INTRODUCTION

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A LARGE BODY of evidence is now available showing that prolonged exposure to moderate ambient heat, heat acclimation, improves the mechanical and metabolic performance of the rat heart (10-13, 19, 20). After acclimation, left ventricular compliance and the generation of systolic pressure are increased and oxygen consumption is lowered. This suggests that the acclimated heart becomes more energetically efficient. Concomitantly, however, the velocity of contraction and relaxation of the acclimated heart is slower than that of the nonacclimated heart (13, 19, 20). Thus, on acclimation, increased efficiency is achieved, at least in part, at the expense of contractile velocity.

Biochemical adaptive modalities provide a mechanistic explanation for some of the adaptations reported above. For example, the greater pressure development is associated with a change in the handling of cytosolic Ca²⁺, as manifested by a larger sarcoplasmic Ca²⁺ pool and greater Ca²⁺ transients on contraction (Ref. 10 and M. Horowitz, O. Cohen, and U. Meiri, unpublished observations). Likewise, the reduced contractile velocity of acclimated hearts is due to a transition from the predominantly fast myosin isoform with high ATPase activity (V₁) in the nonacclimated hearts to a predominance of the slow myosin isoform with low ATPase activity (V₃) in the acclimated heart. The transition to the V₃ myosin isoform, coinciding with a marked decrease in plasma thyroxine level, was blunted when acclimated rats were kept euthyroid. This implies that the redistribution of myosin isoforms occurring on heat acclimation is mediated by the sustained lower plasma concentration of thyroid hormones (12).

Acclimatory features, such as higher pressure production (11, 20) and extensive sarcolemmal Ca²⁺-ATPase activity in the acclimated heart (unpublished data), are difficult to reconcile with the consensus features characterizing hearts from hypothyroid animals: lower pressure generation (4), cardiomyopathy (30) and low ATPase activity (23). Nevertheless, in view of the unequivocal match between the heat acclimation-induced peripheral hypothyroidism and the transition to the V₃ myosin accompanied by a reduction in contractile velocity, it is conceivable that the lower level of thyroid hormone acts to switch on a variety of heat acclimatory responses. This assumption gains support from several studies on both humans and rodents, reporting low circulating plasma thyroxine on heat acclimation/acclimatization (e.g., Refs. 5, 32).

Our knowledge of the influence of thyroid hormones on transcription of critical genes involved in cardiomyocyte contraction and relaxation (e.g., Refs. 14, 17) further substantiates this hypothesis. Some of the "landmark" features associated with hypothyroidism are possibly masked in the acclimated hearts by overriding cues.

In addition to its effect on myosin isoforms (2, 8), a low level of thyroid hormone is known to reduce the expression of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) by controlling the level of phospholamban (PLB), which in the nonphosphorylated form inhibits the SR calcium pumps (1). Kiss et al. (16) showed that the ratio between these two regulatory proteins is a decisive determinant of cardiac relaxation, influencing the Ca²⁺-ATPase affinity to Ca²⁺ and, in turn, Ca²⁺ uptake into the SR pool. Hence, it is likely that changes in the steady-state levels of PLB and SERCA are associated with the negative lusitropic (decreased rate of relaxation) effect occurring on heat acclimation.

In the present study, an attempt was made to substantiate the hypothesis that a reduced thyroid hormone level is associated with the heart's adaptation to prolonged heat. For this purpose, a comparison was made between AC and AC-euthyroid rat hearts. Two aspects were examined: 1) mechanical performance and 2) the steady-state levels of Ca²⁺-ATPase and phospolamban mRNAs and the expression of the Ca²⁺ regulatory proteins SERCA and PLB. Our data indicate that a reduced thyroid hormone level, achieved through heat acclimation, leads to downregulation of Ca²⁺-ATPase mRNA expression and upregulation of phospholamban mRNA expression as well as of their translated proteins. The changes in the concentrations of these proteins coincide with the decreased rate of relaxation in these hearts.

	MATERIALS AND METHODS

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Animals. Male Rattus norvegicus (Zabar strain, albino variant), 80-90 g initial body weight, fed on Ambar laboratory chow and water ad libitum were used. The animals were divided into four groups: 1) control (C); 2) nonacclimated hypothyroid rats (CP); 3) heat-acclimated rats (AC); and 4) euthyroid acclimated rats (AT). The C and CP groups were maintained at an ambient temperature of 24 ± 1°C; the AC and AT groups were kept in a climatic chamber at 34 ± 1°C for 1 mo (9). On termination of the acclimation procedure, the rectal temperature of the heat-acclimated groups was ~0.3-0.5°C higher than that of the normothermic groups (37.5 ± 0.2, 36.12 ± 0.1, 37.8 ± 0.2, and 37.9 ± 0.1°C for C, CP, AC, and AT rats, respectively).

AT rats were obtained by administering 3 ng/ml L-thyroxine (T₄; Sigma) in the drinking water. This dose was chosen after preliminary experiments in which the effect of several concentrations of the hormone in the drinking water on plasma 3,5,3'-triiodothyronine (T₃) and T₄ levels was studied. Hypothyroid rats were obtained by the administration of 0.02% 6-n-propyl-2-thiouracil (PTU) in the drinking water for 1 mo (3).

Left ventricular mechanics. The animals were killed by cervical dislocation. Hearts were rapidly removed and placed in a physiological solution at 4°C. The hearts were then mounted on a Langendorff perfusion apparatus and retrogradely perfused through the aorta at a perfusion pressure of 100 cmH₂O with Krebs-Henseleit bicarbonate buffer containing (in mM) 118 NaCl, 24 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgCl₂, 2.5 CaCl₂, 4.2 KCl, and 5.5 glucose at pH 7.4, maintained at 37°C and bubbled with 95% O₂-5% CO₂ (20). Heart temperature was continuously monitored with a thermocouple (Omega Digicator).

Once perfusion was started, an atrioventricular block was induced by electrical coagulation of the membranous interventricular septum with a fine-tipped soldering iron. A decompressed latex balloon (Hugo Sacks Electronics no. 3 or 4) attached to a Statham P23db pressure transducer (with PE-190 polyethylene tubing) was inserted into the left ventricle via a left atrial incision. The balloon was inflated with saline until the diastolic pressure reached 0 mmHg and then until the systolic pressure was maximal at that diastolic pressure. The inflation volume was thus determined by both left ventricular chamber size and compliance. Although the inflation volume was not the same in all hearts, the left ventricular preload (that at the point of maximal systolic pressure at 0 diastolic pressure) was the same. This technique allowed, therefore, similar preloads among hearts of varying size and compliance. Hearts were paced at 200, 300, and 400 beats/min via stainless steel electrodes with the aid of a Grass S-88 stimulator. Left ventricular pressure (LVP) was recorded using the CODAS data acquisition system (DATAQ) with an IBM/PC computer. All hearts were perfused under these conditions until a steady-state was reached, usually 10-16 min before the experiment was begun.

T₃ and T₄ measurements. During the acclimation period, plasma levels of T₃ and T₄ were measured at 1-wk intervals. For measurement of T₃ and T₄ levels, 0.5-ml blood samples were withdrawn by cardiac puncture and centrifuged. Plasma was kept at 70°C until analysis. Analysis of all samples was performed at the same time. Total plasma T₃ (ng/dl) and T₄ (µg/dl) levels were measured by radioimmunoassay (Coat-A-Count, Diagnostic Products). The sensitivity of the determinations was 7 ng/dl for T₃ and 0.25 µg/dl for T₄.

Semiquantitative determination of Ca²⁺-ATPase and phospholamban mRNA by RT-PCR. To measure transcription of Ca²⁺-ATPase and phospholamban, semiquantitative RT-PCR was performed. Left ventricle tissue from the hearts of five rats in each experimental group was carefully excised, dissected, and homogenized with a polytron (Kinematika, Lucern, Switzerland). Total RNA was extracted with TRI-Reagent (Molecular Research Center). A quantity of 10 µg of total RNA was reverse transcribed in a 50-µl reaction mixture containing 0.5 µg of oligo(dT₁₅) as primer, together with 400 U of MMULV reverse transcriptase, according to the manufacturer's instructions (United States Biochemical, Cleveland, OH). For PCR, 0.3-1 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, 1.5 mM MgCl₂ for Ca²⁺-ATPase, 1 mM MgCl₂ for phospholamban, and 1.5 U of Vent polymerase (USB). We synthesized DNA oligonucleotide primers selected from the published sequence of the Ca²⁺-ATPase gene (21). The sense primer was based on sequence no. 2079-2104 (5'-ATG-AGA-TCA-CAG-CTA-TGA-CTG-GTG-3') and antisense sequence no. 2707-2732 (5'-GCA-TTG-CAC-ATC-TCT-ATG-GTG-ACT-AG-3'). The DNA oligonucleotide primers for phospholamban were selected from the published sequence of the phospholamban gene (24). The sense primer was based on sequence no. 199-220 (5'-TAC-CTT-ACT-CGC-TCG-GCT-ATC-3') and antisense sequence no. 318-339 (5'-CAG-AAG-CAT-CAC-AAT-GAT-GCA-G-3'). The primers were designed to cross introns to avoid confusion between mRNA transcript and genomic DNA. The optimal conditions for each set of primers derived from calibration curves are presented in Fig. 1. Negative and positive controls were included in every run. Each sample was amplified three times in an automated thermal cycler (Perkin Elmer-Cetus, Emeryville, CA). To ensure a fixed amount of initial mRNA, parallel -actin amplification was performed (annealing temp 62°C, 35 cycles) using the following oligonucleotides: 5'-GAG-ACC-TTC-AAC-ACC-CCA-GCC-3' (sense) and 5'-GGC-CAT-CTC-TTG-CTC-GAA-GTC-3' (antisense) (25). PCR products (10 µl each) were separated on 1.5% agarose ethidium-bromide gel, visualized under ultraviolet light, and photographed on high-speed film (Polaroid 667). The prints were scanned by a VISTA 8S scanner (Umax) and the density of the bands was computer analyzed by NIH 1.6 Image software (National Institutes of Health). The relative intensity of bands for each mRNA was divided by the intensity of the band for the internal control, -actin.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Calibration curves for sarcoplasmic reticulum (SR) Ca2+-ATPase and phospholamban primers. A: annealing temperature. B: number of cycles. C: cDNA dilution. D: MgCl2 concentration.

Western immunoblotting. The levels of SERCA and PLB proteins in left ventricle tissue from hearts of rats in each experimental group were obtained by quantitative immunoblotting. Anti-PLB and anti-SERCA monoclonal antibodies were purchased from Affinity Bioreagents.

The PLB and SERCA immunoblots were performed separately on different nitrocellulose membranes. Samples from the same heart homogenates (a quantity of 50 µg cardiac homogenate proteins) were taken in pairs and assigned to either SERCA or PLB immunoblotting. Each sample was separated by 12.5% SDS-PAGE, then transferred to nitrocellulose membranes and reacted with anti-SERCA or anti-PLB monoclonal antibodies at 1:1,000 dilution. Each nitrocellulose membrane included samples from the four groups studied, a group of molecular weight markers, and a control sample prepared from a pooled cardiac homogenate of a known amount of protein, which served as a reference and was immunoblotted with all experimental series. After repeated washings, the membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated rabbit anti-mouse IgG (Sigma), diluted 1:1,000. The membranes were then washed and developed to enhance chemiluminescence (Amersham, Buckinghamshire, UK), and X-ray film was exposed to the membranes. The SERCA and PLB levels were estimated by laser densitometry of the immunoblots. When quantified, the darkness of the band of each experimental sample was normalized to the darkness of the reference sample.

The protein concentration of the myocardial specimens was quantified according to Bradford (Bio-Rad Laboratories kit, Richmond, CA).

Statistics. One-way and two-way ANOVA were employed using commercially available computer software. Treatments were taken as the fixed effects, and the individual hearts were assumed to be random samples from the animal heart population. Values of P < 0.05 were considered to be statistically significant. For comparisons between two groups, this was followed by a Student's t-test using the Bonferroni correction or Dunnett's test. Data are expressed as means ± SE.

	RESULTS

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Plasma thyroid hormones and body and heart weights. Table 1 presents thyroid hormone levels and body and heart weights in rats from the different experimental groups after exposure to the specific treatments for 1 mo. The plasma T₄ level in the C rats (3.97 ± 0.25 µg/dl) concurred with the results of previous studies on plasma T₄ concentration in euthyroid rats (7). In the AC group, T₄ levels were significantly lower than those in the C group (2.62 ± 0.29 µg/dl; P < 0.01), whereas the values obtained for the AT rats resembled those of the C group, suggesting that the AT rats had received the appropriate amount of T₄. The plasma T₃ levels displayed a similar picture. PTU treatment caused a significant reduction in T₄ levels. Plasma T₄ values (<1 µg/dl) in rats belonging to this group were, indeed, the lowest among the experimental groups studied in this investigation.

It is evident that the body weight of the heat-treated and the CP-treated rats was significantly lower than that of the control group (60 and 79%, respectively, P < 0.001). In contrast, the slightly decreased weight noted among the AT rats was not significantly different from that of the C group. The heart weights followed a similar pattern. Nevertheless, the heart weight-to-body weight ratio remained unchanged in all the groups, except for the CP group.

Myocardial performance. The mean LVP developed by AC hearts was significantly greater than that of the C group hearts at all stimulation frequencies. For example, at 300 beats/min (Fig. 2), the pressure generated by the AC hearts was 90.42 ± 20.4 versus 59.62 ± 11.38 mmHg in the C group (P < 0.001). The rates of pressure generation and relaxation (dP/dt · P and dP/dt · P) were slower in the AC than in the C group (dP/dt · P at 300 beats/min was 54.1 ± 5.8 vs. 67.62 ± 6.9; dP/dt · P 31.4 ± 2.8 vs. 35.5 ± 6 mmHg/s · mmHg, respectively; at 400 beats/min, these values were dP/dt · P 61.4 ± 7.2 vs.78.3 ± 8.5 and dP/dt · P 28.4 ± 7.6 vs. 43.3 ± 3.6 mmHg/s · mmHg; P < 0.05), suggesting, as previously shown, that differences in velocity are more pronounced at high beating rates. Individual traces of the isovolumic pressure tracings and the time derivative curves are presented in Fig. 3.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   A: systolic pressure generated by hearts of control (C), hypothyroid (CP), heat-acclimated (AC), and heat-acclimated-euthyroid (AT) rats. B: systolic pressure generated by hearts of nonacclimated, age-matched rats (C) or nonacclimated weight-matched rats (170 g, C1) compared with the CP rats. Pressure was measured at a perfusion pressure of 100 cmH2O at 300 beats/min. Values are expressed as means ± SE; n: C, 14; CP, 9; AC, 14; and AT, 10. * Significant differences from matched controls (P < 0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Representative isovolumic pressure tracings (A and B) and time-derivative curves (C and D) of C and AC hearts. Heart wt, systolic pressure (P), and the (+, ) dP/dt (in mmHg/s) are 1.35 g, 53.3 mmHg, 66.0, and 42.6, respectively, for C and 1.06 g, 99.9 mmHg, 29.0, and 21.5, respectively, for AC.

Administration of thyroxine during the acclimation period to maintain heat-acclimated euthyroid rats (AT group), partially blunted cardiac ability to generate greater pressure. The rates of pressure generation and relaxation in this group (not shown) were similar to these parameters in the C group. The additional group in which intervention in thyroxine level was made, the CP group, showed somewhat unexpected results. CP hearts developed pressures similar to that of the C group. This was confirmed both for the C group and for additional controls (C1) of similar weight to the CP group (160-170 g; Fig. 2). Concomitantly, the rates of pressure relaxation (dP/dt · P) were slower in the CP group than in the matched-weight controls (44.72 ± 4.28 vs. 60.3 ± 6.9 mmHg/s · mmHg; P < 0.005). Values for contractility rates were similar for both groups (70.0 ± 2.59 vs. 65.75 ± 6.3 mmHg/s · mmHg, respectively).

Ca²⁺-ATPase and phospholamban mRNA transcription and SERCA and PLB protein expression. To determine whether changes in the mechanical properties of the AC, AT, and CP hearts were associated with altered expression of SERCA and PLB, the relevant mRNA levels were measured. Representative results of the steady-state mRNA level obtained for Ca²⁺-ATPase and phospholamban mRNAs and the averaged density of the Ca²⁺-ATPase and phospholamban mRNA bands relative to the density of the housekeeping gene -actin are shown in Fig. 4. Heat acclimation resulted in a pronounced decrease (P  0.01) in Ca²⁺-ATPase mRNA level, down to 65% of that of the C group. Maintenance of thyroid hormone levels during acclimation in the AT group abolished the reduction, whereas the decrease in Ca²⁺-ATPase mRNA in the CP group resembled that in the AC group. The phospholamban mRNA levels revealed an opposite trend, with a significant increase in the AC and CP ventricles of up to 188 and 194% (P < 0.005) of the C group, respectively. The mRNA level in the AT group resembled that of the C group.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Semiquantitative RT-PCR analysis of Ca2+-ATPase (A) and phospholamban (B) mRNAs in C, CP, AC, and AT rats. Bar graphs show relative amounts of Ca2+-ATPase (A) and phospholamban (B) mRNAs, normalized to -actin, in the different experimental groups. mRNA of each individual animal was measured independently 3 times. Each bar represents mean ± SE; n: C, AC, and AT, 5; CP, 4. *Significant difference from controls (P < 0.05). Representative sets of bands (10 µl/lane) for each transcript are presented.

The level of SERCA and PLB proteins was measured by quantitative immunoblotting. The AC hearts displayed a significant decrease in SERCA expression, down to 75% of that of the C group (Fig. 5). Maintenance of the thyroid hormone level during acclimation of the AT group abolished this reduction. Interestingly, expression of SERCA in the CP group was elevated and did not follow the positive mRNA-protein relationship usually reported. It concurs, however, with the mechanical properties of these hearts, as discussed below. The PLB levels (Fig. 5) increased in the AC ventricle to 147% of that in the C group. Administration of T₄ to the acclimating rats blunted the change in PLB level observed in the AC rats. In the CP group, similar to the AC group, the level of PLB increased, attaining 143% that of the controls. The ratio of PLB to SERCA (Fig. 6) increased in the AC rats but remained unchanged in the AT and CP groups compared with that in the C group.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Quantification of SERCA (A) and phospholamban (PLB; B) levels in hearts isolated from C, CP, AC, and AT rats. Bar graphs show the SERCA (A) and PLB (B) protein levels in the different groups of rats normalized to control sample. Each heart homogenate was tested 3 times in separate runs. Data are expressed as means ± SE; n: C, AC, and AT, 5; CP, 4. * Significant differences from matched controls (P < 0.05). Representative sets of blots of the various groups (chemoluminesence detection) are presented.

View larger version (56K): [in this window] [in a new window]   Fig. 6.   Percent change in level of PLB and SERCA of CP, AC, and AT rats compared with those of C using the values obtained for C hearts as the baseline (100%). Data are expressed as means ± SE; n: C, AC, and AT, 5; CP, 4. * Significant differences from matched controls (P < 0.05).

In Table 2, a summary of our major results is presented in a qualitative manner.

	DISCUSSION

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Our results strongly support our hypothesis that a sustained chronic low level of plasma thyroid hormones induced by heat acclimation is responsible for the emergence of important cardiac acclimatory responses. Causal evidence is provided that in the heart, the chronic decline, with acclimation, in thyroid hormones leads to decreased Ca²⁺-ATPase and elevated phospholamban mRNA steady-state levels and, in turn, PLB and SERCA expression. It is likely that these opposing responses, leading to an elevated PLB/SERCA are associated at high beating rates with a negative lusitropic effect. The strongest evidence for this causative role is the fact that T₄ supplementation, so as to bring the T₃ and T₄ levels to euthyroidism during acclimation, blunted these changes as well as the augmented pressure production and the reduced rate of pressure generation. These findings, together with those published previously (12), imply a multifaceted effect at the genomic level (Table 3) exerted by the altered T₄/T₃ profile on adaptation to chronic heat.

Mechanical performance of the AC heart. Heat acclimation produced the previously characterized changes in the function of the heart as a pump (19, 20). These included enhanced pressure generation with a concomitant decrease in the rates of contraction and relaxation at high beating frequencies. We previously showed that the enhanced pressure in the AC heart is correlated with an alteration in the function of the SR, leading to a greater recruitment of cytosolic Ca²⁺ (Ca²⁺ transient) on stimulation (10), from a larger sarcoplasmic Ca²⁺ pool (unpublished observations). We also demonstrated that depressed contractile velocity in the AC hearts is correlated with V₃ myosin predominance (12). However, we did not find an explanation for the negative lusitropic effect. Characterization of the steady-state levels of Ca²⁺-ATPase and phospholamban mRNAs and of the protein profile in the present investigation has advanced our understanding in this respect. On heat acclimation, Ca²⁺-ATPase mRNA in the AC hearts was downregulated and the transcription of phospholamban mRNA was upregulated. The concentration of PLB and SERCA, measured by semiquantitative immunoblotting, followed a similar pattern. Thus the relative PLB/SERCA in AC hearts was markedly higher than in the C hearts. Kiss et al. (16) and Luo et al. (22) provided solid evidence that PLB and SERCA are major determinants of cardiac mechanics and showed that an elevated PLB/SERCA is indicative of decreased SERCA pump activity and affinity for Ca²⁺. Hence, the overexpression of PLB and decreased expression of SERCA observed in AC hearts may favor a reduced rate of Ca²⁺ uptake into the SR and a slower rate of relaxation. This was indeed observed in this investigation and in previous ones (20). Our previous data of an enhanced stimulatory effect of isoprenaline in AC vs. C hearts (A. Barak and M. Horowitz, unpublished observations) lend further support to this conclusion: PLB is a prominent mediator of the transduction of cardiac -adrenergic signaling. Accordingly, in PLB-ablated mice the inotropic effect of isoprenaline was attenuated, whereas upregulation of PLB, which is compatible with the condition in the AC heart, enhanced this response (15).

Evidence for the contribution of thyroxine hormone level to the acclimatory mechanical performance response. The partial, although significant, abolition of the greater pressure generated in the hearts of AC rats by maintaining euthyroidism during acclimation provides causal evidence for the intervention of thyroxine profiles in the mechanical properties of the AC heart. In the AT hearts, which developed lower pressure than AC hearts, Ca²⁺-ATPase and phospholamban mRNAs and the expression of the encoded proteins resembled those of the C hearts. This may imply adjustment of Ca²⁺ uptake into its pool at a rate similar to that of the C hearts. Consequently, both the pressure and the rate of relaxation in AT hearts resembled these parameters in C hearts. Hence, the results obtained for the AT group further strengthen our hypothesis regarding the putative role played by the lower thyroxine level in AC cardiac performance. This is compatible with the finding that the transition from the V₁ to the V₃ myosin isoform in AC hearts was blunted when AC rats were kept euthyroid (12).

The preservation and even slight augmentation of pressure generation by the CP hearts, together with the depression in the rates of contraction and relaxation, further support our hypothesis that the lower plasma thyroxine level plays a role in the adaptation of the cardiac contractile machinery to chronic heat. The pressures measured for the CP hearts in this investigation, although compatible with our hypothesis and in agreement with the values obtained by Shroff et al. (29), are at odds with the decrease in pressure reported by Gay et al. (6) and Seppet et al. (28) in hearts of hypothyroid rats. However, the reduction in heart weight in the CP group, although associated with a decrease in wall thickness, appears to be in proportion to the overall decrease in heart size. Mean wall thicknesses in the C and CP groups were 3.7 and 1.98 mm while mean heart weights were 1.31 and 0.69 g, respectively. This is not compatible with "wall thinning" associated with a "dilated cardiomyopathy" picture. On the contrary, it is in agreement with our pressure values.

We assume that differences in the extent of hypothyroidism, leading to a multiplicity of effects of various intensities acting on the host of factors enhancing or interfering with the contractile machinery [e.g., increased L-type calcium channel density (18) and decreased Na⁺-K⁺ pump activity (26), both leading to augmented cytosolic Ca²⁺], could reconcile the discrepancies in our model versus various other experimental models.

The CP rats displayed downregulation of Ca²⁺-ATPase mRNA transcription and overexpression of phospholamban mRNA, together with depressed rates of contraction and relaxation. At the translational level, however, there was increased expression of both SERCA and PLB. Hence, the relative protein ratio remained unchanged in relation to that of the C hearts. This is in accord with similarities in the systolic pressures produced by the two heart populations; however, in our model a higher PLB/SERCA cannot be the explanation for the negative lusitropic effect in the CP group, as suggested for the AC hearts. It should be noted, however, that it is the phosphorylated PLB that is most directly related to relaxation parameters and this was not determined in the present series of experiments. We speculate that the unique relationship between the Ca²⁺-ATPase mRNA level and SERCA expression in the CP rats compared with that in the other experimental groups might stem from a longer half-life due to decreased degradation of this protein accompanying the hypothyroid state.

To conclude, the data of this investigation, together with those published previously, suggest that the low T₃ and T₄ levels accompanying heat acclimation play at least some causative role in the accompanying contractile changes. We also demonstrate in this investigation that the negative lusitropic effect characterizing the diastolic function of the AC heart coincides with an augmented PLB/SERCA, which implies decreased SERCA activity. Another important diastolic feature emerging on acclimation is enhanced compliance (13). These two features appear to be an intrinsic compensatory regulation, allowing greater diastolic filling and stroke volume, despite the slower relaxation.

Perspectives

In a broader sense, the data obtained strengthen sporadic findings that a lowered level of circulating thyroxine might be beneficial to heat endurance and emphasize the need for further studies along this line. Unfortunately, the mechanisms involved have not yet been thoroughly studied.