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Am J Physiol Regul Integr Comp Physiol 292: R844-R851, 2007. First published October 12, 2006; doi:10.1152/ajpregu.00365.2006
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Sex Differences in Renal and Cardiovascular Function: Physiology and Pathophysiology

Hypersensitivity of myofilament response to Ca2+ in association with maladaptation of estrogen-deficient heart under diabetes complication

Ariyaporn Thawornkaiwong, Jantarima Pantharanontaga, and Jonggonnee Wattanapermpool

Faculty of Science, Department of Physiology, Mahidol University, Bangkok, Thailand

Submitted 30 May 2006 ; accepted in final form 5 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The amelioration of cardioprotective effect of estrogen in diabetes suggests potential interactive action of estrogen and insulin on myofilament activation. We compared Ca2+-dependent Mg2+-ATPase activity of isolated myofibrillar preparations from hearts of sham and 10-wk ovariectomized rats with or without simultaneous 8 wk-induction of diabetes and from diabetic-ovariectomized rats with estrogen and/or insulin supplementation. Similar magnitude of suppressed maximum myofibrillar ATPase activity was demonstrated in ovariectomized, diabetic, and diabetic-ovariectomized rat hearts. Such suppressed activity and the relative suppression in {alpha}-myosin heavy chain level in ovariectomy combined with diabetes could be completely restored by estrogen and insulin supplementation. Conversely, the myofilament Ca2+ hypersensitivity detected only in the ovariectomized but not diabetic group was also observed in diabetic-ovariectomized rats, which was restored upon estrogen supplementation. Binding kinetics of beta1-adrenergic receptors and immunoblots of beta1-adrenoceptors as well as heat shock 72 (HSP72) were analyzed to determine the association of changes in receptors and HSP72 to that of the myofilament response to Ca2+. The amount of beta1-adrenoceptors significantly increased concomitant with Ca2+ hypersensitivity of the myofilament, without differences in the receptor binding affinity among the groups. In contrast, changes in HSP72 paralleled that of maximum myofibrillar ATPase activity. These results indicate that hypersensitivity of cardiac myofilament to Ca2+ is specifically induced in ovariectomized rats even under diabetes complication and that alterations in the expression of beta1-adrenoceptors may, in part, play a mechanistic role underlying the cardioprotective effects of estrogen that act together with Ca2+ hypersensitivity of the myofilament in determining the gender difference in cardiac activation.

heart; insulin; beta1-adrenergic receptor; heat shock protein 72

THE WELL-RECOGNIZED GENDER difference in the incidence of cardiovascular disease has led to a number of studies on the influence of female sex hormones on cardiac contractile activation. Suppressed maximum myofibrillar ATPase activity, myofibrillar Ca2+ hypersensitivity, and a significant shift in myosin heavy chain (MHC) toward the beta-MHC isoform have been demonstrated in ovariectomized (OVX) rat hearts (40, 41). Upregulation of beta1-adrenoceptors, which may partly underlie changes in the myofilament Ca2+ activation, was also detected in OVX heart (37). The similarity of Ca2+ hypersensitivity detected in OVX hearts (40, 41) to that in cardiomyopathic hearts (20, 42, 43) previously reported supports the beneficial role of estrogen (E2) in the myocardial activation.

Surprisingly, the cardioprotective effect of estrogen on myocardial function seems to be overcome by diabetes (18, 22, 36). The morbidity and mortality of cardiovascular diseases in diabetic patients appear to be increased in females compared with age-matched males. These gender differences in the incidence of heart disease suggest that deprivation of estrogen and insulin induces interactive effects on the cardiac myofilament response to Ca2+. This notion is indirectly supported by the observation of an additive effect of estrogen deficiency and diabetes on bone mineral density in diabetic-ovariectomized rats (14). Moreover, deficiency of estrogen increases the severity of renal disease in a diabetic rat model in which estrogen replacement is renoprotective by improving renal function and pathology associated with diabetes (29). Despite these reports on the combined effects of estrogen and insulin on various organs, their interactive effects on the cardiac myofilament response to Ca2+ and the underlying mechanism remain unknown.

Alterations in the regulatory effect of beta1-adrenergic stimulation and the protective effect of heat shock proteins (HSP) appear to play important roles in the effects of estrogen and insulin on the cardiac myofilament response to Ca2+. Stimulation of beta1-adrenergic receptors plays a physiologically significant role in enhancing cardiac contractility through modification of Ca2+ flow during the process of excitation contraction coupling and on myofibrillar sensitivity (4). However, chronic or overstimulation of the adrenergic system of the heart causes harmful effects on contractile function (7, 11, 24, 28, 33). We have previously reported that, after 10 wk of ovariectomy, there is an upregulation of beta1-adrenergic receptors in cardiac plasma membrane vesicles compared with sham control, which is completely restored by E2 supplementation (37) or by exercise training (8). Despite controversial data in the expression of beta1-adrenoceptors, a sustained and elevated norepinephrine spillover resulting in chronic stimulation of the receptors has been demonstrated in diabetic hearts (15). There is also evidence of sex hormone-related loss of cardiac protection through reduced expression of HSP72 in OVX hearts (8, 39). This loss of cardioprotective effect was reversed in OVX rats subjected to E2 supplementation (39) or exercise training (8). Similarly, downregulation of HSP72 has been documented in diabetic hearts in which the impaired HSP protection is also offset by endurance exercise (1). Whether deficiency of estrogen and insulin interactively induces a synergistic effect through increased beta1-adrenergic stimulation and/or loss of protective effect via reduced HSP72 expression on the cardiac contractile response to Ca2+ remains to be elucidated.

The present study was designed to evaluate the influence of diabetes on changes in the response to Ca2+ of OVX cardiac myofilaments. We compared the –log free Ca2+ concentration (pCa)-myofilament ATPase relationship of isolated myofibrillar preparations from sham, ovariectomized, diabetic, diabetic-ovariectomized, and diabetic-ovariectomized rat hearts supplemented with estrogen, insulin, or estrogen plus insulin. We also compared the density and binding affinity of cardiac beta1-adrenoceptors in sarcolemmal preparations from these hearts to probe for changes in the effects induced by hormone deficiency. In addition, comparisons of HSP72 content among these hearts were analyzed to probe for changes in this protective factor. Our results showed neither an additive nor synergistic effect of estrogen and insulin on the cardiac contractile response to Ca2+, indicating a similar final common pathway of the hormone action on cardiac contractile activation. Our results also confirm that hypersensitivity of myofilament to Ca2+ is specifically induced in ovarian sex hormone-deprived heart even under diabetes complication and that alterations in expression of beta1-adrenergic receptors account in part for the underlying cardioprotective effects of estrogen that act together with the hypersensitivity of the myofilaments in determining the gender difference on cardiac activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Female Sprague-Dawley rats weighing between 180 and 200 g (8–9 wk old) were randomly divided into seven experimental groups, including sham (SHAM), OVX, diabetic (DM), diabetic-ovariectomized (DM-OVX), and diabetic-ovariectomized supplemented with estrogen (DM-OVX + E2), insulin (DM-OVX + INS), or estrogen plus insulin (DM-OVX + E2 + INS) groups. Rats were first sham-operated or ovariectomized as previously described (8). After surgery (2 days), sham rats were subcutaneously injected with 0.1 ml of corn oil, whereas OVX rats were randomly divided into two groups and subcutaneously injected with either 0.1 ml of corn oil with or without 5 µg of 17beta-estradiol three times per week for 10 wk. After surgery (2 wk), both sham and OVX rats were further randomly divided into nondiabetic and diabetic groups. Diabetes in rats was induced by intraperitoneal injection of freshly prepared streptozotocin (65 mg/kg body wt), whereas nondiabetic groups were injected with citrate buffer. After diabetic induction (3 days), insulin-supplemented rats were subcutaneously injected with 5 units of human insulin on a daily basis for the whole experimental period. We verified diabetic status by determining urinary glucose using a glucose strip on the day after induction and on the day before the rats were killed. Sufficiency of ovariectomy was verified by a decrease in uterine weight. The animal protocol was approved by the Experimental Animal Committee, Faculty of Science, Mahidol University, in accordance with the guidelines of National Laboratory Animal Centre, Thailand.

Cardiac myofibrillar actomyosin Mg2+-ATPase activity. Cardiac myofibrils were prepared from the left ventricles as described by Pagani and Solaro (32). Ca2+-dependent Mg2+-ATPase activity of isolated myofibrils was assayed by determination of inorganic phosphate released in a 10-min linear reaction at 30°C in 2 mM Mg2+, 60 mM imidazole, 5 mM MgATP2–, pH 7.0, ionic strength of 120 mM, and various concentrations of Ca2+ ranging from pCa 7.5 to 4.875. Total concentrations of CaCl2, EGTA, KCl, MgCl2, and ATP were calculated using a computer program generated from the dissociation constants given by Fabiato (13). The concentration of inorganic phosphate was measured by the method of Carter and Karl (10).

Cardiac membrane preparation. Cardiac membrane was prepared from the left ventricle by the method of Baker and Potter (2) with modifications. In brief, the left ventricle was homogenized in ice-cold 10 mM Tris·HCl, pH 8.0, and then incubated in 1 M KCl to dissolve the myofilament proteins. Subsequently, the homogenate was filtered through several layers of cheesecloth, and the filtrate was then centrifuged at 43,900 g, 4°C for 20 min. The pellet was resuspended in the Tris buffer, homogenized, and sedimented. The pellet was dispersed in ice-cold 50 mM HEPES buffer, pH 8.0, in a Teflon glass homogenizer and was immediately used for receptor binding assay after determining the protein content by the Bradford protein assay kit (Bio-Rad).

beta1-Adrenergic receptor binding assay. Binding assay for beta1-adrenergic receptor was performed under equilibrium conditions in various concentrations of [3H]dihydroalprenolol (sp act 92 Ci/mmol; Amersham Pharmacia Biotech) as previously described (37). Nonspecific binding was analyzed in a parallel set of experiment with the addition of (–)-alprenolol, a specific antagonist of beta1-adrenergic receptor. The saturation binding was determined from the relationship between specific binding and free ligand using nonlinear least-square regression analysis. Binding parameters, including the density and dissociation constant of the receptors, were determined from a linear transformation of data to the Scatchard plot of bound/free to bound form.

General methods and statistics. The amounts of beta1-adrenergic receptor and HSP72 were determined by Western blot analysis of left ventricular homogenates using polyclonal antibody against beta1-adrenergic receptor (Affinity Bioreagents, Golden, CO) and HSP72 (Stressgen, Victoria, British Columbia, Canada) and horseradish peroxidase-labeled secondary antibody, with visualization by ECL (Amersham Pharmacia). MHC isoforms of left ventricular trabeculae were separated electrophoretically as previously described (30). Bands from Western blots were quantified using a GS800 densitometer (Bio-Rad). Data are presented as means ± SE. All curve fittings were performed using GraphPad Inplot (ISI Software, San Diego, CA). The significance of differences among groups was analyzed by one-way ANOVA followed by the Student-Newman-Keuls test for multiple comparisons. P < 0.05 was set for the significant difference among the groups.

Materials. Chemicals were purchased from Sigma Chemical (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA), human insulin was from Eli Lilly (Indianapolis, IN), and glucose strips were from Roche (Indianapolis, IN).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Table 1 summarizes body, heart, and uterine weights of all seven experimental groups, namely, sham, OVX, DM, DM-OVX, and DM-OVX supplemented with E2, INS, or E2 + INS. As expected, uterine weights were significantly decreased in OVX and DM-OVX groups compared with sham controls and increased upon E2 supplementation. Uterine weights of DM rats were also significantly lower than shams but in a smaller magnitude compared with ovarian sex hormone-deficient groups. Although significant increases in both heart and body weight were demonstrated in OVX rats, significant decreases in both heart and body weight were detected in DM rats. A decrease in body weight was still observed in the DM-OVX rats without insulin supplementation. Similarly, hypertrophy of the heart represented by an increased heart weight-to-body weight ratio was demonstrated in DM and DM-OVX groups without insulin supplementation.


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  		Table 1. Body weight, heart weight, uterine weight, and %heart weight/body weight

 
pCa-myofilament ATPase relationships were compared with evaluate the interactive effect of E2 and INS deprivation on the cardiac myofilament response to Ca2+. As shown in Fig. 1 and summarized in Table 2, maximum myofibrillar ATPase activity of OVX and DM groups was significantly depressed to the same degree (22.7 and 32.9%, respectively) compared with shams. Maximum myofibrillar ATPase activity was also depressed in DM-OVX rats to a similar degree (29.43%), indicating a lack of additive effect when compared with OVX or DM rats, and ATPase activity was completely restored upon supplementation with both E2 and INS (Fig. 2). On the other hand, the leftward shift in the pCa-myofilament ATPase activity relationship, representing an increase in myofilament sensitivity to Ca2+ (reported as pCa50), was only detected in OVX but not in DM rats when compared with sham controls (Fig. 3 and Table 2). The myofilament Ca2+ hypersensitivity detected in sex hormone-deficient hearts was also observed in DM-OVX rat hearts (Fig. 3) and reversed upon E2 or E2 + INS supplementation (Fig. 4 and Table 2). In all cases, there are no significant differences in the Hill coefficient of the pCa-myofilament ATPase relationship among all groups of animals (Table 2). Analysis of MHC isoforms demonstrated significant reductions in the relative amount of {alpha}-MHC in both OVX (~28%) and DM (~65%) rat hearts from shams, with a more pronounced suppression in the DM group (Fig. 5A). The same pronounced degree of {alpha}-MHC suppression detected in DM rats was also observed in the DM-OVX group (Fig. 5A), which was completely restored only by E2 and INS supplementation (Fig. 5B). These results indicate that estrogen and insulin affect the cardiac contractile activation partly through a common final pathway of myosin isoform expression, whereas deficiency of E2 induces an adaptive response of the myofilament to become more sensitive to Ca2+ even under diabetes complication.


Figure 1
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  		Fig. 1. A: –log free Ca2+ concentration (pCa)-myofibrillar ATPase activity relation. B: comparison of the maximum Ca2+-dependent actomyosin Mg2+-ATPase activities in cardiac myofibrillar preparations from sham (SHAM), ovariectomized (OVX), diabetic (DM), and diabetic-ovariectomized (DM-OVX) rats. Data are means ± SE from 7–8 preparations. *P < 0.05, significant difference from SHAM group using Student-Newman-Keuls test after ANOVA.

 

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  		Table 2. Maximum ATPase activity, pCa50, and Hill coefficient

 

Figure 2
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  		Fig. 2. A: pCa-myofibrillar ATPase activity relation. B: comparison of the maximum Ca2+-dependent actomyosin Mg2+-ATPase activities in cardiac myofibrillar preparations from DM-OVX rats with estrogen (E2) and/or insulin (INS) supplementation. Data are means ± SE from 7–8 preparations. #P < 0.05, significant difference from DM-OVX group using Student-Newman-Keuls test after ANOVA.

 

Figure 3
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  		Fig. 3. A: pCa-%maximum ATPase activity relation. B: comparison of –log of the Ca2+ concentration producing half-maximal activation (pCa50) from SHAM, OVX, DM, and DM-OVX rat hearts. Data are means ± SE from 7–8 preparations. *P < 0.05, significant difference from SHAM group using Student-Newman-Keuls test after ANOVA.

 

Figure 4
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  		Fig. 4. A: pCa-%maximum ATPase activity relation. B: comparison of pCa50 from DM-OVX rats with estrogen and/or insulin supplementation. Data are means ± SE from 7–8 preparations. #P < 0.05, significant difference from DM-OVX group using Student-Newman-Keuls test after ANOVA.

 

Figure 5
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  		Fig. 5. Myosin heavy chain (MHC) region of SDS gels and comparison of the relative amount of {alpha}-MHC (as percentage of total MHC) of left ventricular trabeculae from SHAM, OVX, DM, and DM-OVX rats (A) and from DM-OVX hearts with estrogen and/or insulin supplementation (B). Data are means ± SE from 8 hearts. P < 0.05, significant difference from SHAM (*), OVX ({ddagger}), DM-OVX (#), and DM-OVX with insulin supplementation ({dagger}) groups, respectively, using Student-Newman-Keuls test after ANOVA.

 
To determine whether alterations in the beta1-adrenergic receptor in these hearts were associated with changes in the myofilament response to Ca2+, we measured the density, binding affinity, and protein content of beta1-adrenergic receptor in these hearts using a sarcolemmal preparation and left ventricular homogenate. Similar to the increased Ca2+ sensitivity of the myofilament, a significant increase in the beta1-adrenoceptor density was observed only in OVX (~23%) but not in DM rats compared with shams (Fig. 6A). Moreover, a similar magnitude of enhancement in beta1-adrenoceptor density was also observed in the DM-OVX group (~17%), which was completely restored upon E2 supplementation (Fig. 6B). In agreement with data of receptor density, results from Western blot analysis using a specific anti-beta1-adrenergic receptor antibody demonstrated a significant increase in beta1-adrenergic receptor content in OVX (~48%) and DM-OVX (~49%) groups, and the receptor upregulation in DM-OVX rat hearts could be completely reversed by E2 supplementation (Fig. 7). In all groups, there were no differences in the binding affinity of beta1-adrenergic receptor among the groups, as summarized in Fig. 8. Thus alterations in beta1-adrenoceptor expression underlie the protective role of estrogen in the cardiac contractile response to Ca2+.


Figure 6
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  		Fig. 6. A: comparison of the density (Bmax) of cardiac beta1-adrenergic receptor in left ventricular membrane preparations from SHAM, OVX, DM, and DM-OVX rats. B: comparison of Bmax of cardiac beta1-adrenergic receptor in left ventricular membrane preparations from DM-OVX hearts with estrogen and/or insulin supplementation. Data are means ± SE of 8–10 hearts. P < 0.05, significant difference from SHAM (*) and DM-OVX (#) groups, respectively, using Student-Newman-Keuls test after ANOVA.

 

Figure 7
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  		Fig. 7. A: immunoblot analysis of beta1-adrenergic receptor (beta1-AR) proteins and comparison of the relative band intensity of left ventricular homogenates from SHAM, OVX, DM, and DM-OVX rats. B: immunoblot analysis of beta1-AR proteins and comparison of the relative band intensity of left ventricular homogenates from DM-OVX rats with estrogen and/or insulin supplementation. Data are means ± SE of 8 hearts. P < 0.05, represents significant difference from SHAM (*) and DM-OVX (#) groups, respectively, using Student-Newman-Keuls test after ANOVA.

 

Figure 8
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  		Fig. 8. A: comparison of the dissociation constant (Kd) of beta1-adrenergic receptor of left ventricular membrane preparations from SHAM, OVX, DM, and DM-OVX rats. B: comparison of the Kd of beta1-adrenergic receptor of left ventricular membrane preparations from DM-OVX rats with estrogen and/or insulin supplementation. Data are means ± SE of 8–10 hearts.

 
To further investigate whether changes in the myofilament response to Ca2+ were associated with loss in the cardioprotective effect through expression of HSP72, we determined the amount of this factor using immunoblot analysis. As shown in Fig. 9A, the same magnitude of decrease in HSP72 content was demonstrated in OVX (~27%) and DM (~25%) hearts compared with shams. Similarly, the expression of HSP72 in DM-OVX rats was suppressed to a comparable degree (~22%) compared with shams and increased upon supplementation with both E2 and INS (Fig. 9B). These results demonstrated that loss in cardioprotective effect through decreased HSP72 expression in sex hormone- or insulin-deficit heart parallels the suppression of maximum myofibrillar ATPase activity but not the hormone-associated hypersensitivity of myofilament to Ca2+.


Figure 9
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  		Fig. 9. A: immunoblot analysis of heat shock protein 72 (HSP72) and calsequestrin (CQ) and comparison of the band intensity expressed as a ratio of HSP72 to CQ of left ventricular homogenates from SHAM, OVX, DM, and DM-OVX rats. B: immunoblot analysis of HSP72 and CQ and comparison of the band intensity expressed as a ratio of HSP72 to CQ of left ventricular homogenates from DM-OVX rats with estrogen and/or insulin supplementation. Data are means ± SE of 8 hearts. P < 0.05, significant difference from SHAM (*) and DM-OVX (#) groups, respectively, using Student-Newman-Keuls test after ANOVA.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study is the first to report of a dominant effect of ovariectomy in inducing an enhanced Ca2+ sensitivity of cardiac myofilament under diabetes complication, emphasizing the significance of enhanced myofilament Ca2+ sensitivity in the pathophysiology of the heart in ovariectomy. We also observed the mechanistic adaptation underlying the cardioprotective effects of estrogen that involves elevated expression of beta1-adrenergic receptor and loss of protective factor, HSP72. Moreover, estrogen and insulin physiologically regulate and protect cardiac contractile function by an interactive action of the hormones on the maximum myofibrillar ATPase activity but not the myofilament Ca2+ sensitivity.

In ovariectomy, the close relation between enhanced myofilament sensitivity to Ca2+ and increased beta1-adrenoceptor expression in the heart, with or without complication of diabetes, confirms the adaptation of the contractile response of the heart in a pathological direction. Previous demonstrations that in ovariectomy there is a decrease in intracellular cardiac Ca2+ concentration (34) and sarcoplasmic reticulum Ca2+ uptake activity (9) suggest that both myofilament Ca2+ hypersensitivity and upregulation of beta1-adrenoceptors are likely maladaptive responses induced after sex hormone depletion. The increased adrenergic drive either through upregulation of beta1-adrenoceptors or increase in signaling process is known to be toxic to the heart (7, 11, 24, 28, 33). In a transgenic mouse model, overexpression of human beta1-adrenergic receptors in the heart produces a short-lived improvement of cardiac function but ultimately leads a cardiomyopathic phenotype characterized by dilation and depressed contractile functions (5, 12). This harmful compensatory mechanism of the heart induced by chronic adrenergic stimulation has provided the fundamental basis for the use of anti-adrenergic agents in treatment of chronic heart failure (6, 21, 27, 31). Although the sequential induction between changes in the myofilament response to Ca2+ and in beta1-adrenoceptors in ovariectomy remains unclear, parallel changes in these factors even with diabetes complication provide evidence for a high potential of cardiomyopathy induction in sex hormone-deficient hearts. Moreover, although interactive effects of E2 and insulin on the ovariectomy-associated increase in the myofilament response to Ca2+ are absent, E2 demonstrates a cardioprotective effect over insulin in preventing Ca2+ hypersensitivity of myofilaments. This absence of hormone interaction confirms that Ca2+ hypersensitivity of myofilaments is a specific maladaptive response of the heart induced by E2 deficiency. Physiological suppression of beta1-adrenoceptor expression and stimulation may in part account for the cardioprotective effect of E2 on the cardiac contractile response to Ca2+.

It is not clear how the increase in myofilament response to Ca2+ seen in our study leads to cardiac contractile dysfunction in ovariectomy. Enhanced Ca2+ sensitivity of the myofilament is a common feature in most cardiomyopathy patients (16) and heart failure models (20, 42, 43). Increased myofilament response to Ca2+ is the cellular mechanism proposed for alterations in the Starling force of the heart (35) and could provide a therapeutic approach in the search for Ca2+-sensitizing agents for the heart (23). Elevated Ca2+ regulation of cardiac muscle activation has been shown to be the primary mechanism contributing to pathogenesis of troponin T-linked familial hypertrophic cardiomyopathy (19). Increased affinity of Ca2+ bound to myofilament occurring with mutant cardiac troponin I could also cause a threat for arrhythmic activity associated with cardiomyopathy (26). Moreover, a chronic increase in the cardiac myofilament response to Ca2+ could cause hypertrophic induction in association with mutations in sarcomeric proteins (16). Besides the reported shift in cardiac MHC isoforms in ovariectomy (8, 41) that is more likely to underlie the suppressed maximum myofibrillar ATPase activity, evidence for changes in other sarcomeric proteins that subsequently alter the myofilament response to Ca2+ awaits future studies.

Differential effects of E2 and insulin interaction on cardiac contractile function despite the presence of both receptors in the myocardium (17, 38) suggest that different mechanisms exist for the hormones on the cardiac contractile response to Ca2+. In contrast to other organs, the reversal of maximum cardiac myofibrillar ATPase activity and {alpha}-MHC expression in ovariectomy combined with diabetes only results when both E2 and insulin treatment are given, reflecting an interaction of the hormones in activating myofilament function. On the other hand, the absence of interaction of the hormones on the ovariectomy-associated increase in cardiac myofilament Ca2+ sensitivity confirms that Ca2+ hypersensitivity of myofilament is a specific maladaptive response of the heart induced by sex hormone deficiency. How the hormones act on cardiac contractile function is not known at present.

Inasmuch as the stability and quality control of protein folding after translation in cardiomyocytes are accounted for by the action of a biological chaperone, HSP72 (3), parallel changes in HSP72 level and maximum myofibrillar ATPase activity (Figs. 1, 2, and 9) provide a potential common target for E2- and insulin-controlling process, namely via HSP72 function. There are reports showing that both E2 and insulin control HSP72 expression via phosphorylation of the same transcription heat shock factor-1 (1, 25, 44).

Although homeostatic balance of beta1-adrenergic receptor and protective factor HSP72 is physiologically regulated by E2, it is likely that only protective factors are regulated by insulin. Our data confirm the physiologically cardioprotective function of E2 on the contractile response to Ca2+ even under diabetes complication. These results provide further support for the beneficial use of E2 and beta1-blocker in preventing maladaptation of the heart to estrogen deficiency, thereby lowering the incidence of heart failure in postmenopausal women.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported partly by a Mahidol University Grant (to J. Wattanapermpool) and the Thailand Research Fund (to J. Wattanapermpool). A. Thawornkaiwong received support from the PhD/MD Program, Mahidol University.


   ACKNOWLEDGMENTS
 
We thank Dr. Prapon Wilairat and Dr. Nateetip Krishnamra for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Wattanapermpool, Dept. of Physiology, Faculty of Science, Mahidol Univ., Rama 6 Road, Bangkok 10400, Thailand (e-mail: tejwt{at}mahidol.ac.th)

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
 

  1. Atalay M, Oksala NKJ, Laaksonen DE, Khanna S, Nakao C, Lappalainen J, Roy S, Hänninen O, Sen CK. Exercise training modulates heat shock protein response in diabetic rats. J Appl Physiol 97: 605–611, 2004.[Abstract/Free Full Text]
  2. Baker SP, Potter LT. Purification and partial characterization of cardiac plasma membranes rich in beta-adrenoreceptors. Membr Biochem 3: 185–205, 1980.[Web of Science][Medline]
  3. Benjamin IJ, McMillan DR. Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res 83: 117–132, 1998.[Abstract/Free Full Text]
  4. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198–205, 2002.[CrossRef][Medline]
  5. Bisognano JD, Weinberger HD, Bohlmeyer TJ, Pende A, Raynolds MV, Sastravaha A, Roden R, Asano K, Blaxall BC, Wu SC, Communal C, Singh K, Colucci W, Bristow MR, Port DJ. Myocardial-directed overexpression of the human beta1-adrenergic receptor in transgenic mice. J Mol Cell Cardiol 32: 817–830, 2000.[CrossRef][Web of Science][Medline]
  6. Bristow MR. beta-Adrenergic receptor blockade in chronic heart failure. Circulation 101: 558–569, 2000.
  7. Bristow MR, Minobe W, Rasmussen R, Larrabee P, Skerl L, Klein JW, Anderson FL, Murray J, Mestroni L, Karwande SV, Fowler M, Ginsburg R. beta-Adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms. J Clin Invest 89: 803–815, 1992.[Web of Science][Medline]
  8. Bupha-Intr T, Wattanapermpool J. Cardioprotective effects of exercise training on myofilament calcium activation in ovariectomized rats. J Appl Physiol 96: 1755–1760, 2004.[Abstract/Free Full Text]
  9. Bupha-Intr T, Wattanapermpool J. Regulatory role of ovarian sex hormones in calcium uptake activity of cardiac sarcoplasmic reticulum. Am J Physiol Heart Circ Physiol 291: H1101–H1108, 2006.[Abstract/Free Full Text]
  10. Carter SG, Karl DW. Inorganic phosphate assay with malachite green: an improvement and evaluation. J Biochem Biophys Methods 7: 7–13, 1982.[CrossRef][Web of Science][Medline]
  11. Chakraborti S, Chakraborti T, Shaw G. beta-Adrenergic mechanisms in cardiac diseases: a perspective. Cell Signal 12: 499–513, 2000.[CrossRef][Web of Science][Medline]
  12. Engelhardt S, Hein L, Wiesmann F, Lohse MJ. Progressive hypertrophy and heart failure in beta1-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA 96: 7059–7064, 1999.[Abstract/Free Full Text]
  13. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 157: 378–417, 1988.[Web of Science][Medline]
  14. Fukuharu M, Sato J, Ohsawa I, Oshida Y, Nagasaki M, Nakai N, Shimomura Y, Hattori M, Tokudome S, Sato Y. Additive effects of estrogen deficiency and diabetes on bone mineral density in rats. Diabetes Res Clin Pract 48: 1–8, 2000.[CrossRef][Web of Science][Medline]
  15. Ganguly PK, Beamish RE, Dhalla KS, Innes IR, Dhalla NS. Norepinephrine storage, distribution, and release in diabetic cardiomyopathy. Am J Physiol Endocrinol Metab 252: E734–E739, 1987.[Abstract/Free Full Text]
  16. Gomes AV, Potter JD. Molecular and cellular aspects of troponin cardiomyopathies. Ann NY Acad Sci 1015: 214–224, 2004.[CrossRef][Web of Science][Medline]
  17. Grohé C, Kahlert S, Löbbert K, Stimpel M, Karas RH, Vetter H, Neyses L. Cardiac myocytes and fibroblasts contain functional estrogen receptors. FEBS Lett 416: 107–112, 1997.[CrossRef][Web of Science][Medline]
  18. Gustafsson I, Brendorp B, Seibaek M, Burchardt H, Hildebrandt P, Køber L, and Torp-Pedersen C. Influence of diabetes and diabetes-gender interaction on the risk of death in patients hospitalized with congestive heart failure. J Am Coll Cardiol 43: 771–777, 2004.[Abstract/Free Full Text]
  19. Harada K, Potter JD. Familial hypertrophic cardiomyopathy mutations from different functional regions of troponin T result in different effects on the pH and Ca2+ sensitivity of cardiac muscle contraction. J Biol Chem 279: 14488–14495, 2004.[Abstract/Free Full Text]
  20. Heyder S, Malhotra A, Ruegg JC. Myofibrillar Ca2+ sensitivity of cardiomyopathic hamster hearts. Pflugers Arch 429: 539–545, 1995.[CrossRef][Web of Science][Medline]
  21. Hunt SA, Abraham WT, Chin MH, Feldman AM, Francis GS, Ganiats TG, Jessup M, Konstam MA, Mancini DM, Michl K, Oates JA, Rahko PS, Silver MA, Stevenson LW, Yancy CW, Antman EM, Smith SC Jr, Adams CD, Anderson JL, Faxon DP, Fuster V, Halperin JL, Hiratzka LF, Jacobs AK, Nishimura R, Ornato JP, Page RL, Riegel B. ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the American College of Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by the Heart Rhythm Society. Circulation 112: e154–e235, 2005.
  22. Kannel WB, Hjortland M, Castelli WP. Role of diabetes in congestive heart failure: the Framingham study. Am J Cardiol 34: 29–34, 1974.[CrossRef][Web of Science][Medline]
  23. Kass DA, Solaro RJ. Mechanisms and use of calcium-sensitizing agents in the failing heart. Circulation 113: 305–315, 2006.
  24. Kaye DM, Lefkovits J, Jennings GL, Bergin P, Broughton A, Esler MD. Adverse consequences of high sympathetic nervous activity in the failing human heart. J Am Coll Cardiol 26: 1257–1263, 1995.[Abstract]
  25. Knowlton AA, Sun L. Heat-shock factor-1, steroid hormones, and regulation of heat-shock protein expression in the heart. Am J Physiol Heart Circ Physiol 280: H455–H464, 2001.[Abstract/Free Full Text]
  26. Kobayashi T, Solaro RJ. Increased Ca2+ affinity of cardiac thin filaments reconstituted with cardiomyopathy-related mutant cardiac troponin I. J Biol Chem 281: 13471–13477, 2006.[Abstract/Free Full Text]
  27. Lechat P, Packer M, Chalon S, Cucherat M, Arab T, Boissel JP. Clinical effects of beta-adrenergic blockade in chronic heart failure: a meta-analysis of double-blind, placebo-controlled, randomized trials. Circulation 98: 1184–1191, 1998.
  28. Lefkowitz RJ, Rockman HA, Koch WJ. Catecholamines, cardiac beta-adrenergic receptors, and heart failure. Circulation 101: 1634–1637, 2000.
  29. Mankhey RW, Bhatti F, Maric C. 17beta-Estradiol replacement improves renal function and pathology associated with diabetic nephropathy. Am J Physiol Renal Physiol 288: F399–F405, 2005.[Abstract/Free Full Text]
  30. Martin AF, Phillips RM, Kumar A, Crawford K, Abbas Z, Lessard JL, de Tombe P, Solaro RJ. Ca2+ activation and tension cost in myofialments from mouse hearts ectopically expressing enteric {gamma}-actin. Am J Physiol Heart Circ Physiol 283: H642–H649, 2002.[Abstract/Free Full Text]
  31. Packer M, Coats AJS, Fowler MB, Katus HA, Krum H, Mohacsi P, Rouleau JL, Tendera M, Castaigne A, Roecker EB, Schultz MK, DeMets DL. Effect of carvedilol on survival in severe chronic heart failure. N Engl J Med 344: 1651–1658, 2001.[Abstract/Free Full Text]
  32. Pagani ED, Solaro RJ. Method for measuring functional properties of sarcoplasmic reticulum and myofibrils in small samples of myocardium. In: Methods in Pharmacology, edited by Schwartz A. New York: Plenum, 1984, vol. 5, p. 44–61.
  33. Post SR, Hammond HK, Insel PA. beta-Adrenergic receptors and receptor signaling in heart failure. Annu Rev Pharmacol Toxicol 39: 343–360, 1999.
  34. Ren J, Hintz KK, Roughead ZKF, Duan J, Colligan PB, Ren BH, Lee KJ, Zeng H. Impact of estrogen replacement on ventricular myocyte contractile function and protein kinase B/Akt activation. Am J Physiol Heart Circ Physiol 284: H1800–H1807, 2003.[Abstract/Free Full Text]
  35. Rice JJ, de Tombe PP. Approaches to modeling crossbridges and calcium-dependent activation in cardiac muscle. Prog Biophys Mol Biol 85: 179–195, 2004.[CrossRef][Web of Science][Medline]
  36. Sowers JR. Diabetes mellitus and cardiovascular disease in women. Arch Intern Med 158: 617–621, 1998.[Abstract/Free Full Text]
  37. Thawornkaiwong A, Preawnim S, Wattanapermpool J. Upregulation of beta1-adrenergic receptors in ovariectomized rat hearts. Life Sci 72: 1813–1824, 2003.[CrossRef][Web of Science][Medline]
  38. Velloso LA, Carvalho CRO, Rojas FA, Folli F, Saad MJA. Insulin signalling in heart involves insulin receptor substrates-1 and -2, activation of phosphatidylinositol 3-kinase and the JAK 2-growth related pathway. Cardiovasc Res 40: 96–102, 1998.[Abstract/Free Full Text]
  39. Voss MR, Stallone JN, Li M, Cornelussen RNM, Knuefermann P, Knowlton AA. Gender differences in the expression of heat shock proteins: the effect of estrogen. Am J Physiol Heart Circ Physiol 285: H687–H692, 2003.[Abstract/Free Full Text]
  40. Wattanapermpool J. Increase in calcium responsiveness of cardiac myofilament activation in ovariectomized rats. Life Sci 63: 955–964, 1998.[CrossRef][Web of Science][Medline]
  41. Wattanapermpool J, Reiser PJ. Differential effects of ovariectomy on calcium activation of cardiac and soleus myofilaments. Am J Physiol Heart Circ Physiol 277: H467–H473, 1999.[Abstract/Free Full Text]
  42. Wolff MR, Buck SH, Stoker SW, Greaser ML, Mentzer RM. Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies: role of altered beta-adrenergically mediated protein phosphorylation. J Clin Invest 98: 167–176, 1996.[Web of Science][Medline]
  43. Wolff MR, Whitesell LF, Moss RL. Calcium sensitivity of isometric tension is increased in canine experimental heart failure. Circ Res 76: 781–789, 1995.[Abstract/Free Full Text]
  44. Yu HP, Shimizu T, Choudhry MA, Hsieh YC, Suzuki T, Bland KI, Chaudry IH. Mechanism of cardioprotection following trauma-hemorrhagic shock by a selective estrogen receptor-beta agonist: up-regulation of cardiac heat shock factor-1 and heat shock proteins. J Mol Cell Cardiol 40: 185–194, 2006.[CrossRef][Web of Science][Medline]



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