these groups of cells. In other experiments, (a) the extent of rest potentiation of the twitch amplitude in rat myocytes was also limited by the occurrence of spontaneous Ca2+ release, and (b) in both rat and rabbit myocytes continuously stimulated in a given Cao, the twitch amplitude after the addition of ouabain saturated when spontaneous contractile waves first appeared between stimulated twitches. A mathematical model that incorporates this interaction between action potential-mediated SR Ca2+ release and the occurrence of spontaneous Ca2+ release in individual cells predicted the shape of the Cao-twitch relationship observed in other studies in intact muscle. Thus, the occurrence of spontaneous SR Ca2+ release is a plausible mechanism for the saturation of the inotropic response to Ca2+ in the intact myocardium.