INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

NORMAL ADULT AGING in mammals is associated with altered myocardial structure and function. Whereas systolic function at rest appears to be unaffected by aging, many studies in animals and humans have shown that left ventricular (LV) diastolic function deteriorates with aging (13, 22). In fact, LV diastolic dysfunction is a common condition in the elderly and manifests as exercise intolerance, dyspnea with exertion, and fatigue (29). Previous studies have shown that LV relaxation time is prolonged in senescent rat cardiac muscle (23, 32), and one possible mechanism is a decrease in the rate of Ca²⁺ removal from the contractile elements. The sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA2a) and its inhibitory subunit, phospholamban, are important regulators of SR Ca²⁺ resequestration. SERCA2a, which couples the hydrolysis of one ATP molecule to actively transport two calcium ions into the SR, removes Ca²⁺ from the cytosol to facilitate relaxation of the heart (3, 20). Phospholamban is a regulatory protein of SERCA2a. Dephosphorylated phospholamban inhibits SERCA2a activity, whereas phospholamban phosphorylation, by either calcium/calmodulin- or cAMP-dependent protein kinases, relieves this inhibition (20). Decreased SERCA2a pumping rate, mRNA, and protein levels (2, 7, 12) and decreased phospholamban protein levels (7) have been reported in senescent rat and human myocardium. In contrast, others have reported no change in mRNA and protein levels of SERCA2a with age (6, 18). Another important protein for cytosolic Ca²⁺ removal, the sarcolemmal Na⁺/Ca²⁺ exchanger, is reportedly decreased in senescent rat hearts with no change in mRNA levels (2). The rate of cytosolic Ca²⁺ removal can be enhanced by -adrenergic stimulation, which induces phosphorylation of myofilament and membrane proteins, including phospholamban, and results in disinhibition of SERCA2a and increased SR Ca²⁺ uptake (3, 20). Studies relating myocardial relaxation, Ca²⁺-handling proteins, and -adrenergic stimulation in aging mouse models are lacking.

The inotropy-frequency relationship has been studied in adult isolated mouse hearts (5), papillary muscles (16), and closed-chest anesthetized mice (30); however, the effect of age and heart rate on lusitropy in isolated mouse hearts has not yet been established. Basal heart rates in adult conscious mice range from 550 to 620 beats/min (19), and mice therefore must possess a mechanism for rapid cytosolic Ca²⁺ removal. To our knowledge, data on age-related differences in heart rate in the conscious mouse are not available; consequently, in aging mouse hearts, it is not known if cytosolic Ca²⁺ removal is impaired at heart rates in the in vivo range.

Thus, in this study, we hypothesized that lusitropy is impaired in aging mouse hearts and correlates with an age-related decrease in cytosolic Ca²⁺ removal. To this end, we utilized the Langendorff red blood cell-perfused isolated mouse heart model to investigate the effects of age on the lusitropy-frequency relationship and to assess how increasing the rate of SR Ca²⁺ uptake via -adrenergic stimulation influences this relationship. Furthermore, we assessed correlations between functional lusitropic parameters and age-related alterations in myocardial protein levels of SERCA2a, phospholamban, and Na⁺/Ca²⁺ exchanger.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal model. Adult (5 mo old), old (24 mo old), and senescent (34 mo old) B6C3F1 hybrid mice were provided by the National Institute on Aging (NIA). According to animal survival curves provided by the NIA, the probability of survival is 90% for old and 35% for senescent mice. One week after arrival, the mice were restrained without anesthesia and tail-cuff systolic blood pressure and heart rate measurements were obtained with the use of a computerized tail-cuff system (BP-2000 Visitech Systems), described elsewhere (25). All mice used in the study were males with body weights between 30 and 45 g. The present study was performed in accordance with the guidelines of the Animal Care and Use Committee of the Boston University School of Medicine and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Whole heart perfusion study. To characterize changes in LV function with age, studies were performed in Langendorff-perfused isolated isovolumically beating hearts, as previously described (10). Briefly, mice were injected intraperitoneally with heparin (10, 000 U/kg) and subsequently anesthetized intraperitoneally with a mixture of ketamine (150 mg/kg) and xylazine (15 mg/kg). The thorax was rapidly opened, the heart excised, and a short perfusion cannula inserted into the aortic root to initiate retrograde perfusion. The perfusate consisted of bovine red blood cells at a final hematocrit of 40% in modified Krebs-Henseleit buffer (in mM: 118 NaCl, 4.7 KCl, 2.0 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 26.6 NaHCO₃, 5.5 glucose, 1.0 lactate, and 0.4 palmitic acid and 4 g% BSA). The perfusate was equilibrated with 20% O₂-3% CO₂-77% N₂ to achieve a PO₂ of 120-140 mmHg and pH 7.4. A thin cannula was pierced through the apex of the LV to vent thebesian drainage. A small balloon, custom-made from polyvinyl chloride film and connected to a polyethylene tube, was inserted into the LV through the mitral valve via an incision in the left atrium. A 1.4-Fr high-fidelity microtip transducer (Millar) was guided through the polyethylene tube and positioned inside the balloon. The balloon was inflated with saline to adjust the end-diastolic pressure (EDP) at 5 mmHg, and the balloon volume was held constant for the duration of the experiment. Hearts were paced (Grass Instruments) through platinum wires placed on the epicardial surface of the right ventricle. Coronary perfusion pressure (CPP) was monitored via a sidearm of the aortic cannula connected to a pressure transducer (Gould). An inline ultrasonic flow probe (Transonics Systems) was positioned immediately above the aortic cannula to measure coronary blood flow (CF). LV pressures, CPP, and CF were collected on-line at rates of 400, 20, and 20 samples/s, respectively, with the use of a commercially available data acquisition system (MacLab, ADInstruments). Maximum rate of contraction (+dP/dt) and time constant () of isovolumic pressure decay (described in Inotropic and lusitropic indexes) were calculated off-line.

Experimental protocol. To examine the effects of age and heart rate on contractile and relaxation indexes, a pacing protocol was performed on adult (n = 9), old (n = 10), and senescent (n = 11) mouse hearts. During 20 min of stabilization, hearts were maintained at 37°C at a CPP of 80 mmHg and paced at 5 Hz. The pacing rate was subsequently increased in 1-Hz increments every 3 min, up to 10 Hz.

6

Inotropic and lusitropic indexes. Systolic pressure (SP) and maximum +dP/dt were used as inotropic indexes of systolic function in this study. Diastolic function, or lusitropy, was assessed by changes in isovolumic LVEDP and isovolumic relaxation rates. Isovolumic relaxation can be quantified with the use of the time constant  by fitting the time course of the isovolumic pressure decay with a monoexponential equation (15). Assuming a zero pressure asymptote,  can be calculated using the following equation
where P_t is the LV pressure at time t and P_o is the LV pressure at maximum dP/dt. In whole animal studies and human subjects, maximum dP/dt has been shown to be a reliable indicator of aortic valve closure, and at 5 mmHg before EDP it has been used to denote the onset of mitral valve opening (15). In our isovolumically beating heart model we followed this same method for assessing isovolumic relaxation and measured LV pressure every 2.5 ms from the time of maximal dP/dt to a level 5 mmHg above the EDP of the next beat. The correlation coefficient of each exponential curve fit always exceeded 0.998.  is relatively load insensitive and was selected over other indexes of relaxation such as maximum dP/dt and duration of isovolumic relaxation, because these are more dependent on peak SP than on the rate of ventricular pressure decay (15).

Calculation of systolic circumferential wall stress. To determine whether aging is associated with changes in systolic circumferential stress (), we calculated  from the relationship described by Mirsky (27). A spherical LV chamber model was assumed in which the LV cavity is of radius R_i, and a volume equal to that of the balloon when empty was added to the actual balloon volume to reflect total LV chamber volume (V_T). Thus
and
The total heart volume is equal to the sum of V_T and V_wall, where V_wall is the volume of the LV wall (V_wall = LV wt/1.05, the specific gravity of myocardium). This volume is contained in a sphere of radius R_i + h, where h is the wall thickness. Therefore
and
Peak systolic circumferential wall stress was then derived from
where SP is LV systolic pressure.

Preparation of LV membrane proteins. To determine whether age differences in contractile and relaxation indexes were associated with age-related changes in Ca²⁺-handling proteins, we measured SERCA2a, phospholamban, and Na⁺/Ca²⁺ exchanger protein content from crude membrane preparations. Four additional mice in each group were anesthetized according to the procedure described earlier, the hearts were quickly excised, and the LV was carefully separated and frozen in liquid nitrogen and stored at 70°C until use. For isolation of crude membrane proteins, LV tissue samples were homogenized in 7 vols (vol/wt) of lysis buffer (20 mM Tris · HCl, 1 mM EDTA, 1 mM dithiothreitol, 1 µM leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4) and centrifuged at 200 g for 5 min. The supernatant was recovered and centrifuged at 100,000 g for 60 min. The resulting pellet was resuspended in lysis buffer and homogenized, and its protein concentration was determined according to the method of Bradford (4) using BSA as standard. The yield of total membrane protein was 22.82 ± 2.44, 26.96 ± 1.11, and 31.81 ± 3.75 mg/g LV wet weight in adult, old, and senescent hearts, respectively (no significant differences among groups).

Western blot analysis. The protein homogenates were solubilized in 0.5 M Tris · HCl, pH 6.8, 10% glycerol, 5% SDS, 5% 2--mercaptoethanol, and 0.5% bromophenol blue. Equal amounts of protein extract from each sample (20 µg protein/lane) were subjected to SDS-PAGE with the use of 4-20% gradient gels for SERCA2a and Na⁺/Ca²⁺ exchanger and a 15% gel for phospholamban. After electrophoresis, proteins were transferred to polyvinylidene difluoride membrane blots (Bio-Rad, Richmond, CA) at 4°C for 2 h at 100 V. Blots were blocked in 5% nonfat milk diluted in Tris-buffered saline (TBS; 20 mM Tris · HCl and 500 mM NaCl, pH 7.5) for 1 h at room temperature (RT) and washed in Tween-TBS (TTBS; 0.3% Tween-20 diluted in TBS). The blots were incubated at 4°C overnight in primary antibodies to Na⁺/Ca²⁺ exchanger (SWant, Bellinzona, Switzerland), SERCA2a (Affinity Bioreagents, Golden, CO), or phospholamban (Affinity Bioreagents) diluted in antibody buffer (5% nonfat milk diluted in TTBS). Only the pentameric form of phospholamban was quantified. The blots were then washed in TTBS and incubated in secondary antibody solution diluted in antibody buffer for 1 h at RT. The blots were washed in TTBS, incubated in enhanced chemiluminescence reagent (Bio-Rad) for 5 min, and exposed to X-ray film (Hyperfilm MP, Amersham) for 30 s to 8 min. The band densities were quantified using a commercially available densitometer (PDI).

Statistical analysis. Data are reported as means ± SE. Group differences of a single measurement were tested by ANOVA using the Bonferroni/Dunn method. Data acquired sequentially in an individual group of hearts were tested by repeated ANOVA with a multiple-comparison test of least significant differences. Group comparisons were performed using a two-way ANOVA with a multiple-comparison test of least significant differences. Analyses before and after isoproterenol treatment were performed using paired t-test comparisons. A P value <0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal characteristics and LV morphology. The results presented in Table 1 compare animal characteristics and LV dimensions for all three groups. Systemic blood pressure was lower in old compared with adult mice (P < 0.0005), and resting heart rate was greater in senescent and old mice compared with adult mice (P < 0.005). Differences in blood pressure and heart rate, however, may also reflect the animal's response to restraint and handling.

Inotropic indexes. The relationship between SP and heart rate is shown in Fig. 1A. SP peaked at 6 Hz for senescent hearts and at 7 Hz for adult and old hearts, followed by a decline in all groups at higher heart rates. Relative to baseline at 5 Hz, peak SP increased by 11.7 ± 2.9% in adult, 17.0 ± 4.2% in old, and 8.1 ± 1.6% in senescent hearts (P < 0.05 for 5 Hz vs. 7 Hz in adult and old groups and for 5 Hz vs. 6 Hz in senescent group). At 10 Hz, SP declined from peak SP by 16.8 ± 2.7% in adult, 13.2 ± 1.7% in old, and 20.7 ± 1.7% in senescent hearts (P < 0.005, all groups). The decrease from peak SP to 10 Hz was greater in senescent hearts (P < 0.05) compared with old hearts (Fig. 1A). The relationship between heart rate and +dP/dt is shown in Fig. 1B. For adult and senescent hearts, +dP/dt peaked at 7 Hz, and for old hearts it peaked at 8 Hz; however, +dP/dt in adult hearts reached a plateau at higher heart rates, whereas that in old and senescent hearts declined. Compared with baseline, peak +dP/dt increased 27.6 ± 4.9% in adult, 36.9 ± 6.2% in old, and 21.1 ± 3.0% in senescent hearts (P < 0.0001 for 5 Hz vs. 7 Hz in adult and senescent groups and for 5 Hz vs. 8 Hz in old group). At 10 Hz, +dP/dt declined from peak +dP/dt by 11.9 ± 4.0% in adult (P = nonsignificant), 21.0 ± 3.6% in old (P < 0.01), and 27.4 ± 2.6% in senescent hearts (P < 0.0001). The decline in +dP/dt was significantly greater in senescent hearts (P < 0.05) compared with adult and old hearts (Fig. 1B). These results show that, regardless of age, the inotropy-frequency relationship in mice is characterized by an ascending limb at low heart rates followed by a descending limb at higher heart rates.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Systolic pressure (SP; A), maximal rate of pressure rise (+dP/dt; B), and peak midwall systolic stress (C) of isovolumic contractions in isolated mouse hearts at increasing pacing rates before (open symbols) and during (filled symbols) 106 M isoproterenol administration at 5 mo (circles), 24 mo (triangles), and 34 mo (squares) of age. Data are expressed as means ± SE. * P < 0.05, 2-way ANOVA between groups indicated.  P < 0.05, paired t-test comparison before and during isoproterenol for 5-mo-old hearts;  P < 0.05 for 24-mo-old hearts; and § P < 0.05 for 34-mo-old hearts.

6

Lusitropic indexes. With increasing heart rate, EDP was significantly increased in all three groups (Fig. 2A). In senescent hearts, the increased EDP was greater and occurred at lower heart rates than in old and adult hearts. Also, the increased EDP in old hearts was greater than that in adult hearts. Relative to baseline, EDP at 10 Hz increased 10.1 ± 1.9 mmHg in adult, 16.1 ± 1.6 mmHg in old, and 22.1 ± 1.8 mmHg in senescent hearts (P < 0.0001, all groups). The relationship between heart rate and  is shown in Fig. 2B.  was significantly prolonged in senescent and old hearts compared with adult hearts, suggestive of a decreased LV relaxation rate with age. Interestingly, in all groups, increasing heart rate from 5 to 8 Hz caused  to decrease. Relative to baseline at 5 Hz,  at 8 Hz decreased 3.85 ± 0.64 ms in adult, 3.66 ± 0.66 ms in old, and 3.00 ± 0.61 ms in senescent hearts (P < 0.05, all groups). At heart rates >8 Hz, there was a statistically insignificant trend toward an increase in  in all groups.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   End-diastolic pressure (EDP; A), time constant (; B), and coronary flow normalized to left ventricular (LV) weight (CF; C) in isolated mouse hearts at increasing pacing rates before (open symbols) and during (filled symbols) 106 M isoproterenol administration at 5 mo (circles), 24 mo (triangles), and 34 mo (squares) of age. Data are expressed as means ± SE. * P < 0.05, 2-way ANOVA comparison between groups indicated.  P < 0.05, paired t-test comparison before and during isoproterenol for 5-mo-old hearts;  P < 0.05 for 24-mo-old hearts; and § P < 0.05 for 34-mo-old hearts.

6

Coronary flow. Baseline coronary flow normalized for LV weight (CF) was similar among groups (Fig. 2C). With increasing heart rate, CF increased significantly in all groups. Relative to baseline, CF at 10 Hz increased 72.4 ± 24.4% in adult (P < 0.01), 55.0 ± 17.8% in old (P < 0.05), and 75.7 ± 25.1% in senescent hearts (P < 0.005). After isoproterenol was administered, CF was significantly increased only in adult hearts (P < 0.05).

Ca²⁺-handling proteins. Western immunoblots against SERCA2a, phospholamban, and Na⁺/Ca²⁺ exchanger are shown in Fig. 3A, and corresponding results are shown in Fig. 3B for the three groups. Protein levels of the Na⁺/Ca²⁺ exchanger were reduced by 36.6 ± 6.1% in old and 57.6 ± 7.1% (P < 0.01) in senescent hearts relative to levels in adult hearts. There was no significant difference in SERCA2a protein levels among any of the groups. Phospholamban protein levels were increased by 158.1 ± 42.8% in old (P < 0.005) and 340.1 ± 20.7% in senescent hearts (P < 0.0001) relative to levels in adult hearts. Because phospholamban inhibits SERCA2a, it has been suggested that SERCA2a activity is determined by the ratio of these two proteins (7, 21, 26). Relative to adult hearts, the ratio of SERCA2a and phospholamban was reduced by 67.5 ± 5.5% (P = 0.0005) in old and 69.4 ± 2.7% (P = 0.0005) in senescent hearts.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   A: original Western immunoblots against Na+/Ca2+ exchanger (NCX), sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), and pentameric form of phospholamban (PLB) in 5-, 24-, and 34-mo-old hearts. Identical amounts (20 µg) of membrane proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, probed with SERCA2a-, PLB-, or NCX-selective antibodies, and then visualized by autoradiography. B: Western immunoblot quantification results in 5-mo-old (filled bar), 24-mo-old (open bar), and 34-mo-old hearts (shaded bar). Ratio of SERCA2a to PLB (SERCA2a/PLB) was used as an index of SERCA2a activity. All data are expressed relative to 5-mo-old hearts as means ± SE. * P < 0.05 relative to 5-mo-old hearts;  P < 0.05 relative to 24-mo-old hearts.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The main findings of the present study were that aging was associated with 1) preserved contractile performance but 2) an impaired lusitropy-frequency relationship that was completely reversible with -agonist stimulation and 3) reduced protein levels of Na⁺/Ca²⁺ exchanger and increased phospholamban relative to SERCA2a.

Inotropy-frequency relationship and aging. The mechanisms involved in enhanced contractility with increasing heart rate are associated with increased Ca²⁺ availability to the contractile proteins. This occurs as a result of an increase in transsarcolemmal Ca²⁺ influx, resulting from an increased number of action potentials per unit time, coupled with reduced time for diastolic efflux of Ca²⁺ via the Na⁺/Ca²⁺ exchanger, leading to a rise in SR Ca²⁺ storage and augmentation of subsequent Ca²⁺ transients (3, 20). Normal hearts of large mammals including humans generally exhibit a positive inotropy-frequency relationship (11, 17). In smaller mammals the inotropy-frequency relationship is less apparent: in rat cardiac muscle (28) and isolated mouse hearts (5) the relationship was shown to be negative, whereas in mouse cardiac muscle (16) and in the conscious mouse the relation was shown to be positive (30). In our study, isolated mouse hearts exhibited a positive inotropy-frequency response followed by a negative response, regardless of age. The cause of a negative inotropy-frequency relationship is not clear and could be due to impaired Ca²⁺ release from the SR at high heart rates (3, 20). The present data show a steeper decline of the inotropy-frequency relationship in senescent mice. In contrast, other studies in rats and rabbits show no age-related impairment of the inotropy-frequency relationship (9, 23). The discrepancy could be due to differences in experimental conditions, species, age, and pacing rates selected. The critical importance of the latter in unmasking age-related differences in contractile performance is underscored by our observations and those by others (18) that a significant decrease in contractile performance occurs only at high heart rates. Thus the negative inotropy-frequency response seen in isolated mouse hearts becomes more pronounced with age, and apparently this is only manifested at heart rates close to the physiological range in mice (550-620 beats/min).

Lusitropy-frequency relationship and aging. It is well documented that aging hearts exhibit prolonged LV relaxation rates (23, 32). This was seen in our study as a prolonged  and has been mainly attributed to a reduction in the rate of cytosolic diastolic Ca²⁺ removal (discussed below). Our results show that the lusitropy-frequency relationship is impaired with age, as evidenced by a progressive increase in EDP as heart rate increased. Despite prolonged  in aging hearts, increased heart rate enhanced LV relaxation (observed as a decrease in ) up to 8 Hz. The decrease in , however, was not adequate to compensate for the decreased diastolic duration at high heart rates, as evidenced by elevation in EDP with increasing heart rate for all groups.

Reversal of impaired lusitropy-frequency relationship with -adrenergic stimulation. To test the hypothesis that pharmacologically enhancing LV relaxation could reverse the age-related lusitropy-frequency impairment, hearts were exposed to the -agonist isoproterenol. -Adrenergic stimulation increases lusitropy by phosphorylation of troponin I and phospholamban, leading to faster release of Ca²⁺ from the myofilaments and enhanced reuptake of Ca²⁺ into the SR (3, 20). Administration of isoproterenol resulted in complete recovery of EDP at high heart rates in aging hearts. The prolonged  in aging hearts was dramatically shortened after isoproterenol stimulation and could not be distinguished from that in adult hearts. A decrease in -adrenergic sensitivity with adult aging has been widely documented in the rat and in humans as well (18, 22). Our goal, however, was not to specifically test for -adrenergic desensitization; rather, we wanted to determine whether impaired relaxation was at all reversible with isoproterenol. These results demonstrate that the lusitropic reserve is preserved in aging mouse hearts and thus is capable of accelerating myocardial relaxation when the -adrenergic system is sufficiently stimulated.

Ca²⁺-handling proteins and aging. Consistent with a previous report (2), we observed reduced expression of the Na⁺/Ca²⁺ exchanger in aging hearts. In contrast, SERCA2a levels were not significantly altered, a finding that is in agreement with some (6, 18) but not other studies that show a reduction in SERCA2a levels with age (2, 7). The discrepancy could be due to differences in species, strain, age, and tissue (ventricular vs. atrial) used in the experiments. Phospholamban levels progressively increased with age in our study, whereas senescent human atrial tissue has been reported to have decreased levels (7). However, if the stoichiometry of phospholamban to SERCA2a determines the level of SERCA2a activity (7, 21, 26), aging hearts exhibit a marked depression of SERCA2a activity. The functional significance of increased phospholamban levels relative to SERCA2a is reinforced by a recent report, which showed that increasing the phospholamban-to-SERCA2a ratio prolonged myocyte relaxation, whereas reduction of the ratio shortened relaxation (26). Despite increased phospholamban levels in senescent relative to old hearts, the present finding of similar SERCA2a-to-phospholamban ratios in both groups is consistent with little or no change in relaxation properties (i.e., ) between these two groups. Whether the increase in phospholamban with age in mice is simply due to decreased protein degradation or an adaptive response remains unknown. It is possible that, due to high heart rates in mice, SERCA2a is preserved late in life and an increase in phospholamban serves to conserve energetics in the resting state without compromising maximal SERCA2a activity during times of stress. The present data do not exclude the possibility that the intrinsic SERCA2a pumping rate is decreased in aging hearts (12), but a reduction in SERCA2a activity by increased phospholamban inhibition together with reduced levels of Na⁺/Ca²⁺ exchanger would be consistent with impaired Ca²⁺ removal from the cytosol and correlates with greater passive and dynamic diastolic dysfunction observed in the aging mouse hearts. Targeting of SERCA2a may be an important strategy to improve diastolic function in the elderly. Investigators have already successfully overexpressed SERCA2a via adenoviral gene transfer in senescent rat hearts and were able to show significant improvements in diastolic function (31). Although these data are strongly suggestive of impairment in Ca²⁺ regulation with age, we cannot exclude other possible mechanisms of age-impaired lusitropy such as alterations in myofilament properties, e.g., troponin I.