INTRODUCTION

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IN MAMMALIAN MYOCARDIUM it is widely accepted that contraction of the heart is triggered when the action potential causes a small amount of calcium to cross the sarcolemma. This in turn induces the release of a much larger amount of calcium from the sarcoplasmic reticulum (SR). The myocardium is activated to contract by calcium binding to the myofilaments. Relaxation occurs when calcium dissociates from the myofilaments and is removed from the cytoplasm, either through sequestration by the SR or extrusion by the sarcolemmal Ca²⁺-ATPase and the Na⁺/Ca²⁺ exchanger. Therefore the availability of calcium is an important determinant of the ultimate force of myocardial contraction.

Cellular mechanisms of excitation-contraction coupling (E-C coupling), although studied extensively in mammalian myocardium, are less well understood in amphibians and fish (9). Although teleost myocardium contains all the cellular components for E-C coupling seen in mammalian myocardium (i.e., SR calcium stores, L-type calcium channels, contractile elements, Na⁺/Ca²⁺ exchanger, and sarcolemmal Ca²⁺-ATPase), the exact interaction and role played by each in force generation are still undetermined. More particularly, the relative importance of the sources of calcium for contractile activation in teleosts (transsarcolemmal calcium flux vs. mobilization from the SR) has not been defined clearly.

Not only is the source of calcium for contraction not well established in the fish heart, but there is also evidence that the calcium source may vary with temperature (5, 12, 15, 35). This is further compounded by discrepancies in the literature regarding contractile activation in fish ventricular myocardium using different species, different test parameters, and ranges of acclimation temperatures (7, 12, 13, 19, 28, 32, 34).

To address some of these issues, we elected to study a commonly investigated species of fish, the rainbow trout (Oncorhynchus mykiss). We performed our experiments in vitro at 10°C, as well as at two additional test temperatures of 4 and 18°C. Because the function of cardiac muscle is critically dependent on intracellular calcium concentration ([Ca²⁺]_i), we studied how extracellular calcium concentration ([Ca²⁺]_o) and modulation of sarcolemmal L-type calcium channels affected force generation and time course of contraction in trout ventricular myocardium. In addition, agents known to affect SR function were used to reveal the contribution of intracellular calcium stores to fish cardiac contraction.

The goals of the present work were 1) to clarify the source of intracellular calcium in trout ventricle by analyzing peak force as well as contraction and relaxation phases of the isometric twitch under experimental conditions that modify calcium availability and 2) to determine the effect of acute temperature changes, within the range of environmental temperatures experienced by this species, on the source of [Ca²⁺]_i and the contractile response. Furthermore, we sought to support our prior experimental observations using the perforated patch technique that changes in calcium current activity could indeed affect mechanical properties of the myocardium (10).

	MATERIALS AND METHODS

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Rainbow trout (Oncorhynchus mykiss), 8-10 inches in length and of both sexes, were obtained from the Mohawk Trout Hatchery (Sunderland, MA). The fish were raised outdoors in open tanks maintained at 10°C by a continuous flow of fresh well water and were transferred indoors to an aquarium containing aerated, dechlorinated water (pH 7.0, O₂ 7.5 parts/million) at 10°C for at least 3 days before euthanasia. Fish were stunned with a blow to the head, and the spinal cord was severed. The heart was rapidly excised and placed in ice-cold Ringer solution, where either one or two ventricular strips were dissected free (8 mm long, 1 mm wide), or the sinus venosus and atrium were removed from the heart and the ventricle was opened medially (34). Ventricular strips were used for force-frequency and dose-response experiments ([Ca²⁺]_i and Bay K 8644) where the thinner preparation allowed for faster diffusion. The intact ventricle was used for recording of action potentials. Fish Ringer solution contained (in mM) 127 NaCl, 5.1 KCl, 12 NaHCO₃, 5.55 glucose, 1.6 CaCl₂, and 0.93 MgSO₄ and bubbled with 95% O₂-5% CO₂, pH 7.4 (36).

The tissues were attached to a force transducer in temperature-regulated baths set at the acclimation temperature of 10°C. The tissue was perfused with Ringer solution, which was oxygenated throughout the experiments. During the hour before testing the optimum length-tension for each strip was obtained by stretching the strip in stepwise increments to a length that elicited maximum twitch force in response to a threshold voltage at 0.2 Hz (Grass S88 and S5 Stimulator, Grass Medical Instruments; Quincy, MA). The responses were recorded on a Grass model 78 chart recorder (Grass Medical Instruments). Starting at the acclimation temperature of 10°C, the strips were brought to the experimental temperatures of either 4 or 18°C over a 5- to 10-min period and then held at each temperature for at least 10 min before testing.

Peak force was normalized to cross-sectional area (calculated as weight/length), assuming a cylindrical form and a specific gravity of 1.00; the twitch force is reported in milliNewtons per squared millimeter. For each muscle, force was normalized to the maximum force, and data from all hearts were pooled and averaged (average ± SE). Time to peak tension (TPT) was taken from the initiation of contraction to the maximum peak force. Time to 80% relaxation (t₈₀) was determined as the time from peak force to 80% relaxation (23).

Calcium concentration response. Ventricular strips in 1.6 mM CaCl₂ were stimulated at 0.2 Hz. The concentration of calcium in the bath solution was changed to 0.16 mM (low calcium) and aliquots of 1 M CaCl₂ were mixed into the bath to provide a range of [Ca²⁺]_o from 0.16 to 16 mM. No precipitation was observed at these calcium concentrations. The effects of increasing [Ca²⁺]_o on the force of contraction of the strips were determined at the three experimental temperatures. No response was observed until 1.6 mM [Ca²⁺]_o, and therefore the results begin at that dose. Data were calculated relative to the force seen originally in standard Ringer solution (1.6 mM Ca²⁺).

Force-frequency response. To observe the effects of temperature on the force-frequency relationship of the ventricular strips, isolated muscles were tested over a physiological range at frequencies of 0.2-1.0 Hz, where frequency was increased in incremental steps of 0.2 Hz. Preparations were first stimulated at 0.2 Hz at 10°C, and the contractile force was allowed to reach a new steady state at each test frequency. The temperature was then increased to 18°C or decreased to 4°C over a 5- to 10-min period, held at each temperature for at least 10 min until a new steady state was attained, and the frequency response relationship was repeated.

Action potentials. Transmembrane action potentials were recorded from the most superficial layer of muscle cells from intact ventricles using conventional microelectrode techniques. Excised ventricles were placed in a tissue bath and superfused with Ringer solution. Glass microelectrodes filled with 3 M KCl, which had tip resistances in the range of 20-40 M, were connected to differential preamplifiers with input impedance of 10⁶ M (WPI, KS-700). Amplifier output was monitored and processed with a digital oscilloscope (MacLab; AD Instruments) and then stored on disk for later retrieval and analysis. Test temperatures were maintained with a PolyScience 900 refrigerated circulator. Action potentials were recorded at the three test temperatures and in response to increasing [Ca²⁺]_o and 10 µM Bay K 8644 (Bay K).

Pharmacological treatments. Various pharmacological agents were used to investigate sources of intracellular calcium for muscle activation at the three experimental temperatures. The contribution of sarcolemmal calcium channels was investigated using channel modulators. The cumulative concentration response relationship of Bay K {1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl) phenyl]pyridine-3-carboxylic acid methyl ester}, a dihydropyridine (DHP) L-type calcium channel agonist, to contraction was studied from 0.03 to 10.0 µM. Responses to single doses of compounds that act at the sarcolemmal calcium channel were also studied, i.e., nicardipine (6 µM), a 1,4-DHP antagonist, and cadmium (0.25 mM), an inorganic calcium channel blocker. In addition, the responsiveness of cardiac tissue to pharmacological excitation was tested with 300 nM isoproterenol. The concentrations cited were selected based on the response of whole cell calcium currents in isolated trout myocytes studied under similar conditions (15). Because the SR has been reported as the site for sequestration and release of calcium that controls contractile activation (12, 13), we used agents that block and activate calcium release from the SR, i.e., ryanodine (10 µM) and caffeine (10 mM), respectively, to monitor the contribution of this compartment to piscine cardiac contraction at different temperatures.

Concentrations reported for all pharmacological agents are final bath concentrations. The drugs were allowed to equilibrate until a steady-state response was achieved. Twitch force parameters were recorded at steady state. Unless indicated otherwise, chemicals were obtained from Sigma (St. Louis, MO) and were of the highest analytic grade.

Statistical analysis. Data are means ± SE. Student's t-test was used to compare the means of the different variables given in single doses. For large scale experiments involving a range of treatments at three different temperatures, statistical differences were assessed using ANOVA followed by a Scheffé multiple comparison test. P < 0.05 was considered statistically significant.

	RESULTS

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Acute temperature effects. Because the heart rate affects the balance of calcium in the intracellular space, we tested the contractile force over a range of cardiac pacing. Hearts were initially paced at frequencies ranging between 0.2 and 1.0 Hz at 10°C (acclimation temperature). Pacing itself did not cause any significant changes in either contractile force (Fig. 1), TPT, or t₈₀ (Table 1). Moreover, the acute effects of warmer (18°C) and colder (4°C) test temperatures on the contractile force of trout ventricular myocardium during the frequency series were not significant (Fig. 1, see also controls in Figs. 3 and 5). However, there was a trend for TPT and t₈₀ to be significantly slower at the in vitro test temperature of 4°C and faster at 18°C compared with the acclimation temperature of 10°C at all pacing frequencies (Fig. 1 and Table 1). These differences were consistent and were statistically significant in subsequent experiments (see Tables 3, 5, and 6).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effects of frequency on ventricular force of trout myocardium. Hearts were paced at frequencies ranging from 0.2 to 1.0 Hz at 10°C (acclimation temperature) and 4 and 18°C. Each point is mean ± SE. , 10°C, n = 8. , 18°C, n = 8. , 4°C, n = 5. Inset: representative contractile response of ventricular strip to increasing frequency ranges. Note consistent time course at all experimental temperatures (no vertical scaling).

Spontaneous ventricular action potentials were recorded at the acclimation temperature and at each of the two test temperatures (Fig. 2). In all experiments, action potential duration at 25 and 90% of full repolarization (APD₂₅ and APD₉₀, respectively) were significantly prolonged at colder temperatures relative to warm ones (P < 0.001). For example, APD₉₀ at 4°C was 1,175 ± 36 vs. 695 ± 20 ms at 10°C and 438 ± 25 ms at 18°C. Also APD₂₅ more than doubled as experimental temperatures were lowered from 18 to 4°C (Table 2). Resting membrane potential and action potential amplitude were not significantly affected by the temperature changes.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Effects of temperature on action potential profile in rainbow trout ventricular myocardium. 0, Zero membrane potential.

Role of sarcolemmal calcium channels. The effects of increasing concentrations of [Ca²⁺]_o on the force of contraction of the myocardium were studied at the acclimation temperature (10°C), as well as at the two test temperatures (4 and 18°C) (Fig. 3). At 0.16 mM Ca²⁺ (shown as zero in Fig. 3), virtually no contraction was observed at all test temperatures. Increasing calcium concentrations beyond 1.6 mM elicited an increase in contractile force at all temperatures. In all cases, the response at 16.0 mM was significantly greater than the respective control at 1.6 mM Ca²⁺ (P < 0.05). Activation of the myocardium at 18°C was consistently greater at the higher calcium concentrations compared with that at 4 or 10°C.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Ventricular contraction of rainbow trout myocardium in response to increasing concentrations of calcium at different test temperatures. Hearts were paced at 0.2 Hz, and effects of increasing calcium (0.16-16.0 mM) on contractile force were observed at 10°C (acclimation temperature) and 4 and 18°C. Data were calculated relative to force seen in standard Ringer solution (1.6 mM Ca2+). Only data between 1.6 and 16.0 mM are presented. Each point is mean ± SE. , 10°C, n = 9. , 18°C, n = 5. , 4°C, n = 5. Zero intercept, 0.16 mM Ca2+ at all temperatures. * Significant difference from response at 1.6 mM Ca2+ at respective temperature.  Significant difference from peak force at 4 and 10°C at respective calcium concentration (P < 0.05). Inset: representative contractile response of ventricular strip to increasing extracellular calcium concentration ([Ca2+]o).

As seen with the force of contraction, neither TPT nor t₈₀ was significantly affected by a change in temperature at 1.6 mM Ca²⁺ (Table 3). In addition, raising the [Ca²⁺]_o did not alter these parameters in ventricular tissue at either the acclimation temperature of 10°C or at the elevated temperature of 18°C. However, at 4°C, raising [Ca²⁺]_o from 1.6 to 3.4 mM significantly prolonged both TPT and t₈₀ (P < 0.05). No additional increments were seen with further increases in [Ca²⁺]_o. There were no significant differences in TPT and t₈₀ among the different temperature groups, although there appeared to be a trend toward slower responses at colder temperatures (4°C) vs. faster responses at warmer temperatures (18°C) as mentioned previously.

The effects of elevated calcium concentration on APD were studied at the acclimation temperature (10°C) (Fig. 4). APD was initially recorded at 1.6 mM Ca²⁺ and then at each of the higher concentrations used previously. There was a significant concentration-dependent decrease in both APD₂₅ and APD₉₀ as [Ca²⁺]_o was increased. The response was particularly pronounced at the level of the plateau; APD₂₅ was reduced from 512 ± 31 ms at 1.6 mM Ca²⁺ to 425 ± 12 ms at 3.4 mM (P < 0.001), falling to 348 ± 18 ms at 7.4 mM Ca²⁺ and 263 ± 27 ms at 16.0 mM. Resting membrane potential was not significantly affected by alterations in [Ca²⁺]_o (Table 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Action potential configuration of rainbow trout ventricular myocardium at 10°C in response to increasing concentrations of calcium in bath. 0, Zero membrane potential.

Calcium channel agonists and antagonists. To assess further the contribution of extracellular calcium to ventricular contraction Bay K, an L-type DHP calcium channel agonist, was applied to the myocardium in concentrations ranging from 0.03 to 10.00 µM (Fig. 5). At the acclimation and test temperatures (10 and 18°C) Bay K induced a concentration-dependent increase in peak twitch force with a significant difference at 10 µM Bay K at 10°C and 3 µM at 18°C (P < 0.05). In contrast, at the test temperature of 4°C, a concentration-dependent decrease in peak twitch force was observed (P < 0.05 at 1, 3, and 10 µM). Bay K did not induce a change in TPT or t₈₀ within a given experimental temperature (Table 5). However, under these experimental conditions, there were significant differences in TPT and t₈₀ (P < 0.05) between the experimental temperatures of 4 and 18°C compared with the acclimation temperature of 10°C. The lower temperature caused a longer TPT and t₈₀, whereas the higher test temperature produced a shorter TPT and t₈₀.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Ventricular contraction of rainbow trout myocardium in response to increasing doses of Bay K 8644 (Bay K). Hearts were paced at 0.2 Hz and increasing concentrations of Bay K (0.03-10.00 µM) were added to baths at 10, 4, and 18°C. Data were calculated relative to force at control (no drug treatment). Each point is mean ± SE. , 10°C, n = 8. , 18°C, n = 8. , 4°C, n = 5. * Significant difference from respective control (no drug treatment). Inset: representative contractile response of ventricular strip to 30 nM Bay K.

Action potential responses to Bay K (10 µM) were also monitored in isolated trout hearts at the acclimation temperature, as well as at 4°C (Fig. 6). At this concentration, APD₉₀ was not significantly affected by the calcium channel agonist at either of the temperatures tested. However, plateau duration was slightly, although significantly, increased at 4°C in the presence of the drug (P < 0.05). APD₂₅ was increased from 771 ± 27 to 823 ± 19 ms at 4°C but was not significantly altered by Bay K at 10°C. Bay K did not affect resting membrane potential.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Effects of Bay K (10 µM) on rainbow trout ventricular action potential at 4°C. 0, Zero membrane potential.

Other agents that affect [Ca²⁺]_i. As demonstrated in Fig. 7 there were no temperature-sensitive differences in the inotropic responses of the ventricular myocardium to any of the other pharmacological agents tested (Fig. 7, A-C). Caffeine (10 mM), which liberates Ca²⁺ from the SR, substantially enhanced peak force by ~800% at all temperatures studied (P < 0.05) compared with the untreated control at a given temperature. Similarly, the -receptor agonist isoproterenol significantly enhanced contractility of the fish ventricle by ~600-800% at the three temperatures studied (P < 0.05). Ryanodine, a compound known to inhibit the release of calcium from the SR, significantly reduced the peak twitch force by about 10% but only at 10°C (Fig. 7B, P < 0.05). Ryanodine did not suppress the force of contraction at either 4 or 18°C (Fig. 7, A and C). Nicardipine, a DHP calcium channel antagonist, significantly decreased the peak twitch force at the acclimation temperature by 50% compared with the untreated control, and cadmium (0.25 mM), an inorganic calcium channel blocker, had a significantly greater negative inotropic effect than that of nicardipine at 10°C (80% decrease; Fig. 7B; P < 0.05). In contrast, at 4 and 18°C the negative effects of ryanodine, nicardipine, and cadmium were not statistically significant compared with the untreated control at the acclimation or respective control temperature (Fig. 7, A and C).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effects of caffeine (10 mM), isoproterenol (300 nM), ryanodine (10 µM), nicardipine (6 µM), and cadmium (0.25 mM) on peak force of contraction. Trout ventricular muscles were stimulated at 0.2 Hz, and pharmacological agents were added directly to baths at 4°C (A), 10°C (B; acclimation temperature), and 18°C (C). + Significant difference compared with control at acclimation temperature of 10°C, P < 0.05. * Significant difference compared with untreated control at given temperature (baseline), P < 0.05. # Significant difference between responses to cadmium and nicardipine, P < 0.05. Bars are means + SE; n = 3-6.

As shown in Table 6 none of the drugs altered TPT at a given temperature. However, at 18°C there was a significant prolongation of t₈₀ with caffeine and ryanodine. The overall effects of temperature on TPT and t₈₀ were not significantly different but demonstrated the same trends seen earlier, that is, a prolongation of the time courses at 4°C and a shortening of the time courses at 18°C.

	DISCUSSION

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The present study is the second in a series of electrophysiological and functional investigations aimed at determining the effects of calcium availability and acute temperature changes in the myocardium of a single teleost species. Our fish were continuously maintained at a carefully regulated environmental temperature of 10°C throughout the entire duration of their life and study. This therefore avoids potential adaptive or maladaptive changes in E-C coupling parameters, which may occur in response to variations in environmental temperature.

Acute temperature effects in fish ventricular myocardium. We found that acutely raising or lowering the temperature in vitro had the most prominent effect on the prolongation of the contraction and relaxation phases at 4°C. These results are similar to those reported for mammalian cardiac tissue in vitro, where relaxation time is significantly prolonged by a drop in temperature (24). In this case the contractile response varied depending on the mammalian species. We found that the trout myocardium peak contractile force, in contrast to the duration of contraction, did not demonstrate a consistent difference in response to acute changes in temperature. This suggests that cardiac function in this species can adapt to such changes. Because there is evidence that ion channels and transport systems responsible for regulating [Ca²⁺]_i are affected by temperature change (1-3, 15, 18), such an adaptation would require a new mechanism for intracellular balance of Ca²⁺ entry and removal to maintain consistent [Ca²⁺]_i for continued contractile performance.

The slower contraction time at the lower temperature also suggests a modification in the rates of Ca²⁺ entry and/or removal. Because acute cooling has been shown to induce greater SR calcium release and to depress SR Ca²⁺-ATPase (1, 2), the lower test temperature should result in slower SR calcium reuptake and increased calcium release. If the increased calcium from the SR is balanced by decreased calcium influx via sarcolemmal calcium channels (15) coupled with a slower rate of calcium removal, then the rate of contraction and relaxation might be prolonged. However, the force of contraction could remain constant if the total available calcium was similar. A decreased rate of calcium removal is supported by the prolonged duration of the action potential in trout at 4°C, a response similar to that observed in other teleost species (6). These results led us to further investigate the effects of acute temperature transitions on cardiac contractility and intracellular calcium availability in trout.

Force-frequency response. Because heart rate and cardiac output are known to vary in response to environmental stresses such as temperature variation, we investigated the adaptive responses in contractility as the heart was paced at different rates. At the acclimation temperature of 10°C, the baseline contractile force in trout at 1.60 mM Ca²⁺ and 0.2 Hz was 3.1 ± 0.5 mN/mm². This value is comparable to those reported by Hove-Madsen and Gesser (13) and Shiels and Farrell (28). However, the former investigators reported a positive force-frequency relationship in the rainbow trout kept in fresh water tanks at 10-15°C (13), whereas we found no significant change in the force of contraction over a physiological range of frequencies (0.2-1.0 Hz). Our data are consistent with the findings of Driedzic and Gesser (4) in teleost preparations but is in marked contrast with data from elasmobranchs, which demonstrate a positive inotropic response as the contraction frequency is increased (5). We also found a flat force-frequency relationship when the temperature was adjusted to either 4 or 18°C from the acclimation temperature of 10°C. For mammalian myocardium such a range of temperatures (±5°C) would result in significant shifts on the calcium-force axis and myofilament calcium binding, thereby affecting peak twitch force (10). Our data suggest that Oncorhynchus mykiss can maintain myocardial contractility over a wide range of temperatures at varying heart rates.

We next focused on the availability of calcium from both extra- and intracellular sources. Trout do not have an extensive SR to supply calcium to the cytoplasm (28) nor T tubules to activate the SR (25, 26); therefore, sarcolemmal calcium channels are probably an important source of calcium for contraction. In earlier studies with carp, ryanodine (10 µM), a blocker of SR calcium channels, had no effect on the force of contraction in fish acclimated at either 2 or 22°C; therefore, it was concluded that the SR was not directly involved in the activation of contraction in this species (34). Moreover, the importance of these two compartments has been shown to vary at different temperatures. For example, ryanodine is reported to be ineffective in decreasing contraction at 10°C in rainbow trout but potent at 18°C (12, 14).

Role of sarcolemmal calcium channels in contraction. Calcium entry through the sarcolemma is primarily dependent on L-type calcium channels (15), and the importance of this route of calcium entry was determined by altering [Ca²⁺]_o and by enhancing or blocking the L-type channels with Bay K or nifedipine and cadmium, respectively. At the three experimental temperatures increasing concentrations of extracellular calcium enhanced the peak twitch force, demonstrating that contractile force in fish myocardium is partially dependent on calcium entry through the sarcolemma. The contractile response of myocardium exposed to the acclimation temperature (10°C) and cold temperature (4°C) was significantly less than that at 18°C at the higher calcium concentrations, suggesting a decreased influx of calcium (4) via the channels at the lower temperature. Earlier work (15) investigating calcium currents in trout myocytes supports a temperature effect on calcium influx, because there were parallel but small changes in current with decreasing temperature. Another possibility is a decreased sensitivity of the myofilaments to calcium at the lower temperatures, as reported in frog and mammalian species (10, 11). However, the contractile force in trout was similar at 4 and 10°C with increasing [Ca²⁺]_o, and the apparent dissociation constant appears to be similar, indicating that there is no change in the sensitivity to calcium. A similar insensitivity of Ca²⁺ binding to myofilaments with lowered temperature was found in catfish myocardium using skinned fiber preparations (18). In contrast, using crucian carp, Vornanen (34) reported that the myocardium of trout subjected to long-term cold acclimation was more sensitive to calcium than that from warm-acclimated fish. Whereas we observed a change in the force of contraction in trout with increasing [Ca²⁺]_o, there was no change in contraction time course within a given temperature series.

If calcium entry via L-type calcium channels were enhanced with increasing extracellular calcium we might expect to see a prolonged plateau in the action potential. However, the reverse was true. High calcium significantly shortened the plateau phase and the APD. Intracellular calcium has been shown to regulate APD in amphibians and mammals, in part through activation of K⁺ currents (34). Our findings suggest the activation of a calcium-dependent K⁺ current in trout myocardium, which would bring about repolarization sooner as the [Ca²⁺]_i was increased.

Calcium channel agonist and antagonists. To assess further the importance of L-type calcium channels as a route of calcium entry for myocardial contraction in trout, we modified the function of the channels by using specific agonists and antagonists. We have demonstrated that L-type calcium currents in isolated trout myocytes were responsive to these agents at the given doses (15). To further test whether transsarcolemmal calcium influx contributes to contractile activation and its temperature sensitivity, we investigated the effect of Bay K, a DHP calcium channel agonist, on myocardial contractility and action potential configuration. At the acclimation temperature (10°C) and at 18°C, there was a significant increase in peak twitch force with increasing concentrations of Bay K. These data probably reflect a simple increase in [Ca²⁺]_i with no alteration in SR calcium mobilization and are similar to our results with altered [Ca²⁺]_o. These results agree with our previous findings in perforated patch-clamp studies; that is, there was an increase in peak calcium currents as temperature increased from 4 to 18°C in the presence of Bay K (15). Unexpectedly, at 4°C in isolated muscle preparations, Bay K caused a decrease in peak twitch force. This reduced contractile force at 4°C is partially explained by the fact that 80% of the muscles studied at 4°C developed contractile alternans and an increase in resting force in response to an increase in Bay K concentration. In mammalian hearts, which are dependent on calcium from the SR, the appearance of contractile alternans indicates a disruption of E-C coupling if the cell is overloaded with calcium (16). An inhibitory effect of excess [Ca²⁺]_i is unlikely, however, because we saw only enhanced contraction with very large increases in [Ca²⁺]_i (i.e., with isoproterenol or caffeine). It may be that Bay K has other physiological effects in trout. Bay K did not affect TPT or t₈₀ at any concentration at a given temperature.

Because Bay K prolongs the open state of voltage-dependent calcium channels, thereby allowing greater transsarcolemmal influx of calcium, we would also expect Bay K to induce a prolongation of the plateau phase of the action potential. Indeed the plateau was significantly longer, but only at 4°C, resulting in a prolongation in the early phase of repolarization (APD₂₅) but not the later phase (APD₉₀). This suggests that the influx of calcium at 4°C has been increased by Bay K and further confounds our interpretation of the reduction in contractile force under these conditions. Bay K has been shown previously to prolong the action potential plateau in flounder heart at 10°C (17).

Although Bay K enhanced contractile force at 10 and 18°C, the L-type calcium channel antagonist nicardipine and the calcium channel blocker cadmium significantly reduced the contractile force only at 10°C. However, there was a trend toward reduced peak force at 4 and 18°C as well. The apparently greater negative effect of cadmium is a reflection of its more effective blockade of L-type calcium channels, as reported in isolated trout myocardium (15). These data again indicate a direct relationship between transsarcolemmal calcium influx and myocardial contractility in trout myocardium. Although the cadmium effect did not appear to be significantly different at the three experimental temperatures in vitro, Vornanen reported that cadmium had a greater effect on contractility of trout hearts that had been acclimated over a period of time to cold rather than warm environments (33). Whereas cadmium and nicardipine, which would decrease [Ca²⁺]_i, did not induce changes in the time course of the isometric twitch, neither did increased [Ca²⁺]_o and Bay K, which would increase [Ca²⁺]_i.

-Adrenergic receptor stimulation. Isoproterenol acts by phosphorylating L-type calcium channels via an increase in cAMP and has been shown to significantly increase the calcium current in trout myocytes (15). Therefore, treatment with isoproterenol is another means of increasing the transsarcolemmal availability of [Ca²⁺]_i. As expected, isoproterenol had a positive inotropic effect on cardiac contractile force at all temperatures. We have shown previously that isoproterenol not only increases the magnitude of the calcium current in trout myocytes at all temperatures but also results in a continuous rate of influx during prolonged depolarization (15). Because isoproterenol increases SR calcium reuptake as well as release (22), it probably induces an altered balance between calcium entry and removal. However, Gesser (7) demonstrated that the positive inotropic effect seen in teleosts with adrenaline was not affected by the addition of ryanodine, a blocker of SR Ca²⁺ release, and therefore appeared not to depend on the SR. Again, the increased entry of calcium via L-type calcium channels did not significantly affect the time course of the isometric twitch at a given temperature, in agreement with the data of Gesser (7) and Vornanen (32), in which activation of -receptors had no effect on the time course of relaxation in trout myocardium. This is unlike mammalian myocardium, where adrenaline and isoproterenol result in significant phosphorylation of phospholamban, resulting in enhanced reuptake of calcium by the SR and abbreviation of the time course of relaxation. Again, these data suggest a simple increase in [Ca²⁺]_i for myofilament activation and lend further support to the important role of L-type calcium channels in providing [Ca²⁺]_i from the extracellular compartment to control contractility in the trout myocardium (28, 30).

Intracellular calcium mobilization. The contribution of the SR to the pool of [Ca²⁺]_i during muscle contraction in trout was assessed by both promoting and blocking Ca²⁺ release. Caffeine is an agent that directly stimulates the SR to release large quantities of calcium into the intracellular compartment. At all experimental temperatures, caffeine induced a similar eightfold increase in peak contractile force. Raising or lowering the in vitro temperature did not alter the magnitude of the caffeine-induced release. These responses suggest that so much calcium was released by the SR that any effects of temperature on calcium removal via SR calcium uptake or in the contractile response as a result of alterations in myofilament calcium sensitivity were not apparent. The magnitude of the contractile response indicates that the trout myocardium has the potential to contract vigorously when exposed to increasing intracellular concentrations of calcium. The fact that a similar strong contractile response was also found with isoproterenol stimulation suggests that the quantity of calcium is the important criterion for increased force, rather than the source. In addition, it suggests that both calcium sources, i.e., influx via the sarcolemma or release from the SR, have the potential to substantially increase [Ca²⁺]_i in trout. Moreover, caffeine did not significantly impact TPT or t₈₀ relaxation in hearts beyond that seen with temperature alone. A similar observation was seen in crucian carp at 22°C, where caffeine (5 mM) improved contractility without affecting muscle relaxation (34).

Ryanodine is a neutral plant alkaloid that inhibits SR Ca²⁺ release and enhances SR calcium leak. Because low experimental temperatures might affect the binding affinity of ryanodine (12, 21, 27, 29), we used a relatively high concentration (10 µM). Ryanodine did not impact peak force of contraction at 4 or 18°C but did so at the acclimation temperature in which case peak force was significantly decreased (11%). At 4 and 18°C, similar to a report in crucian carp hearts acclimated at 2 or 22°C (34), 10 µM ryanodine did not change twitch force. These data suggest that at 4 and 18°C the SR does not have a significant role in the contraction response in agreement with previous findings (12, 28). However, at the acclimation temperature, the SR may have a small contributory role in the E-C coupling.

Relationship among force, time course of isometric twitch, and calcium. We have observed changes in the amplitude of the isometric twitch from trout myocardium to gain insight into cellular E-C coupling in this species. Twitch force is determined by calcium binding to the myofilaments and the time course of calcium availability. Relaxation occurs when calcium dissociates from the myofilaments and is resequestered into the SR or extruded from the cell and thus removed from the cytosol. The correlation between intracellular calcium mobilization and contractile force has been demonstrated in mammalian myocardium (8, 37) and is thought to apply to cardiac function in general. The time course of intracellular calcium mobilization is regulated by the balance between transsarcolemmal calcium influx, release of calcium from the SR, and the subsequent reuptake as well as extrusion from the cell by the sarcolemmal Ca²⁺-ATPase and the Na⁺/Ca²⁺ exchanger. Thus one would anticipate that interventions that alter the time course of intracellular calcium mobilization might change the time course of the associated isometric twitch, as well as peak amplitude. A decrease in the affinity of troponin C to calcium would result in a shorter time course of contraction. Conversely, an increase in the affinity of troponin C for calcium should result in a slower rate of contraction and relaxation (20). Interventions that increase SR calcium loading and release without affecting the time course of intracellular calcium mobilization should result in an increase in peak twitch force with no change in the time course of contraction and relaxation, e.g., changes in extracellular calcium, Bay K, caffeine, and isoproterenol. On the other hand, conditions that increase the rate of calcium uptake by the SR would be expected to shorten the time course of the intracellular calcium transient, thereby abbreviating the time course of the contraction unless balanced by increased calcium entry, e.g., higher temperatures. Similarly, conditions that decrease the rate of calcium uptake by the SR would be expected to lengthen the time course of the intracellular calcium transient, e.g., lower temperature, resulting in a prolongation of the contractile response and relaxation. Therefore, it is recognized that changes in the time course of the isometric twitch correlate with changes in [Ca²⁺]_i, which in turn, is a reflection of modifications in calcium mobilization and removal (8, 37).

In conclusion, the relationship between temperature and E-C coupling in trout cardiac tissue is not fixed. The present study suggests that transsarcolemmal calcium influx supports force development in ventricular trout muscle. There is little indication in our study that the SR contributes significantly to E-C coupling between 4 and 18°C in Oncorhynchus mykiss. However, it has been reported that at 25°C in this species, force is correlated with SR function, which appears to contribute significantly to E-C coupling (12). Our data suggest that this observation may be dependent on the thermal acclimation of the fish studied. Nevertheless, the function of myocardial SR in the living trout remains unclear primarily because environmental temperature, which can range from 2 to 25°C, can induce varied and complex adaptive changes in E-C coupling. Furthermore, our data suggest that the function of the trout heart is not adversely affected by acute changes in temperature within 6-8°C of the 10° acclimation temperature. Part of the adaptability is the relative insensitivity to acute temperature changes of many components of the system, e.g., L-type calcium channels (15), Na⁺/Ca²⁺ exchanger (31), myofilament affinity for calcium, SR calcium release, and response to -adrenergic agonists, as shown in the present work. This suggests that whereas temperature may adversely affect one component of calcium availability in trout, it is balanced by a compensatory change. The result is sufficient [Ca²⁺]_i to maintain cardiac contractility.

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