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Am J Physiol Cell Physiol 295: C692-C700, 2008. First published July 9, 2008; doi:10.1152/ajpcell.00287.2008
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Hypothermia increases the gain of excitation-contraction coupling in guinea pig ventricular myocytes

Robin H. Shutt2 and Susan E. Howlett1

1Department of Pharmacology and Division of Geriatric Medicine, Dalhousie University, Halifax, NS, Canada B3H 1X5; 2Jim and Pat Calhoun Cardiology Center, University of Connecticut Health Center, Farmington, Connecticut

Submitted 2 June 2008 ; accepted in final form 9 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Components of excitation-contraction (EC)-coupling were compared at 37°C and 22°C to determine whether hypothermia altered the gain of EC coupling in guinea pig ventricular myocytes. Ca2+ concentration (fura-2) and cell shortening (edge detector) were measured simultaneously. Hypothermia increased fractional shortening (8.3 ± 1.7 vs. 2.6 ± 0.3% at 37°C), Ca2+ transients (157 ± 33 vs. 35 ± 5 nM at 37°C), and diastolic Ca2+ (100 ± 9 vs. 60 ± 6 nM at 37°C) in field-stimulated myocytes (2 Hz). In experiments with high-resistance microelectrodes, the increase in contractions and Ca2+ transients was accompanied by a twofold increase in action potential duration (APD). When voltage-clamp steps eliminated changes in APD, cooling still increased contractions and Ca2+ transients. Hypothermia increased sarcoplasmic reticulum (SR) Ca2+ stores (83 ± 17 at 37°C to 212 ± 50 nM, assessed with caffeine) and increased fractional SR Ca2+ release twofold. In contrast, peak Ca2+ current was much smaller at 22°C than at 37°C (1.3 ± 0.4 and 3.5 ± 0.7 pA/pF, respectively). In cells dialyzed with sodium-free pipette solutions to inhibit Ca2+ influx via reverse-mode Na+/Ca2+ exchange, hypothermia still increased contractions, Ca2+ transients, SR stores, and fractional release but decreased the amplitude of Ca2+ current. The rate of SR Ca2+ release per unit Ca2+ current, a measure of EC-coupling gain, was increased sixfold by hypothermia. This increase in gain occurred regardless of whether cells were dialyzed with sodium-free solutions. Thus an increase in EC-coupling gain contributes importantly to positive inotropic effects of hypothermia in the heart.

positive inotropy; calcium transient; contraction; calcium current; sarcoplasmic reticulum calcium stores

THE PROCESS by which excitation of the myocyte leads to cardiac contraction is known as excitation-contraction (EC) coupling (4). For the past several decades, most investigations of the mechanisms involved in EC coupling in the mammalian heart have been conducted in isolated cardiac myocytes. Interestingly, many seminal studies of cardiac EC coupling have been conducted in isolated myocytes studied at room temperature (1, 12, 14–16, 18, 23, 24, 36, 40). However, mammalian core temperatures generally are in the range of 35–39°C (8). Therefore, studies of mammalian cells conducted at room temperature (20–24°C) have explored cardiac function under hypothermic conditions. It has long been established that hypothermia is a potent positively inotropic stimulus that causes a marked increase in the magnitude of cardiac contraction (6). For example, when temperature is reduced from 37°C to 25°C, the force produced by rabbit ventricular muscle is increased by more than 400% (37). Furthermore, the amplitudes of contractions recorded from rabbit, cat, and ferret ventricular myocytes increase markedly when temperature is reduced from 35°C to 25°C (32). Hypothermia also increases the amplitudes of Ca2+ transients recorded from rat and guinea pig ventricular myocytes (30). These observations indicate that temperature can affect mechanisms that are important in cardiac EC coupling.

Previous studies have shown that hypothermia influences many aspects of cardiac EC coupling. Cooling increases action potential duration (APD) in cardiac myocytes and ventricular muscle preparations from rabbit hearts (34, 37). In addition, cooling reduces peak magnitude of L-type Ca2+ current (ICa-L) and decreases the rate of ICa-L inactivation in rabbit and guinea pig ventricular myocytes (9, 34). Studies have shown that hypothermia slows the rate of Na+/Ca2+ exchange in ventricular myocytes from rabbit, ferret, cat, and guinea pig hearts (32, 41), and cooling inhibits contractions initiated by Ca2+ entry via Na+/Ca2+ exchange (42). Hypothermia also increases the open probability of cardiac sarcoplasmic reticulum (SR) Ca2+-release channels from sheep hearts (39). Interestingly, cooling reduces the activity of the SR Ca2+-ATPase but increases SR Ca2+ load in rabbit, ferret, and cat myocytes (32). Myofilament Ca2+ sensitivity increases in response to cooling in intact rabbit hearts (26, 31) but declines with cooling in chemically skinned rabbit ventricular muscle (21, 22). Thus temperature has a marked influence on many components of the EC-coupling pathway in the mammalian heart.

Ca2+ influx via ICa-L triggers Ca2+ release from the SR, a process known as Ca2+-induced Ca2+ release (CICR; 4). As hypothermia reduces the magnitude of peak ICa-L (9, 34), cooling might actually inhibit CICR. However, hypothermia also increases the open probability of SR Ca2+-release channels (39) and increases SR Ca2+ load (32). These effects of cooling would be expected to increase the amount of Ca2+ released per unit current and thereby increase the gain of EC coupling. However, earlier studies did not record transmembrane current, Ca2+ transients, and contractions simultaneously at either physiological or hypothermic temperatures. Thus it is difficult to predict the effect of temperature on the gain of EC coupling from the results of previous studies. Furthermore, previous studies also used myocytes isolated from different species, utilized different experimental techniques, and examined different temperature ranges to evaluate the effect of temperature on various components of cardiac EC coupling. In the present study, we evaluated components of EC coupling in guinea pig ventricular myocytes at 37°C and 22°C, under otherwise similar experimental conditions, to determine whether hypothermia alters the gain of cardiac EC coupling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Myocyte isolation. All experiments were performed according to the guidelines published by the Canadian Council on Animal Care (CCAC; Ottawa, Ontario: vol. 1, 2nd ed., 1993; vol. 2, 1984), and the Dalhousie University Committee on Laboratory Animal Care approved all animal protocols. Adult guinea pig ventricular myocytes were isolated acutely, as described previously (38, 43). Briefly, guinea pigs were anesthetized with pentobarbital sodium (120 mg/kg ip). Anesthetic was coinjected with heparin (3,300 IU/kg). The heart was cannulated and perfused with a nominally Ca2+-free isolation buffer (in mM: 135.5 NaCl, 4 KCl, 1.2 KH2PO4, 1.2 MgSO4, 10 HEPES, and 12 glucose; pH to 7.4 with NaOH), followed by a digestion buffer of the same composition but with the addition of collagenase (16–23 mg/50 ml, Worthington Type II) and protease (4 mg/50 ml, Sigma). The digested ventricles were minced in high K+ buffer (in mM: 45 KCl, 3 MgSO4, 50 L-glutamic acid, 30 KH2PO4, 20 taurine, 0.5 EGTA, 10 HEPES, and 10 glucose; pH to 7.4 with KOH), and cells were dissociated by gentle agitation. Isolated myocytes were incubated at room temperature in the dark with 5 µM fura-2 AM (fura-2 AM stock solution in anhydrous DMSO) for 15 to 20 min. Fura-2-loaded myocytes then were transferred to an experimental chamber installed on the stage of an inverted microscope (Nikon TE200).

Experimental protocols. Myocytes were superfused with a buffer of the following composition (in mM): 145 NaCl, 10 HEPES, 10 glucose, 4 KCl, 1 MgCl2, and 1 CaCl2 either at physiological temperature (37°C) or at room temperature (22°C). Myocytes were exposed either to buffer at room temperature, or to buffer at physiological temperature, but not both. Lidocaine (0.3 mM) was included in the buffer solution in voltage-clamp experiments to inhibit sodium current. In some experiments, myocytes were field stimulated at 2 Hz through silver wire electrodes connected to a stimulator (Grass Technologies, West Warwick, RI), and contraction and Ca2+ transients were recorded simultaneously. Unloaded cell shortening (contraction) was recorded at 120 Hz with a video edge detector (Crescent Electronics, Sandy, UT). Ca2+ transients were recorded with a PTI (Photon Technology International, Birmingham, NJ) fluorescence recording system as described below. In other experiments, myocytes were current or voltage clamped with either high-resistance microelectrodes (15–25 M{Omega}; 2.7 M KCl filled; physiological intracellular Na+ levels) or with patch pipettes (2–3 M{Omega}) filled with a Na+-free solution of the following composition: 0 mM NaCl, 70 mM KCl, 70 mM potassium aspartate, 4 mM MgATP, 1 mM MgCl2, 2.5 mM KH2PO4, 0.12 mM CaCl2, 0.5 mM EGTA, 10 mM HEPES, and 50 µM 8-bromo-cAMP (pH 7.2, KOH). Transmembrane currents, contractions, and Ca2+ transients were recorded simultaneously. Cells were voltage clamped with discontinuous single-electrode voltage-clamp techniques (5–8 kHz) with an Axoclamp 2B current and voltage-clamp amplifier (Molecular Devices, Sunnyvale, CA) and ClampEx software (version 8.1, Molecular Devices, Sunnyvale, CA). Current-clamp experiments were performed with the same amplifier. In current clamp experiments, cells were stimulated with a train of stimuli delivered at a frequency of 2 Hz before measurement of responses. In voltage-clamp experiments, trains of ten 200-ms conditioning pulses preceded test steps. Conditioning pulses were made from –80 to 0 mV and were delivered at a rate of 2 Hz.

To estimate SR Ca2+ load, a rapid solution switcher was used to rapidly expose voltage-clamped cells to buffer containing 10 mM caffeine. The solution switcher allowed us to switch between different solutions while the solution temperature was maintained at either 22°C or 37°C. Caffeine was applied for 1 s in a nominally Ca2+- and Na+-free solution to inhibit extrusion of Ca2+ from the cytosol by Na+/Ca2+ exchange (in mM: 140 LiCl, 4 KCl, 10 glucose, 5 HEPES, 4 MgCl2, 10 caffeine, and 0.3 lidocaine; pH 7.4 with LiOH). Under these experimental conditions, caffeine application did not induce inward Na+/Ca2+ exchange current, as reported in previous studies (e.g., 10, 25). In nominally Ca2+- and Na+-free solution, the caffeine-induced Ca2+ transient does not begin to decay until the end of the caffeine exposure. Changes in Ca2+ concentrations induced by caffeine were measured and compared at the two temperatures. In some experiments, cells were exposed to Ca2+- and Na+-free solution before and during caffeine application. Estimates of SR Ca2+ stores were the same, regardless of whether the switch to caffeine was preceded by application of Ca2+- and Na+-free solution.

Fluorescence recording. Fura-2 fluorescence was recorded and measured as previously described (38). Briefly, fura-2 was excited at 340 and 380 nm, and fluorescence emission was measured at 510 nm. Fluorescence emission was measured at sample rate of 100 points/s for each excitation wavelength. Background fluorescence was subtracted from the measured emissions. The ratio of fluorescence emission recorded during excitation at 340 and 380 nm was determined. Emission ratios were converted to Ca2+ concentration with an in vitro calibration curve determined from known concentrations of Ca2+. The methodology used to generate the calibration curve was adapted from Grynkiewitz et al. (20). Different Ca2+ concentrations were generated by the addition of different volumes of Ca2+-containing, EGTA-buffered solution to a Ca2+-free, EGTA-buffered solution. Since temperature can influence the binding of Ca2+ to fura-2, separate fura-2 calibration curves were generated at 37°C and 22°C. As altering the temperature of EGTA-buffered solutions alters the free Ca2+ (21), the free Ca2+ concentration at each temperature was determined using a computer-based free ion concentration calculator WinMAXC Maxchelator (Chris Patton, Stanford University, Pacific Grove, CA). By altering free Ca2+ concentration, temperature also can influence the binding of hydrogen ions by EGTA and therefore influence pH (21). Therefore, when the parallel calibrations at 37°C and 22°C were undertaken, separate adjustments of pH also were made. The fura-2 fluorescence ratio at 37°C was shown to be equivalent to Ca2+ concentrations that were ~72% of those for the same ratio at 22°C. This relationship was tested for free Ca2+ concentrations, which were representative of the intracellular Ca2+ concentrations reported in previous experiments in guinea pig ventricular myocytes (38). The intracellular Ca2+ concentrations reported here were adjusted for the difference in fura-2 calibration between the two temperatures.

Data analyses. Contraction amplitudes and Ca2+ current amplitudes were normalized to compensate for possible differences in cell size. Contractions were expressed as percentage change in diastolic cell length to provide an estimate of fractional shortening. Currents were normalized by cell capacitance to compensate for differences in cell size. Note that values of capacitance were similar at the two temperatures (164 ± 12 vs. 160 ± 10 pF at 22°C and 37°C, respectively; n = 7–11 cells/group; not significant). Contractions, membrane voltage, and currents were recorded with ClampEx 8.1 (Molecular Devices, Sunnyvale, CA) software and analyzed with ClampFit 8.1 (Molecular Devices) software. Sigmaplot 2001 (Sysstat Software, Point Richmond, CA) was used to construct graphs. Statistical analyses were performed with either Sigmaplot 2001 or Sigmastat version 2.03 (Sysstat Software). Mean data were expressed as means ± SE; all comparisons between 37°C and 22°C were made with unpaired Student's t-tests or ANOVA where appropriate and differences between means were considered significant if P < 0.05.

Chemicals. Lidocaine, HEPES buffer, MgCl2, anhydrous DMSO, and caffeine were purchased from Sigma Aldrich Canada (Oakville, ON). Invitrogen (Burlington, ON) was the supplier for fura-2 AM. All other chemicals were purchased from BDH (Toronto, ON).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We first examined the effects of hypothermia on Ca2+ transients and contractions recorded simultaneously from guinea pig ventricular myocytes that were field stimulated at 2 Hz. Myocytes were loaded with fura-2. Recordings from individual myocytes were made at either physiological (37°C) or hypothermic (22°C) temperatures. Figure 1, A and B, shows representative Ca2+ transients (top traces) and contractions (bottom traces) elicited by field stimulation at 37°C and 22°C. At physiological temperature, Ca2+ transients and contractions were small and contraction amplitude was proportional to Ca2+ transient amplitude (Fig. 1A). Hypothermia produced large Ca2+ transients and large contractions that were proportional to the size of the Ca2+ transients (Fig. 1B). Figure 1C shows mean measurements of peak systolic Ca2+ and diastolic Ca2+ levels at 37°C and 22°C. Peak systolic Ca2+ and diastolic Ca2+ concentrations were significantly elevated by hypothermia (Fig. 1C). Mean amplitudes of Ca2+ transients (Fig. 1D) and contractions (Fig. 1E) also were markedly increased by hypothermia (Ca2+ transient amplitude 157 ± 33 vs. 35 ± 5 nM at 37°C; fractional shortening 8.3 ± 1.7 vs. 2.6 ± 0.3% at 37°C; n = 7–10 cells per group; P < 0.05).


Figure 1
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  		Fig. 1. Hypothermia increased the amplitudes of contractions and Ca2+ transients in field-stimulated myocytes. Representative Ca2+ transients (top) and cell shortening (bottom) recorded simultaneously from guinea pig ventricular myocytes that were field stimulated at 2 Hz. Cell shortening is shown as downward deflections in the recordings. Myocytes were superfused with a buffer either warmed to 37°C (A) or maintained at 22°C (B). C: mean systolic and diastolic Ca2+ concentrations were significantly increased by hypothermia. D: mean amplitudes of Ca2+ transients were greater at 22°C than at 37°C. E: contraction amplitudes were expressed as percentage change in cell length to indicate the degree of fractional shortening. Hypothermia significantly increased the mean magnitude of contraction. n = 10 cells at 22°C and 7 different cells at 37°C; *P < 0.05, unpaired t-test.

 
To determine whether the positive inotropic effect of hypothermia was accompanied by changes in action potential configuration, we made simultaneous recordings of action potentials along with contractions and Ca2+ transients. Cells were stimulated with current pulses delivered at a frequency of 2 Hz. Figure 2A shows a representative action potential, contraction, and Ca2+ transient recorded simultaneously at 37°C. Similar recordings from a different cell at 22°C are shown in Fig. 2B. Under hypothermic conditions, the APD was prolonged (Fig. 2, A and B). The Ca2+ transient and contraction elicited by this prolonged action potential at 22°C were both longer and larger than those recorded from myocytes at physiological temperature (Fig. 2, A and B). Under hypothermic conditions (Fig. 2C), the mean APD at 90% repolarization (APD90) was more than two times longer (P < 0.05; values for APD90 were 123 ± 23 vs. 255 ± 42 ms at 37°C and 22°C, respectively; n = 11–15 cells/group). APD at 50% repolarization (APD50) also was significantly prolonged by hypothermia (values for APD50 were 98.0 ± 21.4 vs. 198.1 ± 37.6 ms at 37°C and 22°C, respectively; n = 11–15 cells/group; P < 0.05). As shown in the field stimulation data, fractional shortening (Fig. 2D), peak systolic Ca2+ (Fig. 2E), and the amplitude of the Ca2+ transient (Fig. 2F) all were significantly elevated at 22°C compared with responses recorded from myocytes at 37°C. Thus hypothermia prolonged APD and increased the amplitudes of contractions and Ca2+ transients.


Figure 2
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  		Fig. 2. Action potential duration (APD) was prolonged, and contraction and Ca2+ transient amplitudes were increased at 22°C compared with 37°C. Representative examples of action potentials (top), Ca2+ transients (middle), and contractions (bottom) recorded in response to a depolarizing current pulse delivered at a rate of 2 Hz recorded at 37°C (A) or 22°C (B). APD was prolonged, and Ca2+ transient and contraction amplitudes were increased by hypothermia. C: mean APD at 90% repolarization (APD90) was significantly prolonged at 22°C compared with physiological temperature. This was paralleled by significant increases in the mean amplitudes of contractions (D), peak systolic intracellular Ca2+ levels (E), and Ca2+ transient amplitudes (F). n = 11–15 cells at 22°C and 11–13 different cells at 37°C; *P < 0.05 by unpaired t-test.

 
To determine whether the increased size of contractions and Ca2+ transients was due to increased APD, we stimulated cells with rectangular voltage-clamp pulses of fixed duration to eliminate temperature-dependent changes in action potential configuration. High-resistance microelectrodes and voltage-clamp methods were used to elicit and simultaneously record Ca2+ transients, contractions, and ICa-L. The voltage-clamp protocol is shown at the top of Fig. 3. Ten conditioning pulses from –80 to 0 mV were delivered before each test step from –40 to 0 mV. Representative recordings of ICa-L, Ca2+ transients, and contractions at 37°C and 22°C are shown in Fig. 3, A and B. In contrast to recordings at 37°C, ICa-L recorded from myocytes superfused with buffers at 22°C achieved a smaller peak current amplitude (Fig. 3, A and B). Conversely, Ca2+ transient and contraction recorded under hypothermic conditions appeared much larger than responses elicited at 37°C (Fig. 3, A and B). These examples were representative of the mean data. Peak ICa-L density (pA/pF) was significantly reduced at 22°C when compared with 37°C (Fig. 3C; 1.3 ± 0.4 and 3.5 ± 0.7 pA/pF, respectively, n = 7–11 cells/group; P < 0.05). In addition, fractional shortening (Fig. 3D), diastolic Ca2+ (Fig. 3E), peak systolic Ca2+ (Fig. 3E), and Ca2+ transient amplitude (Fig. 3F) all were markedly and significantly elevated under hypothermic conditions compared with responses elicited at 37°C. The hypothermia-induced increases in Ca2+ transient amplitude and fractional shortening were proportional, both nearly fivefold. Thus, even when the duration of depolarization was similar at the two temperatures, Ca2+ transients and contractions were larger at 22°C than at 37°C. Therefore, prolongation of the action potential was not required for the positive inotropic effects of hypothermia to occur.


Figure 3
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  		Fig. 3. Mean amplitudes of L-type Ca2+ current (ICa-L) were reduced by hypothermia, even though contraction and Ca2+ transient amplitudes were increased. The voltage-clamp protocol is shown at the top of the figure. A: representative voltage-clamp recordings of ICa-L (top), Ca2+ transients (middle), and cell shortening (bottom) from an individual myocyte at 37°C. B: representative recordings of ICa-L, Ca2+ transient, and contraction recorded at 22°C. Hypothermia decreased the amplitude of ICa-L and increased the amplitudes of the Ca2+ transient and the contraction. C: mean amplitudes of ICa-L elicited by a test step from –40 to 0 mV were significantly reduced at 22°C compared with 37°C. D: hypothermia increased mean amplitudes of contraction when compared with responses at physiological temperature. E and F: mean systolic and diastolic Ca2+ concentrations as well as Ca2+ transient amplitudes were elevated at 22°C compared with 37°C. n = 9–11 myocytes at 22°C and 7–11 different myocytes at 37°C; *P < 0.05, unpaired t-test.

 
To evaluate whether hypothermia altered SR Ca2+ load in our model, we compared SR Ca2+ load at hypothermic and physiological temperatures. Myocytes were voltage clamped, stimulated at 2 Hz with conditioning pulses from –80 to 0 mV, and repolarized to –40 mV, as shown in the schematic at the top of Fig. 4. SR Ca2+ load was estimated by a rapid application of 10 mM caffeine for 1 s. Caffeine was applied in a nominally Na+- and Ca2+-free solution to minimize extrusion of Ca2+ by Na+/Ca2+ exchanger. Figure 4, A and B, shows representative recordings of caffeine-induced Ca2+ transients recorded at 37°C and 22°C. The caffeine-induced Ca2+ transient was much larger at 22°C than at 37°C. Mean data show that caffeine-releasable SR Ca2+ stores were significantly elevated at 22°C compared with caffeine-induced SR Ca2+ release at 37°C (Fig. 4C; 212 ± 50 and 83 ± 17 nM, respectively; n = 7 cells/group; P < 0.05). We also calculated fractional SR Ca2+ release at the two different temperatures by dividing the amplitude of the Ca2+ transient elicited by a test step from –40 to 0 mV by the peak caffeine-induced transient elicited from a holding potential of –40 mV as described previously (11). We found that hypothermia caused a significant (greater than twofold) increase in fractional SR Ca2+ release (Fig. 4D). Thus, under hypothermic conditions, SR Ca2+ load and fractional SR Ca2+ release are increased.


Figure 4
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  		Fig. 4. Hypothermia increased caffeine-releasable SR Ca2+ stores in voltage-clamped myocytes. The voltage-clamp protocol is shown at the top of the figure. A 1-s caffeine application was preceded by 10 conditioning pulses delivered at a rate of 2 Hz; caffeine was applied 300 ms after repolarization to –40 mV. A and B: representative examples of caffeine-induced Ca2+ transients at 37°C (left) and 22°C (right). Arrow denotes application of 10 mM caffeine. C: mean data show that caffeine-releasable SR Ca2+ stores were significantly elevated at 22°C compared with responses at physiological temperature. D: cooling significantly increased the fractional release of sarcolplasmic reticulum (SR) Ca2+. n = 7 myocytes at 22°C and 7 different myocytes at 37°C; *P < 0.05, unpaired t-test.

 
We minimized the potential impact of Ca2+ loading via reverse-mode Na+/Ca2+ exchange in our experiments by dialyzing myocytes at the two temperatures with identical nominally Na+-free patch pipette solutions. ICa-L, Ca2+ transients, contractions, and SR Ca2+ load were examined in myocytes dialyzed with patch pipettes with the same voltage-clamp protocols as described previously. Representative examples of recordings and a schematic of the voltage-clamp protocol are shown in Fig. 5. A voltage-clamp step from –40 to 0 mV at 37°C induced a large rapid inward ICa-L (Fig. 5A). The test step also elicited a small Ca2+ transient and contraction (Fig. 5A). The amplitude of ICa-L was smaller at 22°C compared with the physiological temperature (Fig. 5, A and B). The amplitudes of Ca2+ transients and contractions elicited by the test step were larger at 22°C than at 37°C (Fig. 5, A and B). The mean amplitude of peak ICa-L was significantly smaller at 22°C than at 37°C (Fig. 5C). The average degree of fractional cell shortening (Fig. 5D) and the mean amplitudes of Ca2+ transients (Fig. 5E) were significantly greater under hypothermic conditions when compared with responses elicited at physiological temperatures. Diastolic Ca2+ also was increased at 22°C compared with 37°C (Fig. 5F).


Figure 5
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  		Fig. 5. Amplitudes of contractions and Ca2+ transients were increased by hypothermia in myocytes dialyzed with patch pipettes that contained 0 mM intracellular Na+. Top, voltage-clamp protocol. A: representative examples of ICa-L (top), Ca2+ transient (middle), and contraction (bottom) recorded simultaneously from a single myocyte at 37°C. B: representative recordings of ICa-L, a Ca2+ transient, and a contraction recorded from a myocyte at 22°C. Hypothermia reduced the amplitude of ICa-L but increased the size of the Ca2+ transient and contraction compared with responses at 37°C. C: mean amplitudes of ICa-L elicited by a step from –40 to 0 mV were reduced by hypothermia when cells were dialyzed with 0 mM Na+. D: mean contraction amplitudes recorded from myocytes dialyzed with 0 mM Na+ pipette solution were increased by hypothermia. E: mean Ca2+ transient amplitudes were increased at 22°C compared with 37°C when cells were dialyzed with 0 mM intracellular Na+. F: average diastolic Ca2+ levels also were increased by hypothermia when myocytes were dialyzed with 0 mM Na+ pipette solution. n = 13–16 myocytes at 22°C and 8–9 different myocytes at 37°C; *P < 0.05, unpaired t-test.

 
SR Ca2+ content also was assessed by rapid application of caffeine in myocytes voltage clamped with patch pipettes and dialyzed with Na+-free intracellular solution. Caffeine was applied in Na+- and Ca2+-free solution as described in the methods. The caffeine-induced Ca2+ transient was much smaller at 37°C than at 22°C (Fig. 6, A and B). Figure 6C shows that caffeine-releasable SR Ca2+ load was significantly elevated at 22°C in myocytes dialyzed with patch pipette solutions compared with cells investigated at 37°C (160 ± 29 and 75 ± 19 nM, respectively; n = 8–16 cells/group; P < 0.05). Fractional SR Ca2+ release also was increased 150% by hypothermia in these experiments (Fig. 6D). These results show that hypothermia had a positive inotropic effect on isolated guinea pig ventricular myocytes, even when cells were dialyzed with Na+-free intracellular solutions.


Figure 6
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  		Fig. 6. SR Ca2+ stores were augmented by hypothermia in myocytes dialyzed with patch pipettes that contained 0 mM intracellular Na+. Top, voltage-clamp protocol. A and B: representative caffeine-induced Ca2+ transients recorded at physiological (left) and hypothermic (right) temperatures. Arrow denotes application of 10 mM caffeine. C: mean amplitudes of caffeine-induced Ca2+ transients. SR Ca2+ load was larger at 22°C compared with 37°C. D: cooling also increased fractional release of SR Ca2+. n = 13–16 myocytes at 22°C and 8–9 different myocytes at 37°C; *P < 0.05, unpaired t-test.

 
To determine whether cooling affected the gain of EC coupling, we first examined the effects of hypothermia on the rate of SR Ca2+ release. Mean results are shown in Fig. 7. The rate of SR Ca2+ release was measured as the rate of rise of the Ca2+ transient elicited by a step from –40 to 0 mV. The rate of rise was calculated as the time taken for the Ca2+ transient to increase to its peak systolic level, from the starting diastolic level (expressed in nM/s). This technique has been used previously to estimate the gain of EC coupling (11, 19), although direct sarcolemmal Ca2+ influx also may contribute to this Ca2+ transient. The rate of rise of the Ca2+ transient was significantly greater under hypothermic conditions, regardless of whether recordings were made with high-resistance electrodes (Fig. 7A) or with patch pipettes to dialyze the intracellular milieu (Fig. 7B). We also compared the impact of hypothermia on the gain of EC coupling. EC-coupling gain was measured as the rate of SR Ca2+ release per unit Ca2+ current. Figure 7, C and D, shows that EC-coupling gain was significantly increased, approximately sixfold in hypothermic myocytes when compared with cells investigated at 37°C. Values of gain reported here are intermediate between those reported previously for sheep and mouse ventricular myocytes (11, 19). This temperature-dependent increase in gain occurred when recordings were made with high-resistance electrodes (Fig. 7C) and when recordings were made with patch pipettes (Fig. 7D). Thus hypothermia markedly increased the gain of cardiac EC coupling under the experimental conditions used in this investigation.


Figure 7
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  		Fig. 7. Gain of SR Ca2+ release was significantly increased by hypothermia, in both cells that contained full intracellular Na+ and in cells dialyzed with 0 mM Na+ patch pipette solution. A: mean rates of rise of Ca2+ transients recorded from undialyzed myocytes. The rate of rise of the Ca2+ transient was increased significantly by hypothermia. B: mean rate of rise of the Ca2+ transient recorded from myocytes dialyzed with 0 mM Na+ patch pipette solution also was elevated by hypothermia. C: mean gain of SR Ca2+ release in undialyzed myocytes was increased at 22°C compared with 37°C. D: mean gain of SR Ca2+ release also was increased under hypothermic conditions in myocytes dialyzed with 0 mM Na+-filled patch pipettes. n = 11–16 cells at 22°C and 9–10 different cells at 37°C; *P < 0.05, unpaired t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study evaluated components of EC coupling in guinea pig ventricular myocytes at 37°C and 22°C to determine whether hypothermia altered the gain of EC coupling. Hypothermia increased the amplitudes of contractions and Ca2+ transients in field-stimulated myocytes. Cooling also increased diastolic Ca2+. When action potentials were recorded simultaneously with contractions and Ca2+ transients, the increased size of contractions and Ca2+ transients was accompanied by an increase in APD. However, cooling increased contractions and Ca2+ transients even when cells were activated with voltage-clamp steps of fixed duration to eliminate changes in APD. Hypothermia also reduced peak ICa-L but increased SR Ca2+ content and fractional SR Ca2+ release. When cells were dialyzed with identical patch pipette solutions to minimize temperature-related differences in intracellular composition, hypothermia still decreased peak ICa-L and increased contractions, Ca2+ transients, and SR stores. Hypothermia also increased the gain of EC coupling, measured as the rate of SR Ca2+ release per unit Ca2+ current. This increase in gain occurred regardless of whether cells were dialyzed with patch pipette solutions or whether high-resistance microelectrodes were used to minimize disruption of the internal milieu. These observations suggest that an increase in the gain of cardiac EC coupling contributes importantly to positive inotropic effects of hypothermia in the heart.

A major finding in this study was the observation that hypothermia caused a marked increase in the gain of cardiac EC coupling. To our knowledge, this observation has not been reported previously. Our voltage-clamp studies showed that peak ICa-L was significantly smaller at 22°C than at 37°C. However, despite the decrease in peak ICa-L in response to hypothermia, we found that the amplitudes of Ca2+ transients and contractions remained significantly larger at 22°C compared with physiological temperature. Although a decrease in the amplitude of ICa-L in response to cooling has been reported previously (9, 34), earlier studies did not record currents, Ca2+ transients, and contractions simultaneously at physiological and hypothermic temperatures. Therefore, previous studies did not determine the influence of hypothermia on the gain of EC coupling. We measured the rate of SR Ca2+ release per unit Ca2+ current to estimate gain at 37°C and 22°C and found that hypothermia dramatically increased EC-coupling gain. These findings suggest that an increase in the amount of Ca2+ released per unit current contributes importantly to the positive inotropic effects of hypothermia in isolated ventricular myocytes.

The increase in EC-coupling gain caused by hypothermia could be related to an increase in SR Ca2+ load and/or an increase in the open probability of Ca2+ release channels, as these factors can increase EC-coupling gain (3). Indeed, we found that cooling increased SR Ca2+ load in guinea pig ventricular myocytes, as reported in mouse, rabbit, ferret, and cat myocytes (17, 32). Hypothermia also increased fractional SR Ca2+ release to between 60% and 93% of total SR Ca2+ in our study, as reported other mammalian models when SR Ca2+ load is high (3). It is likely that this increase in SR Ca2+ load with cooling contributes to the increase in EC-coupling gain observed at 22°C. However, hypothermia also increases the open probability of ryanodine receptors in SR membranes isolated from sheep hearts (39). Individually, each of these hypothermia-induced changes in SR Ca2+ handling could increase the gain of SR Ca2+ release. Together, they are likely contributors to the increase in the gain of SR Ca2+ release at 22°C demonstrated in our study.

One mechanism that might contribute to the increase in SR Ca2+ load observed at 22°C is inhibition of the Na+ pump by reduced temperature (13). Inhibition of the Na+ pump should increase cytosolic Na+ levels and favor Ca2+ influx via reverse-mode Na+/Ca2+ exchange (27). SR Ca2+ load would be increased by SR Ca2+ uptake through the SR Ca2+-ATPase, which is more efficient than the Na+ pump under hypothermic conditions (13). However, we also investigated the impact of cooling on myocytes voltage clamped with patch pipettes containing a nominally Na+-free solution. This would inhibit Ca2+ uptake into the cytosol by reverse-mode Na+/Ca2+ exchange. Yet, when reverse-mode Na+/Ca2+ exchange was inhibited by Na+-free solutions, SR Ca2+ load was elevated at 22°C, and the degree of elevation was similar to that seen when recordings were made with high-resistance microelectrodes. Furthermore, hypothermia still increased the size of contractions and Ca2+ transients and reduced peak Ca2+ current under these conditions. It is possible that dialysis with Na+-free solution may not restrict rapid changes in subspace intracellular Na+, which could promote reverse-mode Na+/Ca2+ exchange, and directly contribute to EC coupling (28). The majority of reverse-mode Na+/Ca2+ exchange occurs in response to the inward Na+ current at the beginning of the action potential (3, 33). However, we blocked Na+ current with lidocaine and test steps from –40 mV, so the influence of subspace Na+ on Na+/Ca2+ exchange should be minimal in our voltage-clamp experiments. Thus our results suggest that, although SR Ca2+ loading by reverse-mode Na+/Ca2+ exchange may contribute to increased SR Ca2+ load and positive inotropy under hypothermic conditions, reverse-mode Na+/Ca2+ exchange is not required on a beat-to-beat basis for positive inotropy to occur.

It also is possible that the increase in SR Ca2+ load that occurs under hypothermic conditions may be due to the hypothermia-induced decrease in amplitude of ICa-L. The amplitudes of Ca2+ transients are regulated through a system of negative feedback (40). When the magnitude of ICa-L increases, Ca2+ transient size initially increases, producing a reduction in SR load, which in turn reduces Ca2+ transient amplitude. Thus the myocyte reregulates around a new, lower gain, level of CICR. Trafford et al. (40) also suggested that the converse is true. If so, then the reduction in peak ICa-L reduce Ca2+ transient amplitude temporarily, producing an increase in SR Ca2+ load. The elevation of SR Ca2+ load would increase the gain of Ca2+ release and lead to a re-regulation of SR load and Ca2+ transient amplitude around a new, higher norm.

Another mechanism that may increase SR Ca2+ load under hypothermic conditions is inhibition of Ca2+ efflux mechanisms by cooling. Interestingly, a recent study in guinea pig myocytes showed that Ca2+ efflux via sarcolemmal Ca2+-ATPase is much slower at 22°C than at physiological temperature (30). Cooling also reduces Ca2+ efflux via the Na+/Ca2+ exchanger in myocytes from rabbit, ferret, cat, and guinea pig hearts (30, 32, 41). In contrast, Ca2+ sequestration by the SR Ca2+-ATPase is much less temperature sensitive than efflux mechanisms (30). Thus inhibition of Ca2+ efflux mechanisms by hypothermia could lead to the increase in SR Ca2+ content observed in our study. This is supported by our observation that diastolic Ca2+ was markedly increased by hypothermia compared with diastolic Ca2+ levels in cells at 37°C.

The observation that hypothermia caused a marked increase in contraction amplitude is supported by many other studies (32, 34, 36). Some investigators have suggested that an increase in myofilament Ca2+ sensitivity is responsible, at least in part, for the increase in contraction amplitude associated with hypothermia (26, 30). However, we found that the hypothermia-induced increase in contractions was accompanied by a proportional increase in the size of Ca2+ transients. Puglisi et al. (32) also reported that amplitudes of contractions and Ca2+ transients increased in parallel under hypothermic conditions. Together, these findings suggest that the temperature-related increase in contraction amplitude is primarily attributable to an increase in availability of activator Ca2+ rather than a change in myofilament Ca2+ sensitivity. This conclusion is supported by previous studies that suggest changes in myofilament Ca2+ sensitivity with cooling likely reflect changes in Ca2+ buffering by EGTA due to the effect of temperature on buffering equilibria and to actual changes in myofilament Ca2+ sensitivity (21, 22).

Hypothermia also increased APD in guinea pig myocytes, as in other mammalian species (34, 36). Prolongation of the action potential by hypothermia would prolong depolarization of the membrane and extend voltage-gated channel openings, including openings of the L-channel (3, 7, 34, 35). Thus increased APD alone has marked positive inotropic effects (7, 35) and may contribute to the positive inotropic effects of hypothermia in field-stimulated and current-clamped guinea pig ventricular myocytes. However, we found that hypothermia also increased the amplitudes of contractions and Ca2+ transients even when we eliminated changes in APD with voltage-clamp pulses of fixed duration. Thus, although prolongation of the action potential is likely to play a role in the positive inotropic effects of hypothermia, our results show that hypothermia affects components of EC coupling other than APD to augment cardiac contraction.

One of the key observations in this study is that the gain of EC coupling is increased by hypothermia. These findings are particularly important when one considers the large number of seminal studies of cardiac EC coupling where recordings have been made from myocytes at subphysiological temperatures. As a result, much of our understanding of EC coupling in the heart is based on studies conducted under hypothermic conditions, where SR Ca2+ load and the gain of SR Ca2+ release are artificially elevated. Experiments designed to increase SR Ca2+ load or increase the gain of SR Ca2+ release may produce less change in the size of the Ca2+ transient or contraction at room temperature than at physiological temperature. Furthermore, the high gain and elevated SR Ca2+ load produced by hypothermia may mask important pathophysiological changes in EC coupling in models of heart disease. The high gain that we have reported at 22°C also may obscure the effects of factors with subtle effects on EC coupling, which would alter the gain of EC coupling at more physiological temperatures.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by grants from the Canadian Institutes of Health Research and from the Heart and Stroke Foundation of Nova Scotia. During this study R. Shutt received studentship support from the Heart and Stroke Foundation of Canada.


   ACKNOWLEDGMENTS
 
The authors thank Peter Nicholl, Steven Foster, Cindy Mapplebeck, and Dr Jiequan Zhu for excellent laboratory and technical assistance. The authors are grateful for discussions of some of the data in this paper with the late Dr. Gregory Ferrier.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. E. Howlett, Dept. of Pharmacology, 5850 College St., Sir Charles Tupper Medical Bldg., Dalhousie Univ., 5850 College St., Halifax, NS, Canada B3H 1X5 (e-mail: susan.howlett{at}dal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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