Compared to sham-operated controls, myofilaments from hearts of ovariectomized (OVX) rats demonstrate an increase in Ca2+ sensitivity with no change in maximum tension (Wattanapermpool J and Reiser PJ. Am J Physiol 277: H467–H473, 1999). To test the significance of this modification in intact cells, we compared intracellular Ca2+ transients and shortening of ventricular myocytes isolated from sham and 10-wk OVX rats. There was a decrease in the peak Ca2+ transient with prolonged 50% decay time in OVX cardiac myocytes without changes in the resting intracellular Ca2+ concentration. Percent cell shortening was also depressed, and relaxation was prolonged in cardiac myocytes from OVX rats compared with shams. Ovariectomy induced a sensitization of the myofilaments to Ca2+. Hypercapnic acidosis suppressed the shortening of OVX myocytes to a lesser extent than that detected in shams. Moreover, a larger compensatory increase in %cell shortening was obtained in OVX myocytes during prolonged acidosis. The elevated compensation in cell shortening was related to a higher amount of increase in the amplitude of the Ca2+ transient in OVX myocytes. However, these differences in Ca2+ transients and %cell shortening were no longer evident in the presence of 1 μM cariporide, a specific inhibitor of Na+/H+ exchanger type 1 (NHE1). Our results indicate that deprivation of female sex hormones modulates the intracellular Ca2+ concentration in cardiac myocytes, possibly via an increased NHE1 activity, which may act in concert with Ca2+ hypersensitivity of myofilament activation as a determinant of sex differences in cardiac function.
- sarcomeric proteins
- proton exchanger
sex steroid hormones exert significant control over cardiac physiology, but the molecular and cellular mechanisms for these effects remain unclear. Understanding these mechanisms is crucial for translation to hormone therapy (30). Scheuer and colleagues (23, 24) first demonstrated a suppression of function in isolated heart preparation in both gonadectomized male and female rats. Moreover, these investigators reported that the decreases in ejection fraction, peak systolic pressure, and cardiac output in the hearts from gonadectomized rats could be normalized with hormone supplementation. These results, which pointed to an important role of both male and female steroid hormones in regulating cardiac function, fit with the hypothesis of a cardioprotective role of female sex hormones. Epidemiological studies also supported this hypothesis by demonstrating that men have a higher incidence of heart disease than premenopausal women (13). Echocardiographic assessment of cardiac function in premenopausal women also demonstrated enhanced contractile function compared with postmenopausal women (19). Evidence of improved prognosis in female heart failure patients taking estrogen compared with those off estrogen also supports the hypothesis of the importance of sex hormones in heart diseases (15, 20).
Although, antiapoptotic effects of estrogen have been reported (18), depression in Ca2+ fluxes to and from the myofilaments appears to account in part for the depression of contractility in hearts of ovariectomized (OVX) rats. Ren et al. (21) reported that myocytes from 6-wk OVX rat hearts demonstrated slowing of relaxation and contraction kinetics associated with a slower decay and depressed peak amplitude of Ca2+ transients. Bupha-Intr and Wattanapermpool (3, 4) reported a depression in Ca2+ uptake rate and ATPase activity by vesicles of the cardiac sarcoplasmic reticulum (SR) derived from hearts of rats 10 wk following ovariectomy, compared with sham controls. Depression in expression of sarco(endo)plasmic reticulum Ca2+-ATPase at the protein and mRNA levels appeared to account for this depression in myocyte function and Ca2+ fluxes (4). There is also evidence of sex-related differences in fluxes through the Na+/Ca2+ exchanger (NCX). Sugishita et al. (28) reported that metabolic inhibition induced a relatively large increase in intracellular Ca2+ in myocytes of transgenic male vs. female mouse hearts overexpressing NCX. Treatment of the males with 17β-estradiol (E2) significantly attenuated this difference. Whether these effects on intracellular Ca2+ are due directly to NCE activity or indirectly through modification of the activity of the Na+/H+ exchanger (NHE1) remains unclear. There is, however, evidence of acute effects of E2 on regulation of NHE1 activity. In demonstrating that acute treatment with E2 improved recovery of hearts stressed by normoxic acidification, Anderson et al. (1) proposed a mechanism involving, in part, promotion of NO synthesis that increased NHE1 activity.
Alterations in myofilament response to Ca2+ also appear to play an important role in effects of estrogen on cardiac function. There is a reduction in ATPase rate in myofilaments isolated from hearts of OVX rats, which is attributable to a shift in myosin isoform population from one predominant in the alpha-myosin heavy chain to one containing significant amounts of beta-myosin heavy chain (32, 35). Moreover, compared with preparations from shams, ATPase activity (33) and tension (32) of myofilaments from OVX rat hearts were more sensitive to Ca2+. These changes in myofilament response to Ca2+ were reversed in preparations from OVX rats supplemented with E2 (35) or in animals subjected to exercise training (3).
In experiments reported here, we have extended our studies of the relationship between sex hormones and myofilament response to Ca2+ with the objective of understanding the relevance of this effect in isolated cardiac myocytes. We think this understanding is important in view of evidence that an increase in myofilament response to Ca2+ is a common functional consequence of mutations in sarcomeric proteins that are linked to familial hypertrophic myopathies (9). Moreover, the role of myofilament response to Ca2+ has not been generally considered in the cardio-protective effects of E2 in acidosis (1) and metabolic inhibition (28), even though there is evidence that altered myofilament response to Ca2+ is a dominant mechanism responsible for the fall in tension associated with acidosis (26). We, therefore, compared the effects of hypercapnic acidosis on shortening and intracellular Ca2+ of myocytes isolated from sham, OVX, and OVX + E2 rat hearts. We also compared tension-pCa relations in detergent extracted fiber bundles from these hearts. To probe for changes in myofilament response to Ca2+, we compared responses at physiological pH to responses at acidic pH. Our results confirm that cardiac myofilaments functioning in intact cardiac myocytes from OVX rat hearts are more sensitive to Ca2+ than in control myocytes. Our data also indicate that chronic deprivation of female sex hormones modulates the intracellular Ca2+ concentration in cardiac myocytes via an increase in NHE1 activity, which may act in concert with the hypersensitivity of myofilament activation as a significant determinant of sex differences in cardiac function.
MATERIALS AND METHODS
Our studies used female Sprague-Dawley rats that were OVX or sham operated at 8 wk of age by the supplier (Harlan, Indianapolis, IN) and killed at 18 wk of age. We randomly divided the OVX rats into two groups, and injected them subcutaneously with 0.1 ml of corn oil with or without 5 μg of 17β-estradiol. We did these injections three times per week for 10 wk, as previously described (4). We verified deficiency of ovarian sex hormones by the absence of ovarian tissues and by the shrinkage of uterine mass (4).
Cardiac myocyte isolation.
At the 10-wk time point, we heparinized (1,000 unit/kg body wt) the rats, and after 30 min, we anesthetized them with an intraperitoneal injection of pentobarbital sodium (25 mg/kg body wt). Left ventricular myocytes were isolated as previously described (30, 39, 40). The heart was rapidly removed, and the aorta was cannulated and perfused with nominally oxygenated Ca2+-free Tyrode solution until complete cessation of beating. The perfusion solution was then switched to 25 μM CaCl2 Tyrode solution containing 1 mg/ml BSA, 160 unit/ml collagenase type II (Boehringer Mannheim), and 0.1 mg/ml protease type XIV (Sigma, St. Louis, MO). The hearts were then perfused for 16.5 min/g wet wt with the above solution. At the end of the perfusion period, the hearts were removed from the cannula and placed into a petri dish containing 50 μM CaCl2 Tyrode-BSA buffer. Ventricles were cut into small pieces and gently triturated with a pipette and filtered through a cell collector. The cell suspension was placed into centrifuge tubes and gradually increased the extracellular CaCl2 concentration to a final level of 0.5 mM CaCl2. Myocytes were studied within 6 h after isolation.
Intracellular Ca2+ transients, pH, and cell shortening.
Myocytes were loaded with 5 μM Fura-2 acetoxymethyl ester (Fura-2-AM) for 15 min in 0.5 mM CaCl2 Tyrode buffer with 1 mg/ml BSA and 5% FBS for 15 min. After washing off the excess Fura-2-AM, the myocytes were electrically stimulated at a frequency of 0.5 Hz for 10 to 15 min or until the shortening was stable with perfusion of 2 mM CaCl2 Tyrode buffer at room temperature. Fura-2 fluorescence (340:380 ratio) and cell shortening were monitored simultaneously, as described in detail by Wolska et al. (38).
To test the effect of acidic pH on intracellular Ca2+ transients and cell shortening, we perfused the myocytes with control Krebs-Henseleit buffer (KH) saturated with 95% O2-5% CO2 for 15 min, with KH saturated with 85% O2-15% CO2 for 20 min, and then with control KH. In these experiments, we also determined intracellular pH with the aid of 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, as previously described (37), and we made the measurements in the presence and absence of 1.0 μM cariporide, an inhibitor of the NHE1 (11).
Force measurements on skinned fiber bundles.
We determined the relationship between pCa (–log of the molar-free Ca2+) and tension (force/cross-sectional area) in detergent (Triton X-100) extracted fiber bundles prepared from left ventricular papillary muscles, as previously described (34, 38, 39). The fiber bundles (∼150–200 μm in diameter and 4–5 mm long) were cut in ice-cold high relaxing (HR) buffer: 10 mM EGTA, 2 mM free Mg2+, 5 mM MgATP2−, 79.2 mM KCl, 12 mM creatine phosphate, 20 mM MOPS, pH 7.0, ionic strength 0.15 M, 2.5 μg/ml pepstatin A, 1 μg/ml leupeptin, and 50 μM PMSF. After mounting the fiber bundles in an apparatus for determination of force, sarcomere length was set at 2.2 μm, as determined from a laser diffraction pattern in HR solution and cross-sectional area determined. Isometric force was recorded on a chart recorder at different pCa at room temperature. CaCl2 concentrations in HR were varied to achieve a range of Ca2+ concentrations as computed using a computer program and binding constants summarized by Godt and Lindley (8). Only fiber bundles that developed 85–90% of their initial maximum tension at the end of experiment were retained for analysis. We plotted isometric tension as a function of pCa, fitted the curves to the A. V. Hill equation, as previously described (41), and expressed the relations as a percent of maximum tension. pCa50 (half maximally activating pCa values) was computed from individual Hill fits of each pCa-tension relation and then averaged.
Expression of NHE1.
To determine the level of expression of the NHE1 gene, real-time, one-step quantitative RT-PCR with SYBR Green detection (Qiagen,Valencia, CA) was performed using a LightCycler thermocycler (Roche Diagnostics), as previously described (1). Briefly, total RNA was isolated from ventricular tissue with the RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA). The RNA was reverse transcribed, and the cDNA used to derive a PCR product for NHE1 and ribosomal protein L7. The PCR product was cloned into the vector, pBlue-Topo (Invitrogen, Carlsbad, CA). The plasmid underwent amplification, DNA purification, and in vitro transcription. The resulting RNA was recovered, quantified, and used as standards in the RT-PCR reaction. Gene expression was normalized to ribosomal protein L7. The following primers were used: NHE1 (forward) 5′-TTTCCCCgATTTCCTTCTCT-3′; NHE1 (reverse) 5′-gCgTgTAAgACCTgggACAT-3′; Ribosomal protein L7 (forward) 5′-gAAgCTCATCTATgAgAAggC-3′; Ribosomal protein L7 (reverse) 5′-CAgACggAgCAgCTgCAgCAC-3′. We also determined the levels of NHE1 by Western blot analysis of left ventricular proteins separated by 8% SDS-PAGE with anti-NHE1 antibody (Chemicon, 1:1,000), using an HRP-labeled secondary antibody and visualization using ECL (Amersham Pharmacia, Piscataway, NJ).
Data are presented as means ± SE. All curve fittings were achieved using GraphPad Prism (San Diego, CA). One-way ANOVA was used for statistical analysis and Student-Newman-Kuels test for multiple comparisons. P < 0.05 was considered the significant difference among groups.
Data in Table 1 summarize heart, body, and uterine weights of sham, OVX, and OVX +E2 animals. Compared with the shams, OVX rats demonstrated a significant increase in both heart and body weight but no change in heart weight/body wt ratio. As expected, uterine weight was significantly lower in the OVX rats compared with shams and increased significantly upon E2 supplementation. However, uterine weight did not return fully to control levels with E2 supplementation. We think this is likely due to a requirement for other hormones in the regrowth of the uterus.
Cell shortening and intracellular calcium transients in myocytes.
Table 2 summarizes data comparing mechanics of ventricular myocytes from sham, OVX, and OVX + E2 rat hearts. OVX induced a change in all parameters. The percent cell shortening was significantly depressed by ∼22%, as was +dL/dt, the maximum velocity of shortening by ∼30%, and −dL/dt, the maximum velocity of relengthening by ∼50%. The prolongation of the time to peak amplitude of shortening and the time to 80% full relaxation (T80) also reflected a significant alteration in myocyte shortening and relengthening kinetics. These changes in mechanical properties were only partially restored by E2 supplementation. Percent cell shortening and T80 were both significantly different from OVX myocytes, but other parameters were not. As indicated by the data in Table 3, which summarizes our measurements of intracellular Ca2+ transients, the depression in percent cell shortening induced by OVX was associated with a significant fall in the peak amplitude of the Ca2+ transients with no change in diastolic levels of Ca2+ or the time to peak amplitude. The time to 50% decay of the Ca2+ transient (T50) was significantly prolonged by OVX and restored to control levels by E2 supplementation. Moreover, in contrast to the findings from measurements of percent cell shortening, the reduction in amplitude of Ca2+ transients and prolonged time of Ca2+ decay in OVX myocytes could be completely restored with E2 supplementation.
Previous studies demonstrated that compared with controls, ATPase rate (32) and force (33) of myofilaments isolated from hearts of OVX rats are more sensitive to Ca2+. To determine whether the increase in myofilament sensitivity occurred in the myocytes from OVX hearts, we performed a set of experiments, in which we varied extracellular Ca2+ to generate a range of increasing levels of the Ca2+ transient. We determined the relationship between the peak amplitude of these Ca2+ transients (expressed as 340:380 ratio) and the extent of cell shortening. Figure 1 summarizes the data for the level of Ca2+-inducing half-maximal activation of shortening in myocytes from shams, OVX, and OVX + E2 hearts. The value of the peak amplitude of Ca2+ transient that gave rise to 50% of the maximum cell shortening was significantly diminished in OVX myocytes (Fura-2 ratio = 2.52 ± 0.05 in sham controls, and Fura-2 ratio = 2.11 ± 0.04 in OVX myocytes) and was partially restored in the myocytes from OVX + E2 (Fura-2 ratio = 2.30 ± 0.04). These results indicated that the previous in vitro data, indicating an enhanced myofilament response to Ca2+ associated with OVX also occurs in the intact cardiac myocytes.
Acidosis, myofilament tension, and myocyte function.
In view of the cardioprotective effects of E2 in acidosis (1) and metabolic inhibition (28) and inasmuch as altered myofilament response to Ca2+ is a dominant mechanism responsible for the fall in tension associated with acidosis (26), we compared the effects of pH on pCa-tension relations of skinned fiber bundles isolated from sham, OVX, and OVX + E2 rat hearts. We also determined the relative effects of hypercapnic acidosis on shortening and intracellular Ca2+ of myocytes isolated from sham, OVX, and OVX + E2 rat hearts.
Figure 2A shows the relations between pCa and relative tension at pH 7.0, pH 6.8, and pH 6.5 for skinned fiber bundles from the three groups of rat hearts. These results demonstrate the well-known effect of acidic pH to desensitize cardiac myofilaments to Ca2+. Data in Fig. 2B summarize the half-maximal activating pCa values (pCa50). In agreement with previous results (33), the skinned fibers from OVX rat hearts demonstrated a significant increase in sensitivity to Ca2+ at pH 7.0 (pCa50 = 5.84 ± 0.02) compared with fibers from shams (pCa50 = 5.77 ± 0.02). Skinned fibers from hearts of OVX rats supplemented with E2 had a sensitivity to Ca2+ essentially the same as controls (pCa50 = 5.75 ± 0.03). However, at pH 6.8 and 6.5, there were no significant differences in the pCa50 among the fiber bundles from the three groups. In all cases, there were no significant differences in the Hill coefficient among the fiber groups.
We also determined the relative effects of hypercapnic acidosis on shortening and intracellular Ca2+ of myocytes isolated from sham, OVX, and OVX + E2 rat hearts. Intracellular pH fell from a value of pH 7.25 ± 0.08 in control conditions to pH 6.65 ± 0.07 during perfusion with KH buffer saturated with 15% CO2-85% O2. The top three records in Fig. 3A show representative traces of myocyte shortening before, during, and after these changes in pH. In myocytes from sham rat hearts, about 3 min after introduction of the hypercapnic KH buffer, the extent of cell shortening fell significantly before rising to a steady-state value after about 5 min. This steady-state level of extent of shortening remained significantly lower (56.4 ± 3.9% reduction) than the value before acidosis and abruptly returned to this control level with an overshoot after reintroduction of the control KH buffer. However, although myocytes from OVX hearts demonstrated the initial fall in extent of shortening similar to that of the sham control myocytes, extent of shortening rose to a steady-state level 99.8 ± 5.4% of the value in the control KH buffer. This resistance to hypercapnic acidosis was partially reversed (72.0 ± 2.8% reduction in cell shortening) in myocytes from hearts of the OVX + E2 rats but remained significantly higher (P < 0.01) than during the control period. The lower 3 traces in Fig. 3A show typical records of myocyte shortening obtained in the presence of cariporide, an inhibitor the NHE1 exchanger (11). Regardless of the source, the myocytes demonstrated a lack of biphasic response and essentially the same extent of severe reduction in steady-state shortening with hypercapnic acidosis. Figure 3B summarizes the change in extent of shortening at 5 min, 10 min, and with recovery from acidosis. These data show that the significant protection of OVX myocytes from acidosis was not present in the myocytes treated with cariporide. To determine whether cariporide might affect myofilament response to Ca2+, we compared pCa-tension relations in the presence and absence of 1 μM cariporide. These experiments (data not shown) demonstrated that cariporide does not exert any direct effects on myofilament response to Ca2+.
Data in Fig. 4 summarize the percent change in peak amplitude of intracellular Ca2+ transients in the cardiac myocytes from the three groups in the absence and presence of cariporide before, at 5 min and at 10 min of acidosis, and during recovery after hypercapnic acidosis. In the absence of cariporide, the Ca2+ transient increased significantly at 10 min in myocytes from the OVX and OVX +E2 rat hearts. However, there were no changes in the peak amplitudes of the Ca2+ transients at any time in the presence of cariporide. The data in Figs. 3 and 4 indicate that the contribution of the activity of the NHE1 and NCX in regulation of intracellular Ca2+ was relatively high in myocytes from OVX rat hearts compared with shams. We, therefore, determined the level of expression of NHE1 message and protein in the hearts from the three groups of rats. Figure 5A shows results of experiments, in which we quantified the level of expression of the NHE1 gene employing real-time quantitative RT-PCR with SYBR Green detection. The data showed no significant differences. This was also true at the protein level, as illustrated in Fig. 5B, which shows a representative Western blot, indicating that there was no significant difference in expression of NHE1 among the sham, OVX, and OVX + E2 hearts. This determination was made three times with no difference in protein level detected.
Our study is the first to report an effect of ovariectomy to enhance Ca2+-sensitivity of myofilaments functioning in the intact cardiac myocyte. An important new finding with regard to this altered response to Ca2+ is that although it appears to occur at pH 7.0, the sensitization did not occur at pH 6.8 or pH 6.5. This result adds new insights with regard to the interpretation of the significance of the altered myofilament response to Ca2+ in the physiology and pathology of the myocardium. Our data are also the first to demonstrate a significant role of enhanced NHE1 activity in altering the response of heart cells from OVX rats to the stress of acidosis.
There is now general agreement that altered myofilament response to Ca2+ is a significant aspect of the integrated biology of cardiac function (27). A mechanism involving sarcomere length-dependent alterations in myofilament response to Ca2+ remains the dominant theory for the cellular basis of Starling's Law of the heart (22). Moreover, there is substantial evidence for the functional significance of myofilament protein phosphorylation as a significant factor controlling cardiac dynamics (25). As illustrated by the data presented here, depressed cardiac function in acidosis also reflects an influence of intracellular environment to depress myofilament response to Ca2+, a mechanism that is dominant in determining cellular function. For example, Figs. 4 and 5 summarized data showing a severe depression in cell shortening with acidosis in the presence or absence of cariporide with little or no change in the peak amplitude of the Ca2+ transient. Chronic increases in cardiac myofilament response to Ca2+, which our previous (32, 33) and present data indicate occur with ovariectomy, are now also recognized to be of major significance as a cause of hypertrophy associated with mutations in sarcomeric proteins (9, 10). The increased Ca2+ sensitivity of OVX cardiac myofilaments is also similar to that reported in cardiomyopathic and heart failure models (10, 36). The elevated myofilament response to Ca2+ may induce diastolic defects leading to cell stretch and elaboration of signals promoting hypertrophic growth (7).
The effects of chronic increases in myofilament response to Ca2+ are, however, complex inasmuch as there is evidence of the beneficial effects of therapeutic agents, which induce myofilament Ca2+ sensitization (12). Moreover, hearts of transgenic mouse models with constitutive myofilament Ca2+-sensitization associated with modification of troponin I demonstrate remarkable resistance to acidosis (32, 35), sepsis (15), and ischemia/reperfusion (2, 6). An important distinction is that compared with controls, the myofilaments containing modified troponin I or treated with Ca2+-sensitizing agents are not only more sensitive to Ca2+ they are resistant to deactivation by acidic pH (26, 39), whereas the myofilaments from OVX hearts are not.
To more clearly understand whether the increase in myofilament response to Ca2+ associated with ovariectomy is an adaptive or a maladaptive response, we stressed myocytes with acidosis. We chose this approach because of the dominance of depressed myofilament Ca2+ responsiveness in acidosis, and because of its relevance to ischemia and ischemia/reperfusion injury (2). We found that the acidosis-induced depression of shortening of myocytes isolated from hearts of OVX rats was blunted compared with the controls. Although this blunting may have been due, in part, to the enhanced sensitivity of the myofilaments to Ca2+, data shown in Fig. 2 indicate that the sensitization is apparent only at pH 7.0.
It is likely that this increase in Ca2+ during acidosis in myocytes from OVX hearts and not in controls occurred as a result of increased Na exchange associated with stimulation of the NHE1 and a resultant decrease in the efflux of Ca2+ through NCX. This could be due either to a reduced Ca2+ efflux or increased influx via reverse mode activity of the NCX. It is also possible that the elevation of intracellular Ca2+ is due, in part, to a concerted depression in Ca2+ uptake by the SR, which was demonstrated in earlier studies to occur in OVX rat hearts (4). Yet, inhibition of the differential effects of acidosis on myocytes from OVX and sham hearts by cariporide demonstrated the critical role of NHE1 in this response. We did not find a difference in expression of NHE1 among the three groups studied. We therefore conclude that compared with controls, the loss of ovarian sex hormones may induce a sensitization of NHE1 activity to pH. This conclusion fits with studies investigating acute effects of E2, which reported administration of E2 to hearts during ischemia, and reperfusion limited the rise in Ca2+ occurring with ischemia and reperfusion (1). By use of inhibitors, these studies provided evidence that acute administration of E2 increased nitric oxide synthase (NOS) activity and nitric oxide (NO) release and thereby depressed the activity of the NHE1, resulting in a decreased intracellular Na and a limited rise in Ca2+ during ischemia/reperfusion. A role for NOS has been documented as a basis for the difference in control of NCX activity between hearts of male and female rats (5, 17). Our data provide evidence that the protective effect of E2 in ischemia and reperfusion occurs during chronic depressions in levels of ovarian sex hormones. In this case, the withdrawal of E2 would release NHE1 from inhibition by NO, resulting in an enhanced activity, which in concert with altered NCX activity, led to elevated intracellular Ca2+. Morimoto et al. (16) reported an increase in mesenteric endothelial NOS expression associated with E2 treatment of OVX rats. An effect of chronic deprivation of ovarian sex hormones on NOS activity is an attractive mechanism, but whether this occurs with ovariectomy awaits further investigation.
This work was supported by National Institutes of Health Grants RO1 HL 64035 (R. J. Solaro), PO1 HL 62426 (R. J. Solaro), and RO1 HL 64209 and 79032 (B. M. Wolska). Dr. Bupha-Intr also received support as a Golden Jubilee predoctoral fellow of Mahidol University, Bangkok, Thailand.
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