AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 286: H2219-H2228, 2004. First published February 12, 2004; doi:10.1152/ajpheart.01096.2003
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Insights into cardioprotection obtained from study of cellular Ca2+ handling in myocardium of true hibernating mammals

Atsuko Yatani,1 Song-Jung Kim,1 Raymond K. Kudej,1 Qian Wang,1 Christophe Depre,1 Keiichi Irie,1 Evangelia G. Kranias,2 Stephen F. Vatner,1 and Dorothy E. Vatner1

1Cardiovascular Research Institute and Department of Cell Biology and Molecular Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103; and 2Department of Pharmacology and Cell Biophysiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267

Submitted 24 November 2003 ; accepted in final form 6 February 2004


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mammalian hibernators exhibit remarkable resistance to low body temperature, whereas nonhibernating (NHB) mammals develop ventricular dysfunction and arrhythmias. To investigate this adaptive change, we compared contractile and electrophysiological properties of left ventricular myocytes isolated from hibernating (HB) woodchucks (Marmota monax) and control NHB woodchucks. The major findings of this study were the following: 1) the action potential duration in HB myocytes was significantly shorter than in NHB myocytes, but the amplitude of peak contraction was unchanged; 2) HB myocytes had a 33% decreased L-type Ca2+ current (ICa) density and twofold faster ICa inactivation but no change in the current-voltage relationship; 3) there were no changes in the density of inward rectifier K+ current, transient outward K+ current, or Na+/Ca2+ exchange current, but HB myocytes had increased sarcoplasmic reticulum Ca2+ content as estimated from caffeine-induced Na+/Ca2+ exchange current values; 4) expression of the L-type Ca2+ channel {alpha}1C-subunit was decreased by 30% in HB hearts; and 5) mRNA and protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), phospholamban, and the Na+/Ca2+ exchanger showed a pattern that is consistent with functional measurements: SERCA2a was increased and phospholamban was decreased in HB relative to NHB hearts with no change in the Na+/Ca2+ exchanger. Thus reduced Ca2+ channel density and faster ICa inactivation coupled to enhanced sarcoplasmic reticulum Ca2+ release may underlie shorter action potentials with sustained contractility in HB hearts. These changes may account for natural resistance to Ca2+ overload-related ventricular dysfunction and point to an important cardioprotective mechanism during true hibernation.

woodchuck; cardiac myocyte; calcium current; sarcoplasmic reticulum; action potential; potassium; exchanger

SIGNIFICANT INSIGHT into the understanding of the pathogenesis of heart failure has been gained from studies of patients with heart disease or animal models (18, 28, 29, 46). Alternatively, insight can also be obtained from animals that face major environmental stresses. For example, naturally hibernating (HB) animals such as woodchucks, hedgehogs, or chipmunks undergo pronounced cardiovascular changes as they enter into hibernation: heart rate and blood pressure decrease, and body temperature decreases from 37°C when awake to 16–10°C (25). Under these conditions, the hearts of nonhibernating (NHB) animals develop serious ventricular arrhythmias or cease contraction (16). However, the hearts of hibernators exhibit remarkable tolerance and resistance to the impact of these stresses. Despite the dramatic adaptation of HB myocardium, the cellular basis for the adaptive mechanisms in HB animals is not well known (49).

A few studies (22–24, 27, 48) compared the source of Ca2+ for contraction in cardiac muscle from HB animals with that in NHB animals. Experimental studies have shown that intact papillary muscles isolated from HB animals exhibit an altered excitation-contraction coupling during hibernation. As in most mammalian NHB species, cardiac muscle contraction of the HB heart is dependent on both transsarcolemmal Ca2+ influx and intracellular Ca2+ stores (7). In contrast, muscle contraction during hibernation is mainly regulated by the sarcoplasmic reticulum (SR) Ca2+ release (16, 22, 23, 27, 48). Furthermore, ryanodine caused a transient positive inotropic effect only in HB animals that was similar to cardiac muscle with increased SR Ca2+ load (6, 30). However, there is no indication of ventricular arrhythmias such as afterdepolarization or aftercontraction that commonly occur in Ca2+-overloaded myocardium (22). These observations suggest that a coordinated remodeling of cellular Ca2+ handling during hibernation may contribute to forceful contraction and the marked resistance to ventricular dysfunction.

In the present study, to examine the cellular basis for the coordinated mechanisms, we compared action potential, L-type Ca2+ current (ICa), K+ channel currents, and Na+/Ca2+ exchange current (INa/Ca) in left ventricular (LV) myocytes isolated from NHB and HB woodchuck hearts. In addition, the profiles of cellular Ca2+ regulatory proteins in LV myocardium from NHB and HB woodchucks were assessed by Western blotting and mRNA analysis.

Our data indicate that myocytes isolated from HB hearts exhibit significantly shorter action potential duration (APD) and higher SR Ca2+ content due to the higher SR Ca2+ uptake function, which may provide a coordinated cardioprotective mechanism to prevent Ca2+ overload in HB animals.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal model. A total of 19 woodchucks (10 NHB and 9 HB animals) were used in this study. Telemetry transmitters (model TL11M2-D70-PCT) were implanted in a subcutaneous pocket in the animal's left flank to measure temperature and ECG. HB woodchucks were killed after displaying deep hibernation, i.e., heart rate <25 beat/min and nonresponse to mild external stimuli. NHB animals consisted of "age-matched controls," i.e., animals having undergone hibernation that were killed between March and April. The animals were maintained and the experiments were approved by the New Jersey Medical School Animal Care Committee.

Electrophysiology. After completion of the in vivo measurements, LV myocytes were isolated from NHB and HB woodchuck hearts using a method described previously (32, 51). Cell contraction and Ca2+ transients were measured as previously described (52). Briefly, isolated LV myocytes were perfused with Tyrode solution that contained (in mmol/l) 120 NaCl, 2.6 KCl, 1.0 CaCl2, 1.0 MgCl2, 11 glucose, and 5 HEPES (pH 7.3) and were field stimulated at 1.0 Hz. Myocyte contractile and relaxation functions were measured using a video motion-edge detector at 32 ± 2°C. For the Ca2+ transient measurements, cells were loaded with 5 µM fura 2 acetoxymethyl ester (AM) at room temperature for 30 min, and intracellular free Ca2+ was monitored as the fura 2 fluorescence ratio at 340- to 380-nm wavelengths using the Photoscan dual-beam spectrofluorophotometer (Photon Technology). The changes in Ca2+ transient were evaluated by direct reading of the fluorescence intensity. The time for 70% decay of Ca2+ transient (TRC70) was also evaluated.

Action potential and membrane currents were measured using the whole cell variation of the patch-clamp technique (20, 32, 33, 51) at room temperature (22 ± 2°C). Membrane capacitance was measured using voltage ramps of 0.8 V/s from a holding potential of –50 mV.

ICa values were recorded with an external solution that contained (in mM) 2 CaCl2, 1 MgCl2, 135 tetraethylammonium chloride, 5 4-aminopyridine, 10 glucose, and 10 HEPES (pH 7.3). The pipette solution contained (in mM) 100 cesium aspartate, 20 CsCl, 1 MgCl2, 2 MgATP, 0.5 GTP, 5 EGTA, and 5 HEPES (pH 7.3). These solutions provided isolation of ICa from other membrane currents such as Na+ and K+ channel currents and also from Ca2+ flux through the Na+/Ca2+ exchanger (NCX). For action potential and K+ current recordings, myocytes were perfused with normal Tyrode solution that contained (in mM) 135 NaCl, 1.8 CaCl2, 1 MgCl2, 5.4 KCl, 10 glucose, and 10 HEPES (pH 7.3). The pipette filling solution for action potential recordings contained 200 µg/ml amphotericin and (in mM) 140 KCl, 2 MgCl2, 10 NaCl, 2 ATP, and 5 HEPES (pH 7.3). For the K+ current measurements, 10 µM nifedipine was added to block ICa, and patch-pipette solution contained (in mM) 110 potassium aspartate, 20 KCl, 2 MgCl2, 2 ATP, 0.5 GTP, 5 EGTA, and 5 HEPES (pH 7.3). For measurements of INa/Ca, the external solution contained (in mM) 150 NaCl, 2 CsCl, 2 MgCl2, 1 CaCl2, 0.001 nifedipine, 0.02 ouabain, and 5 HEPES (pH 7.3). The pipette solution contained (in mM) 20 NaOH, 110 CsOH, 50 aspartic acid, 1 MgCl2, 2 MgATP, 42 EGTA, and 5 HEPES (pH 7.4). The concentration of free internal Ca2+ was adjusted to 67 nM by adding CaCl2 (15). To activate INa/Ca, the cells were held at –40 mV and the external solution was rapidly switched to one in which equimolar LiCl was substituted for NaCl. Exposure of the myocytes to Na+-free solution produced outward Na+ extrusion through the NCX (21, 33).

The SR Ca2+ content was evaluated by a train of 10 conditioning pulses (100 ms at 0.5 Hz, from –70 to +10 mV) to load SR Ca2+ (26, 33, 41, 45). The cell was then superfused rapidly with external solution that contained caffeine (10 mM). The external solution contained (in mM) 150 NaCl, 2 CsCl, 2 MgCl2, 1 CaCl2, and 5 HEPES (pH 7.3). The pipette solution contained (in mM) 150 CsCl, 1 MgCl2, 2 MgATP, 0.1 EGTA, and 5 HEPES (pH 7.3).

Western blotting. Quantitative immunoblotting was performed as previously reported (19, 20). Antibodies to phospholamban (PLB), calsequestrin (CSQ), sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), and the NCX were obtained from Affinity Bioreagents (Golden, CO), and antibodies to PLB were obtained from Cyclacel (Dundee, UK). The LV tissue was homogenized on ice with a Polytron in buffer [50 mM Tris·HCl (pH 7.5) and 10 mM histidine]. The suspension was then diluted in an equal volume of lysis buffer. Protein concentration was measured with bicinchoninic acid (Pierce). The lysates were mixed with Laemmli sample buffer and separated by SDS-PAGE. The resolved proteins were electrotransferred to polyvinylidene difluoride or nitrocellulose membranes. Membranes were incubated in 5% skim milk and Tween- and Tris-buffered saline (TTBS; 20 mM Tris·HCl, 150 mM NaCl, and 0.1% Tween 20) to block the nonspecific binding of the antibody and then rinsed in TTBS. The membranes were first incubated with primary antibody in TTBS that contained 5% milk for 1–2 h or overnight at 4°C, rinsed with TTBS, and then incubated with the appropriate secondary antibody. Equal amounts of soluble proteins from HB and NHB hearts were used for electrophoresis and subsequent blotting. For the immunoblots of cardiac L-type Ca2+ channel {alpha}1-subunits and other Ca2+- handling proteins, ~50 and 4–20 µg of proteins, respectively, were loaded. After the final rinse, the membrane blots were developed with a chemiluminescence reagent (NEN Labs; Boston, MA) and exposed to X-ray film.

Quantitative PCR. Quantitative RT-PCR (7700 Prizm, Perkin-Elmer/ABI) was performed to measure the gene expression of SERCA2a, PLB, CSQ, NCX, and cyclophilin. For each measurement, the mRNA of interest was reverse transcribed with specific primers and subsequently used for quantitative two-step PCR with SYBRGreen fluorescent dye. Owing to variation in sample-to-sample loading, PCR data are reported per number of cyclophilin transcripts measured in each sample.

Statistical analysis. Data are expressed as means ± SE; N represents the number of animals used. For myocyte experiments, multiple cells from individual woodchucks were examined for each protocol, and n represents number of myocytes examined from different animals. Statistical analysis was performed by t-test between NHB and HB hearts or myocytes isolated from HB or NHB hearts with significance imparted at the P < 0.05 level.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In our prior study (25), we found that NHB woodchucks have a body temperature of 36 ± 0.5°C and heart rate of 86 ± 4 beats/min, whereas in HB woodchucks heart rate decreased to 27 ± 8 beats/min and temperature decreased to 13 ± 0.6°C. In the present study, we confirmed these data, i.e., the HB woodchucks had a decreased heart rate (17 ± 5 beats/min) and decreased body temperature (13 ± 1.0°C), which are the conditions whereby the hearts of NHB animals would develop ventricular fibrillation and stop functioning (16).

Figure 1 shows twitch contraction and Ca2+ transients in myocytes isolated from NHB and HB hearts under steady-state conditions (1.0 Hz). The amplitudes of myocyte contraction and Ca2+ transient are similar between the two groups. The pooled data (right) indicate that the maximum rate of contraction (–dL/dt) was similar in NHB and HB myocytes, but relaxation rate (+dL/dt) was slower in HB myocytes; however, the TRC70 time was faster in HB myocytes. These results are consistent with previous muscle contraction data with electrical stimulation on hibernators, which showed that the magnitude of muscle contraction remains at a relatively high level during hibernation (49). Furthermore, the data suggest that the availability of activator Ca2+ for contraction is not altered in HB myocytes.



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  		Fig. 1. Typical examples of myocyte contractions (A) and fura 2 Ca2+ transients (B) recorded in left ventricular (LV) myocytes isolated from hearts of nonhibernating (NHB) and hibernating (HB) woodchucks. Left: contractions and Ca2+ transients were recorded during field stimulation at a frequency of 1.0 Hz. Right: summaries of results. Data are means ± SE of NHB (n = 40) and HB (n = 32) myocytes for contractile parameters on measurements and NHB (n = 30) and HB (n = 24) myocytes from five NHB and five HB woodchucks. *P < 0.05. TRC70, time for 70% decay of Ca2+ transient; –dL/dt, maximum rate of contraction; +dL/dt, relaxation rate.

 
Action potentials. Action potentials were recorded in NHB and HB myocytes in Tyrode solution (Fig. 2). NHB myocytes typically had a rapid upstroke and a plateau before repolarization. The shape of the action potential was similar to that seen in ventricular myocytes from mammalian species such as the guinea pig, rabbit, or dog (35, 51, 53). HB myocytes displayed a shorter action potential compared with NHB myocytes. APD values quantified at 50% and 90% repolarization (APD50 and APD90, respectively) were significantly shorter in HB compared with NHB myocytes (Table 1). There were no significant differences in the resting membrane potentials and overshoot potentials between the two groups.



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  		Fig. 2. Differences in action potential shape and duration (APD) recorded in NHB and HB myocytes. APD values were quantified at 50% and 90% repolarization (APD50 and APD90, respectively). Membrane potential was recorded in the current-clamp mode at 0.2 Hz. APD50 and APD90 values recorded from HB myocytes were significantly shorter compared with NHB myocytes. Data are from four NHB (n = 18 myocytes) and four HB (n = 12 myocytes) woodchucks. *P < 0.05.

 

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  		Table 1. Action potential characteristics recorded from NHB and HB myocytes

 
K+ channel currents. The characteristic shape and duration of an action potential are determined by the intricate balance of the depolarizing and repolarizing currents (6, 39, 50). Woodchuck myocytes are rarely used for electrophysiological studies, and variations in the expression of membrane currents in LV myocytes have not yet been characterized. We found that 4-aminopyridine-sensitive transient outward K+ currents, which could contribute to action potential repolarization (34, 37), are present in woodchuck myocytes (Fig. 3B, inset). The K+ channel blocker tetraethylammonium chloride (10 mM) had no significant effect (not shown). As shown in Fig. 2, there was no significant change in amplitude between the two groups.



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  		Fig. 3. Transient outward currents recorded in NHB and HB myocytes. Representative families of currents were elicited by voltage steps from –60 to +60 mV in 20-mV increments from a holding potential of –80 mV (A). Current-voltage relationships for peak values were normalized to cell capacitance to yield current densities (B). Data are from six NHB (n = 17 cells) and four HB (n = 12 cells) woodchucks. Effects of 10 mM 4-aminopyridine (4-AP) on K+ currents (+60 mV) in HB myocytes are shown (inset).

 
Similarly, there was no significant difference in the density of the inward rectifier K+ currents between NHB and HB myocytes. The mean current densities at –100 mV in NHB and HB myocytes were –10.8 ± 0.6 (n = 15) and –10.1 ± 0.5 pA/pF (n = 12), respectively. Thus it appears that changes in repolarizing currents are not associated with action potential shortening in HB myocytes.

L-type Ca2+ currents. There were no significant changes in K+ channel currents in HB myocytes. Thus a change in the amplitude and/or kinetics of ICa could explain the altered action potential profile that we observed. To evaluate this, we analyzed ICa (Fig. 4, A and B). The peak inward ICa density value (peak ICa amplitude normalized relative to cell capacitance) was ~36% smaller in HB myocytes (4.4 ± 0.2 pA/pF; n = 97) compared with NHB myocytes (6.0 ± 0.3 pA/pF; n = 92). There was no significant change in the current-voltage relationship between the two groups (Fig. 4C).



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  		Fig. 4. A and B: whole cell Ca2+ current (ICa) characteristics in NHB (A) and HB (B) myocytes. Currents were elicited from a holding potential of –50 mV to the indicated test potentials. C: current-voltage relationships in NHB and HB myocytes. ICa was normalized to cell capacitance to obtain current densities. Data are from six NHB (n = 60 cells) and five HB (n = 83 cells) woodchucks. D: Western blot analyses of L-type Ca2+ channel {alpha}1C-subunits for NHB and HB hearts. Averaged values for NHB (N = 6 hearts) and HB (N = 6 hearts) animals are plotted below the immunoblots. *P < 0.01.

 
As shown in Fig. 4D, the expression level of the L-type Ca2+ channel {alpha}1C-subunit, which contains the ion-conducting pore (9) in HB hearts, was reduced by 35 ± 8%; this is consistent with the decreased ICa being due to fewer Ca2+ channels (rather than altered regulation). However, because we were unable to measure protein levels owing to the unavailability of the antibody against the general {beta}-subunit, we cannot exclude the possibility that alterations in the {beta}-subunit levels might influence channel activity.

ICa decay kinetics are also an important parameter for Ca2+ entry. Thus we analyzed whether the time course of inactivation was altered in HB myocytes (Fig. 5). At the peak potential (+10 mV), ICa inactivated rapidly during maintained depolarization in both groups. Despite the smaller ICa value, HB myocytes exhibited significantly faster inactivation compared with NHB myocytes (Fig. 5A). The times to half-decay (t1/2) in HB (n = 60) and NHB (n = 91) myocytes were 24 ± 2 and 37 ± 2 ms, respectively (Fig. 5C). Decreased ICa amplitude is often associated with prolonged inactivation due to less Ca2+-induced channel inactivation (52), therefore, the more rapid inactivation could have been secondary to increased SR Ca2+ loading and release (20, 32, 42). Thus we measured t1/2 in the presence of ryanodine (10 µM). After the application of ryanodine, the rate of ICa inactivation as measured by t1/2 was increased in both groups (Fig. 5A). There was no significant difference in the inactivation rate between the two groups (Fig. 5, B and C), which indicates that Ca2+-dependent inactivation was increased in HB myocytes due to enhanced Ca2+ release from the SR in response to ICa.



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  		Fig. 5. Time course of ICa and the effects of ryanodine on NHB and HB myocytes. A: superimposed current traces before and after ryanodine (10 µM) application. Depolarizing steps to +10 mV were applied from a holding potential of –50 mV. B: traces comparing time courses of ICa recorded from NHB and HB myocytes in the presence of ryanodine. Currents were scaled to the same peak-current amplitude to compare the waveform (A and B). C: average half-decay (T1/2) values of ICa before and after the application of ryanodine were obtained for NHB and HB myocytes. Data are from six NHB (n = 58 myocytes) and five HB (n = 81 myocytes) woodchucks. *P < 0.01.

 
Measurement of INa/Ca. We next examined INa/Ca in myocytes from NHB and HB hearts (Fig. 6). The myocytes were held at –40 mV, and INa/Ca was activated by rapidly reducing the external Na+ concentration (21, 26, 33). When the external Na+ was replaced by Li+, the membrane current shifted to an outward direction in both groups (Fig. 6, A and B). There was no significant difference in the peak amplitude of INa/Ca between NHB and HB myocytes. Under our experimental conditions, the average INa/Ca densities in NHB and HB myocytes were 0.5 ± 0.05 (n = 38) and 0.5 ± 0.06 pA/pF (n = 33), respectively (Fig. 6C). The results indicate that changes in Ca2+ influx and/or efflux through the NCX are unlikely to occur in HB myocytes.



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  		Fig. 6. A and B: Na+/Ca2+ exchange current (INa/Ca) from myocytes isolated from NHB (A) and HB (B) hearts. Representative INa/Ca induced by a rapid solution change from 150 mM Na+ to 150 mM Li+ (indicated at top) at a holding potential of –40 mV. C: average INa/Ca density in NHB and HB myocytes was obtained by dividing peak outward current amplitude by membrane capacitance. Data are from four NHB (n = 10 cells) and four HB (n = 9 cells) woodchucks.

 
SR Ca2+ content measured as caffeine-induced INa/Ca. Taken together, the electrophysiological data suggest that smaller ICa density and enhanced SR Ca2+ release during twitches contribute to the cardiac phenotype observed in HB myocytes. To further test for this, we measured SR Ca2+ content by integrating INa/Ca after a rapid application of 10 mM caffeine (8, 26, 41). Figure 7, A and B, shows the inward currents during caffeine application, which are thought to be the result of a transient increase of intracellular Ca2+ that is accompanied by a transient INa/Ca. In both NHB and HB myocytes, caffeine activated INa/Ca, but the integrated current was significantly larger in HB myocytes (Fig. 7C).



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  		Fig. 7. A and B: comparison of sarcoplasmic reticulum (SR) Ca2+ content measured as caffeine-induced INa/Ca in NHB (A) and HB (B) myocytes. An example of INa/Ca activated by a rapid application of 10 mM caffeine at a holding potential of –70 mV is shown. Bars indicate the period of caffeine application. C: mean integral INa/Ca during exposure of caffeine in myocytes from four NHB (n = 8) and four HB (n = 9) woodchucks. *P < 0.05.

 
Ca2+ regulatory protein and gene expression. To examine changes in mRNA levels of Ca2+ transport proteins during hibernation, we performed RT-PCR analysis on RNA isolated from NHB and HB hearts using specific primers for SERCA2a, PLB, CSQ, and NCX (Fig. 8). The mRNA level of SERCA2a was increased threefold, whereas the transcript that encodes PLB was decreased by 50% in HB hearts compared with NHB hearts. There were no changes in CSQ or NCX gene expression.



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  		Fig. 8. Determination of transcripts for proteins involved in Ca2+ homeostasis in NHB (N = 4) and HB (N = 4) hearts. Expression of the corresponding transcripts for sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), phospholamban (PLB), calsequestrin (CSQ), and the Na+/Ca2+ exchanger (NCX) was normalized per cyclophilin transcript. *P < 0.01.

 
Western blot analysis of NHB and HB hearts revealed a pattern of changes similar to gene expression of SERCA2a, PLB, NCX, and CSQ (Fig. 9): SERCA2a protein levels were increased by 300 ± 35%, whereas PLB levels decreased by 55 ± 5% in HB hearts compared with NHB hearts. Thus the ratio of PLB to SERCA2a was significantly reduced in HB hearts, which indicates that a larger portion of the pumps were in the "uninhibited state" relative to NHB hearts. There were no changes in CSQ and NCX expression levels.



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  		Fig. 9. Western blot analyses of proteins involved in Ca2+ homeostasis in NHB and HB hearts. Averaged values in NHB (N = 4) and HB (N = 4) hearts are plotted below the immunoblots. *P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we found that 1) HB myocytes have significantly shorter APDs compared with NHB myocytes but no change in contractile amplitude; 2) there was no change in K+ channel currents, but ICa density was significantly decreased in HB myocytes; and 3) HB myocytes had enhanced SR Ca2+ uptake capacity but no change in NCX. Thus our data suggest that the significantly enhanced SR Ca2+ uptake capacity and shorter APD that resulted from a reduction of Ca2+ channels contribute to maintain normal diastolic and systolic function during hibernation (16, 25). Because electrophysiological remodeling and defects in cellular Ca2+ handling are a major cause of ventricular arrhythmias and fibrillation in failing hearts (28), data obtained during natural hibernation may provide unique insights into the treatment of patients with heart failure.

Ionic mechanisms of APD shortening. Previous studies of intact papillary muscles isolated from HB chipmunks or ground squirrels have reported an absence of action potential plateau but no data at the cellular level (24, 47). Our data revealed that ventricular myocytes isolated from HB woodchucks have significantly shorter APD50 and APD90 values compared with NHB myocytes. There was no significant difference in the resting membrane potential between the two groups. These data are in sharp contrast to the action potential prolongation that occurs in ventricular myocytes from failing hearts (3, 28, 43).

In cardiac hypertrophy and failure, prolongation of the action potential repolarization is often associated with a reduction in K+ channel currents (17, 37, 43). In the present study, we found that woodchuck ventricular myocytes express inward rectifier and transient outward K+ currents; however, these currents were unchanged during hibernation. Instead, we observed a significant reduction of peak ICa density in HB myocytes. Furthermore, HB myocytes exhibited significantly faster ICa inactivation compared with NHB myocytes. There was no change in the voltage-dependent activation characteristics in HB myocytes. One group has reported (1) that there is a 60% reduction of ICa in myocytes isolated from HB vs. awake ground squirrels. However, that study showed significantly slower ICa inactivation and shifts of the voltage dependence of ICa activation to positive potentials (15–20 mV) in HB myocytes compared with NHB myocytes. The reason for the disparity in the results of that study and the present one is unclear but may be explained by the differences in experimental conditions. Alekseev et al. (1) recorded ICa in a Na+-containing external solution using holding potentials between –60 and –70 mV. Furthermore, recording pipettes were filled with a solution that contained no Ca2+ buffer, such as EGTA, which may significantly influence Ca2+-dependent L-type Ca2+ channel properties (32).

Regarding the ICa inactivation, because Ca2+ entry via the L-type Ca2+ channel promotes Ca2+-induced ICa inactivation in heart, significantly decreased ICa amplitude should be associated with prolonged inactivation, which suggests an additional role for ICa inactivation (31, 32). The difference in the rate of ICa inactivation was abolished when ryanodine was used to suppress Ca2+ release from the SR. These data suggest that the increased Ca2+-dependent inactivation observed in HB myocytes is primarily due to enhanced SR Ca2+ release. Consistent with this notion, we found that caffeine-induced inward INa/Ca, an estimate of the SR Ca2+ content, was increased twofold in HB myocytes compared with NHB myocytes. Notably, the adaptation does not involve altered function of the NCX.

Taken together, our electrophysiological data suggest that the shorter APD values observed in HB myocytes involve cellular Ca2+ handling processes and are different from the predominant abnormalities in K+ channel currents that are found in cardiac hypertrophy and failure. Prolongation of APD has been proposed (17, 35, 37) to provide a substrate for development of ventricular arrhythmias and fibrillation that arise from early afterdepolarization or a variety of oscillatory depolarizations, which is a plausible cause of sudden cardiac death. Thus the shortening of the APD could be one of the cellular mechanisms responsible for the dramatic resistance to arrhythmias and fibrillation expressed in woodchuck myocardium during hibernation.

Changes in Ca2+ regulatory protein expression. The findings of normal contractile amplitude (although Ca2+ influx was reduced in HB myocytes) provide strong evidence that excitation-contraction coupling is altered in myocytes from HB hearts. Because the fractional Ca2+ release is regulated by the amount of trigger Ca2+ (ICa amplitude) and SR Ca2+ content in cardiac myocytes, we expected a smaller amplitude for Ca2+ transients in HB myocytes due to significantly reduced ICa (4, 15, 44). However, HB myocytes appear to have relatively larger SR Ca2+ contents compared with NHB myocytes, which could be the reason that our data demonstrates similar Ca2+ transients between NHB and HB myocytes. These results are consistent with earlier studies in HB papillary muscle (23, 24, 47). Using the L-type Ca2+ channel blockers nifedipine or Cd2+ and the SR Ca2+ store inhibitors caffeine or ryanodine, these studies showed that cardiac muscle contraction of HB animals showed decreased sensitivity to Ca2+ channel inhibition and enhanced sensitivity to SR Ca2+ inhibitors. However, molecular mechanisms for the altered Ca2+ handling during hibernation were unclear.

A faster rate of Ca2+ uptake and a greater level of Ca2+ accumulation in cardiac SR vesicles isolated from HB ground squirrels were previously reported by Belke et al. (5). However, in that study, the high rate of Ca2+ uptake was not associated with enhanced enzymatic activity or total amount of SERCA. In the present study, using Western blotting, we demonstrated that changes in expression levels of Ca2+ regulatory proteins are responsible for changes in Ca2+ homeostasis during hibernation. For example, the simple explanation for the reduced ICa in HB myocytes would be a decrease in the numbers of Ca2+ channels (instead of channel regulation). Similarly, the enhanced ability of Ca2+ uptake by intracellular Ca2+ stores in HB hearts compared with NHB hearts could be explained by a change in the relative ratio of SERCA2a (threefold increase) to PLB (55% decrease). In this regard, we found no other changes such as NCX or the Ca2+-binding protein CSQ in the SR, which increases the Ca2+ storage capacity (40). Thus the origin of activator Ca2+ for contraction is significantly shifted to SR Ca2+ release from extracellular Ca2+ entry in HB myocytes. Increased SERCA and decreased PLB appear to be critical contributors to maintenance of contraction during hibernation. The increased SR Ca2+ uptake could explain the faster rate of Ca2+ decline during twitch observed in HB myocytes.

Importantly, the changes we observed in HB myocytes are directionally opposite to those in myocytes from failing hearts. Most previous studies in cardiac hypertrophy and failure have shown that changes in Ca2+ handling are due to a diminished relative protein expression of SERCA relative to PLB (12). For example, it has been shown (12) that SERCA protein levels were significantly reduced in failing hearts while unchanged protein levels of PLB were demonstrated. These changes are often associated with compensatory upregulation of NCX (13, 14, 36).

In addition, it is well documented that ventricular contractile dysfunction after ischemia and infarction is associated with electrical remodeling and changes in Ca2+ handling at the cellular level (10, 11, 38). An abnormal increase in resting Ca2+ has also been reported in ischemia and hypoxia (2). Increasing SR Ca2+ uptake usually causes SR Ca2+ overload, which is a predisposing factor for the development of arrhythmias, but no ventricular dysfunction or arrhythmias were found in HB animals.

In this study, we have not directly addressed alternative explanations for the maintained contractility during hibernation despite decreased Ca2+ influx. These include enhanced Ca2+ sensitivity of myofibrils that may be related to a slower relaxation time observed in HB myocytes, increased responsiveness of SR Ca2+ release channels, and/or enhanced functional coupling of L-type Ca2+ channels and SR Ca2+ release channels. Future studies could further delineate which cellular processes are critically involved in natural resistance to ventricular fibrillation. However, our results strongly support the hypothesis that SR Ca2+ uptake is markedly enhanced during true hibernation. This coupled with shorter APD associated with downregulation and increased Ca2+-dependent inactivation of ICa may in part provide a coordinated cardioprotective mechanism to prevent intracellular Ca2+ overload and/or metabolic demand associated with intracellular Ca2+ cycling in HB myocytes.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grants HL-61476, HL-59139, HL-33107, RR-16592, AG-14121, and HL-63020.


   ACKNOWLEDGMENTS
 
We thank Dr. R. Honda for technical assistance with this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Yatani, Cardiovascular Research Institute, Dept. of Cell Biology and Molecular Medicine, UMDNJ, New Jersey Medical School, 185 South Orange Ave., G609, Newark, NJ 07103 (E-mail: yataniat{at}umdnj.edu).

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
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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