INTRODUCTION

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IN A PREVIOUS STUDY (26), we found that the availability of L-type Ca²⁺ channels of guinea pig ventricular cells is reduced by G protein-dependent slow recovery kinetics when animals are exposed to cold for >3 wk. The physiological implication of this kinetic change, however, remains unsolved. If the influx of Ca²⁺ via L-type Ca²⁺ channels is decreased, contraction of the papillary muscle dissected from cold-acclimated guinea pigs might be significantly smaller than that of control. Furthermore, the induction of the extraslow inactivation by the cold acclimation has been observed only in the dissociated myocytes, whose current systems might artificially be modified through the dissociation procedure. In the present study, we measured contraction of the intact papillary muscles dissected from control and cold-acclimated guinea pig hearts to address these problems.

Surprisingly, the effect of prolonged cold exposure on cardiac contractile properties of the papillary muscle has not been reported previously. In cardiac hypertrophy (4, 11, 16) or cardiomyopathy (9), the force of contraction is depressed with retardation of contraction and relaxation, possibly due to the dysfunction of sarcoplasmic reticulum (SR). Comparison of the papillary muscle contraction in cold-acclimated guinea pigs with the control may give some insight into the modulation of the contraction pattern through various long-term conditioning programs.

	METHODS

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Preparation and tension measurement. All animal treatments were performed according to the guidelines of Helsinki Declaration, and the experimental protocols used were approved by the animal use and care committee of Kyoto University. Male Hartley guinea pigs aged 4-5 wk were individually caged and kept for >3 wk in a cold (5°C) or modestly warm room (~25°C) before they were used, the conditions of which were the same as those described in our previous study (26). During the period of cold exposure, the body weight of guinea pigs increased from ~200-250 to ~350-450 g, similar to control animals kept at room temperature.

Solutions. The composition of the control Tyrode solution used for recording the developed tension was (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 0.3 NaH₂PO₄, 5.5 glucose, and 5 HEPES; the pH was adjusted to 7.4 with NaOH. Ryanodine (Sigma, St. Louis, MO; 2 µM) was added to the control external solution when appropriate. The composition of the high-K⁺ solution was (in mM) 140 NaCl, 15 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 0.3 NaH₂PO₄, 0.2 BaCl₂, 5.5 glucose, and 5 HEPES; the pH was adjusted to 7.4 with NaOH. In the experiments using ryanodine, contraction was measured 20 min after the application of the drug.

Statistics. Values were expressed as means ± SE as far as available. Statistical difference was examined using paired or unpaired Student's t-test (P < 0.05).

	RESULTS

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Reduction of developed tension of papillary muscle after cold acclimation. The maximum diameter of the papillary muscles from control animals was 0.92 ± 0.07 mm (range 0.8-1.2 mm; n = 14), which was not statistically different from those from cold-acclimated guinea pigs (1.04 ± 0.05 mm, range 0.9-1.3 mm; n = 16, P < 0.05). Similarly, no statistical difference was observed between the length of the papillary muscles from control animals (3.6 ± 0.3 mm, range 1.8-4.8 mm; n = 14) and that from cold-acclimated animals (3.21 ± 0.31 mm, range 2.0-4.9 mm; n = 16, P < 0.05). To compare the contractility of the papillary muscle having different sizes, the amplitude of developed tension was normalized to the cross-sectional area of the muscle (dyne/cm²) that was calculated from the maximum diameter. Figure 1 demonstrates records of the developed tension at different stimulus frequencies, which are representative of the control (A) and cold-acclimated (B) guinea pig, respectively. As evident from the calibration bar, the developed tension was much smaller in the cold-acclimated animal than in the controls at all stimulus frequencies used. Figure 1 summarizes data where at all stimulus frequencies, the difference between the control and the cold-acclimated group was statistically significant (P < 0.05). At the stimulus frequency of 1 Hz, for example, the magnitude of developed tension was 3,021 ± 873 dyne/cm² (n = 11) in the cold-acclimated muscles, whereas the control was 19,201 ± 2,460 dyne/cm² (n = 11) (P < 0.05). It should also be noted that the "positive staircase" was evident in both groups, with increasing stimulus frequency between 0.5 and 3 Hz.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Decrease in developed tension of papillary muscle after cold acclimation. Peak developed tension of right ventricular papillary muscle was normalized by cross-sectional area (dyne/cm2) and is shown as mean ± SE. Typical original traces at 0.1, 1, 2, and 3.3 Hz in control (A) and cold-acclimated (B) guinea pigs are also shown at top. Amplitude was measured at steady state in response to various frequencies of stimulation (0.1, 0.2, 0.33, 0.5, 1, 1.4, 2, 2.5, 3.3, 4, and 5 Hz). Contraction of cold-exposed guinea pigs (, n = 11) was significantly smaller than that of control group (, n = 11) at all frequencies tested (* P < 0.05).

Comparison of twitch time courses. The reduction of the developed tension observed in various pathological conditions is accompanied by slower time courses of contraction, most probably due to dysfunction of SR (7-10). To examine this possibility in the cold-acclimated animals, the time to peak tension and time to 80% reduction from the peak tension were measured in papillary muscles from control and cold-acclimated guinea pigs at various stimulus frequencies (Table 1). The time to peak tension was significantly faster in the cold-acclimated animals than in the controls, whereas the time to 80% regression from the peak tension was not different in either group. These results suggested that the reduction of the developed tension after the cold acclimation is distinguished from those in cardiac hypertrophy (4, 11, 16) or cardiomyopathy (9).

Effects of high-K⁺ depolarization. Our previous study (26) using whole cell patch clamp in dissociated single ventricular cells revealed that cold acclimation of guinea pigs induced an extra inactivation kinetic of the L-type Ca²⁺ channels that was much slower than the usual voltage-dependent inactivation. The recovery from this slow inactivation is so delayed that repetitive action potentials or depolarizing pulses cause accumulation of inactivation. If a significant fraction of L-type Ca²⁺ channels is not available due to this slow inactivation induced by the preceding action potentials, the depression of the developed tension in the cold-acclimated animal might be due to a smaller Ca²⁺ current.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of high-K+ solution on contraction of papillary muscle dissected from control and cold-acclimated guinea pigs. Contraction of papillary muscle was elicited by 1-Hz stimulation in control Tyrode solution, then external solution was switched to high-K+ (27 mM) solution containing 0.2 mM Ba2+ (arrows). Contraction of control guinea pigs was only slightly reduced (A), whereas it was abolished in cold-exposed guinea pigs (B).

Effects of ryanodine on contraction. The increase in Ca²⁺ to trigger the twitch contraction is provided by Ca²⁺ release from SR and also potentially by the reverse mode of the sarcolemmal Na⁺/Ca²⁺ exchange. Ryanodine has been used as a tool to disable the SR Ca²⁺ release and thereby to separate these SR and sarcolemmal mechanisms. The twitch contractions of the papillary muscle before (Fig. 3, ) and after (Fig. 3, ) application of 2 µM ryanodine are superimposed at different stimulus frequencies, and the ryanodine-sensitive component (Fig. 3, thick traces) was obtained as the difference between two records. If it is simply assumed that the ryanodine-sensitive component represents a contraction due to the SR Ca²⁺ release, the smaller magnitude of the ryanodine-sensitive component in the cold-acclimated cells compared with the control is consistent with the lower availability of the L-type Ca²⁺ channel to induce SR Ca²⁺ release after the cold acclimation. Furthermore, the stimulus frequency-dependent increase in magnitude of the ryanodine-sensitive component in cold-acclimated muscle suggests that the SR content of Ca²⁺ was increased by repetitive activation of the L-type Ca²⁺ channels. In contrast, the almost equal amplitudes of the ryanodine-sensitive component in the control cells may suggest that the SR Ca²⁺ store was almost saturated at 0.5 Hz stimulation. The relative peak amplitude of the ryanodine-sensitive component of the control group (n = 4), as normalized by its value at 2-Hz stimulation, was 0.87 ± 0.07 and 0.92 ± 0.09 at 0.5 and 1.0 Hz, respectively. In contrast, the normalized values calculated in cold-exposed guinea pigs (n = 4) were 0.18 ± 0.04 (0.5 Hz) and 0.63 ± 0.08 (1.0 Hz). Thus the results suggest that reverse mode of the Na⁺/Ca²⁺ exchange is predominantly involved in the positive staircase in control animals due to saturation of the SR Ca²⁺ store at low frequency of stimulation.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effects of ryanodine on contraction in control and cold-acclimated guinea pigs. Contractions were recorded in papillary muscle from a control (A) and a cold-acclimated (B) guinea pig without and with 2 µM ryanodine. Twitch contractions of papillary muscle before () and after () application of 2 mM ryanodine. Thick curves indicate ryanodine-sensitive component. Note that ryanodine-sensitive component is not significantly different in control, whereas it increased with higher stimulation. See RESULTS for details.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effects of ryanodine over wide range of frequencies in control and cold-acclimated guinea pigs. Peak tension [, control (Cont); , ryanodine (Ryan)] and diastolic tension (, Cont; , Ryan) were measured with and without ryanodine over a wide range of frequencies in a control (A) and cold-acclimated (B) guinea pigs. Note that peak tension of control guinea pigs is also reduced by ryanodine (Ryan) at very low frequencies. See RESULTS for details.

	DISCUSSION

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It is well established that the magnitude of twitch contraction is in general dependent on the magnitude of the L-type Ca²⁺ current in the cardiac muscle through two mechanisms. 1) The Ca²⁺ influx determines the Ca²⁺-induced Ca²⁺ release in the functional coupling between the sarcolemmal L-type Ca²⁺ channel (dihydropyridine receptor) and the Ca²⁺-releasing channel (ryanodine receptor) on SR (1, 2, 6). 2) The SR Ca²⁺ store is dependent on the Ca²⁺ influx through the L-type Ca²⁺ channel (3, 6, 22, 24). The present study revealed that the contractility of papillary muscle is significantly depressed by cold acclimation of guinea pigs. This finding is consistent with our previous observation (26) that cold acclimation induced an additional slow inactivation of the L-type Ca²⁺ channel and thereby markedly reduced the availability of the L-type Ca²⁺ channel during repetitive membrane excitation. In fact, the time constant for removal of the inactivation is ~2 s at the resting potential ( = 2,200 ms at 80 mV) in the cold-acclimated cells, in contrast to the time constant of 10-20 ms in control myocytes.

The results in the present study suggest that the observation of the extraslow inactivation in the single ventricular myocytes after the cold acclimation (26) is not due to the dissociation procedure using collagenase that might artifactually modify the cell membrane. In the intact papillary muscle, direct measurement of the Ca²⁺ channel inactivation is hampered by the structural complexity. However, the measurement of the twitch contraction in the high-K⁺ solution (Fig. 2) strongly supported the hypothesis that there is slow inactivation by the cold acclimation in the intact papillary muscle. According to the measurement of inactivation in the whole cell voltage clamp, more than one-half of the L-type Ca²⁺ channels are inactivated at 40 mV. Thus it is plausible the slow action potential could not be triggered in the cold-acclimated papillary muscle.

The positive staircase observed in Fig. 1 is usually explained by the accumulation of the SR Ca²⁺ by the more frequent activation of the L-type Ca²⁺ channels (3, 6, 22, 24). However, the peak tension recorded in the presence of ryanodine also showed the frequency-dependent increase (Fig. 3). Furthermore, the amplitude of the ryanodine-sensitive component of contraction was not significantly changed by increasing stimulation rate in control guinea pigs, in contrast to the evident positive staircase of the component in cold-acclimated guinea pigs. If it is assumed that 2 µM ryanodine completely disabled SR, the ryanodine-insensitive component can only be explained by assuming accumulation of Ca²⁺ in the cytosol through the reverse mode of Na⁺/Ca²⁺ exchange during the action potential plateau or through depression of the forward mode of Na⁺/Ca²⁺ exchange in the presence of relatively large Ca²⁺ influx at the plateau potential (12, 18-20, 23). It would be suggested that Na⁺/Ca²⁺ exchange may be largely involved in the positive staircase in guinea pigs without cold acclimation because of the saturation of the Ca²⁺ release from SR at low frequency of stimulation. Direct activation of contractile proteins via Na⁺/Ca²⁺ exchange has been previously indicated by Nuss and Houser (23), who recorded contractions elicited by voltage-clamp depolarizations of single feline ventricular cells in the presence of ryanodine. The reversal potential of the exchange during the plateau potential might vary depending on the intracellular Na⁺ concentration. Repolarization of the membrane terminated the tonic component of contraction by increasing the driving force for the Ca²⁺ extrusion through the Na⁺/Ca²⁺ exchange. The obvious question is how the positive staircase of the ryanodine-insensitive contraction is explained. The electron probe microanalysis by Wendt-Gallitelli et al. (27) revealed that the concentration of Na⁺ within 20 nm of the inner side of the ventricular cell sarcolemma increases to ~40 mM just after 18 repetitive voltage-clamp depolarizations, with a steep gradient of Na⁺ from beneath the membrane to the center of the cell. The increased concentration remained unchanged in the following 8 s. Local Na⁺ accumulation close to membrane by repetitive stimulation was also suggested by Semb and Sejersted (25), who observed that ouabain-sensitive ⁸⁶Rb uptake is increased without significant change in intracellular Na⁺ activity. The stimulus-dependent accumulation of Na⁺ just beneath the membrane may shift the reversal potential of Na⁺/Ca²⁺ exchange current to more negative potentials favoring influx of Ca²⁺ rather than the efflux via Na⁺/Ca²⁺ exchange. Thus the tonic component of contraction during the plateau potential might be increased with increasing stimulus frequency (Fig. 3). The ryanodine suppression of the maximum tension was observed also in control guinea pigs at frequencies <0.5 Hz, suggesting that Ca²⁺-induced Ca²⁺ release from SR is a major source of contraction at very low frequencies. It would be suggested that the accumulation of Na⁺ is less at lower frequencies at which the extrusion of Na⁺ via both Na⁺/Ca²⁺ exchange and Na⁺/K⁺ pump may take place at resting potential.

When animals are exposed to cold, plasma catecholamines are dramatically increased to maintain the body temperature (10, 14). High concentrations of catecholamines are known to induce Ca²⁺ overload in cardiac cells, which is accompanied by an arrhythmic contraction and excitation due to abnormal release of Ca²⁺ from SR (5, 13, 21). The parasympathetic transmitter ACh can attenuate the increased availability of the L-type Ca²⁺ channel (10) in the continuous presence of catecholamines; however, these ACh effects accompany a rebound increase in L-type Ca²⁺ current and twitch tension of cardiac muscle when ACh is removed (8). Such a post-ACh augmentation of catecholamine effects may occur in the hearts of conscious animals, where parasympathetic activity must often change. Thus it would be suggested that the induction of the slow inactivation kinetics of L-type Ca²⁺ channels may be effective in preventing cardiac Ca²⁺ overload under cold exposure.

Perspectives