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Am J Physiol Heart Circ Physiol 288: H1088-H1096, 2005. First published October 21, 2004; doi:10.1152/ajpheart.00746.2004
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Modest actomyosin energy conservation increases myocardial postischemic function

Bradford C. Blunt,2 Yi Chen,2 James D. Potter,1 and Polly A. Hofmann2

1Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida; and 2Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 26 July 2004 ; accepted in final form 14 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have proposed that pharmacological preconditioning, leading to PKC-{epsilon} activation, in hearts improves postischemic functional recovery through a decrease in actomyosin ATPase activity and subsequent ATP conservation. The purpose of the present study was to determine whether moderate PKC-independent decreases in actomyosin ATPase are sufficient to improve myocardial postischemic function. Rats were given propylthiouracil (PTU) for 8 days to induce a 25% increase in {beta}-myosin heavy chain with a 28% reduction in actomyosin ATPase activity. Recovery of postischemic left ventricular developed pressure (LVDP) was significantly higher in PTU-treated rat hearts subjected to 30 min of global ischemia than in control hearts: 57.9 ± 6.2 vs. 32.6 ± 5.1% of preischemic values. In addition, PTU-treated hearts exhibited a delayed onset of rigor contracture during ischemia and a higher global ATP content after ischemia. In the second part of our study, we demonstrated a lower maximal actomyosin ATPase and a higher global ATP content after ischemia in human troponin T (TnT) transgenic mouse hearts. In mouse hearts with and without a point mutation at F110I of human TnT, recovery of postischemic LVDP was 55.4 ± 5.5 and 62.5 ± 14.5% compared with 20.0 ± 2.9% in nontransgenic mouse hearts after 35 min of global ischemia. These results are consistent with the hypothesis that moderate decreases in actomyosin ATPase activity result in net ATP conservation that is sufficient to improve postischemic contractile function.

hypothyroid; troponin T; adenine nucleotides; adenosinetriphosphatase; left ventricular developed pressure; preconditioning

PREISCHEMIC EXPOSURE TO ADENOSINE, opioid, or adrenergic receptor agonists confers a protective effect against myocardial ischemic injury. Pyle et al. (23) demonstrated a 30% increase in postischemic left ventricular developed pressure (LVDP) in isolated rat hearts treated preischemically with a {kappa}-opioid receptor agonist compared with control hearts. Cardioprotective effects are also seen with intermittent, short-duration ischemia. For example, an ischemic preconditioning protocol utilized by Murry et al. (17), four cycles of 5 min of ischemia followed by 5 min of reperfusion, resulted in an area of infarct that was 25% less than under control conditions after 40 min of global ischemia. Mechanisms that have been suggested to be involved in this phenomenon include opening of mitochondrial ATP-sensitive K+ (KATP) channels (2), decreased activity of the Na+/H+ exchanger (10), and conservation of ATP (23, 26).

Many laboratories have demonstrated a link between preconditioning and PKC-{epsilon} activation. PKC-{epsilon} phosphorylates a number of myocardial proteins, including the serine 43/45 sites on troponin I (18), which leads to a 20% decrease in Ca2+-activated actomyosin ATPase activity (23). Actomyosin ATPase accounts for ~70% of ventricular myocyte ATP use (6), and it was theorized that even a modest decrease in actomyosin ATPase activity would cause a significant net conservation of ATP (23). This, in turn, would provide energy to maintain critical ATP-dependent processes and result in continued Ca2+ homeostasis and cell viability.

Although there is support for the hypothesis that energy conservation may be cardioprotective (23, 26), there is some skepticism about the extent of its role and ability to contribute to in vivo cardioprotection. ATP buffering is strictly regulated in the heart through the creatine phosphate system and maintains ATP at constant levels with little variation. Thus the decrease in actomyosin ATPase by PKC-{epsilon} activation may be ineffective with respect to contributing to cardioprotection. In addition, the data from KATP channel studies are quite compelling, supporting their role as a mechanism of ischemic preconditioning (2, 7, 30). However, it seems likely that multiple mechanisms protect the heart from ischemic dysfunction.

In the present study, we decreased actomyosin ATPase in a PKC-independent manner to study the role, if any, of reduced actomyosin ATPase and ATP conservation in cardioprotection. Hoh et al. (11) showed that increases in {beta}-myosin heavy chain ({beta}-MHC) expression lead to decreases in actomyosin ATPase activity in hypophysectomized rats. MHC exists in two isoforms: {alpha}-MHC, which has a fast rate of ATPase activity, and {beta}-MHC, which has a slow rate of ATPase activity. Hoh et al. showed a decrease in {alpha}-MHC concomitant with an increased expression of {beta}-MHC proportional to the level of hypothyroidism in rats. Therefore, we induced moderate levels of hypothyroidism to produce a slight increase in {beta}-MHC expression and a corresponding decrease in actomyosin ATPase. A second model used in our studies consisted of a mouse expressing a human wild-type (WT) cardiac troponin T (HcTnT) transgene or one containing a point mutation at F110I of HcTnT. In vitro studies utilizing the mutant HcTnT-F110I protein reconstituted with rabbit actin and porcine tropomyosin/troponin C/troponin I showed a decreased actomyosin ATPase activity (31). Using these two very distinct models, we tested whether PKC-independent decreases in actomyosin ATPase improve postischemic function and the extent to which any improvement could be correlated to net ATP conservation.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Animal models. All procedures were carried out according to the guidelines set by the Animal Care and Use Committee of the University of Tennessee Health Science Center. Female Sprague-Dawley rats (150–250 g) were allowed food ad libitum and free access to water or water containing propylthiouracil (PTU, 0.8 mg/ml) for 8 days before experiments. This treatment causes a mild hypothyroidism that results in a moderate increase of myocardial {beta}-MHC (11). PTU-treated rats consumed less food and water on days 1 and 2, but intake returned to normal by day 3. Rats were chosen for the PTU study because of the well-documented effects of PTU on rat hearts (1, 11, 15, 19, 22, 24, 29).

Male and female nontransgenic (NTg) and HcTnT transgenic mice were generated as described previously (16). Cardiac-specific overexpression of the human WT or amino acid-substituted (F110I) TnT gene in the mouse allowed for replacement of the endogenous mouse cardiac TnT (cTnT) without increase in total TnT protein (12). Expression levels of HcTnT-WT and HcTnT-F110I were estimated to be ~45–50%. Quantitation of transgenic HcTnT was achieved by Western blot using anti-cTnT polyclonal antibody and anti-HcTnT monoclonal antibody. A linear curve was obtained by mixing an increasing percentage of human to mouse heart protein, and then the standard curve was used to determine the percentage of HcTnT expression in each transgenic line. The total protein was normalized by dividing the optical density of the band immunostained with the anti-HcTnT monoclonal antibody by the density of the band stained with the anti-cTnT polyclonal antibody. F110I designates a point mutation in the TnT gene associated with familial hypertrophic cardiomyopathy, which exhibits a lower maximal Ca2+-activated actomyosin ATPase activity in vitro (31). There was no evidence of cardiomyopathy in these mice at the time of experimentation. All data are from hearts of mice at 9–10 wk of age.

Langendorff-perfused heart preparation. Rats were anesthetized with isoflurane, and hearts were excised and cannulated in ice-cold modified Krebs-Henseleit buffer containing (in mM) 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 11 glucose, 25 NaHCO3, and 1.3 CaCl2, pH 7.4. Hearts were mounted on the Langendorff apparatus and perfused with oxygenated Krebs-Henseleit buffer at 37°C. After left atriotomy, a cellophane balloon attached to a pressure transducer (BLPR, World Precision Instruments, Sarasota, FL) was inserted into the left ventricle and inflated to an initial end-diastolic pressure (EDP) of 5–15 mmHg. Hearts were paced with electrodes at 300 beats/min, and pacing voltage was set at twice the threshold value. LVDP and EDP were measured continuously throughout the experiment.

Rat hearts were subjected to a 20-min baseline equilibration period followed by 30 min of global, normothermic ischemia and 60 min of reperfusion. Pre- and postischemic LVDP were averaged for the final 10 min of baseline equilibration and the final 10 min of reperfusion, respectively. Rat heart data were excluded if LVDP was not maintained between 80 and 140 mmHg during the preischemic equilibration period.

Experiments using mouse hearts were similar to those done using rat hearts with the following exceptions. Mice were anesthetized with pentobarbital sodium (65 mg/kg ip). An open-ended cannula with an external diameter of 1.27 mm was attached to the pressure transducer and inserted into the left ventricle for pressure measurements. A cannula of this external diameter allows for maximum production of developed pressure in mouse hearts (25). Mouse hearts were paced at 360 beats/min. Duration of the global, normothermic ischemia was 35 min. Mouse heart data were excluded if LVDP was not maintained between 40 and 100 mmHg during the preischemic equilibration period.

Myocardial adenine nucleotide and high-energy phosphate content. Langendorff-perfused hearts were assayed for adenine nucleotides before ischemia (rat and mouse), after 5 min of ischemia (rat only), or after 30 (rat) or 35 (mouse) min of ischemia followed by 10 min of reperfusion. Tissue was prepared according to a modification of the method described by Teerlink et al. (32). Briefly, ventricular tissue was frozen in liquid nitrogen, pulverized into a powder, and homogenized in 0.66 M HClO4 for 30 s. After centrifugation, the pellet was reconstituted and assayed for protein content. The supernatant was neutralized with 0.66 M KH2PO4, and the resulting potassium perchlorate precipitate was removed. Samples were frozen at –80°C for later analysis. Reverse-phase HPLC analysis, a modification of the method described by Teerlink et al., was carried out on a Waters Symmetry C18 4.6 x 150-mm 3.5-µm column by gradient elution with 0.2 M KH2PO4 at pH 5.0 and water-methanol-acetonitrile [50:25:25 (vol/vol/vol)]. Flow rate was held constant at 1 ml/min. Total run time for each injection was 16 min. Samples were detected at 210 nm for creatine and creatine phosphate and at 254 nm for ATP, ADP, and AMP. Metabolite content is expressed as nanomoles per milligram of protein.

MHC composition. After Langendorff perfusion, ventricles from rat hearts were removed and homogenized in standard relaxing solution containing (in mM) 100 KCl, 10 imidazole, 1 MgCl2, 2 EGTA, and 4 ATP. Homogenized samples were then diluted 1:1 with urea sample buffer, separated into aliquots, and frozen for later analysis. SDS-PAGE of MHC content was determined according to a modification of the method described by Reiser and Kline (27). Frozen samples were thawed for 30 min and heated for 4 min at 95°C. Samples were diluted 1:100 with urea sample buffer, loaded on a 7% acrylamide, large-format gel, and run at a constant voltage of 200 V for 26 h at 4–8°C. Gels were fixed overnight and silver stained (ProteoSilver stain kit, Sigma, St. Louis, MO) for determination of MHC band density using NIH Image software.

PKC-{epsilon}/PKC-{delta} Western blot analysis. Samples were collected and prepared as described above (see MHC composition). Samples were run on SDS 5% acrylamide stacking and 12% acrylamide resolving gels. Immunoblotting was modified from the protocol of Lester et al. (14). Briefly, gel proteins were transferred to polyvinylidene difluoride membranes, which were incubated for 2 h with monoclonal antibodies to PKC-{epsilon} (1:1,000) or PKC-{delta} (1:100; BD Biosciences, San Diego, CA). Densities of PKC-{epsilon} and PKC-{delta} immunoreactive bands were determined using NIH Image software. Protein levels were normalized to bands on a Coomassie-stained gel.

Creatine kinase activity. Hearts from rats were subjected to 10 min of Langendorff perfusion for removal of all residual blood. The ventricles were removed and frozen in liquid nitrogen. Frozen tissue was pulverized and homogenized at 4°C for 10 s, and 0.1% Triton X-100 was added to homogenates (33). Creatine kinase (CK) activity was measured photometrically on the basis of the method described by Oliver (20) and available as a kit (Equal Diagnostics, Exton, PA). Protein was determined by the biuret method. Enzymatic activity is expressed as micromoles per minute per milligram of protein.

Myofibrillar Ca2+-activated actomyosin ATPase activity. The low-Ca2+, pCa 9.0, ATPase assay buffer contained 25.96 mM KCl, 5.13 mM MgCl2, 3.16 mM Na ATP, 2 mM EGTA, 20 mM imidazole, and 4.86 µM CaCl2, pH 7.0. The maximum-Ca2+, pCa 4.0, ATPase assay buffer contained 23.48 mM KCl, 5 mM MgCl2, 3.22 mM NaATP, 2 mM EGTA, 20 mM imidazole, and 2.15 mM CaCl2, pH 7.0. All other buffers were appropriate mixtures of pCa 9.0 and pCa 4.0. Myofibrils from rat or mouse ventricles were added to ATPase assay buffers at 27°C and incubated for 15 min. The reaction was stopped by addition of 20% trichloroacetic acid. Inorganic phosphate (Pi) levels were measured photometrically on the basis of the method described by Daly and Ertingshausen (5) and available as a kit (Equal Diagnostics). Pi production was linear for time and sample volume at 27°C (data not shown). Protein concentration was determined according to the Lowry method. Enzymatic activity was expressed as nanomoles of Pi per milligram of protein per minute. Samples were excluded if Pi contamination was >25 nmol Pi·mg protein–1·min–1.

Statistical analysis. Values are means ± SE. Statistical significance was assumed for P < 0.05. Data were analyzed by Student's t-test, one- or two-way ANOVA, and Tukey's honestly significant difference or least-significant difference multiple comparison. CK activity was analyzed using a paired t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of hypothyroid model. Characteristics of the hypothyroid rats are shown in Table 1. Decreased food and water consumption was observed in the PTU-treated group on days 1 and 2, but intake returned to normal by day 3. Body weight was slightly but significantly lower in PTU-treated than in control rats after 8 days of treatment. PTU-treated hearts exhibited a 25% increase in {beta}-MHC content compared with control hearts after 8 days of treatment (Fig. 1). There were no statistically significant differences in baseline LVDP between PTU-treated and control hearts.


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  		Table 1. Characterization of control and hypothyroid rats

 


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  		Fig. 1. Representative SDS polyacrylamide gel comparing myocardial {alpha}- and {beta}-myosin heavy chain (MHC) content in rats treated for 8 days with propylthiouracil (PTU) with that in control (Con) rats.

 
Functional recovery of hypothyroid hearts. Time to onset of rigor contracture, defined as >5-mmHg rise in EDP after the start of ischemia, was 20.8 ± 0.9 and 13.6 ± 0.6 min for PTU-treated and control hearts, respectively (Fig. 2A). Postischemic functional recovery was dramatically higher in PTU-treated than in control hearts (Fig. 2B). PTU-treated hearts recovered 57.9 ± 6.2%, whereas control hearts recovered 32.6 ± 5.1%, of preischemic LVDP values. Concomitantly, the preischemic-to-postischemic increase in EDP was significantly lower for PTU-treated than for control hearts (Fig. 2C).



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  		Fig. 2. Time to onset of rigor contracture and recovery of postischemic function in hearts from PTU-treated and control rats. Time to onset of rigor contracture (A) is defined as a >5-mmHg rise in end-diastolic pressure (EDP) after the start of ischemia. B and C: myocardial postischemic recovery, calculated as a percentage of preischemic left ventricular developed pressure (LVDP), and change in postischemic EDP after 30 min of ischemia followed by 60 min of reperfusion. Values are means ± SE; n = 10–11. *P < 0.05 vs. control.

 
Adenine nucleotide content of hypothyroid hearts. Adenine nucleotide content of hearts from PTU-treated and control rats was not significantly different before ischemia and at 5 min of ischemia (Fig. 3, A and B). After 30 min of ischemia and 10 min of reperfusion, ATP and ADP content were higher and AMP content was lower in PTU-treated than in control hearts (Fig. 3C).



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  		Fig. 3. Adenine nucleotide content of hearts from PTU-treated and control rats before ischemia (A), after 5 min of ischemia (B), and after 30 min of ischemia followed by 10 min of reperfusion (C). Values are means ± SE; n = 4–8. *P < 0.05 vs. control.

 
Creatine balance and PKC levels in hypothyroid hearts. CK activity was significantly higher in PTU-treated than in control hearts: 4.6 ± 0.6 vs. 3.2 ± 0.4 IU/mg protein (Fig. 4A). In addition to an increase in CK activity, we observed alterations in creatine and creatine phosphate content in PTU-treated compared with control hearts. Creatine levels were significantly higher at each time point in PTU-treated hearts (Fig. 4, B–D). PTU-treated hearts also showed a trend toward a higher preischemic level of creatine phosphate, and postischemic levels of creatine phosphate were significantly higher than in control hearts (Fig. 4, B–D).



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  		Fig. 4. A: creatine kinase (CK) activity in hearts from PTU-treated and control rats before ischemia. B–D: creatine and creatine phosphate content of hearts from PTU-treated and control rats before ischemia (B), after 5 min of ischemia (C), and after 30 min of ischemia followed by 10 min of reperfusion (D). Values are means ± SE; n = 4–8. *P < 0.05 vs. control.

 
Levels of PKC-{epsilon} expression were similar in hearts treated with PTU for 8 days and in control hearts (Fig. 5). Cumulative PKC-{epsilon} immunoreactive density normalized to protein load was 0.41 ± 0.18 (n = 4) in hearts treated with PTU for 8 days and 0.37 ± 0.10 (n = 3) in control hearts compared with an approximately fivefold increase in hearts treated with PTU for 3 wk. Additionally, levels of PKC-{delta} expression were similar in hearts treated with PTU for 8 days and control hearts [0.72 ± 0.07 (n = 6) and 0.64 ± 0.06 (n = 6)], whereas a heart treated with PTU for 3 wk exhibited a nearly twofold increase in PKC-{delta} immunoreactivity.



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  		Fig. 5. Typical immunoblot of PKC-{epsilon} (A) and PKC-{delta} (B) levels in hearts from rats treated with PTU for 3 wk, rats treated with PTU for 8 days, and control rats. Normalization for protein levels was carried out by comparison with myosin light chain 2 (MLC 2) bands on Coomassie-stained SDS polyacrylamide gel, and data are expressed as density ratio of PKC Western blot to MLC 2 Coomassie-stained gel for each lane.

 
Ca2+-activated actomyosin ATPase activity in hypothyroid hearts. The pCa-ATPase relation was significantly different between PTU-treated and control myofibrils. Maximal Ca2+-activated actomyosin ATPase activity was significantly decreased by 28% in PTU-treated compared with control myofibrils (Fig. 6A). There were no differences in the slope and pCa50 values for relative Ca2+-activated actomyosin ATPase activity between PTU-treated and control hearts (Fig. 6B).



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  		Fig. 6. Cumulative absolute (A) and relative (B) Ca2+-activated actomyosin ATPase activity as a function of Ca2+ concentration in ventricular myofibrils from PTU-treated and control rats. For relative ATPase, values were normalized to pCa 4.0, maximum ATPase of that group. Slope and pCa50 values for relative Ca2+-activated actomyosin ATPase activity (B) were not significantly different. Values are means ± SE; n = 3–4. *P < 0.05 vs. control.

 
Functional recovery of TnT transgenic hearts. Characteristics of the transgenic mice are shown in Table 2. In a separate set of mice, the heart weight-to-body weight ratio was significantly different between HcTnT-WT and HcTnT-F110I mice. HcTnT transgene expression was estimated at 50% and 45% in HcTnT-WT and HcTnT-F110I hearts, respectively. Preischemic LVDP values for NTg, HcTnT-WT, and HcTnT-F110I hearts were not significantly different. Postischemic functional recovery was considerably higher in HcTnT-WT and HcTnT-F110I than in NTg hearts (Fig. 7A). Postischemic recovery was not significantly different between HcTnT-WT and HcTnT-F110I hearts. Preischemic-to-postischemic increases in EDP were not different between any of the groups (Fig. 7B).


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  		Table 2. Characterization of TnT transgenic mice

 


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  		Fig. 7. Recovery of postischemic function in hearts from nontransgenic mice, mice expressing a wild-type human cardiac troponin T (TnT) transgene (HcTnT-WT), and mice expressing a point mutation at F110I of HcTnT (HcTnT-F110I). Myocardial postischemic recovery is calculated as percentage of preischemic LVDP (A) and increase in postischemic EDP (B) after 35 min of ischemia followed by 60 min of reperfusion. Values are means ± SE; n = 4–6. *P < 0.05 vs. nontransgenic.

 
Adenine nucleotide content of TnT transgenic hearts. Preischemic adenine nucleotide content of hearts from NTg, HcTnT-WT, and HcTnT-F110I mice was not significantly different (Fig. 8A). After 35 min of ischemia and 10 min of reperfusion, HcTnT-F110I hearts exhibited a higher ATP content and a trend toward a lower AMP content than NTg hearts (Fig. 8B). There were no significant differences in postischemic adenine nucleotide content between HcTnT-WT and HcTnT-F110I hearts. There were no significant differences in creatine and creatine phosphate between NTg, HcTnT-WT, and HcTnT-F110I hearts (data not shown).



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  		Fig. 8. Adenine nucleotide content of hearts from nontransgenic, HcTnT-WT, and HcTnT-F110I mice before ischemia (A) and after 35 min of global ischemia followed by 10 min of reperfusion (B). No significant differences were observed in preischemic parameters. Values are means ± SE; n = 3–4. *P < 0.05 vs. nontransgenic.

 
Ca2+-activated actomyosin ATPase activity in TnT transgenic hearts. The pCa-ATPase relation was significantly different between NTg and HcTnT-WT and between NTg and HcTnT-F110I hearts. Maximal Ca2+-activated actomyosin ATPase activity was 26% lower in HcTnT-WT and 29% lower in HcTnT-F110I myofibrils than in NTg cardiac preparations (Fig. 9A). There were no significant differences between HcTnT-WT and HcTnT-F110I cardiac preparations. There were no differences in slope and pCa50 values for relative Ca2+-activated actomyosin ATPase activity between NTg, HcTnT-WT, and HcTnT-F110I hearts (Fig. 9B).



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  		Fig. 9. Cumulative absolute (A) and relative (B) Ca2+-activated actomyosin ATPase activity in ventricular myofibrils from nontransgenic (NTg), HcTnT-WT, and HcTnT-F110I mice. For relative ATPase, values were normalized to pCa 4.0, maximum ATPase of that group. Slope and pCa50 values for relative Ca2+-activated actomyosin ATPase activity (B) were not significantly different. Values are means ± SE; n = 3–4. *P < 0.05 vs. NTg.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
PTU-treated rat hearts exhibited an increase in {beta}-MHC, with a subsequent decrease in actomyosin ATPase activity and a higher global ATP content after normothermic ischemia. Furthermore, postischemic functional recovery dramatically increased in PTU-treated rat hearts. TnT transgenic mouse hearts also exhibited a lower maximal rate of actomyosin ATPase activity and a higher global ATP content after ischemia. Concomitantly, TnT transgenic mouse hearts demonstrated a considerable increase in postischemic functional recovery. The data from these two independent models suggest that a moderate decrease in ATP use through slowed actomyosin ATPase leads to a biologically significant net conservation of ATP, which improves postischemic functional recovery. These data are consistent with the hypothesis that {alpha}-adrenergic (3) and opioid receptor agonists (23), which also reduce actomyosin ATPase, are able to improve myocardial postischemic function through a mechanism of ATP conservation.

The PTU-dependent increase in {beta}-MHC, an ~25% increase over control, was expected, given the acute time frame of PTU administration (11). The observed increase in {beta}-MHC resulted in a 28% reduction in actomyosin ATPase activity. This decrease in actomyosin ATPase activity contributed to a higher global ATP and ADP content as well as a lower AMP content after ischemia than in control rat hearts. Furthermore, PTU-treated rat hearts exhibited an increased time to onset of rigor contracture (i.e., the point at which ATP concentration is essentially zero and actomyosin cross bridges freeze in the attached state) compared with control hearts. The observed increase in time until onset of rigor contracture suggests a more favorable energetic balance in PTU-treated hearts and increased availability of high-energy phosphates during early ischemia, allowing for maintenance of vital cellular functions. This is evident from the increase in recovery of postischemic LVDP and decrease in postischemic EDP in PTU-treated rat hearts. Thus a decreased use of ATP via the slower {beta}-MHC actomyosin ATPase contributes to a net conservation of ATP and improved recovery of postischemic function.

CK activity increased in PTU-treated rat hearts compared with control rat hearts. Previous studies demonstrated that CK activity is inversely related to thyroid hormone (28). Therefore, induction of hypothyroidism was expected to and did increase CK activity. Along with the observed increase in CK were alterations in creatine and creatine phosphate handling. PTU-treated rat hearts exhibited higher creatine content before, during, and after ischemia as well as higher creatine phosphate content during reperfusion than control rat hearts. The sarcolemmal Na+/creatine transporter has been shown to be downregulated in response to hyperthyroid conditions in the rat heart, resulting in decreased intracellular levels of creatine and creatine phosphate (24). Therefore, it is possible that its activity and/or protein levels may be increased under hypothyroid conditions, thus contributing to the observed increase in total intracellular creatine content in PTU-treated rat hearts compared with control. Because the creatine phosphate system is the major buffer for ATP in cardiac muscle, these changes may influence the buffering capacity for high-energy adenine nucleotides. However, creatine phosphate stores are equally depleted in <5 min of ischemia (Ref. 8 and present study), and changes in the creatine phosphate system are not sufficient to explain the observed preservation of ATP and subsequent delay of rigor contracture.

Abe et al. (1) observed an increase in postischemic ATP and functional recovery in long-term (>3 wk) PTU-treated rats. These rats would have had a 100% conversion from {alpha}- to {beta}-MHC. More recently, long-term PTU-treated rats were shown to recover ~50% more LVDP than control rats after 30 min of global ischemia followed by reperfusion (22). These hearts exhibited a 1.4-fold increase in PKC-{epsilon} but no change in PKC-{delta} (22). One difference between these previous studies (1, 22) and the present study is that the level of hypothyroidism was considerably increased with long-term PTU treatment. Regulation and/or expression of many myocardial proteins, including {beta}-MHC and PKC-{epsilon}, are altered by severe, prolonged perturbations in thyroid hormone levels. However, in contrast to the high levels of PKC-{epsilon} and PKC-{delta} in a heart treated with PTU for 3 wk, the moderate level of hypothyroidism induced by 8 days of PTU treatment did not result in increased levels of either PKC isoform compared with control in the present study.

In addition to changes in {beta}-MHC and PKC-{epsilon} expression, the fully hypothyroid state is associated with decreased activity and/or levels of Na+-K+-ATPase and sarcolemmal Ca2+-activated ATPase (29). A decrease in the activity or levels of ATP-dependent pumps would conserve ATP and help preserve ionic balance during ischemia, particularly between the arrest of contractile activity and the onset of rigor contracture. However, the ATP savings associated with decreased ATP-dependent pump function are quantitatively much smaller than those associated with decreased actomyosin ATPase activity, which accounts for 70% of ventricular ATP use (6).

Another consequence of severe hypothyroidism is a reduction in O2 consumption and mitochondrial respiration, resulting in decreased H2O2 production and, subsequently, reactive oxygen species (ROS) generation (15). The role of ROS in cardioprotection is somewhat controversial. A modest increase in ROS production during early ischemia has been shown to have beneficial effects (13). Conversely, decreased ROS production at the onset of reperfusion has also been shown to be beneficial (21). It is unknown whether ATP-dependent pump function or ROS generation is altered to any significant extent after only 8 days of PTU treatment.

Fully hypothyroid hearts exhibit decreased contractile function and alterations in Ca2+ handling (19, 29). If these properties were altered after 8 days of PTU administration, then preischemic LVDP should have been significantly decreased compared with control hearts. No differences in baseline contractile function between PTU-treated and control hearts were observed. This further underscores that PTU treatment for 8 days induces only a mild hypothyroid state.

The time points at which ATP conservation occurs may determine the extent to which the myocardium is protected. During early ischemia, the decreased rate of ATP consumed by {beta}-MHC results in substantial conservation of ATP and delays the onset of rigor contracture and subsequent damage. Upon reperfusion, conservation of ATP by the slower actomyosin ATPase provides the necessary energy to support vital ATP-dependent processes, which promote a faster return to homeostasis. This minimizes the damaging effects of Ca2+ overload and, subsequently, improves postischemic function.

Postischemic LVDP recovery increased dramatically in HcTnT-F110I compared with NTg mouse hearts. HcTnT-F110I mouse hearts also exhibited a higher ATP content as well as a trend toward a lower AMP content after ischemia than NTg mouse hearts. This postischemic adenine nucleotide profile was very similar to that seen in the PTU model. In addition, HcTnT-F110I mouse hearts exhibited a 29% lower maximal actomyosin ATPase activity than NTg mouse hearts. Tobacman et al. (34) observed a significant reduction in the binding affinity of the HcTnT-F110I protein for tropomyosin. This may account for the resultant decrease in ATPase activity. The observed lower actomyosin ATPase activity contributes to a net conservation of ATP, as evidenced by the postischemic adenine nucleotide profile.

HcTnT-WT mouse hearts also showed an increase in postischemic LVDP recovery as well as a 26% lower maximal actomyosin ATPase activity than NTg mouse hearts. However, in contrast to HcTnT-F110I mice, ATP content after 35 min of ischemia followed by 10 min of reperfusion was not significantly different between HcTnT-WT and NTg mouse hearts. The variability in the HcTnT-WT HPLC data may account for this discrepancy. In addition, it is quite possible that the adenine nucleotide profile is significantly different from that in NTg hearts at time points during ischemia and reperfusion that were not assessed. The HPLC-determined adenine nucleotide profiles were highly time dependent during reperfusion.

There were no significant differences when data from HcTnT-WT and HcTnT-F110I mice were compared. The observed differences between the transgenic and NTg mice may be due to the properties associated with the incorporation of the human vs. the mouse TnT. HcTnT-WT and HcTnT-F110I mice exhibited a lower actomyosin ATPase activity and improved postischemic functional recovery. Furthermore, HcTnT-F110I mouse hearts had a higher ATP content after ischemia. Together, these data are consistent with the idea that the lower actomyosin ATPase activity of HcTnT transgenic mouse hearts leads to a conservation of ATP, resulting in improved postischemic function.

There were no significant differences in creatine and creatine phosphate content between NTg, HcTnT-WT, and HcTnT-F110I mouse hearts. Therefore, although the changes in the creatine phosphate system could have a potential role in the net conservation of ATP in the PTU model, it has no major role in the transgenic model. Furthermore, there were no significant differences in the slope and pCa50 of the relative actomyosin ATPase activity-pCa curves between any of the groups in either model. This indicates that a change in the Ca2+ sensitivity of tension is not a part of the mechanism accounting for the improved postischemic function.

There has been some controversy over the properties afforded to the myocardium by the F110I mutation of TnT. Szczesna et al. (31), using porcine skinned myofilament preparations reconstituted with various TnT mutations, showed that HcTnT-F110I increased the Ca2+ sensitivity of force development and decreased the maximal rate of ATPase by ~27%. In contrast to these findings, Yanaga et al. (35) observed no change in Ca2+ sensitivity of force development and an increase in the maximal rate of ATPase in rabbit cardiac myofibrils reconstituted with HcTnT-F110I. The reason for this apparent discrepancy is unclear. Using transgenic TnT mouse myofibrils, we found a decrease in the maximal rate of actomyosin ATPase that is comparable to that observed by Szczesna et al. However, as stated above, WT and F110I human cardiac TnT myofibrils exhibit the same decrease in actomyosin ATPase observed in our studies.

Numerous hypotheses have been proposed to explain the cardioprotective effect of pharmacological and ischemic preconditioning. Of these hypotheses, a change in KATP channel activation is one of the most prominent (2). It is known that KATP channel agonists mimic the effects of preconditioning, whereas KATP channel antagonists block these effects. Although there has been some controversy with regard to whether the cardioprotective effects of the KATP channel are of sarcolemmal (30) or mitochondrial (7, 21) origin, evidence supports the activation of KATP channels as effectors of preconditioning. The proposal that opening of mitochondrial KATP channels promotes ATP conservation through a more efficient energetic state is supported by observations that certain KATP channel agonists lead to significant ATP conservation (9). Although these data are quite compelling, they do not negate the hypothesis that ATP conservation also contributes to the effects on preconditioning. Both models in the present study exhibited considerable ATP conservation through KATP channel-independent pathways and resulted in significant cardioprotection. The increased concentration of ATP would actually be expected to inhibit KATP channels in the PTU and TnT transgenic hearts. It seems likely that KATP channel activation and ATP conservation are not mutually exclusive but both contribute to the overall cardioprotective phenomenon under physiological conditions.

Other mechanisms have been proposed to explain cardioprotection by ischemic preconditioning. These include decreased activity of the Na+/H+ exchanger (10), production of ROS and nitric oxide during early ischemia (13), decreased ROS production during reperfusion (21), activation of heat shock proteins (4), and decreased ATP utilization and/or net ATP conservation (23, 26). Although our data suggest that energy conservation plays a significant role in cardioprotection, it does not rule out these other possibilities. In fact, because of the complexity of the signaling cascades within the ischemic myocyte, it is quite possible that multiple pathways are working in conjunction with one another or may act as redundant systems to protect the myocardium from ischemic damage.

The common denominators of the two animal models presented in this study are a decrease in actomyosin ATPase activity, a more favorable postischemic energetic state, and improved postischemic LVDP recovery. This suggests that, in vivo, moderate ATP conservation through a decrease in actomyosin ATPase can improve myocardial postischemic function.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Training Fellowship HL-07641-13 (to B. C. Blunt) and Grants HL-67415 and HL-42325 (to J. D. Potter) and HL-48839 (to P. A. Hofmann).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Hofmann, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (E-mail: phofmann{at}physio1.utmem.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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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