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1Laboratory of Environmental Physiology, Faculty of Dental Medicine, The Hebrew University, Jerusalem, Israel; 2Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, Baltimore; and 3Department of Medicine, Cardiology, Johns Hopkins University, Baltimore, Maryland
Submitted 28 November 2006 ; accepted in final form 26 March 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cardiomyocyte contractility; calcium transients; ryanodine receptors; L-type calcium channel; phospholamban
In its nonphosphorylated form, phospholamban (PLB), a Ca2+ pump regulatory protein, inhibits the cardiac sarcoplasmic reticulum (SR) Ca2+ pump SERCA2 (Ca2+ ATPase type 2) (1). Among the acclimatory responses of the heart are coincident downregulation of SERCA2 mRNA and upregulation of PLB transcription and translation, thereby elevating the PLB/SERCA2 ratio, suggesting that changes in myocyte Ca2+ handling plays a major role in acclimation (10). In favor of this hypothesis are studies on Ca2+ handling in several thermally adapted fish species (32), pigeons (2), and ground squirrels (25), all demonstrating that SR regulatory proteins are important targets of thermal adaptation. Unfortunately, little information is available on calcium handling and the role of calcium in the acclimatory machinery of the mammalian heart. Nevertheless, the wide range of taxonomic groups with these common adaptive targets imply that Ca2+ handling-mediated adaptations are evolutionarily conserved.
In the heart, hyperthermia per se induces negative inotropism accompanied by an increase in the oxygen cost of contractility (29). Faster twitch kinetics further promote the loss of inotropy (17). Given that AC enhances cardiac mechanical and metabolic performance under normothermic conditions (9, 10, 20, 23, 24), we hypothesized that acclimation attenuates the detrimental hyperthermic effects on cardiac muscle.
This investigation had two main goals: 1) to characterize cellular Ca2+ dynamics in cardiomyocytes and their impact on pressure generation in heat-acclimated hearts; and 2) to define the beneficial effects of heat acclimation on cardiac muscle and cardiomyocyte performance during heat stress.
We used isolated hearts to characterize performance with experimental paradigms designed to mimic physiological stimulating conditions and isolated cardiomyocytes to investigate cellular mechanisms. Our results show that AC enhanced contractile force via altered calcium handling at the level of cross-talk between the Ca2+-induced Ca2+ release (CICR) components and SERCA2 regulatory proteins, as well as altering myofilament responsiveness. Hardening of the myofilaments and improved metabolic efficiency were associated with enhanced endurance during hyperthermia.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Male 3-wk-old Rattus norvegicus (Sabra strain, albino variation) initially weighing 80–90 g, fed Ambar laboratory chow and water ad libitum, were randomly assigned to heat-acclimated and normothermic groups. The normothermic groups included untreated animals that served as controls (C). The heat-acclimated groups were divided into fully acclimated rats (AC) and those that only underwent the initial phases of heat acclimation (AC-2d), facilitating the study of adaptive kinetics (18, 19).
All animals were kept in light-cycled rooms (12:12-h light-dark cycle). The C group was kept at an ambient temperature of 24 ± 1°C; heat acclimation was achieved by continuous exposure to 34 ± 1°C and 30–40% relative humidity for 2 days (AC-2d) and 30 days (AC), as previously described (9, 19). Isolated hearts or cardiomyocytes from each group were randomly assigned either to normothermic or to hyperthermic conditions. Given that calcium regulatory proteins and calcium handling are important objectives of thermal acclimation (2, 20), in addition to the characterization of performance profiles at basal Ca2+ levels, cardiac performance was also examined with additional calcium loads. The expression of excitation-contraction (E-C) coupling and Ca2+ regulatory proteins was also measured. All experimental protocols were approved by the Ethics Committee for Animal Experimentation of the Hebrew University.
Experimental Procedures
Cardiac mechanical performance during heat stress before and after heat acclimation. Animals were euthanized by cervical dislocation following ketamine-xylazine anesthesia (8.5 mg/100 g body wt ketamine in 0.5% xylazine). Hearts were rapidly removed and placed in a physiological solution (containing in mM: 118 NaCl, 24 NaHCO3, 1.2 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, 4.2 KCl, and 5.5 glucose at pH 7.4) at 4°C. The hearts were then mounted on a Langendorff perfusion apparatus and retrogradely perfused via the aorta at a perfusion pressure of 100 cmH2O as previously described (23). As soon as the perfusion started, atrioventricular block was induced by electrical coagulation of the membranous interventricular septum using a fine-tipped soldering iron. A deflated latex balloon (Hugo Sacks Electronics no. 3 or 4) attached to a Statham P23db pressure transducer was inserted into the left ventricle and gradually inflated with saline until maximal systolic pressure at 0-mmHg diastolic pressure was recorded. Hence the left ventricular preload was similar for all hearts (10, 23). The hearts were paced at 300–500 beats/min using stainless steel electrodes and a Grass S-88 stimulator. Left ventricular pressure was recorded using a computerized data-acquisition system (MP100, Biopac Systems, Santa Barbara, CA), and rates of pressure development and relaxation were calculated as ±dP/dt/P. All experiments began when a steady state was reached, 10–16 min after the onset of perfusion. After data collection at normothermic temperatures (37°C), the temperature was elevated to 39°C, 41°C, and then returned to 37°C. After each elevation, the heart was allowed to stabilize (as detailed above), and pressure measurements at all pacing rates were repeated. To gain further insight into calcium handling by the heart, a Ca2+ dose-response curve was established using CaCl2 concentrations in the range 0.5–3 mM. To further characterize the role of SR Ca2+ pool (vs. extracellular input) in the acclimatory process, we established the Ca2+ dose-response curves within a range of 2–10 mM by inducing tetani after preinduction of 2.5 M ryanodine during 20 min. After the SR Ca2+ stores were emptied via twitch stimulation at 90 V (27), Ca2+ dose-response curves were established under prolonged stimulation (pulses of 60–70 ms, frequency of 8–12 Hz at 75 V).
Cardiomyocyte contractility and Ca2+ transients in control and heat-acclimating rat hearts. Animals were euthanized, and the hearts were rapidly removed and placed in a physiological solution at 4°C. Cardiomyocytes were isolated using a modification of the technique described by Silverman's group (14). Briefly, the hearts were retrogradely perfused for 3 min with Krebs-Henseleit bicarbonate buffer (KHB), followed by perfusion for 5 min with calcium-free KHB, and then for 20–30 min with calcium-free KHB containing 0.2 mg/ml collagenase (type II, Sigma). The left ventricle was dissociated, and the myocytes were resuspended twice in calcium-free KHB. The myocytes were then suspended in KHB containing 100 mg/ml BSA and 50 µM CaCl2. The cell preparations contained 50%-70% rod-shaped cells. Cell shortening and Ca2+ transients (Ca2+ T) were measured separately at 37°C. The intracellular Ca2+ concentration ([Ca2+]i) was measured by incubating the cells at 37°C with the acetoxymethyl ester form of either fura-2 (3 µM together with 0.075% pluronic acid for 15 min) or indo-1 (2 µM and 0,075% pluronic acid for 20 min) and resuspended in KHB solution containing albumin (100 mg/ml, 1.2 mM Ca2+). The myocyte suspension was examined in a chamber with a quartz base using an inverted epifluorescence microscope (Nikon Diaphot 200) and perfused with KHB solution at 37°C. The myocytes were field-stimulated (0.5 Hz, square waves), and contractions [amplitude systolic motion (ASM)] were measured using a video motion edge detector (Crescent Electronics, Sandy, UT). For fura-2 measurements, the cells were alternately excited at 340 and 380 nm with 510 nm emission; indo-1 cells were excited at 340 nM, and emission was recorded at 405 and 495 nM wavelengths, using a PTI fluorimetric system (Photon Technology International). After steady-state stimulation at 1 Hz (1), the SR Ca2+ content was measured by the rapid application of 20 mM caffeine to induce SR Ca2+ release, as described in Ref. 30. The amplitude of the caffeine-induced Ca2+ T was used as an index of SR Ca2+ content. The responsiveness of the cardiomyocytes to Ca2+ load was assessed by establishing a Ca2+ dose-response curve, with Ca2+ concentrations ranging from 0.5 to 6.0 mM. An additional experimental series was conducted to test myocyte endurance under hyperthermic conditions of 42°C. Cell shortening and Ca2+ Ts were measured as above. During heating, basal Ca2+ levels and cell length under nonstimulating conditions were also measured.
Ca2+ levels were presented either in nanomolar calcium (when fura-2 was used) or as the ratio of fluorescence 340/380 nm and 409/495 nm for fura-2 or indo-1, respectively. Ca2+ level (in nM) was calculated according to Grynkiewicz et al. (15). Calibration was conducted at 37°C using Ca2+-free and Ca2+-containing solutions up to 100 µM [Ca2+]. For basal information on cardiomyocyte performance, we calculated fractional shortening (as a percentage of resting cell length), Ca2+ T amplitude, the instantaneous rate of contraction and relaxation (±dL/dt and ±dL/dt/Lmax-shortening, where Lmax-shortening is length at maximal shortening), and Ca2+ fluxes (±dCa2+ T/dt and ±dCa2+ T/dt/Ca2+ Tmax, where Ca2+ Tmax is maximum Ca2+ T).
Protein and mRNA analyses. Protein and mRNA levels of L-type Ca2+ channel [dihydropyridine receptor (DHPR)] and ryanodine receptors (RyRs), two key proteins in the CICR cascade and nonphosphorylated PLB and phosphorylated PLB [PLBs (phosphorylation at the serine site) and PLBtr (phosphorylation at the threonine site)] were measured before and after heat acclimation using Western immunoblotting and RT-PCR, respectively. Calsequestrin was stained and analyzed.
Western blot analysis and protein staining. For preparing whole cell lysates for measuring RyRs, PLB, and phosphorylated PLB, the left ventricle was homogenized with Tris buffer (1 M, pH 7.5) glycerol (50%), SDS (20%), and 0.1 mM PMSF. The homogenate was then centrifuged for 20 min at 14,000 rpm at 4°C. For DHPR, membranal fractions were prepared. The left ventricular tissue was homogenized with Tris (50 mM, pH 7.4), sucrose (0.25 M), and PMSF (0.1 mM). The homogenate was centrifuged at 3,500 g, and the supernatant then underwent centrifugation at 45,000 g (12). Total protein was measured using the Bradford assay (Bio-Rad, Richmond, CA). For fractionation by electrophoresis, 50 µg of protein was loaded into each lane. For RyRs and DHPR, 5% polyacrylamide gels were used under denaturing conditions, and then the separated proteins were electrotransferred onto nitrocellulose membranes (38). For total PLB (PLBt), PLBs, and PLBtr, 12.5% polyacrylamide gels and polyvinylidene difluoride (PVDF) membranes were used. Membranes were probed with specific primary antibodies [anti-mouse monoclonal RyRs, DHPR 1:1,000 (ABR, Golden, CO, USA) and polyclonal PLB, 1:5,000 (PhosphoProtein Res Elfodlea, UK)] followed by a second detector antibody (horseradish peroxidase-conjugated rabbit anti-mouse IgG) (Jackson Immune Research Laboratory) diluted 1:1,000 as previously described (9, 10, 26). Specific antibody binding was detected using enhanced chemiluminescence (Amersham) and visualized by exposing an X-ray film to the membrane (10, 26). For calsequestrin, 12.5% polyacrylamide gel was used, fixed for 20 min with glutaraldehyde (2%), and stained with 25% isopropanol, 10% foramide, 15 mM Tris, and 0.005% Stains-All (Sigma). Stains-All binds to calsequestrin, yielding a blue color (Ref. 7; and Shoshan Bar-Matz V, personal communication). The density of the scanned protein bands was calculated using Tina software (Raytest, Straubenhardt, Germany). For all proteins studied, each heart sample was tested four times, in separate runs. The scanned protein bands were normalized to a control pool sample.
RT-PCR.
Changes in mRNA transcripts were detected using semiquantitative RT-PCR, as previously described (10, 26). Briefly, total RNA was extracted from the left ventricle homogenate, using TRI-REAGENT (Molecular Research Center). Total RNA (10 µg) was reverse transcribed in a 50 µl reaction mixture containing 0.5 µg of oligo(dT)15 as primer, together with 400 U of MMLV reverse transcriptase, according to the manufacturer's instructions [U.S. Biochemical (USB), Cleveland, OH]. For the PCR, 5 µl of the cDNA mixture was added to 50 µl of a master mix containing 200 µM of each dNTP, 100 pM of each specific primer, and 1.5 units of Vent polymerase (USB). We synthesized DNA oligonucleotide primers for RyRs and DHPR. The oligonucleotide sequences were as follows: for DHPR, sense 5'-TCC-TGA-ACT-CCA-TA-TCA-AGG-C-3', antisense 5'-CTG ATA CAC TGG AAC ACC GTC-3'; and for RyRs, sense 5'-TGG AAA CTT GGA GTC GTGTTC AC-3' and antisense 5'-GTG TCA CCA TCC TCG CTT TTA TTG-3'. All sequences were prepared with (Macvector software using the sequence #M67516 and alignment of U95157
[GenBank]
and AF130880
[GenBank]
for DHPR and RyRs, respectively). To ensure equal amounts of initial mRNA, we performed parallel 
-actin amplifications (annealing temperature 62°C, 30 cycles). The PCR products were resolved on 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light. Band density was analyzed using Tina software (Raytest).
Statistics
A commercial statistics package (SigmaStat 2.03, SPPS) was used to determine statistical significance. Treatments were taken as the fixed effects, and individual samples were assumed to be random samples from the population. Two-way ANOVA for repeated measures followed by multiple comparisons was used to look for significant differences between the treatment groups. Student's unpaired t-test was used for individual matched-group comparisons. The data are expressed as means ± SE unless otherwise stated; P < 0.05 was considered statistically significant
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RESULTS |
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The results presented in Figs. 1 and 2 clearly show that pressures generated by heat-acclimated hearts were markedly higher than those of nonacclimated hearts, although the rates of pressure development and relaxation were slower (Table 1, row 1). The results are similar to our previous findings (9, 10, 20, 23, 24). The novel findings reported here are related to cardiac endurance during hyperthermic or calcium loads.
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Effects of temperature on developed pressure. Figure 2 shows that in C hearts, pressure generation diminishes as temperature increases, whereas AC hearts enhance pressure generation, reaching peak values at 41°C and 39°C for beating rates of 300 and 500 beats/min, respectively. The changes in the developed pressure with temperature elevation at basal and high beating rates, as well as the return to normothermic temperature (at 37°C), are depicted in Fig. 2B. The C hearts showed a rundown of pressure generation and failed to restitute pressure to that of preheating conditions upon recovery at normothermic temperature. The AC hearts, in contrast, resumed preheat stress pressure on recovery to normothermic temperatures at all pacing rates. Notably, the hyperthermic response was frequency dependent; increased beating rate exacerbated the temperature effect; namely it interfered with pressure generation. In C hearts at 500 beats/min, the decrease in the developed pressure was more pronounced than that at the resting-pace rate, whereas AC hearts demonstrated a peak pressure at 39°C rather than at 41°C (Fig. 2B), as measured for basal beating rate. An additional interesting finding observed in this experimental series was the greater accelerating effect of temperature on the velocity of relaxation in AC compared with C hearts (Table 1). At 41°C, this increase at 300 beats/min amounted to 58.0% and 38.8% (P < 0.05) for AC and C hearts, respectively.
Mechanical Performance of Isolated Cardiomyocytes
To gain insight into the mechanism underlying the differences in pressure generation between AC and C hearts, we studied the contractile responses in isolated cardiomyocytes during the evolution of acclimatory homeostasis and when it was achieved. Intracellular calcium was measured under the various experimental conditions to determine whether the changes in contractile force resulted from altered calcium handling. The peak contractile force in myocytes of AC rats under normothermic conditions, shown as the percent shortening of the myocyte (ASM), depicted in Fig. 3, was almost twice that of controls (Fig. 3, A and D). Noteworthy is the delayed onset of contraction (with respect to the onset of Ca2+ signal) of the AC myocyte (AC 36.2 ± 2.9 ms, C 25.3 ± 3 ms; P < 0.01). A greater contractile force in AC vs. C myocytes with delayed response was also seen on the second day of acclimation. (Fig. 3D). Concomitantly, the amplitude of the intracellular Ca2+ T was elevated in AC and AC-2d myocytes by 147% and 148%, respectively (Fig. 3E). Similar results were observed using both fura-2 and indo-1 (Fig. 3, B and C). To test whether greater Ca2+ T correlated with a larger SR-Ca2+ caffeine pool, the Ca2+ SR pool was measured following exposure of the cardiomyocytes to two Ca2+ loads (1.2 and 3.6 mM). No differences in Ca2+ caffeine pool size were found between the AC vs. the C groups (%change: +10.6 ± 10% and +12.1 ± 9.7% for 1.2 and 3.6 mM Ca2+, respectively; nonsignificant). In contrast, the AC-2d myocytes showed a significantly higher Ca2+ SR caffeine pool (+76%, P < 0.003 and +65%, P < 0.01 for 1.2 and 3.6 mM Ca2+, respectively). Given that our previous data showed changes in twitch kinetics in long-term AC hearts (10), we calculated this parameter in C and AC cardiomyocytes. The kinetics of transients and twitch development were different at both acclimation phases compared with those of the controls. This difference is exemplified in C and AC myocytes by the time elapsed between the contraction and relaxation phases at 50% of peak force amplitude and the rates of Ca2+ flux and force generation during contraction and relaxation (Fig. 3F). Whereas no change in the Ca2+ T time profile was recorded, the twitch time profile was elongated in the AC myocytes. Table 2 presents the positive and the negative time derivatives of the contractile responses before and after normalization to peak force and Ca2+ T amplitudes. The derivative ratios imply that to produce forces similar to those of C myocytes, greater Ca2+ flux is required in AC myocytes. In an additional experimental series, the performance of myocytes subjected to progressively increasing calcium loads was studied. AC myocytes (both short- and long-term phases) endured markedly higher intracellular Ca2+ levels, yet greater Ca2+ Ts were required in the AC and the AC-2d myocytes to produce similar forces, suggesting a decreased responsiveness of the contractile elements (Fig. 4).
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Prior studies from our laboratory provided evidence that an altered PLB/SERCA2 ratio in C vs. AC heart is associated with greater pressure generation (10). In this study, we found different Ca2+ T kinetics; therefore, we studied additional proteins associated with the calcium-induced calcium release pathway. No changes were detected between C vs. AC groups in calsequestrin (–5.46 ± 3.4%, nonsignificant). The data are congruous with the similarity in SR-caffeine calcium pool size among these groups. In contrast, significant changes were detected in the levels of L-type calcium channels (DHPR) and RyRs (Fig. 6, A and B). The AC hearts demonstrated significant upregulation in the expression of both DHPR and RyRs (70%, P < 0.05 and 30%, P < 0.05, for DHPR and RyRs, respectively). Concomitantly, a downregulation of the DHPR and RyRs transcripts (data not shown) suggested reciprocal relations between each transcript and its expressed proteins. Similar to our earlier results (10), PLBt in AC hearts was elevated, in turn leading to a decreased PLBs/PLBt ratio (Fig. 6C). At that acclimation phase, no significant change was detected in PLBtr. In the AC-2d group, neither DHPR nor RyRs changed compared with C hearts. In contrast, PLBs was significantly upregulated, and PLBtr decreased by 82.5 ± 7.3% (P < 0.01) (Fig. 6, A–C).
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DISCUSSION |
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An additional important finding is that heat acclimation augments pressure development during severe hyperthermia at a normal range of beating rates and protects its decline at very high frequencies. Such enhanced endurance was also displayed in isolated myocytes, whereas in contrast, nonacclimated hearts failed to restitute pressure above 39°C. The desensitization of the myofilament response found in this investigation, together with enhanced metabolic efficiency (24), play important roles in the hyperthermic endurance of the AC heart.
Contractile Force and Ca2+ Handling by Acclimated Myocytes
Heat-acclimated cardiomyocytes developed significantly greater Ca2+ T and, in turn, contractile force. The different kinetics of the Ca2+ T, in terms of the rate of SR efflux and resequestration, suggest different Ca2+ handling by the myocyte, leading to a profoundly greater Ca2+ T amplitude.
During cardiac E-C coupling, brief openings of L-type Ca2+ channels activate RyRs by the mechanism of Ca2+-induced Ca2+ release (3, 11) to generate Ca2+ T that activate contraction. In turn, L-type Ca2+ channels are inactivated (5, 37) and terminate SR Ca2+ release, thus allowing the SERCA2 and the sarcolemmal Na+/Ca2+ exchanger (NCX) to resequester or extrude Ca2+ from the cytosol to cause relaxation. Within this frame, the results obtained here, together with our previous data on SR calcium regulatory proteins, provide the rationale to theorize about the mechanism underlying the variations in E-C coupling between acclimated and nonacclimated hearts.
Several models of E-C coupling (5, 37) suggest that a RyRs/DHPR ratio of 6–7/1 is required for RyRs activation in the rat heart. Altered cross-talk between these two components in several pathological conditions is responsible for changes in myocyte contractility. For example, the upregulation of the RyRs vs. DHPR is beneficial in the failing heart because it increases the Ca2+ T amplitude (13). In contrast, diltiazem, an L-type channel inhibitor, prevented cardiomyopathy in an MHC transgenic mouse model by enhancing RyRs phosphorylation and SR Ca homeostasis (31). In the experiments described here, the ability of 10 µM ryanodine to equalize pressure generation in AC and C hearts, over a wide [Ca2+] range, demonstrated the important role of RyRs in the generation of higher pressure in AC hearts. In an additional experimental series, we measured a significant upregulation in the expression of both L type Ca2+ channels and RyRs, at a ratio of 
2.3 (for details, see Fig. 6). These findings further emphasize the principal role of RyRs-DHPR cross-talk in the acclimated state. Increased protein expression alone does not explain the activation profiles of the channels but rather suggests that compensatory processes can maintain CICR to generate optimal pressure in the acclimated heart. As the inhibition of RyRs eliminates differences between the C and the AC heart, DHPR upregulation and enhanced RyR responsiveness are likely to compensate for a lower activation of the channel (DHPR). Although a direct study of these issues is beyond the scope of the present study, observations from our laboratory (22) demonstrating lower inward Ca2+ current in AC vs. C cardiomyocytes favor both compensatory upregulation of DHPR and enhanced RyR responsiveness. The resemblance of calsequestrin level and caffeine calcium pool size in C and AC cardiomyocytes, implying a greater Ca2+ T, independent of the SR Ca pool, agrees with several other reports showing changes in Ca2+ T amplitude despite similar calsequestrin levels and SR calcium pool (33, 36).
In contrast to Eynan et al. (10), who reported a higher nonphosphorylated PLB/SERCA2 ratio and lower SERCA2 levels in AC hearts, our results showed lower PLBs/PLBt in AC hearts. Given that PLB serine 16 is the dominant site for a protein kinase A-mediated PLB relaxant effect (6, 35), the results further support lower SERCA2 activity and, in turn, lower Ca2+ reuptake to the SR pool in AC hearts. This decrease could contribute to both the Ca2+ amplitude and the negative lusitropic effect reported in AC hearts (10, 23). Theoretically, enhanced NCX possibly contributes to the maintenance of diastolic Ca2+ level as well (4). Although this aspect was not studied in this investigation, the global genomic analyses of AC hearts using gene chip technology demonstrated a upregulation of 5 orders of magnitude in the NCX3 transcript, implying activation of this pathway (Kodesh E and Horowitz M, unpublished observations).
An emerging issue in this investigation was the lower responsiveness of the contractile proteins to Ca2+ load, demonstrated by 1) the Ca2+ T-ASM relations curve (Fig. 5), suggesting that a greater Ca2+ T is required to generate similar forces in the AC cardiomyocyte; and 2) the higher dCa2+T/ dt-to-dF/dt ratio in AC vs. C myocytes (Table 2). Coincidentally, greater endurance to extracellular and intracellular Ca2+ load both in isolated hearts and cardiomyocytes of AC animals (Figs. 1 and 4) complements the requirements for increased force generation. Notably, a similar response found in heat-acclimated pigeons (2) implies uniformity in the acclimatory response. We could speculate that improved endurance of the heart to calcium overload enables the heart to function properly under adverse environmental conditions (27a) or impaired homeostasis, as when Ca2+ overload leads to deleterious effects in ischemia or hypoxia (16). Heat-acclimation ischemia-reperfusion cross-tolerance has been well established in the AC heart (20).
Acclimation Dynamics
Although changes in Ca2+ T amplitude and ASM were already seen after two acclimation days in AC-2d and AC groups, long-lasting changes in EC coupling and calcium regulatory proteins, leading to greater Ca2+ T, were observed only after long-term heat acclimation. This finding suggests that the increased Ca2+ T measured on two acclimating days occurred by a different mechanism than that for the long-term acclimated heart. Our finding of greater Ca2+ SR pool, greater PLBs, and a drop in PLBtr levels in AC-2d hearts implies an enhanced rate of Ca2+ cycling, in and out of the SR, at that acclimation phase. This temporal profile fits with our previously described biphasic acclimation model (9), in which desensitized adrenergic signaling in the heart is dominant during short-term acclimation (2–5 days), whereas upon long-term acclimation (30 days and onward), augmented adrenergic inotropic responses (via PLB elevation) and a decreased velocity of relaxation have been reported (9, 10). The predominance of the slow myosin isoform V3 following AC (21) contributes to the slower force generation at that acclimation phase. Sustained low thyroxine levels when acclimatory homeostasis is achieved causally associates with these responses (9, 10, 20, 21). In all likelihood, at the onset of acclimation, rapid transient mechanisms are recruited, whereas to achieve long-term acclimatory homeostasis, a reprogramming of gene expression and posttranscriptional changes are involved (e.g., Refs. 9, 10, 19, 20, 26; and this investigation).
Cardiac Performance During Heat Stress
Using two experimental paradigms, we substantiated that heat acclimation enhances mechanical performance and confers cardiac protection under hyperthermic conditions, both at resting and accelerated beating rates. In the whole heart, at the resting beating rate, pressure generation in AC hearts increased over a range of 37–41°C and resumed prehyperthermic levels when the temperature returned to 37°C. With increased pacing, the peak pressure of AC hearts was achieved at 39°C and recovered prehyperthermic pressure following a return to normothermic conditions. In contrast, hyperthermic C hearts showed a slightly decreased pressure generation when maintained at a resting beating rate but malfunctioned (Fig. 2) and did not recover at high beating rates. Hiranandani et al. (17) demonstrated reduced peak force development in rat trabeculae, whereas the force-frequency relation shifted rightward to a higher frequency optimum. That finding does not agree with our results in the isolated perfused heart. This dissimilarity, however, could stem from differences in the experimental model, including the lower glucose concentration in our perfusate. This explanation fits with Saeki et al. (29), who demonstrated increased O2 consumption and decreased contractile efficiency during hyperthermia. We suggest that an improved metabolic efficiency in AC vs. C hearts (18, 20, 23) underlies, at least partially, the profound enhanced endurance of cardiac muscle during hyperthermia.
In contrast to isolated hearts, isolated C cardiomyocytes showed increased contractility upon hyperthermia. The AC myocytes maintained their advantage over the C myocytes, but unlike C hearts, remained unaffected by higher temperatures. The significant elevation of Ca2+ Ts, however, in both C and AC myocytes, suggests a dichotomy between calcium handling and myofilament response, with hardening of the myofilament responsiveness to Ca2+ in the hyperthermic AC cardiomyocytes (Fig. 5, A vs. B). An additional notable difference, emphasizing the benefits of AC myocytes, was the attenuation of Ca2+ overload and, in turn, the resting tension vs. C myocytes. Collectively, the results support a protection of AC cardiomyocytes against the detrimental effects of hyperthermia. The discrepancy in the safety temperature margin between the isolated C heart and the isolated C myocytes is likely to occur because of the difference in the pacing rate and the duration of stimulation.
The investigation presented here focuses primarily on the phenomenology of hyperthermic effects on AC and C hearts. Nevertheless, integrating our knowledge of cardiac acclimatory responses with the data available on hyperthermic, heat stroke-mimetic effects on the cardiac contractile response allows us to theorize about the protective pathways that develop during heat acclimation against hyperthermia. Everett et al. (8) and Nath et al. (28), using both resting tension and fluorescence changes of fluo-3 AM dye as indirect and direct indicatives, respectively, of cytosolic free calcium concentration in guinea pig papillary muscles, provided evidence that hyperthermia causes a reversible increase in resting tension at temperatures between 45 and 50°C and irreversible contracture above these temperatures. Such changes were due to altered SR rather than to sarcolemmal malfunction. The leakiness of RyRs, leading to Ca2+ overload under pathological hyperthermic conditions, also supports this notion (34). The increased resting basal cytosolic Ca2+ and tension of the hyperthermic C myocytes observed here is in accordance with the findings of Everett et al. and Nath et al. (8, 28). The AC myocytes demonstrated desensitization of this effect.
In conclusion, this study shows that long-term heat acclimation produces changes in the mechanical performance of the heart via the reprogramming of gene expression. We demonstrated that greater Ca2+ T generation and decreased calcium responsiveness play a major role in greater force/pressure development. Desensitization of the myofilaments to hyperthermic conditions enhances endurance under these conditions.
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FOOTNOTES |
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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.
* H. Kanana and R. Zoizner contributed equally to this work. 
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AR profile and heart responsiveness to isoproterenol and calcium. Isr J Zool 49: 219–231, 2003.[Web of Science]This article has been cited by other articles:
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  A. Tetievsky, O. Cohen, L. Eli-Berchoer, G. Gerstenblith, M. D. Stern, I. Wapinski, N. Friedman, and M. Horowitz Physiological and molecular evidence of heat acclimation memory: a lesson from thermal responses and ischemic cross-tolerance in the heart Physiol Genomics, June 1, 2008; 34(1): 78 - 87. [Abstract] [Full Text] [PDF]
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Significant difference between C and AC hearts (P < 0.0001) when temperature, beating rates, and AC were taken as the independent variables. B: effects of progressively increasing temperature (37°C–41°C) and recovery at normothermic conditions (37°C) on pressure generation at basal (300 beats/min) and rapid (500 beats/min) beating rates. The data (means ± SE, n = 6–9) are plots of the results presented in A. The pressure developed in AC hearts was significantly higher than in C hearts in all pacing frequencies at all temperatures. *Significant differences between AC and C hearts at each bath temperature point (P < 0.05).
