INTRODUCTION

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A WELL-RECOGNIZED GENDER difference in the incidence of cardiovascular diseases has been hypothesized to be due in part to the influence of steroid sex hormones on myocardial function. However, the significance of these steroid hormones in cardiac myofibrillar function is not well understood. Schaible et al. (25) studied whole heart preparations from adult rats that were ovariectomized before puberty and found decreases in cardiac output, peak systolic pressure, and ejection fraction at all preloads, compared with sham-operated controls. Moreover, Ca²⁺-myosin ATPase activity was significantly reduced and the myosin isoenzymes were shifted from a predominant V₁ pattern to a predominance of V₃ isoenzyme. Rats ovariectomized after the postpubertal period also demonstrated the same changes in cardiac function that can be prevented by replacement with estrogen or testosterone (26). Recently, a possible modulating effect of ovarian sex hormone deficiency on the Ca²⁺ responsiveness of cardiac myofilament activation by induction of myofilament Ca²⁺ hypersensitivity and suppression of maximum myofibrillar ATPase activity has been reported (31).

There is a depression in the maximum Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity of cardiac myofilaments in several types of heart failure (1, 20, 30). Moreover, studies of myofibrillar mechanical properties in canine experimental heart failure (36), in genetically cardiomyopathic hamster hearts (11), as well as in human dilated cardiomyopathies (35) indicate an increase in Ca²⁺ sensitivity of the myofilaments without any change in maximum isometric tension. It has been suggested that strong cross-bridge interactions of myosin with thin filaments are an important determinant of the level of myofilament activation in both normal and myopathic hearts (30). Furthermore, changes in thin filament proteins in association with heart failure also appear to induce alterations in myofilament regulation (2, 12, 34). Alterations in thin filament proteins in relation to myocardial dysfunction may include shifts in the isoform population (2, 12), breakdown of specific proteins (34), and covalent modulations of proteins by phosphorylation (29). A shift in the myosin isoenzyme expression pattern from predominantly V₁ to V₃ occurs in ovariectomized rats (25, 26). However, no detectable isoform shift or breakdown of any thin filament proteins following ovariectomy has been reported. This suggests the possibility that the increased myofilament response to Ca²⁺ after ovariectomy (31) may result from other forms of thin filament modulation. Among thin filament proteins, troponin I (TnI) plays a key role in the control and modulation of myofilament activity (29). In addition, it is the cardiac isoform of TnI that is specifically phosphorylated by protein kinase A which then induces a decrease in myofilament Ca²⁺ sensitivity of the heart under -adrenergic stimulation (27). This modulation of thin filament proteins by phosphorylation does not appear in skeletal muscles in which the slow or fast skeletal isoform of TnI was present. It was, therefore, of interest to investigate whether ovariectomy induces similar changes in the Ca²⁺ responsiveness of skeletal muscle myofilaments also, especially of soleus fibers which contain the same isoform of troponin C (TnC) as in cardiac tissue.

This study focused on two questions. 1) Is the reported increase in Ca²⁺ sensitivity of myofilaments from ovariectomized hearts (31) associated with an alteration of myofibrillar mechanical properties? 2) Does long-term deprivation of ovarian sex hormones affect the mechanical properties of soleus fibers in the same manner? We used glycerinated skinned ventricular trabeculae and single soleus fibers from ovariectomized rats to answer these questions. The tension-pCa relationships of trabeculae and soleus fibers from ovariectomized and control rats were compared. The results of our studies support the possibility that modulation of myofilament regulatory proteins, especially cardiac TnI (cTnI), may occur in response to long-term reduction in ovarian sex hormones that results in increased cardiac myofilament Ca²⁺ sensitivity.

	MATERIALS AND METHODS

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Animal preparation. Female Sprague-Dawley rats weighing between 150 and 200 g (8-10 wk old) were anesthetized with ketamine (60 mg/kg) and xylazine (5 mg/kg) intraperitoneally. Ovariectomy or a sham operation was performed through bilateral skin incisions in the flank area of the lower back. The skin incision was closed with sterile nylon sutures, and all animals were then fed with estrogen-free rat food and water ad libitum for the remainder of the study. On the basis of a previous study (31) on changes in biochemical properties of cardiac myofilaments at 10 wk after ovariectomy, 10 and 14 wk were chosen as the postovariectomy period for the present study. Adequacy of ovariectomy was determined by uterine mass on the day the rats were killed.

Skinned fiber studies. Ten or fourteen weeks after surgery, the rats were killed with inhalation of CO₂. The heart and soleus muscles were rapidly removed, weighed, and prepared for skinned trabeculae and soleus fibers, as previously described (33). Skinned trabeculae were dissected from the left ventricular wall and placed in relaxing solution containing 2% Triton X-100 for 2 min. Trabeculae were mounted between the arms of a direct-current torque motor (model 300H; Cambridge Technology, Cambridge, MA) and an isometric tension transducer (model 403, Cambridge Technology) in the experimental chamber containing relaxing solution. The torque motor was used to introduce slack during steady activation to obtain an accurate measurement of developed tension. The motor and transducer were set on three-way positioners that could be moved to adjust the sarcomere length, which was measured from Polaroid photographs. For soleus experiments, a single fiber segment was carefully isolated from a bundle and treated as described above for trabeculae. The width and depth of trabeculae and soleus fibers used in this study were 50-180 µm. Fiber cross-sectional area (CSA) was calculated from the depth and width measurements by assuming an elliptical fiber circumference.

Gel electrophoresis. Myosin heavy chain (MHC) isoforms of individual trabeculae and single soleus fibers used for mechanical measurements were electrophoretically separated as described earlier (22), using fiber or trabecula volumes of ~1.0 nl as sample loads. Combined right and left atrial samples and transmural pieces (~20 mg) of the right and left ventricular free walls from ovariectomized and sham-operated rats were also analyzed for MHC isoform composition. The silver-stained gels were scanned by a GS300 scanning densitometer (Hoefer Scientific). The relative amounts of -MHC and -MHC in a sample were calculated from the scan results.

Statistical analyses. The tension-pCa relations were fitted to the Hill equation {relative activity = [Ca²⁺]ⁿ/(K + [Ca²⁺]ⁿ), where K is pCa₅₀, the pCa corresponding to 50% of maximal tension development, and n is the Hill coefficient} using nonlinear least-squares regression analysis (GraphPad InPlot, ISI software, version 4) to derive the pCa₅₀ and Hill coefficient. Data are given as means ± SE. The unpaired t-test was used to determine the significance of differences between sham-operated and ovariectomized groups of the same duration of study (i.e., 10 or 14 wk), except where noted otherwise.

Chemicals. All chemicals were purchased from Sigma (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA).

	RESULTS

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Adequacy of ovarian sex hormone deficiency in ovariectomized rats was verified by significant reductions of uterine mass (P < 0.005) in these animals when compared with sham-operated rats (Table 1). Body mass also increased significantly (P < 0.05) after ovariectomy. However, there were no changes in the ratios of heart mass and soleus mass to brain mass at 10 and 14 wk after ovariectomy. Brain mass was utilized for normalization of heart and soleus masses to avoid a possible increase in body mass due to increased fat content after ovariectomy. Fat is relatively sparsely vascularized and, as such, has a relatively small effect on hemodynamic load. The relative amount of -MHC increased (P < 0.01) in both the right and left ventricles (RV and LV, respectively) after ovariectomy (Fig. 1). The -MHC levels in the RV and LV were 5.4- and 2.7-fold, respectively, greater in the ovariectomized (RV, 30.0 ± 6.0%; LV, 40.0 ± 2.8%) compared with the sham-operated (RV, 5.6 ± 1.4%; LV, 14.8 ± 2.1%) rats (10-wk and 14-wk samples combined; n = 6 hearts for both groups). -MHC was not detected in any of the atrial samples from either ovariectomized or sham-operated rats. Moreover, silver-stained electrophoretic gels of individual trabeculae used in the tension experiments demonstrated significant decreases in the relative amount of -MHC (i.e., increases in the relative amount of -MHC) in trabeculae from ovariectomized hearts in both periods of study (Fig. 2).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Myosin heavy chain (MHC) region of SDS gels. A: gels on which were loaded samples of combined right and left atria (A), right ventricle (RV), and left ventricle (LV) from sham-operated control (Sham) and ovariectomized rats (OVX). B: gel on which single trabeculae from hearts of sham-operated (S) or ovariectomized (O) rats were loaded. Note relatively greater levels of -MHC in trabeculae from ovariectomized rats.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Relative amount of -MHC (as a percentage of total MHC) of trabeculae from 10- and 14-wk groups of OVX and Sham rats. Data represent means ± SE from 7-9 preparations. * P < 0.05, ** P < 0.005, significantly different from Sham group of same period of study.

To test for a possible modulation of tension development by ovarian sex hormones, the tension-pCa relations of skinned trabeculae from 10- and 14-wk ovariectomized rats were studied. Sarcomere length was set to 1.9-2.1 µm. As shown in Fig. 3, A and B, the maximum tension developed by trabeculae from ovariectomized rats was not significantly different from sham-operated controls in both periods of the study. Furthermore, there was no correlation, as tested with linear regression, between percent -MHC and the maximum tension among the individual trabeculae from either ovariectomized or sham-operated rats (correlation coefficient = 0.16 and 0.03, respectively). When comparisons were made between the relative tension-pCa relationships of trabeculae from ovariectomized rats and those from sham-operated controls, shifts of the curve to the left were demonstrated in both 10- and 14-wk experiments as shown in Fig. 3, C and D, respectively. As a result, calcium sensitivities of trabeculae were significantly increased (P < 0.01) from pCa₅₀ values of 5.65 ± 0.02 and 5.66 ± 0.02 in sham-operated trabeculae of 10- and 14-wk groups, respectively, to 5.74 ± 0.02 in ovariectomized hearts of both groups. However, there was no significant change in the slope or Hill coefficient of the tension-pCa relations in these trabeculae. These results demonstrate an increase in Ca²⁺ sensitivity of tension development in cardiac myofibrillar preparations of rats 10 and 14 wk after ovariectomy.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Tension-pCa relations of trabeculae from Sham and OVX rats. Isometric tension generated by trabeculae from 10-wk (A) and 14-wk (B) groups was measured in various randomized activating solutions ranging from pCa 7.0 to 4.0 at pH 7.0 for each preparation. Relative tensions developed in trabeculae from 10-wk (C) and 14-wk (D) groups were derived by dividing absolute tension at any given pCa by peak absolute tension (Po) developed by same preparation at pCa 4.0. Data represent means ± SE from 8-10 preparations. CSA, cross-sectional area.

To test the possibility that thin filament proteins are the site of action for the increase in Ca²⁺ sensitivity of activation after ovariectomy, we compared the effect of ovariectomy on tension development in trabeculae to that in single soleus fibers. Inasmuch as the same TnC isoform is present in both trabeculae and soleus fibers, the only difference in troponin subunit isoforms between these muscle types is in TnI and troponin T (TnT). The tension-pCa relations of single skinned soleus fibers from sham-operated and ovariectomized rats were studied with sarcomere length set at 2.2-2.4 µm. Only the soleus fibers of 14-wk ovariectomized rats demonstrated a significant (P < 0.05) increase in CSA, compared with that of control fibers (data not shown). As shown in Fig. 4, the maximum tension was significantly lower in soleus fibers from ovariectomized rats compared with sham-operated controls in both 10-wk (~19%, P < 0.01, Fig. 4A) and 14-wk experiments (~20%, P < 0.001, Fig. 4B). However, there were no changes in the Ca²⁺ sensitivity or Hill coefficient of tension development or in V_max in soleus fibers after ovariectomy (Table 2). All of the single soleus fibers used for tension measurements, from both ovariectomized and sham-operated rats, contained only the slow type I MHC isoform (data not shown). Moreover, no changes in the isoform expression pattern of both thick and thin filament proteins in soleus muscles after ovariectomy were detected with SDS-PAGE (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Tension-pCa relations of single soleus fibers from Sham and OVX rats. Isometric tension generated by soleus fibers from 10-wk (A) and 14-wk (B) groups were measured in various randomized activating solutions ranging from pCa 7.0 to 4.0 for each preparation. Data represent means ± SE from 7 or 8 preparations. * P < 0.05, ** P < 0.005, significantly different from Sham at same pCa and same period of study.

	DISCUSSION

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Results presented here extend our understanding of how long-term ovarian sex hormone deprivation modulates myofilament activation. Although it has been proposed that an influence of steroid sex hormones on myocardial function can account in part for a well-recognized gender difference in the age of onset and mortality rate from cardiovascular disease in humans, the exact effects of these hormones on heart functions, especially at the myofilament level, are not yet clear. Wattanapermpool (31) recently reported a possible modulating effect of ovarian sex hormones on the Ca²⁺ responsiveness of cardiac myofilaments by inducing an increase in the Ca²⁺ sensitivity but reducing the maximum myofibrillar ATPase activity of isolated myofibrillar preparations from 8- to 10-wk ovariectomized rat hearts. In the present study, an increase in Ca²⁺ sensitivity of isometric force development in skinned trabeculae from ovariectomized hearts was detected. There was no significant change in the maximum force of contraction in these ovariectomized rat hearts, as measured in skinned trabeculae, even though there was a significant change in MHC isoform expression.

Effects of ovariectomy on cardiac functions, i.e., suppression of maximum ATPase activity but increased myofibrillar Ca²⁺ sensitivity as shown in this study, resemble many recent reports on cardiomyopathy and heart failure models (11, 35, 36). Heyder et al. (11) found a higher pCa₅₀ in skinned muscle fiber preparations from ventricles of genetically cardiomyopathic Syrian hamsters. Based on the hypothesis of Malhotra (16) that troponins may be altered in cardiomyopathic hamster hearts, Heyder et al. (11) extracted and replaced endogenous TnI of the skinned muscle fiber preparations from RV and LV with bovine cardiac troponin. After replacement, the Ca²⁺ sensitivity of fibers was similar to that of bovine heart. This finding indicates that the type of troponin (skeletal vs. cardiac) is a strong determinant of myofibrillar Ca²⁺ sensitivity. Moreover, incubation of fiber bundles with the catalytic subunit of cAMP-dependent protein kinase (PKA) normalized the enhanced Ca²⁺ responsiveness of fibers from diseased hamsters (11). Wolff et al. (36) studied myocyte-sized myofibrillar preparations from permeabilized myocardium of a canine model of dilated cardiomyopathy produced by chronic rapid pacing. They demonstrated an increase in the calcium sensitivity of isometric tension in failing myocardium that could be reversed by treatment with PKA. Similar results were obtained by Wolff et al. (35) using permeabilized myocyte preparations from human dilated cardiomyopathies. They suggested that the increased calcium sensitivity of isometric tension in cardiomyopathy may be due, at least in part, to a reduction in -adrenergically regulated proteins such as TnI and/or C protein. This possibility is supported by a recent report of Bodor et al. (3) in which a reduced extent of cardiac TnI phosphorylation by PKA was demonstrated in preparations from failing adult human hearts. The similarity in the calcium responsiveness between ovariectomized hearts and failing myocardium suggests a possibly similar mechanism, at least in part, for the effects of long-term deprivation of ovarian sex hormones on cardiac myofilaments. Further characterizations of other changes in functions of sarcolemmal and sarcoplasmic reticulum membranes after ovariectomy should provide a more complete assessment of the impact of changes in ovarian sex hormone levels on the etiology of heart failure.

Our results suggest that the alteration of myofibrillar Ca²⁺ sensitivity after ovariectomy is cardiac specific; that is, there was no change in the Ca²⁺ sensitivity of isometric tension but a significant reduction (~20%) in the maximum force of contraction in soleus fibers from ovariectomized rats. It is not known at this point how ovarian sex hormone deprivation induces a decrease in the maximum force of contraction in soleus fibers in which only MHC type I was detected. However, this decrease in force could involve either a decrease in the number of cross-bridge attachments to actin and/or a decrease in the force generated per cross bridge. Measurements of fiber stiffness, an estimate of the relative number of cross bridges interacting with actin, should provide additional insight.

The observed cardiac-specific change in myofibrillar Ca²⁺ sensitivity suggests possible alterations in the thin filament regulatory complex of ovariectomized hearts. Many studies have demonstrated that alterations of thin filament proteins in cardiac and skeletal muscles are associated with altered Ca²⁺ sensitivity (11, 33). Alterations may include isoform shifts, modifications of the protein without a shift in isoform expression, and/or degradation of proteins. As reported earlier (31), there were no detectable changes or loss of any thin filament components in ovariectomized rat hearts. This suggests a possible modification of thin filament proteins in these hearts which then affected TnC Ca²⁺ binding upon activation. Soleus and cardiac thin filaments contain the same cTnC (cardiac or slow skeletal TnC) isoform; however, isoforms of TnI and TnT in these two muscle types are different. Inasmuch as fetal TnT isoform reexpression was detected in the failing human heart (2, 35), there was no correlation between the fraction of this isoform alteration and myofibrillar calcium sensitivity of isometric tension (35). Most evidence suggests that TnI is responsible for the increased Ca²⁺ sensitivity in the heart. The cTnI isoform expressed in cardiac tissue, but not slow skeletal TnI in soleus fibers, can be specifically phosphorylated by PKA at the NH₂-terminal extension (32), which then induces a decrease in myofilament sensitivity to Ca²⁺ without affecting maximal ATPase activity. It is this cTnI phosphorylation by PKA that has been defined as the basis for the decrease in Ca²⁺ myofilament sensitivity following sympathetic stimulation on -adrenergic receptors both in vivo (28) and in vitro (19).

Many sympathetic alterations, including decreased norepinephrine content of sympathetic terminals, -receptor downregulation, and desensitization of adenylate cyclase stimulation, have been reported in both human heart failure (5, 9, 14) and animal models of heart failure (7, 8, 18). These sympathetically impaired hearts could have a lower level of phosphorylated cTnI with consequently increased myofilament sensitivity to calcium, as discussed. It is not known at present whether ovarian sex hormones induced any change in sympathetic control of the heart that may lead to alterations in myofibrillar protein phosphorylation and, therefore, myofilament Ca²⁺ sensitivity. However, combined administrations of estrogen and progesterone in ovariectomized rats caused an increase in the receptor densities of muscarinic and -adrenergic receptors as well as a decrease in the binding affinity of -adrenergic receptors in vivo (15). Moreover, Malhotra et al. (17) measured myocardial catecholamine stores and reported an unaltered content after ovariectomy. These results imply possible effects of ovarian sex hormone deficiency on sympathetic modulations at receptor and/or postreceptor levels of the heart. Because the level of TnI phosphorylation was not measured in the present study, it is not possible to conclude whether this contributed to the observed change in Ca²⁺ sensitivity. Furthermore, phosphorylation of other myofibrillar proteins, including thick filament proteins, or the change in MHC isoform expression may also have contributed to the altered Ca²⁺ sensitivity following ovariectomy.

The presence of functional estrogen receptors on cardiac myocytes and the fact that estrogen exposure could modulate gene expression in these cells support the hypothesis that the heart is a target organ for sex steroid actions (10). Johnson et al. (13) studied estrogen receptor gene knockout mice and found that the membrane density of cardiac L-type Ca²⁺ channels is regulated by physiological levels of estrogen through the action of estrogen receptors. A decrease in estrogen levels may then lead to abnormal cardiac excitability and increased risk of cardiovascular diseases through an increase in the number of Ca²⁺ channels. Recent data from rabbit myocardium also show an increase in L-type Ca²⁺ channel density after ovariectomy (21). However, a study in ovariectomized rats demonstrated opposite changes in Ca²⁺ channel expression (4). The interactions between increased or decreased Ca²⁺ entry and potentially altered activation level with increased myofilament Ca²⁺ sensitivity after ovariectomy deserve further investigation. Additional studies may also lead to a better understanding of potential targets and effective hormone supplemental regimens for therapeutic and preventive approaches for cardiovascular diseases in women.