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Am J Physiol Heart Circ Physiol 291: H2362-H2370, 2006. First published May 26, 2006; doi:10.1152/ajpheart.00070.2006
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Cardiac excitation-contraction coupling is altered in myocytes from aged male mice but not in cells from aged female mice

Scott A. Grandy and Susan E. Howlett

Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada

Submitted 17 January 2006 ; accepted in final form 19 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study characterized age-related alterations in excitation-contraction (EC)-coupling in ventricular myocytes and investigated whether these alterations are affected by the sex of the animal. Voltage-clamp experiments were conducted in myocytes from young adult (~7 mo) and aged (~24 mo) male and female mice. Intracellular Ca2+ concentrations and unloaded cell shortening were measured at 37°C with fura-2 and a video edge detector. Fractional shortening and Ca2+ current density were significantly reduced in aged male myocytes compared with those in young adult male cells. In addition, Ca2+ transients were significantly smaller in aged male myocytes. Sarcoplasmic reticulum (SR) content, assessed by rapid application of 10 mM caffeine, declined with age in male myocytes. However, EC coupling gain and fractional release of SR Ca2+ were similar in young adult and aged male cells. In contrast to results in male animals, fractional shortening and Ca2+ current densities were similar in young adult and aged myocytes isolated from female hearts. Furthermore, Ca2+ transient amplitudes were unaffected by age in female cells. Interestingly, SR Ca2+ content was elevated in aged female myocytes, and fractional SR Ca2+ release declined with age in females. However, the gain of EC coupling was not different in myocytes from young adult and aged female mice. These data demonstrate that age-related alterations in EC coupling are more prominent in myocytes from male hearts than in cells from female hearts and suggest that it is important to consider sex as a variable in studies of the effects of aging on cardiac EC coupling.

senescence; sex; sarcoplasmic reticulum; calcium transients; calcium-induced calcium release.

EVEN IN THE ABSENCE OF overt cardiovascular disease, mammalian hearts show important age-related changes in contractile function (20). Whereas contractility at rest is not altered substantially by age, contractile function during exercise is compromised in the aging heart (20). In addition, the duration of contraction is prolonged and relaxation is incomplete in the aging heart (20, 21). Interestingly, alterations in contractile function have also been identified in isolated ventricular myocytes from aged animals (16, 23, 33). For example, the ability to increase contraction amplitude in response to either catecholamines or increased stimulation frequency is impaired in aging myocytes (23, 33). Furthermore, contraction times are prolonged in aged myocytes compared with those in myocytes isolated from younger animals (23, 33). Similar changes are observed when Ca2+ transients are measured in ventricular myocytes from aged hearts (16, 23). These observations suggest that defects in contractile function originate at the cellular level in the aging cardiac myocyte.

Cardiac contraction is triggered by action potentials, which depolarize the cell and lead to a transient rise in intracellular free Ca2+ (2). In mammalian heart, most of the Ca2+ required to activate contraction is released from internal stores in the sarcoplasmic reticulum (SR) (2). The process whereby excitation of the sarcolemma leads to SR Ca2+ release and contraction is called excitation-contraction (EC) coupling. It is widely believed that EC coupling is initiated by a small influx of extracellular Ca2+ through L-type Ca2+ channels, which triggers the release of a much larger amount of Ca2+ through SR Ca2+ release channels, a process known as Ca2+-induced Ca2+ release (CICR) (9–11). As previous studies (16, 23) have shown that Ca2+ transients are disrupted in aging myocytes, these observations suggest that one or more of the processes involved in EC coupling are altered in the aging heart.

Previous studies of EC coupling in aging myocytes have been conducted in cells isolated from male animals only (33), female animals only (7), male and female animals combined (16), or from animals where the sex has not been specified (23). However, several lines of evidence suggest that there are important sex-related differences in EC coupling in cardiac ventricular myocytes. In cardiac myocytes isolated from young adult female rats, contractions and Ca2+ transients are smaller and slower than in cells from age-matched male rats (6, 22). However, Ca2+ current density is increased in myocytes from young adult female rats compared with that in males (30). SR Ca2+ stores are similar in myocytes from male and female rats (5), and Na+/Ca2+ exchange is not altered by sex in murine myocytes (29). However, myocytes from male rats show greater increases in intracellular Ca2+ in response to catecholamines or increased stimulation frequency than cells from females (5, 22). These findings suggest that there are key sex-related differences in EC coupling in cardiac myocytes from young adult hearts. However, whether aging affects EC coupling differently in myocytes from male and female hearts has not been investigated.

The objectives of this study were 1) to explore the cellular bases for age-related alterations in EC coupling in cardiac ventricular myocytes and 2) to determine whether aging affects EC coupling differently in myocytes from male and female hearts. Studies compared contractions, Ca2+ transients, Ca2+ current density, and SR Ca2+ content in voltage-clamped myocytes isolated from young adult (~7 mo) and aged (~24 mo) male and female mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation. All experiments were performed in accordance with guidelines published by the Canadian Council on Animal Care and were approved by the Dalhousie University Committee on Animal Care. Experiments were conducted on ventricular myocytes from male and female young adult (~7 mo) and aged (~24 mo) mice. Young B6SJLF1/J mice (4–6 wk) were obtained from Jackson Laboratories (Bar Harbour, ME) and raised in the Dalhousie University Animal Care facility until they reached the appropriate age. Myocytes were isolated as described previously (12). Briefly, animals were weighed and anesthetized with pentobarbital sodium (200–300 mg/kg ip) and coinjected with heparin (100 U) to decrease blood coagulation. Hearts were cannulated in situ and perfused retrogradely through the aorta (2 ml/min) with oxygenated (100% O2, 37°C) Ca2+-free solution of the following composition: (in mM) 130 NaCl, 5 KCl, 25 HEPES, 0.33 NaH2PO4, 1.0 MgCl2, 20 glucose, 3.0 Na-pyruvate, and 1.0 lactic acid (pH 7.4 with NaOH). Hearts were then perfused for 10 min with Ca2+-free solution containing collagenase (24 mg/30 ml, Worthington Type I, 244 U/mg), protease (9.9 mg/30 ml, Boehringer-Mannheim), and trypsin (30 mg/30 ml, Sigma) supplemented with 50 µM Ca2+. The ventricles were next cut into a beaker filled with a high K+ buffer of the following composition: (in mM) 50 glutamic acid, 30 KCl, 30 KH2PO4, 20 taurine, 10 HEPES, 10 glucose, 3 MgSO4, and 0.5 EGTA (pH 7.4 with KOH). The ventricles were then minced in the high K+ buffer. Myocytes were released from the tissue by gentle agitation; the cell suspension was filtered through a 225-µm polyethylene filter (Spectra/Mesh).

General methods. Myocytes were loaded with cell permeant fura-2 AM (5 µM; stock solution prepared in anhydrous DMSO) and incubated for 30 min in the dark at room temperature as described in a previous study (12). An aliquot of cells was placed in a 0.75-ml chamber on the stage of an inverted microscope. Cells were allowed to settle for 15 min and then 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 CaCl2, and 1 MgCl2 (pH 7.4 with NaOH). The buffer solution was supplemented with 4 mM 4-aminopyridine to block transient outward current and 0.3 mM lidocaine to block Na+ current. All experiments were conducted at 37°C.

Young adult and aged myocytes were voltage clamped with discontinuous single electrode voltage-clamp techniques (5–8 kHz). Cells were impaled with high-resistance microelectrodes (18–26 M{Omega}, filled with 2.7 M KCl) to minimize cell dialysis. Voltage clamp was conducted with an Axoclamp 2B amplifier (Axon Instruments) and ClampEx software (version 8.1, Axon Instruments, Foster City, CA). Cells were held at –80 mV. All test steps were preceded by 10 conditioning pulses (50 ms) from –80 to 0 mV. After each conditioning pulse, cells were repolarized for 450 ms to achieve a stimulation frequency of 2 Hz. Stimulation with a train of 50-ms voltage-clamp pulses was used to simulate a train of murine action potentials (34) and thereby provide a consistent activation history. After the last conditioning pulse, cells were repolarized to –40 mV for 450 ms and then depolarized to 0 mV for 250 ms to activate Ca2+ current and contraction. Unloaded cell shortening was measured with a video edge detector (Crescent Electronics, Sandy, UT). Cell images were visualized with a closed-circuit television camera (model TM-640, Pulnix America) and displayed on a television monitor.

Ca2+ concentrations. Fura-2 fluorescence was recorded and measured as described previously (12). Briefly, fluorescence was measured with a DeltaRam fluorescence system (Photon Technology International) and Felix software (version 1.4, Photon Technology International). Fura-2 was excited at 340 and 380 nm, and emission of photons, excited by each wavelength, was measured at 510 nm. Fluorescence emission was measured at a rate of 100 samples/s for each of the 340 and 380 excitation wavelengths. The ratio of the emissions corresponding to 340/380 nm was converted to intracellular Ca2+ concentration with an in vitro calibration curve. The calibration curve was determined with the same light path used experimentally as described in our previous studies (12, 26, 27). Contractions, transmembrane currents, and fluorescence were recorded simultaneously. This was accomplished 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.

Caffeine-induced Ca2+ transients were used as an index of SR Ca2+ content (2). In these experiments, cells were voltage clamped and stimulated with a train of 50-ms pulses from –80 to 0 mV, delivered at a frequency of 2 Hz. The conditioning pulses ensured that each cell had a comparable history of regular activation before caffeine application. After the conditioning pulse train, cells were held at –60 mV. Caffeine (10 mM) was applied 500 ms after the last conditioning pulse. A rapid solution switcher was used to apply caffeine for 1 s. The rapid switcher is a computer-controlled device that allows complete change of the solution bathing the myocyte in <0.5 s, while maintaining temperature at 37°C (14). Caffeine was applied in a solution of the following composition: (in mM) 10 caffeine, 140 LiCl, 4 KCl, 10 glucose, 5 HEPES, 4 MgCl2, 4 4-aminopyridine, and 0.3 lidocaine. Caffeine was used to release SR Ca2+ and thus estimate SR Ca2+ content. Na+ and Ca2+ were not included in the caffeine solution to minimize extrusion of Ca2+ by the Na+/Ca2+ exchanger (19).

Data analysis. Contraction amplitude was measured as peak shortening with respect to cell length immediately before cell shortening. Aged ventricular myocytes are larger than young adult cells, as described in RESULTS and as described previously (7, 13, 23, 24, 31). Therefore, contractions were normalized as a percentage of resting cell length to allow comparison of fractional shortening between young adult and aged groups. Time-to-peak contraction was measured as time between the initiation of contraction and maximal cell shortening, and time to half relaxation was the time required for contraction to relax by 50%. Diastolic [Ca2+] (the concentration immediately preceding the test step from –40 to 0 mV), peak systolic [Ca2+], and Ca2+ transient amplitude (the difference between systolic and diastolic [Ca2+]) were measured. The rate of rise of the depolarization-induced Ca2+ transient was also measured. The time constant of decay of the caffeine-induced Ca2+ transient was determined by fitting a single exponential function to the decay phase of the transient. The amplitude of peak inward Ca2+ current was measured as the difference between peak inward current and a reference point at the end of the voltage step. Cell membrane area was determined by integrating capacitive transients with pCLAMP software; Ca2+ current was normalized by cell capacitance and expressed as Ca2+ current density. Ca2+ current inactivation was measured with Clampfit 8.1 (Axon Instruments, Union City, CA). A single exponential function was fit to the decay phase of the current recordings to calculate the time constant (tau) for inactivation. The gain of CICR was calculated as the ratio of the rate of rise of the Ca2+ transient per unit current.

Statistical analyses. Sigmaplot (version 8.0) was used to construct all graphs. Differences between means for young adult and aged myocytes were assessed with two-way ANOVA, with sex and age as main factors. A Student-Newman-Keuls post hoc test with a Bonferroni correction was used to determine specific differences between groups. ANOVA was performed with SigmaStat version 2.03 (Jandel). All data are presented as means ± SE. Differences were considered significant when P < 0.05.

Chemicals. Lidocaine, HEPES buffer, EGTA, MgCl2, anhydrous DMSO, 4-aminopyridine, and caffeine were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Fura-2 AM was purchased from Invitrogen (Burlington, ON, Canada). All other chemicals were purchased from BDH (Toronto, ON, Canada). Stock solutions of fura-2 AM were prepared by dissolving 50 µg of fura-2 AM in 20 µl of anhydrous DMSO. All other chemicals were dissolved in deionized water.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ages and body weights were compared in young adult and aged animals (Table 1). Results showed that aged male and female animals were significantly older than young adult animals. Body weights were significantly greater in aged female mice compared with those in young adult female mice (Table 1). In contrast, body weights were similar in young adult and aged male animals. Young female mice also weighed significantly less than young male mice, although this sex difference was not observed in the aged group (Table 1). To determine whether myocyte size increased with age in the mouse heart, we also compared myocyte length and cell capacitance in young adult and aged animals (Table 1). Mean cell length was significantly greater in aged male myocytes compared with that in young adult male myocytes. Cell capacitance, which provides an estimate of cell membrane area, was also significantly greater in aged male myocytes compared with young adult cells (Table 1). In contrast, cell length and cell capacitance measurements were similar in young and aged myocytes from female hearts. To compensate for differences in cell size, Ca2+ current and contraction data were normalized to cell capacitance and cell length, respectively, in all experiments.


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  		Table 1. Physical characteristics

 
The next series of experiments compared and characterized fractional shortening and Ca2+ current density in young adult and aged myocytes from male and female hearts. Figure 1 shows representative examples of contractions and Ca2+ currents recorded from young adult and aged myocytes from animals of each sex. The voltage protocol used to activate Ca2+ currents and contractions is described in detail in MATERIALS AND METHODS and is shown schematically in Fig. 1A. Myocytes were stimulated with 10 conditioning pulses (50 ms) administered at a frequency of 2 Hz before test steps. Cells were repolarized to –40 mV after the last conditioning pulse, and test steps were made to 0 mV to activate Ca2+ current and contraction (Fig. 1A). Representative current and contraction recordings for young adult and aged male myocytes are illustrated in Fig. 1, B and C, respectively. In this example, the peak amplitude of Ca2+ current and the magnitude of contraction were smaller in the aged male cell compared with those in the younger cell. Representative recordings of Ca2+ currents and contractions from young adult and aged female myocytes are illustrated in Fig. 1, D and E. In contrast to those from males, Ca2+ current and contraction amplitudes were similar in these recordings from young adult and aged myocytes from female mice.


Figure 1
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  		Fig. 1. Representative examples of contractions and Ca2+ currents recorded from myocytes isolated from young adult and aged male and female mice. A: schematic of voltage-clamp protocol used in these studies. After a train of conditioning pulses, cells were depolarized from –40 to 0 mV to elicit contractions and Ca2+ currents. B and C: representative contractions (top) and Ca2+ currents (bottom) recorded from young adult and aged myocytes isolated from male hearts. D and E: representative contractions (top) and currents (bottom) recorded from ventricular myocytes isolated from young adult and aged female mice.

 
Mean characteristics of contractions recorded from young adult and aged myocytes are shown in Fig. 2. Fractional shortening was significantly lower in cells from aged male mice compared with that in cells from young adults (Fig. 2A). In contrast, fractional shortening was similar in young adult and aged myocytes from female mice (Fig. 2A). Thus fractional shortening was reduced in myocytes from aging male hearts but not in cells from female hearts. We also compared the time courses of contraction in young adult and aged myocytes from male and female hearts. Results showed that the time-to-peak contraction was significantly prolonged in aged male myocytes compared with that in young adult cells (Fig. 2B). However, time-to-peak contraction was similar in young adult and aged myocytes isolated from female hearts (Fig. 2B). Half-relaxation times were similar in young adult and aged myocytes for both males and females (Fig. 2C). Contraction times were significantly longer in young female myocytes compared with those in young adult male cells (Fig. 2, B and C), although this sex difference was not observed in the aged group. Thus aging prolongs contraction time only in myocytes from male animals.


Figure 2
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  		Fig. 2. Mean fractional shortening, time-to-peak contraction, and half-relaxation time in myocytes from young adult and aged male and female mice. A: mean fractional shortening in aged male and female myocytes compared with that in young adult cells. Values were 6.9 ± 1.1 vs. 3.6 ± 0.9% for young adult and aged males and 4.9 ± 1.4 vs. 3.8 ± 0.6% for young adult and aged females. B: mean time-to-peak contraction in aged myocytes compared with that in young adult cells. Values were 37.6 ± 5.5 vs. 51.9 ± 3.6 ms for young adult and aged males and 52.6 ± 4.6 vs. 58.7 ± 4.2 ms for young adult and aged females. C: mean half-relaxation times in young adult and aged male and female myocytes. Values were 32.0 ± 2.5 vs. 38.1 ± 5.5 ms for young adult and aged males and 47.5 ± 5.9 vs. 43.2 ± 3.6 ms for young adult and aged females. *P < 0.05; young adult males, n = 13; aged males, n = 14; young adult females, n = 11; and aged females, n = 20.

 
Mean Ca2+ current densities recorded from young adult and aged myocytes are shown in Fig. 3. In male myocytes, Ca2+ current density was significantly lower in aged cells compared with that in young adult cells (Fig. 3A). However, Ca2+ current density was similar in young adult and aged myocytes from female mice (Fig. 3A). We also determined whether the rate of inactivation of Ca2+ current was altered by age in male and/or female myocytes. The time constant for inactivation of Ca2+ current (tau) was calculated by fitting a single exponential function to the decay phase of the current. Results showed that the time constant for inactivation of Ca2+ current was significantly prolonged in aging male myocytes compared with that in younger cells (Fig. 3B). In contrast, the time constant for inactivation of Ca2+ current was similar in young adult and aged female cells (Fig. 3B). These results show that Ca2+ current density is reduced and the rate of inactivation of Ca2+ current is prolonged in myocytes from aging male hearts but not in cells from female hearts.


Figure 3
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  		Fig. 3. Mean Ca2+ current density and time course of Ca2+ current inactivation (tau) in myocytes from young adult and aged male and female mice. A: mean Ca2+ current densities in aged male and female myocytes compared with those in young adult myocytes. Values were –6.0 ± 0.4 vs. –3.2 ± 0.9 pA/pF for young adult and aged males and –4.6 ± 0.4 vs. –3.5 ± 0.5 pA/pF for young adult and aged females. B: mean time constants (tau) for inactivation of Ca2+ current in myocytes isolated from aged mice compared with those in cells from younger animals. Values were 9.0 ± 0.4 vs. 14.5 ± 1.5 ms for young adult and aged males and 11.9 ± 0.7 vs. 12.1 ± 0.6 ms for young adult and aged females. *P < 0.05; young adult males, n = 13; aged males, n = 13; young adult females, n = 11; and aged females, n = 20.

 
As the degree of fractional shortening is dependent on SR Ca2+ release, we also compared systolic and diastolic Ca2+ levels and amplitudes of Ca2+ transients in young adult and aged cells from male and female hearts. The voltage-clamp protocol used in these experiments is illustrated in Fig. 4A. Figure 4, B and C, shows representative Ca2+ transients recorded from young adult and aged myocytes isolated from male hearts. In these examples, the Ca2+ transient amplitude was smaller in the aged cell than in the young adult cell. In addition, both systolic and diastolic Ca2+ levels were lower in the aged cell compared with those in the young adult cell. We also compared systolic and diastolic Ca2+ levels and amplitudes of Ca2+ transients in young adult and aged cells from female hearts. Figure 4, D and E, shows representative Ca2+ transients recorded from young and aged female myocytes. These examples showed that systolic and diastolic Ca2+ levels and Ca2+ transient amplitudes did not differ dramatically between young adult and aged female cells (Fig. 4, D and E).


Figure 4
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  		Fig. 4. Representative Ca2+ transients recorded from cells isolated from young adult and aged male and female mice. A: schematic of voltage-clamp protocol used in these studies. Cells were stimulated with a train of conditioning pulses and then depolarized from –40 to 0 mV to activate Ca2+ transients. Protocol is described in detail in the MATERIALS AND METHODS. B and C: representative Ca2+ transients from young adult and aged male myocytes. D and E: representative Ca2+ transients recorded from young adult and aged female myocytes. [Ca2+]i, intracellular Ca2+ concentration.

 
Figure 5 shows mean systolic Ca2+ concentrations, mean diastolic Ca2+ concentrations, and mean amplitudes of Ca2+ transients recorded from young and aged myocytes isolated from male and female animals. Mean systolic and diastolic Ca2+ concentrations were significantly lower in aged male myocytes compared with those in younger male cells (Fig. 5, A and B). In addition, the mean Ca2+ transient amplitudes were significantly smaller in aged male myocytes compared with those in young adult cells (Fig. 5C). In contrast to results in male cells, mean systolic and diastolic Ca2+ concentrations and Ca2+ transient amplitudes were similar in young adult and aged female myocytes (Fig. 5, A–C). These results showed that systolic and diastolic Ca2+ concentrations and Ca2+ transient amplitudes were smaller in aged male myocytes than in younger cells. Interestingly, there also were sex differences in the aged group, because systolic and diastolic Ca2+ levels were significantly greater in aged female cells than in aged male cells (Fig. 5, A and B).


Figure 5
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  		Fig. 5. Systolic Ca2+ concentrations, diastolic Ca2+ concentrations, and Ca2+ transients in cells from young adult mice compared with those in cells from aged mice. A: mean systolic Ca2+ concentrations in aged male and female myocytes compared with those in young adult myocytes. Values were 438.3 ± 39.9 vs. 206.1 ± 54.1 nM for young adult and aged males and 419.8 ± 59.1 vs. 343.5 ± 26.5 nM for young adult and aged females. B: mean diastolic Ca2+ concentrations in young adult and aged male and female myocytes. Values were 270.8 ± 24.5 vs. 123.1 ± 33.6 nM for young adult and aged males and 279.1 ± 39.0 vs. 231.5 ± 14.9 nM for young adult and aged females. C: mean Ca2+ transient amplitudes in aged male and female myocytes compared with those in younger cells. Values were 167.4 ± 22.4 vs. 83.0 ± 23.2 nM for young adult and aged males and 140.7 ± 23.5 vs. 112.0 ± 16.2 nM for young adult and aged females. *P < 0.05; young adult males, n = 10; aged males, n = 7; young adult females, n = 9; and aged females, n = 21.

 
We also measured the maximum rate of rise of the Ca2+ transient to estimate SR Ca2+ release flux as in previous studies (7), and we compared release flux in young adult and aged myocytes from male and female mice. The average maximum rate of rise of the Ca2+ transient was significantly lower in aged male myocytes compared with that in cells from young adult males (Fig. 6A). However, the mean rate of rise of the Ca2+ transient was similar in myocytes from young adult and aged female mice (Fig. 6A). Thus SR Ca2+ release was reduced in aged male but not female myocytes compared with that in younger animals. We used these estimates of release flux to determine whether the amount of Ca2+ released per unit Ca2+ current density, an estimate of the gain of CICR (7), was altered by age in male or female myocytes. Figure 6B shows that the gain of CICR was similar in young adult and aged myocytes, regardless of the sex of the animal. Thus, even though SR Ca2+ release was reduced in cells from aged males, the gain of CICR was not altered by age in either male or female myocytes.


Figure 6
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  		Fig. 6. Rate of rise of Ca2+ transient and gain of Ca2+-induced Ca2+ release (CICR) in cells from young adult and aged male and female mice. A: rate of rise of the Ca2+ transient in myocytes from young and aged male and female mice. Values were 4,167 ± 667 vs. 1,606 ± 258 nM/s for young adult and aged males and 3,652 ± 1,267 vs. 2,654 ± 381 nM/s for young adult and aged females. B: gain of CICR, expressed as rate of rise of Ca2+ transient (nM/s) per unit Ca2+ current density (pA/pF), compared in young adult and aged male and female myocytes. Values were 828.0 ± 180.9 vs. 715.2 ± 237.3 nM·s–1·(pA/pF)–1 for young adult and aged males and 859.6 ± 272.3 vs. 909.5 ± 145.4 nM·s–1·(pA/pF)–1 for young adult and aged females. *P < 0.05; young adult males, n = 9; aged males, n = 7; young adult females, n = 9; and aged females, n = 21.

 
The next series of experiments determined whether SR Ca2+ content differed between young adult and aged myocytes. In these experiments, cells were loaded with fura-2, and SR Ca2+ content was assessed by rapid application of 10 mM caffeine (0 mM Na+, 0 mM Ca2+) as described in MATERIALS AND METHODS. The voltage-clamp protocol used in these experiments is illustrated in Fig. 7A. Cells were stimulated with conditioning pulses (2 Hz) and then repolarized to –60 mV. Caffeine was applied with a rapid solution switcher for 1 s, 500 ms after the last conditioning pulse. Representative caffeine-induced Ca2+ transients from young adult and aged male myocytes are shown in Fig. 7B. In this example, the amplitudes of caffeine-induced Ca2+ transients were similar in myocytes isolated from young adult and aged male mice (Fig. 7B). In contrast, Fig. 7C shows representative examples of caffeine-induced Ca2+ transients in female cells. The caffeine-induced Ca2+ transient was larger in the aged female myocyte compared with that in the younger cell (Fig. 7C). Figure 7D shows mean amplitudes of caffeine-induced Ca2+ transients in all groups. Results showed that there was no significant effect of either age or sex on SR Ca2+ content. However, there was a significant age-sex interaction, such that SR Ca2+ content decreased with age in cells from male mice and increased with age in the female cells (Fig. 7D). We also measured the decay kinetics of the caffeine-induced Ca2+ transients as described in MATERIALS AND METHODS. Values were 650 ± 122 vs. 921 ± 191 ms (n = 11–12) for young adult and aged males, respectively, and 869 ± 217 vs. 886 ± 107 ms (n = 10–19) for young adult and aged females, respectively. There was no significant effect of either age or sex on the decay rate of the caffeine transient. We also calculated the fractional release of Ca2+ from the SR by dividing the amplitude of the Ca2+ transient by the amplitude of the caffeine transient as described previously (7). Fractional release was similar in young adult and aged male myocytes, although fractional release of SR Ca2+ actually decreased with age in cells from female mice (Fig. 7E). Thus, although SR Ca2+ content declined with age in the male group, fractional release of SR Ca2+ was not affected by age in male myocytes. In contrast, SR Ca2+ stores increased with age in the female group, yet fractional release of SR Ca2+ declined with age in females.


Figure 7
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  		Fig. 7. Sarcoplasmic reticulum (SR) Ca2+ content from cells isolated from young adult and aged male and female mice. A: schematic of voltage-clamp protocol used in these experiments. Cells were stimulated with a train of conditioning pulses and then held at a potential of –60 mV. Caffeine was applied for 1 s to release SR Ca2+. B: representative caffeine-induced Ca2+ transients from young adult (top) and aged (bottom) male cells. C: caffeine-induced Ca2+ transients recorded from young adult (top) and aged (bottom) female myocytes. D: mean amplitudes of caffeine-induced Ca2+ transients recorded from young adult and aged myocytes. Interaction between age and sex is statistically significant. Values were 243.3 ± 34.7 vs. 166.9 ± 25.6 nM for young adult and aged males and 164.0 ± 23.8 vs. 231.1 ± 19.3 nM for young adult and aged females. E: mean values for fractional release (Ca2+ transient divided by the caffeine transient) in young adult and aged male and female myocytes. Values were 68.8 ± 9.2 vs. 49.7 ± 13.9% for young adult and aged males and 85.8 ± 14.4 vs. 48.6 ± 7.0% for young adult and aged females. *P < 0.05; young adult males, n = 10; aged males, n = 7; young adult females, n = 9; and aged females, n = 21.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We examined the cellular bases for age-related alterations in cardiac EC coupling in murine ventricular myocytes and investigated whether aging affected EC coupling differently in cells from male and female hearts. We found that fractional shortening, Ca2+ current density, and Ca2+ transients were decreased in aged male myocytes compared with those in cells from younger animals. In addition, systolic and diastolic Ca2+ concentrations were lower in aged male myocytes. The time course of inactivation of Ca2+ current was slowed, and the time course of contraction was prolonged in cells from aged male mice. SR Ca2+ content decreased with age in male myocytes. However, neither the gain of CICR nor fractional release of SR Ca2+ was affected by age in male cells. Very different results were obtained in cells from female hearts. Fractional shortening, Ca2+ current density, and Ca2+ transients were similar in myocytes from young and aged female mice, and systolic and diastolic Ca2+ concentrations did not differ between groups. Furthermore, neither the time course of contraction nor the rate of Ca2+ current inactivation were affected by age in female cells. SR Ca2+ content was elevated in aged female myocytes. However, CICR gain was similar in young and aged female cells, whereas fractional release of SR Ca2+ declined with age in female cells. We also observed sex differences in cardiac EC coupling. Contraction duration was prolonged in young female cells compared with that in young male cells, whereas systolic and diastolic Ca2+ levels were higher in aged female cells than in aged male cells. Thus there are age- and sex-related changes in cardiac EC coupling, although age-related changes are more evident in male cells than in female cells.

A major finding in the present study is that age-associated changes in cardiac EC coupling are more prominent in myocytes from male mice than in cells from female animals. To our knowledge, this observation has not been reported previously. Earlier studies of the cellular basis for changes in EC coupling in aging have used myocytes from male animals only (13, 33), female animals only (7), combined data from male and female animals (16), or did not identify the sex of the animals and may therefore have combined data from male and female animals (23). Our results demonstrate that it is very important to consider sex as a variable in studies of the effects of aging on cardiac EC coupling and suggest that differences between males and females are important to consider in studies of the biological mechanisms of aging.

A number of previous studies of cardiac EC coupling in aging have used ventricular myocytes isolated from male rats. These studies showed that contraction amplitudes are similar in young adult and aged rat myocytes when the cells are field stimulated (13, 33). However, action potential duration is prolonged in aged male rat myocytes (4, 24, 31, 32). Therefore, in field-stimulation experiments the duration of depolarization would differ between young and aged cells. Interestingly, Janczewski et al. (17) provided evidence that aging rat myocytes utilize prolongation of the action potential to preserve SR Ca2+ stores and reduce deficits in EC coupling. This mechanism may account for the absence of differences in contractions and Ca2+ transients in field-stimulated myocytes from young and aged male rats (13, 33).

In the present study, we explored effects of age and sex on EC coupling in murine ventricular myocytes, and we controlled the duration of depolarization with rectangular voltage-clamp steps to eliminate possible differences in action potential configuration. In cells stimulated at a frequency of 2 Hz, we found that fractional shortening and Ca2+ transients were smaller in myocytes from aged male mice than in cells from younger animals, whereas responses were similar in cells from young and aged females. These findings generally agree with results of previous studies in murine myocytes. Isenberg et al. (16) showed an age-related reduction in amplitudes of Ca2+ transients initiated by voltage-clamp pulses delivered at frequencies of 4 Hz or greater in murine ventricular myocytes. In addition, contractions and Ca2+ transients are smaller in rapidly paced (>4 Hz), field-stimulated cells from aged mice compared with cells from younger animals (23). In the present study, we observed differences in the amplitudes of contractions and Ca2+ transients in male myocytes at lower stimulation rates than previously reported. Results from male and female myocytes were combined in one previous study (16) and may have been combined in another study (23). Therefore, we may have detected a difference in contractions and Ca2+ transients at lower stimulation frequencies because we separated data from male and female mice. The results of the present investigation suggest that the age-related reductions in amplitudes of contractions and Ca2+ transients reported previously in mouse myocytes (16, 23) may result from changes in cells from male animals. In contrast to our results and those reported previously in rodent myocytes (13, 16, 33), Dibb et al. (7) found that Ca2+ transients initiated by voltage-clamp steps actually were larger in ventricular myocytes from aged female sheep compared with those in younger animals. It is difficult to directly compare results from myocytes isolated from 8-yr-old sheep to myocytes from 2-yr-old mice. However, the study by Dibb et al. (7) demonstrates that marked age-related changes in EC coupling can occur in ventricular myocytes from female animals.

The present study showed that time-to-peak contraction was prolonged in cells from aged males but not in cells from aged females. Other studies (13, 32) have also found that time-to-peak contraction is prolonged in ventricular tissues and myocytes from aged male rats. Wei et al. (32) suggested that the increased action potential duration in aged rat myocytes might increase the time-to-peak contraction by altering the rate at which intracellular Ca2+ increases. However, this cannot explain the increase in time-to-peak contraction observed in aged male myocytes in the present study where cells were voltage clamped. On the other hand, as discussed in detail below, we did find that the rate of inactivation of the Ca2+ current was slower in aged male myocytes compared with that in younger cells, as shown previously in aged myocytes from male rat hearts (18, 24, 31). Thus the prolongation of Ca2+ current could contribute to the increase in time-to-peak contraction observed in aged male myocytes in the present study.

Our results showed that the rate of inactivation of the Ca2+ current was prolonged in myocytes from aged male mice, as shown previously in cells from male rats (24, 31). In addition, the time constant for inactivation of ensemble-averaged Ca2+ currents is prolonged in aged rat myocytes (18). It has been suggested that the slowed rate of Ca2+ current inactivation may be due to a decrease in intracellular Ca2+ in aging myocytes, which could lead to less Ca2+-dependent inactivation of Ca2+ current (31). The results of the present study support this idea. We found that SR Ca2+ release and intracellular Ca2+ concentrations were lower in aged male myocytes than in young adult cells. Thus a decrease in intracellular Ca2+ release could alter Ca2+-dependent inactivation of Ca2+ current and thereby decrease its rate of inactivation in aged male myocytes.

We also found that Ca2+ current density was smaller in myocytes from aged males than in cells from younger mice. However, there was no age-related difference in Ca2+ current density in cells from female mice. Several previous studies have compared whole cell Ca2+ current in ventricular myocytes from young adult and aged animals, and results have been contradictory. Isenberg et al. (16) reported that Ca2+ current density was unchanged in young and aged mouse ventricular myocytes. Furthermore, some studies (31, 33) in male rat myocytes have shown that Ca2+ current density is similar in cells from young adult and aged animals, whereas others (24) have shown an age-associated reduction in Ca2+ current density. On the other hand, Dibb et al. (7) found that Ca2+ current density was increased in myocytes from older female sheep. Taken together with our results, these observations suggest that age-related changes in Ca2+ current density may vary with different experimental conditions and with the sex and species of the animal investigated.

The reduction in Ca2+ current density in myocytes from aged male mice might, in theory, be due to inactivation of Ca2+ current by elevated intracellular Ca2+ (2, 8). However, our results showed that SR Ca2+ content, intracellular Ca2+ levels, and SR Ca2+ release were reduced in aged male myocytes compared with those in younger cells. Thus it is unlikely that Ca2+ inactivation of Ca2+ current accounts for the reduced Ca2+ current density in aged male myocytes. However, radioligand binding studies (15) have shown that the density of dihydropyridine receptors, and thus of putative L-type Ca2+ channels, is reduced in membranes from aged male hamster hearts compared with that in membranes from younger hearts. Therefore, it is possible that a reduction in L-type Ca2+ channel density contributes to the decrease in peak Ca2+ current density in myocytes from aged male mice.

It is likely that reduced Ca2+ current density contributes importantly to the decrease in SR Ca2+ release observed in cells from aged male mice. It is well established that the amplitude of Ca2+ current grades the magnitude of SR Ca2+ release (3, 25). Therefore, a reduction in peak Ca2+ current density in aging would be expected to reduce SR Ca2+ release, and fractional shortening would decrease accordingly. Indeed, this is what we observed in cells from aged male animals. We also found that SR Ca2+ content was decreased in myocytes from aged male mice. Because the magnitude of SR Ca2+ release is influenced by SR Ca2+ content (1), this reduction in SR Ca2+ content could, in theory, reduce the amount of Ca2+ released from the SR during CICR. However, we found that the gain of CICR, which reflects the amount of Ca2+ released from the SR per unit current, was unaffected by age in cells from male animals. Therefore, the reduction in SR Ca2+ content does not appear to decrease the gain of EC coupling in aged mouse myocytes. Together, our observations suggest that the reduction in amplitude of trigger Ca2+ current in aged male myocytes releases less SR Ca2+, which reduces fractional shortening in aged male myocytes.

In contrast to our findings in male mice, there were few age-related alterations in cardiac EC coupling in cells from female mice. Ca2+ transients, Ca2+ current density, and fractional shortening were similar in myocytes from young and aged female mice. We did observe an increase in SR Ca2+ content in cells from aged female hearts. However, the increase in SR Ca2+ content did not lead to an increase in the gain of CICR in aged female myocytes. Furthermore, because Ca2+ transients were similar in young and aged female myocytes, the fractional release of SR Ca2+ actually was reduced by age in cells from female mice. These observations suggest that preservation of Ca2+ current density and increase in SR Ca2+ content may preserve SR Ca2+ release and maintain fractional shortening in aging female cells.

The results of the present study demonstrate that cell length and cell capacitance increase with age in ventricular myocytes from male mice but not in cells from female mice. Ventricular myocyte hypertrophy has been reported previously in a number of different models of aging (7, 13, 23, 24, 31). Importantly, an increase in myocyte cell length, with no corresponding increase in cell width, has been reported previously in murine ventricular myocytes, although the study (23) did not specify the sex of the mice. Only one previous study (28) has examined the effects of age and sex on myocyte cell size. Olivetti et al. (28) examined human hearts and found that aging was associated with a loss of ventricular myocytes and an increase in the size of the remaining cells in males but not in females (28). These results suggest that the stimulus for myocyte hypertrophy in the hearts of male mice might be a decrease in the number of myocytes with age, although this has not yet been examined experimentally.

Our study also provided evidence for sex differences in components of cardiac EC coupling in murine myocytes. We found that contraction duration was prolonged in myocytes from young female mice compared with cells from young males. These findings agree with results of previous studies (6, 22) in rat myocytes, which showed that contractions and Ca2+ transients are prolonged in female myocytes compared with those in male cells. However, we also found that contraction amplitudes, Ca2+ transient amplitudes, and Ca2+ current densities were similar in young male and female cells. These findings differ from results of previous studies in rats, which reported that contractions and Ca2+ transients are smaller in young female rat cells than in male cells (6, 22) and that Ca2+ current density is increased in female cells compared with that in males (30). Differences in experimental conditions, e.g., temperature, age, and type of animals used, may account for differences in results between our study and previous studies.

There are some limitations to the present study. The results presented here apply to voltage-clamped, unloaded murine ventricular myocytes paced at a frequency of 2 Hz and investigated at physiological temperature. Further experiments will be required to determine whether results can be extrapolated to intact hearts or to cardiac myocytes investigated under different experimental conditions. It is also possible that age or sex could affect the buffering of intracellular Ca2+ by the fluorescent probe. Finally, SR Ca2+ stores were measured by application of caffeine to cells held at a potential of –60 mV after the conditioning pulse train, whereas Ca2+ transients were measured in cells held briefly at –40 mV after the conditioning pulse train. It is possible that the difference in holding potential could affect SR Ca2+ stores differently depending on the age or sex of the animal. Additional experiments will be required to address these issues directly.

In summary, this study demonstrates that age-related changes in cardiac EC coupling in murine ventricular myocytes are influenced markedly by the sex of the animal. We found that fractional shortening, Ca2+ current density, Ca2+ transients, and SR Ca2+ stores were significantly reduced in aged male myocytes compared with those in cells from younger animals. However, neither fractional release of SR Ca2+ nor the gain of CICR was altered by age in male myocytes. In contrast, fractional shortening, Ca2+ current density, and Ca2+ transients were similar in young adult and aged myocytes from female hearts. Interestingly, SR Ca2+ content was elevated in aged female cells and fractional SR Ca2+ release decreased, although the gain of CICR was not affected by age in these cells. Our observations show that age-related changes in cardiac EC coupling are prominent in myocytes from male but not female hearts and suggest that it is important to consider sex as a variable in studies of the effects of aging on cardiac EC coupling.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by grants from The Canadian Institutes of Health Research and the Heart and Stroke Foundation of Nova Scotia, and S. A. Grandy was supported by a graduate studentship from those institutions.


   ACKNOWLEDGMENTS
 
We thank Peter Nicholl, Dr. Jiequan Zhu, and Steve Foster for excellent laboratory technical support and for assistance in preparation of illustrations; and the late Dr. Greg Ferrier for helpful discussions of the data presented in this study. Present address of S. A. Grandy: Institut de Cardiologie de Montreal, 5000 est rue Belanger, Montreal, QC, H1T 1C8, Canada


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. E. Howlett, Dept. of Pharmacology, 5850 College St., Sir Charles Tupper Medical Bldg., Dalhousie Univ., Halifax, NS, B3H 1X5, Canada (e-mail: susan.howlett{at}dal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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