We evaluated effects of age on components of excitation-contraction (EC) coupling in ventricular myocytes from male and female rats to examine sex differences in mechanisms responsible for age-related contractile dysfunction. Myocytes were isolated from anesthetized young adult (∼3 mo) and aged (∼24 mo) Fischer 344 rats. Ca2+ concentrations and contractions were measured simultaneously (37°C, 2 Hz). Fractional shortening declined with age in males (6.7 ± 0.6% to 2.4 ± 0.4%; P < 0.05), as did peak Ca2+ transients (47.7 ± 4.6 to 28.1 ± 2.1 nM; P < 0.05) and Ca2+ current densities (−7.7 ± 0.7 to −6.2 ± 0.5 pA/pF; P < 0.05). Although sarcoplasmic reticulum (SR) Ca2+ content was similar regardless of age in males, EC coupling gain declined significantly with age to 55.8 ± 7.8% of values in younger males. In contrast with results in males, contraction and Ca2+ transient amplitudes were unaffected by age in females. Ca2+ current density declined with age in females (−7.5 ± 0.5 to −5.1 ± 0.7 pA/pF; P < 0.05), but SR Ca2+ content actually increased dramatically (49.0 ± 7.5 to 147.3 ± 28.5 nM; P < 0.05). Even so, EC coupling gain was not affected by age in female myocytes. Age also promoted hypertrophy of male myocytes more than female myocytes. Age and sex differences in EC coupling were largely maintained when conditioning pulse frequency was increased to 4 Hz. Contractions, Ca2+ transients, and EC coupling gain were also smaller in young females than in young males. Thus age-dependent changes are more prominent in myocytes from males than females. Increased SR Ca2+ content may compensate for reduced Ca2+ current to preserve contractile function in aged females, which may limit the detrimental effects of age on cardiac contractile function.
- calcium release
- calcium transients
in humans, aging causes significant changes in contractile function in the heart, even in the absence of cardiovascular disease (33). Studies in healthy humans with no risk factors for cardiovascular disease have shown that cardiac contractile function is relatively well preserved at rest regardless of age (32, 33). However, the ability to increase contractile force in response to increased demand is compromised in older adults and myocardial relaxation is slowed (32, 33). Many of these changes are also seen in hearts from animal models of aging. In isolated cardiac tissues and intact hearts, peak contractions are unaffected by age at low pacing rates but contractions are smaller and slower in aged muscles than in younger muscles at rapid rates or when β-adrenergic receptors are stimulated by catecholamines (2, 5, 6, 18, 23, 31, 36, 43, 47, 48, 57). This deficit in contractile function arises, at least in part, because the ability of individual ventricular myocytes to contract deteriorates with age. Both peak contractions and the underlying Ca2+ transients decrease with age, especially when myocytes are paced at rapid rates or stimulated with catecholamines (27, 37, 58). These findings indicate that cellular modifications in the events that link excitation to release of sarcoplasmic reticulum (SR) Ca2+ and contraction, a process known as excitation-contraction (EC) coupling, occur in the aging heart.
Historically, perhaps due to concerns about the estrus cycle, virtually all investigations of the impact of age on cardiac contractile function have either used hearts and cells from male animals (e.g., 1, 2, 5, 6, 18, 22, 23, 31, 36, 43, 47, 48, 51, 57, 58) or have not specified the sex of the animals used (27, 37). Thus whether age affects cardiac contractile function in females remains to be clarified. Previous studies in young adult hearts have shown that there are sex differences in cardiac contractile function, and these differences are present at the level of the individual ventricular myocyte. Contractions and Ca2+ transients are smaller and slower in myocytes from young adult female rats when compared with males, and these sex differences are exacerbated by stimuli that increase demand, such as rapid pacing rates (8, 12, 34, 55). Importantly, we recently discovered that the impact of age on cardiac EC coupling is markedly influenced by the sex of the animal. We compared young adult (5–7 mo) and aged (24 mo) mice of both sexes and found that peak contractions and Ca2+ transients decline with age in ventricular myocytes from male mice but not in cells from female mice (21). These observations suggest that age-related changes in EC coupling in murine ventricular myocytes are influenced markedly by the sex of the animal.
These sex differences are potentially very important, since modifications in cardiac contractile function are certain to interact with disease in the aging heart. Furthermore, a growing body of evidence suggests that age-related changes in the heart may be more prominent in men than in women. For example, studies with echocardiography (11, 45) or cardiac catheterization (24) have shown that left ventricular contractility is greater in older women than in older men. These sex differences in contractile function may help explain why older men and women are susceptible to different cardiac diseases (13). Still, few studies have used aged female animals, so relatively little is known about the impact of age on specific components of cardiac EC coupling. Furthermore, whether the protective effect of female sex on cardiac contractile function applies only to aged murine myocytes or whether it can be generalized to other models of aging is not known.
The primary objective of this study was to evaluate the impact of age and sex on cellular mechanisms of EC coupling in ventricular myocytes from a commonly used animal model of aging, the Fischer 344 rat. These investigations compared amplitudes of contractions, Ca2+ transients, peak Ca2+ current density, and SR Ca2+ content in voltage-clamped ventricular myocytes isolated from young adult (∼3 mo) and aged (∼24 mo) Fischer 344 rats of both sexes. Studies were conducted at physiological temperature in cells paced with conditioning pulse trains at frequencies of 2 and 4 Hz.
MATERIALS AND METHODS
All animal protocols in this study conformed to the guidelines published by the Canadian Council on Animal Care (Vol. 1, 2nd edition, 1993; Vol. 2, 1984), and the Dalhousie University Committee on Laboratory Animals approved all protocols. Fisher 344 rats of both sexes were obtained from either Charles River Laboratories or the National Institute on Aging (Baltimore, MD). Young adult rats were ∼3 mo old, and aged rats were ∼24 mo old. Animals were housed in microisolator cages in the Animal Care Facility at Dalhousie University. The animals were maintained on a 12-h:12-h light/dark cycle, with free access to food and water.
Rat ventricular myocytes were isolated by enzymatic dissociation as described previously (41). Animals were weighed and injected with heparin (3,000 U/kg ip) 30 min before they were anesthetized with pentobarbital sodium (220 mg/kg ip). The aorta was cannulated in situ, and hearts were perfused at 18–20 ml/min for 5 min with perfusion buffer that contained (in mM) 135.5 NaCl, 4 KCl, 10 HEPES, 1.2 MgSO4, 1.2 KH2PO4, 12 glucose, and 200 μM CaCl2 (pH 7.4, gassed with 100% O2, 37°C). The heart was next perfused for an additional 5 min with nominally Ca2+-free perfusion buffer. Hearts were then perfused with the same buffer supplemented with protease dispase II (Roche Diagnostics; 0.10–0.14 mg/ml), collagenase type 2 (240 U/mg; Worthington, Lakewood, NJ; 0.3–0.6 mg/ml), and 50 μM Ca2+ for 15–20 min. The ventricles were next isolated, minced in a high potassium buffer of the following composition (in mM): 80 KOH, 30 KCl, 3 MgSO4·7H2O, 30 KH2PO4, 50 L-glutamic acid, 20 taurine, 0.5 EGTA, 10 HEPES, and 10 glucose (pH 7.4 with KOH) and filtered through a 225-μm polyethylene filter. Experiments used only quiescent, rod-shaped myocytes with clear striations and no visible membrane damage. The yield of viable myocytes was between 40% and 60% for young adult hearts and 30–50% for aged hearts as in previous studies of young adult and aged rat ventricular myocytes (17, 35, 41).
Young adult or aged myocytes were loaded with fura-2 AM (5 μM) and incubated in the dark for 30 min at room temperature. An aliquot of cell suspension was placed in a 0.75-ml chamber on the stage of an inverted microscope. After 15 min, the cells were superfused at 3 ml/min with a buffer solution of the following composition (in mM): 145 NaCl, 10 glucose, 10 HEPES, 4 KCl, 1.8 CaCl2, and 1 MgCl2 (pH 7.4 with NaOH). In all experiments reported here, the buffer solution contained 4-aminopyridine (2 mM) to inhibit transient outward current and lidocaine (0.3 mM) to inhibit sodium current. All experiments were performed at 37°C.
Contractions and Ca2+ transients were recorded simultaneously. Contractions were recorded as unloaded cell shortening at 120 Hz with a video edge detector (Crescent Electronics, Sandy, UT). Cell images were viewed with a charge-coupled device (CCD) camera (model TM-640; Pulnix America) and displayed on a television monitor. At the start of each experiment, resting cell length and width were measured with the edge detector. Ca2+ transients were recorded with a PTI (Photon Technology International, Birmingham, NJ) fluorescence system as described below. Cells were voltage-clamped with high resistance microelectrodes (15–25 MΩ; filled with filtered 2.7 M KCl) to minimize disruption of the intracellular milieu. Experiments used discontinuous single electrode voltage clamp techniques (5–8 kHz) with an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA) and ClampEx software (version 8.2; Molecular Devices, Sunnyvale, CA). Cells were held at −80 mV. All test steps were preceded by 10 50-ms conditioning pulses from −80 to 0 mV delivered at either 2 or 4 Hz as described in results. Following each conditioning pulse, cells were repolarized for either 450 or 200 ms to achieve stimulation frequencies of 2 or 4 Hz, respectively. After the last conditioning pulse, cells were repolarized to −40 mV and then depolarized to 0 mV for 250 ms to simultaneously activate contractions, Ca2+ transients, and Ca2+ currents.
Fura-2 fluorescence was recorded and measured as described previously (41). Briefly, fura-2 was excited at 340 and 380 nm and fluorescence emission was measured at 510 nm with a DeltaRam fluorescence system (PTI) and Felix software (version 1.4; PTI). Fluorescence emission was measured at a sampling interval of 5 ms for each excitation wavelength. Emission ratios corresponding to 340/380 nm were converted to Ca2+ concentration with an in vitro calibration curve determined with known concentrations of Ca2+, as in our previous studies (e.g., 41). Contractions and fluorescence were recorded simultaneously, by splitting the light from the microscope with a dichroic cube. The dichroic cube sent the red light to the television camera and edge detector system and sent the remaining light to the photomultiplier tube for fluorescence measurement.
A rapid solution switcher was used to briefly expose myocytes to buffer solution supplemented with 10 mM caffeine to estimate SR Ca2+ load. The solution switcher maintained the solution temperature at 37°C throughout experiments. In these experiments, cells were voltage-clamped and stimulated with conditioning pulses delivered at frequencies of 2 or 4 Hz as described above. Following the conditioning pulse train, cells were repolarized to −40 mV, and after 500 ms, caffeine was applied for 1 s in a buffer solution of the following composition (in mM): 10 caffeine, 140 LiCl, 4 KCl, 10 glucose, 5 HEPES, 4 MgCl2, 2 4-aminopyridine, and 0.3 lidocaine. Under these experimental conditions, caffeine application did not induce inward Na+-Ca2+ exchange current, as shown previously (14, 29).
Contraction amplitude was measured as the difference between systolic and diastolic cell length. Contractions were normalized as a percentage of resting cell length to account for differences in cell length between groups. The time between initiation of contraction and maximal cell shortening was recorded as the time-to-peak contraction. The time required for contraction to relax by 50% from the peak of the contraction was recorded as the half-relaxation time. Ca2+ transient amplitudes were measured as the difference between systolic and diastolic Ca2+ concentrations for depolarization and caffeine-induced contractures (called the contracture Ca2+). The rates of rise, times-to-peak, and 90% decay times of depolarization-induced Ca2+ transients also were measured. Fractional release of Ca2+ from the SR was calculated by dividing the amplitude of the Ca2+ transient by the amplitude of the contracture Ca2+. Peak inward Ca2+ current was measured as the difference between peak inward current and a reference point at the end of the voltage step. In some experiments, we confirmed that the current measured under our experimental conditions was Ca2+ current. We recorded currents in the absence and presence of a rapid switch to cadmium (200 μM), to block Ca2+ current. We found that cadmium reduced peak Ca2+ current from −1.60 ± 0.53 nA to −0.10 ± 0.06 nA (n = 4) in myocytes from young adult males. Similar results were obtained in cells from young adult females and in cells from aged animals of both sexes; cadmium blocked more than 90% of the Ca2+ current in all groups. Cell capacitance was determined by integrating capacitive transients with pCLAMP software and Ca2+ currents were normalized by cell capacitance to account for differences in cell size between groups. Ca2+ current inactivation was measured with Clampfit 8.1 (Axon Instruments, Union City, CA). The time constant (tau) for inactivation of the Ca2+ current was calculated from a single exponential function that was fitted to the decay phase of the current recordings. The gain of EC coupling was calculated for each cell from paired measures of the rate of rise of the Ca2+ transient (in nM/s) and the peak normalized Ca2+ current (in pA/pF). Gain was expressed as the rate of rise of the Ca2+ transient per unit Ca2+ current.
Statistical analyses were performed with SigmaStat (version 3.1; Systat Software). Differences between means for young adult and aged myocytes were evaluated by two-way ANOVA, with sex and age as main factors. Specific differences between groups were determined by means of a Student-Newman-Keuls post hoc test with a Bonferroni correction. All graphs were constructed with Sigmaplot (version 8.0; Systat Software). Data are presented as means ± SE. Differences were considered significant if P < 0.05.
Lidocaine, HEPES buffer, EGTA, MgCl2, anhydrous dimethyl sulfoxide (DMSO), 4-aminopyridine and caffeine were purchased from Sigma-Aldrich Canada. (Oakville, ON, Canada). Fura-2 AM was obtained from Invitrogen (Burlington, ON, Canada). All other chemicals were purchased from BDH (Toronto, ON, Canada). Stock solutions of Fura-2 AM were prepared in anhydrous DMSO, with a final concentration of 0.2% DMSO, and stored at −20°C until use. All other chemicals were dissolved in deionized water.
Physical characteristics of the animals used in this study are shown in Table 1. Young adult animals were ∼3 mo of age, whereas aged animals were ∼24 mo old. There was no significant difference in age between males and females in either the young adult or the aged groups (Table 1). Aged animals were heavier than young adult animals, and male rats were heavier than females, regardless of age (Table 1). Physical characteristics of the ventricular myocytes used in this study also are shown in Table 1. Cell length increased with age in both groups, but it increased significantly more in males than in females. Cell width increased with age in myocytes from males, but not females. Table 1 also shows that cell area increased with age in males (from 2557.1 ± 96.9 to 4081.9 ± 147.6 μm2; P < 0.05) and in females (from 2403.1 ± 127.3 to 2906.8 ± 114.8 μm2; P < 0.05), although this increase was considerably greater in males than in females. In contrast with cell size, there was no significant increase in cell capacitance with age (Table 1). To compensate for differences in cell size between groups, we normalized contractions by cell length in all experiments. We also normalized currents by cell capacitance.
To evaluate the impact of sex on age-related changes in EC coupling, we compared characteristics of contractions, Ca2+ transients, and Ca2+ currents in voltage-clamped ventricular myocytes from young adult and aged rats of both sexes. Ca2+ transients, contractions, and Ca2+ currents were activated by the voltage-clamp protocol shown at the top of Fig. 1. Figure 1A shows representative examples of Ca2+ transients (top), contractions (middle), and Ca2+ currents (bottom) recorded from young adult and aged myocytes isolated from male rats. Similar recordings from female cells at both ages are shown in Fig. 1B. Mean values for peak contractions, Ca2+ transients, and Ca2+ currents are presented in Fig. 1 and are summarized in Table 2. For mean data, contractions were normalized to cell length to calculate fractional shortening in all groups (Fig. 1C). Figure 1C shows that mean fractional cell shortening decreased markedly with age in cells from males. Fractional shortening declined from 6.7 ± 0.6% in young adult males to 2.4 ± 0.4% in aged males. In contrast, fractional cell shortening did not change with age in myocytes from females. Values were 4.9 ± 0.7% versus 4.9 ± 0.5% in young adult and aged animals, respectively (Fig. 1C and Table 2). There also were sex differences in fractional shortening. Contractions were smaller in young adult female cells than in young adult males, but larger in aged females compared with aged males (Fig. 1C and Table 2). Mean amplitudes of Ca2+ transients declined with age from 47.7 ± 4.6 nM to 28.1 ± 2.1 nM in myocytes from males (Fig. 1D and Table 2; P < 0.05). In contrast, peak Ca2+ transients were not affected by age in cells from females (Fig. 1D). There were no other age- or sex-associated differences in the size of Ca2+ transients between groups. Mean amplitudes of Ca2+ currents are shown in Fig. 1E. Ca2+ currents were normalized by cell capacitance to compensate for differences in cell size between groups. Mean data show that Ca2+ current density was smaller in aged cells than in young adult cells, regardless of sex (Fig. 1E and Table 2).
We also compared the time constant for inactivation of Ca2+ current (tau) between groups by fitting a single exponential function to the decay of the current. The mean time constant for inactivation of the Ca2+ current was prolonged by age in male myocytes (values were 9.5 ± 0.4 ms vs. 11.4 ± 0.5 ms for 18 young adult and 15 aged male cells, respectively; P < 0.05). In contrast, the values of tau were similar in young adult and aged female cells, although inactivation was prolonged in young adult females compared with young adult males (values were 11.1 ± 0.6 ms vs. 11.5 ± 0.5 ms for 13 young adult and 13 aged female cells, respectively). Thus Ca2+ current density declined with age regardless of the sex of the animal, but inactivation of the Ca2+ current was prolonged only in myocytes from aged males.
We determined whether there were age and sex differences in the time courses of contraction and Ca2+ transients in these studies. Figure 2 shows mean data for the time courses of contraction (Fig. 2, A and B) and Ca2+ transients (Fig. 2, C and D). There were few age- or sex-related differences in the time course of contraction. Time-to-peak contraction decreased slightly with age in cells from males (Fig. 2A), although half-relaxation times were similar in all groups (Fig. 2B). Time-to-peak Ca2+ transient was prolonged in young adult female cells compared with males (Fig. 2C). Ca2+ transients also decayed more slowly in cells from aged males compared with young adult males (Fig. 2D). Mean values for the 90% decay times were 136.5 ± 17.2 ms vs. 203.5 ± 17.7 ms for 19 young adult and 23 aged male cells, respectively (P < 0.05). In addition, Ca2+ transients decayed significantly more slowly in aged male myocytes than in aged female cells (Fig. 2D). These results demonstrate that aging increased the duration of Ca2+ transients in myocytes from males but not females.
To determine whether differences in the amount of SR Ca2+ available for release contributed to age- and sex-differences in Ca2+ release, we assessed SR Ca2+ stores by rapid application of 10 mM caffeine as described in methods. Representative examples of contracture Ca2+ recordings are shown in Fig. 3A. Figure 3B shows that mean amplitudes of contracture Ca2+ were not affected by age in cells from males. In contrast, the size of the contracture Ca2+ transient increased dramatically with age in cells from females (Fig. 3B). Values for contracture Ca2+ increased from 49.0 ± 7.5 nM in young females to 147.3 ± 28.5 nM in aged females (Table 2; P < 0.05). Figure 3C shows the fraction of Ca2+ released from the SR per beat in each of the four groups examined in this study. Fractional release was calculated as the peak Ca2+ transient (as in Fig. 1D) divided by the peak contracture Ca2+ for each cell. Results showed that fractional release declined with age in both males and females (Fig. 3C). Fractional release declined with age from 71.6 ± 5.2% to 48.7 ± 4.3% of SR Ca2+ in males and 65.5 ± 5.1% to 37.3 ± 5.2% of SR Ca2+ in females (Table 2; P < 0.05). Diastolic Ca2+ levels also were measured in these experiments (Fig. 3D). Diastolic Ca2+ was measured at −80 mV, just before the conditioning pulse train. We found that diastolic Ca2+ levels were not affected by age, although diastolic Ca2+ was lower in young adult females than in young adult males (Fig. 3D). These observations indicate that fractional release of SR Ca2+ declined with age in all groups. This occurred because even though the size of the Ca2+ transient did not decrease with age in females, the amount of Ca2+ available for release in the SR increased markedly with age in the female group.
To determine whether the amount of SR Ca2+ released per unit Ca2+ current was affected by the age and sex of the animal, we next calculated the gain of EC coupling (15). The maximum rate of rise of the Ca2+ transient was first calculated to examine and compare SR Ca2+ release flux between groups. The mean rate of rise of the Ca2+ transient decreased markedly with age in male cells (Fig. 4A). Furthermore, the rate of rise of the Ca2+ transient was lower in young female myocytes when compared with young males (Fig. 4A). We then calculated the gain of EC coupling by dividing the rate of rise of the Ca2+ transient by Ca2+ current density (Ca2+ current data shown in Fig. 1E). Figure 4B shows that the gain of EC coupling declined with age in cells from males, but not in cells from females. Gain declined with age to 55.8% of the values observed in young adult males (Table 2; P < 0.05). EC coupling gain was also lower in young female myocytes than in young male cells (Fig. 4B). These results demonstrate that both SR Ca2+ release flux and the gain of EC coupling were reduced in cells from aged males. Ca2+ release flux and EC coupling gain were also much lower in young adult female myocytes when compared with age-matched males.
To determine whether differences in the voltage dependence of EC coupling parameters might account for the age- and sex-differences observed in our study, in some experiments we activated Ca2+ transients and Ca2+ currents by test steps to +20 and +40 mV. The results of these experiments are presented in Table 3. Our results show that peak Ca2+ current activated by a test step to +20 mV declined with age in both males and females, whereas the associated Ca2+ transient declined with age only in males (Table 3). The gain of EC coupling at +20 mV was higher in young males than in aged males, although this effect was not statistically significant (Table 3). When Ca2+ transients and Ca2+ currents were activated by test steps to +40 mV, the size of the peak responses declined in all groups and differences between groups were no longer statistically significant (Table 3). EC coupling gain was also not significantly different between groups in these experiments. These findings indicate that the age and sex-dependent differences in Ca2+ transients and Ca2+ currents that we observed when cells were depolarized to 0 mV are present at +20 mV, although the effect on EC coupling gain is not.
Since previous studies have shown that contractile dysfunction in aging is exacerbated by rapid pacing (e.g., 37), we determined whether a deficit in contractile function would emerge in female myocytes when pacing frequency was increased from 2 to 4 Hz. A frequency of 4 Hz is close to the physiological heart rate in a rat. Figure 5A shows representative examples of Ca2+ transients, contractions, and Ca2+ currents recorded from myocytes isolated from young adult and aged male rats. Similar recordings from female cells at both ages are shown in Fig. 5B. Mean amplitudes of contractions declined with age in cells from male rats but not in myocytes from female animals (Fig. 5C). Contractions also were larger in young adult males than in young females (Fig. 5C). Ca2+ transients declined with age in males and not in females (Fig. 5D). These results are virtually identical to the results we obtained when cells were paced at 2 Hz (Fig. 1, C and D). Figure 5E shows that peak Ca2+ current amplitude declined with age in females but not in males.
To determine whether pacing frequency would affect age-dependent changes in caffeine-induced Ca2+ release, we compared amplitudes of contracture Ca2+ recordings after a 4-Hz conditioning pulse train as shown at the top of Fig. 6. Figure 6A shows examples of contracture Ca2+ recordings from young adult and aged myocytes isolated from male and female hearts. Mean amplitudes of contracture Ca2+ transients were not affected by age in cells from male animals (Fig. 6B). In contrast, peak contracture Ca2+ transients were significantly larger in aged female cells compared with young adult females (Fig. 6B). These observations demonstrate that SR Ca2+ stores increased with age in female myocytes, regardless of pacing frequency. We also calculated the gain of EC coupling in these experiments. Figure 6 shows that the average Ca2+ release flux (Fig. 6C) and the mean gain of EC coupling (Fig. 6D) declined with age in myocytes from male rats but not in cells from female rats when the stimulation frequency was increased to 4 Hz. These findings show that age-related contractile deficits are more prominent in ventricular myocytes from males than females, even when the stimulation frequency is increased from 2 to 4 Hz.
The effects of stimulation frequency on key parameters measured in this study are shown in Fig. 7. Data from males are shown on the left, and data from females are presented on the right. Increasing stimulation frequency from 2 to 4 Hz had no significant effect on peak contractions or Ca2+ transients, regardless of the age or sex of the animal used (Fig. 7, A–D). Ca2+ current density declined as stimulation frequency increased in young adult males (Fig. 7E) and the overall effect of frequency on peak Ca2+ current density was significant in males. In contrast, increasing stimulation frequency had no effect on Ca2+ current density in females (Fig. 7F). The overall effect of stimulation frequency on EC coupling gain was significant in males (Fig. 7G), although gain declined with age in males at both frequencies tested. In contrast, gain increased as stimulation frequency was increased in females regardless of age (Fig. 7H). SR Ca2+ content increased when pacing frequency was increased in young adult and aged males (Fig. 7I). However, stimulation frequency had no significant effect on SR Ca2+ content in females, although age increased SR stores at both 2 and 4 Hz (Fig. 7J).
This study determined whether the impact of age on cellular mechanisms of cardiac EC coupling differed between the sexes in rat ventricular myocytes. Results showed that there were marked sex differences in the effect of age on cardiac EC coupling. In myocytes from male rats, age promoted cellular hypertrophy. Still, peak contractions and the underlying Ca2+ transients were significantly smaller in cells from aged males compared with younger males and Ca2+ transients decayed more slowly in male myocytes. Ca2+ current density also was lower in aged males compared with younger males. The amount of Ca2+ available for release in the SR was not affected by age in cells from male rats. Despite this, EC coupling gain declined with age in males. In contrast with our findings in male myocytes, we found that female myocytes showed only a modest degree of age-related cellular hypertrophy. What is more, peak contractions and Ca2+ transients were unaffected by age in female myocytes. Ca2+ current density did decline with age in females, but SR Ca2+ content actually increased dramatically. Despite the increase in SR Ca2+ content, the gain of EC coupling was not affected by age in cells from females. Generally, similar effects of age and sex on components of EC coupling were observed when conditioning pulse frequency was increased from 2 to 4 Hz. Our study also showed that there were sex differences in EC coupling in cells from young adult rats; contractions and Ca2+ transients were smaller and the gain of EC coupling was lower in cells from young females compared with males. These observations demonstrate that components of EC coupling are modified by the sex of the animal and indicate that the impact of age on cardiac EC coupling is more prominent in myocytes from males than in cells from females.
The impact of age on mechanisms of cardiac EC coupling.
Most previous studies of the impact of age on contractile function in ventricular myocytes have used cells from male animals or have not specified sex (27, 37, 56). Our study demonstrated clearly that the ability of ventricular myocytes to contract deteriorated with age in cells from male rats, but not in cells from female rats. These observations agree with results of our previous study in ventricular myocytes from young adult (5–7 mo) and aged (24 mo) mice of both sexes (21). Our study also provides evidence for cellular mechanisms that underlie this age-related deficit in contractile function in male myocytes. We found that peak Ca2+ current declined with age in male cells, as reported previously in aged rat and mouse myocytes investigated at physiological temperatures (21, 38). It is well established that Ca2+ influx through L-type Ca2+ channels triggers the release of a much larger amount of Ca2+ from the SR, a process known as Ca2+-induced Ca2+ release (4). Studies also have shown that Ca2+ release is proportional to the magnitude of the Ca2+ current (4). Therefore, it is likely that a smaller Ca2+ current triggered a proportionally smaller release of SR Ca2+ and activated a smaller contraction in aged male cells.
Our study demonstrated that contractile function did not deteriorate with age in myocytes from female rats. Interestingly, preservation of cardiac contractile function in aged female myocytes has been reported previously in myocytes from mice (21). In addition, contractile function has been reported to actually increase with age in myocytes from female sheep (15). The results of the present study also suggest a mechanism by which cardiac contractile function is preserved in myocytes from aged female rats. As with our results in males, we found that peak Ca2+ current declined with age in cells from females. This would be expected to trigger a smaller release of Ca2+ from the SR and activate a smaller contraction. However, this did not occur. Therefore, it is possible that the increase in SR Ca2+ content observed in aged female myocytes compensates for reduced Ca2+ current and preserves SR Ca2+ release to maintain contraction. Interestingly, although we previously found that SR Ca2+ content increased with age in cells from female mice, peak Ca2+ current did not decline (21), which suggests that the impact of age on mouse and rat cells is similar but not identical.
We also found that the gain of EC coupling declined with age in males. This indicates that the amount of Ca2+ released per unit Ca2+ current is reduced with age in male myocytes. Gain is regulated by SR Ca2+ content and by the activity of the SR Ca2+ release channels (4, 30, 49). Since we found that SR Ca2+ content was unaffected by age, reduced SR Ca2+ availability is unlikely to account for reduced gain in the aging male heart. However, it is possible that aging affects SR Ca2+ release channels and thereby reduces EC coupling gain in the aging heart. SR Ca2+ is released in discrete release units known as Ca2+ sparks (9). Previous studies have shown that the aging process modifies the occurrence and characteristics of spontaneous Ca2+ sparks (26, 60). Indeed, spark amplitude, duration, and width have been shown to decline with age in rat ventricular myocytes, although whether these studies used male or female rats was not specified (60). Thus it is possible that a decrease in unitary Ca2+ release in aging accounts for the decrease in gain observed in aged males in our study. In contrast with our findings in male rats, SR Ca2+ stores increased with age in male mice and therefore gain did not decline with age in mice (21). These findings indicate that age-dependent modifications in EC coupling are similar but not identical in myocytes from mice and rats.
We found that age affected the fraction of Ca2+ released from the SR, independent of the sex of the animals. Indeed, fractional release of SR Ca2+ declined with age in both males and females, although the reasons for the decrease in fractional release differed between the sexes. The decrease in fractional release with age in male cells likely resulted from the age-dependent decrease in trigger Ca2+ influx, which diminished Ca2+-induced Ca2+ release and reduced the magnitude of the Ca2+ transient. Fractional release declined with age in females because, even though the magnitude of the Ca2+ transient did not decline, the amount of Ca2+ in the SR increased markedly with age. Thus the amount of Ca2+ released with each beat in an aged female myocyte represents a smaller proportion of the available SR Ca2+, even though the released Ca2+ activates a full-sized Ca2+ transient.
The present study also found that contractions were significantly smaller in cells from aged males compared with aged females, but Ca2+ transients were similar in the two groups. These findings suggest that myofilament sensitivity to Ca2+ may decline with age in males. Indeed, one previous study has provided evidence that myofilament Ca2+ sensitivity declines with age in male rat hearts (40), although this has not been reported in all studies (47). Still, the results of our study suggest that a decline in myofilament Ca2+ sensitivity contributes to the age-related decrease in contractile function in males, although further experiments will be required to address this possibility experimentally.
Age also affected the time courses of contractions and Ca2+ transients recorded in our study. Time-to-peak contraction was significantly prolonged in young adult males compared with aged males. This difference is surprising, as there is an age-related shift from the faster α-myosin heavy chain to the slower β-isoform in cardiac muscles from male rats (5). We also showed that the decay rate of Ca2+ transients slowed with age in male cells, but not in female cells. Prolongation of the decay of the Ca2+ transient with age has been reported previously in voltage-clamped mouse ventricular myocytes, although the sex of the animals used in these experiments was not specified (27). Our findings extend this observation to demonstrate that slowing of Ca2+ transients with age occurs in cells from males but not females. An increase in Ca2+ influx via reverse-mode Na-Ca2+ exchange could explain the increased duration of Ca2+ transients in aged myocytes. However, there is no evidence for increased reverse mode exchange in aging rat ventricular myocytes (39). Indeed, others have attributed the age-related prolongation of Ca2+ transient decay to a decrease in sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) activity in the aging heart (27). Although the decay of the Ca2+ transient was prolonged by age in males, there was no corresponding increase in the half-relaxation time for contraction. One explanation for these observations is that the age-related decrease in myofilament Ca2+ sensitivity in male myocytes (40) accounts for the absence of contraction during the decay phase of the Ca2+ transient in cells from aged males.
Sex differences in EC coupling in myocytes from young adult rats.
Our study also provides evidence for sex differences in EC coupling in ventricular myocytes from young adult rats. We found that peak contractions were smaller in cells from young adult females when compared with males, as reported in previous studies of field-stimulated ventricular myocytes from young adult rats (12). However, our study also showed that peak Ca2+ transients were similar in the two groups. One explanation for these observations is that myofilaments in cells from male rats may be more sensitive to Ca2+ than myofilaments in females. There is some evidence to support this idea, since cells from ovariectomized female rats have higher myofilament Ca2+ sensitivity than cells from intact females (Ref. 56 but also Ref. 44). Our results also showed that the rate of rise of the Ca2+ transient was slower and Ca2+ transients were prolonged in young females compared with young males, as reported previously in field-stimulated cells from young adult rats (12). The decrease in rate of Ca2+ release in female myocytes did not prolong the time-to-peak contraction. This suggests that the slowed rate of rise of the Ca2+ transient in female myocytes was not sufficient to affect the ability of the myofilaments to generate contraction.
In addition, we found that peak Ca2+ currents were similar in young adult rats regardless of the sex of the animal, as reported previously in rat ventricular myocytes (Ref. 34 but also Ref. 53). Unlike previous studies, we measured Ca2+ transients and Ca2+ currents simultaneously; thus we were able to calculate the gain of EC coupling in young male and female cells. We found that gain was much smaller in young females than in young males. This novel finding suggests that there may be sex differences in unitary Ca2+ release in the young adult heart, although this has not yet been investigated.
The impact of stimulation frequency on age- and sex-dependent changes in EC coupling.
Previous studies have shown that the age-related decline in peak contractions and Ca2+ transients is augmented when myocytes are paced at rapid rates (27, 37). Therefore, we increased the pacing frequency from 2 to 4 Hz to determine whether age-related deficits in contractile function would be augmented by an increase in stimulation frequency. We found that the contractile deficit in aged male cells was similar at the two pacing frequencies. Furthermore, deficits in contractile function did not emerge in aged female myocytes even when stimulation frequency was increased.
The increase in stimulation frequency did affect some EC coupling parameters measured in this study. We found that the increase in pacing rate caused a marked increase in SR Ca2+ content in male cells but not in female cells, regardless of the age of the animal. This agrees with results of a previous study in cats, which showed that rapid pacing did not increase SR Ca2+ load in young adult females but did increase SR Ca2+ load in young adult males (44). There are several reasons why the SR may not load with Ca2+ as effectively in female hearts as in males. Our study indicates that sex differences in the magnitude of the Ca2+ current are an unlikely explanation. However, expression of the Na+-Ca2+ exchanger is increased in female hearts compared with males (10). This could result in more Ca2+ extrusion and less Ca2+ loading in the SR. Sex differences in SERCA activity and/or Ca2+ leak from the SR also could contribute, although there is little evidence for sex differences in the expression of these proteins (8, 10). Our results also showed that the magnitude of the Ca2+ current was smaller at 4 Hz than at 2 Hz in both young adult and aged male cells. This may reflect Ca2+ inactivation of the Ca2+ current at higher pacing rates (4). Why this occurred only in males is not clear, although increased Na+-Ca2+ exchange activity in females (10) could promote Ca2+ extrusion and limit Ca2+ inactivation of the Ca2+ current. Finally, our study showed that increased pacing frequency increased the gain of EC coupling in males and females, regardless of age. EC coupling gain is regulated by SR Ca2+ load and by the activity of the SR Ca2+ release channels (4, 30, 49). The increase in SR Ca2+ content may account for the increase in gain in male cells paced at 4 Hz. Other factors, such as the activity of the SR Ca2+ release channels, may be involved in the increase in gain with pacing frequency in female myocytes.
The peak Ca2+ transients and contracture Ca2+ transients recorded in this study are low when compared with values reported some other studies (e.g., 12, 15). This is likely due, at least in part, to the fact that many previous studies of EC coupling have investigated myocytes at room temperature, whereas the present study investigated cells at 37°C. It is well established that temperature has profound effects on Ca2+ handling and homeostasis in myocytes from various mammals (e.g., 4). Indeed, we previously showed that peak Ca2+ transients, SR Ca2+ load, and EC coupling gain increase dramatically as cells are cooled from 37°C to 22°C (50). The peak Ca2+ transients and caffeine-induced responses recorded in our previous study at 37°C were similar in magnitude to responses recorded in the present study.
Age and biological sex affect ventricular myocyte morphology.
Previous studies of isolated ventricular myocytes in rat models have established that aging is accompanied by a decrease in the total number of myocytes and hypertrophy of the remaining cells (7, 17, 28, 38, 54, 58). These studies used male rats, although one previous study that reported no effect of age on myocyte size used female rats (3). In our study we directly compared ventricular myocytes from young adult and aged male and female rats and found that age-related cellular hypertrophy was much more pronounced in myocytes from male rats when compared with cells from females. A recent study in isolated myocytes from monkeys also showed that myocyte hypertrophy occurred in male hearts but not in female hearts (59), and similar results have been reported in aged mice (21). Furthermore, postmortem histological analysis of the impact of age on myocytes in human hearts showed that cell loss and reactive hypertrophy occurred in hearts from older men but not in hearts of older women (42). Together with the results of our studies, these data indicate that sex differences in the degree of age-related myocyte hypertrophy occur in a number of different mammals including humans. Our study also showed that cell width increased with age in myocytes from males but not females. Increased myocyte width is believed to increase ventricular wall thickness (19). Thus the difference in cell width between the sexes reported in our study may explain why left ventricular wall thickness increases with age in males but not in females (20, 59). Although we found that cell area increased with age in myocytes from males and females, cell capacitance did not increase significantly with age in either group. This suggests that t-tubule area may not increase with age in proportion to the increase in cell size as reported previously in a tachycardia-induced model of myocyte hypertrophy (25). Because the increase in cell size was especially prominent in cells from aged males, this potential decrease in t-tubule area might contribute to the decrease in gain observed in this group, although additional experiments will be required to confirm this possibility.
This study demonstrates that age-associated deficits in contractile function are much more prominent in ventricular myocytes from male rats than in cells from female rats. These observations are consistent with results of our previous study in mice (21). This is an important observation, which shows that the protective effect of female sex on cardiac contractile function at the cellular level is conserved in at least two different species. Although few studies have investigated the impact of sex on age-dependent changes in cardiac contractility, there is some evidence that these sex differences occur in various mammals including humans. In vivo studies have shown that β-adrenergic stimulation causes a smaller increase in contractility in aged male monkeys than in younger males, but responses to β-adrenergic stimulation are not affected by age in females (52). Fractional shortening also is lower in aged male rats compared with aged females (16). In addition, a recent study used echocardiography and showed that left ventricular contractility was greater in older women than in older men (11). These sex differences in contractile function are important, since modifications in cardiac contraction are certain to interact with disease in the aging heart. For example, our findings may help explain why heart failure with normal ejection fraction is common in older women, whereas heart failure with reduced ejection fraction occurs in men (46).
Our study showed that age-related changes in ventricular myocyte dimensions and function are much more pronounced in males than in females. Aged male cells showed marked cellular hypertrophy, whereas female cells did not. Peak contractions, Ca2+ transients, and the gain of EC coupling declined with age in males but not females. Ca2+ currents did decline with age in both sexes. In contrast, SR Ca2+ content was unaffected by age in males, but increased dramatically with age in females. These sex differences were observed regardless of whether cells were paced at 2 or 4 Hz. We also found the contractions and Ca2+ transients were smaller and EC coupling gain was lower in cells from young females compared with young males. These observations show that components of EC coupling are modified by the sex of the animal and demonstrate that age-related contractile dysfunction is more prominent in myocytes from males compared with females. The age-related increase in SR Ca2+ content in female cells may compensate for the decrease in Ca2+ current to maintain contraction. This female advantage may limit detrimental effects of age on cardiac EC coupling.
This study was supported in part by grants from the Canadian Institutes for Health Research and the Heart and Stroke Foundation of Nova Scotia.
No conflicts of interest are declared by the author(s).
We express appreciation for excellent technical assistance provided by Dr. Jiequan Zhu, Peter Nicholl, and Cindy Mapplebeck. We thank Dr. Kenneth Rockwood for valuable comments on an earlier draft of this article.
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