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Influence of age and run training on cardiac Na⁺/Ca²⁺ exchange

¹Department of Integrative Physiology, University of Colorado at Boulder, Boulder, Colorado 80309-0354; ²University of Vermont, Burlington, Vermont 05405; and ³Weis Research Center, Danville, Pennsylvania 17822

Submitted 23 May 2003 ; accepted in final form 21 July 2003

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Effects of age and training on myocardial Na⁺/Ca²⁺ exchange were examined in young sedentary (YS; 14-15 mo), aged sedentary (AS; 27-31 mo), and aged trained (AT; 8- to 11-wk treadmill run training) male Fischer Brown Norway rats. Whole heart performance and isolated cardiocyte Na⁺/Ca²⁺ exchange characteristics were measured. At the whole heart level, a small but significant slowing of late isovolumic left ventricular (LV) relaxation, which may be indicative of altered Na⁺/Ca²⁺ exchange activity, was seen in hearts from AS rats. This subtle impairment in relaxation was not observed in hearts from AT rats. At the single-cardiocyte level, late action potential duration was prolonged, resting membrane potential was more positive, and overshoot potential was greater in cardiocytes from AS rats than from YS rats (P < 0.05). Training did not influence any of these age-related action potential characteristics. In electrically paced cardiocytes, neither shortening nor intracellular Ca²⁺ concentration ([Ca²⁺]_i) dynamics was influenced by age or training. Similarly, neither age nor training influenced the rate of [Ca²⁺]_i clearance via forward (Na_in⁺ /Ca_out²⁺) Na⁺/Ca²⁺ exchange after caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum or cardiac Na⁺/Ca²⁺ exchanger protein (NCX1) expression. However, when whole cell patch-clamp techniques combined with fluorescence microscopy were used to evaluate the ability of Na⁺/Ca²⁺ exchange to alter cytosolic [Ca²⁺] ([Ca²⁺]_c) under conditions where membrane potential (V_m) and internal and external [Na⁺] and [Ca²⁺] could be controlled, we observed age-associated increases in forward Na⁺/Ca²⁺ exchange-mediated [Ca²⁺]_c clearance (P < 0.05) that were not influenced by training. The age-related increase in forward Na⁺/Ca²⁺ exchange activity provides a hypothetical explanation for the late action potential prolongation observed in this study.

caffeine; NCX1; Fisher Brown Norway rat; treadmill; heart; cardiocyte; calcium; sodium

From studies of enriched sarcolemmal vesicles or muscle strips isolated from rat, Na⁺/Ca²⁺ exchange activity has been reported to be decreased (13), increased (9), or unaffected (1) in aged myocardium. The basis for this lack of consensus is not known, but it may be related to methodological differences in membrane vesicle preparation or experimental models. Specifically, factors that influence Na⁺/Ca²⁺ exchange activity, including membrane potential (V_m) and transsarcolemmal [Ca²⁺] and [Na⁺] gradients, or other cytosolic modulators of Na⁺/Ca²⁺ exchange activity may not be functionally operational or observable in studies of membrane vesicles and muscle strips.

Consequently, we sought to determine the effects of advanced age on Na⁺/Ca²⁺ exchange activity in left ventricular (LV) cardiocytes isolated from the rat. By using isolated cardiocytes, Na⁺/Ca²⁺ exchange activity could be assessed under conditions in which V_m and/or transsarcolemmal [Ca²⁺] and [Na⁺] gradients could be controlled and in a setting in which the most basic levels of cellular organization were still intact. Whole heart performance and action potential characteristics were assessed to determine whether a correlation exists between age-related changes in Na⁺/Ca²⁺ exchange activity and myocardial relaxation or repolarization. In addition, exercise training has been shown to improve mechanical relaxation in aged myocardium (12, 34, 37, 39), whereas it does not appear to reverse age-related action potential prolongation (12). Our working hypothesis was that Na⁺/Ca²⁺ exchange activity would be reduced in ventricular cardiocytes isolated from aged rats compared with their young sedentary counterparts and that endurance training would normalize (increase) Na⁺/Ca²⁺ exchange. These alterations would mirror the age- and training-related alterations in myocardial relaxation. An alternative to our working hypothesis is that advanced age would bring about an increase in myocardial Na⁺/Ca²⁺ exchange activity that would be unaffected by endurance training and that these alterations would be associated with changes in the duration of the later phase of action potential repolarization. Contrary to our working hypothesis, the results of our studies indicate that advanced age increased forward Na⁺/Ca²⁺ exchange in LV cardiocytes and run training did not significantly alter Na⁺/Ca²⁺ exchange in aged myocardium.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Animal model. Young sedentary (YS; 14-15 mo), aged sedentary (AS; 27-31 mo), and aged trained (AT) male Fisher Brown Norway (FBN) rats were used in this study. This rat strain was used for its relatively well-known and nonpathological response to advancing age (22, 43). All animals were maintained on a 12:12-h light-dark cycle and provided food and water ad libitum. Animal protocols received prior approval from the Institutional Animal Care and Use Committee at the University of Colorado at Boulder and were conducted under the guidelines accepted by the American Physiological Society. The run training protocol was conducted over a minimum of 10 wk on motorized treadmills kept at a 10% grade. AT rats were initially introduced and familiarized to the treadmill with a single 5-min walk (<14 m/min). As presented in Fig. 1, the speed, duration, or both were incrementally increased each day for 2 wk (5 days/wk). After this introductory period, the daily training protocol consisted of a 5-min warm-up at 14 m/min, followed by 40 min at 17.5 m/min, 5 days/wk, for at least 8 and no more than 11 wk.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Treadmill introduction and training protocol for aged trained (AT) rats. Gray areas indicate the warm-up period (14 m/min), black areas the training period (17.5 m/min), and white areas the cool-down period (14 m/min). The rats were introduced to and familiarized with treadmill running for 2 wk. The speed, duration, or both were incrementally increased each day during this 2-wk introductory period on a 5 days/wk schedule. After the introductory period, the daily training protocol consisted of a 5-min warm-up at 14 m/min, followed by 40 min at 17.5 m/min, up a 10% grade, 5 days/wk for at least 8 but not more than 11 wk.

Isovolumic left heart experiment. Hearts from YS (n = 9), AS (n = 10), and AT (n = 10) rats were isolated and instrumented to assess isovolumic LV pressure dynamics as previously described (20). Briefly, Langendorff-perfused hearts were instrumented with a custom-made, highly compliant, fluid-filled latex balloon sized to fit the LV cavity, which was coupled to a pressure-transducing catheter. Hearts were electrically paced at 5 Hz. Before data collection, fluid was injected into the latex balloon to yield a steady-state minimum LV pressure of 10 mmHg. Experiments were performed at 35°C. The free [Ca²⁺] of the normal Krebs-Henseleit perfusate was 1.25 mM.

Cardiocyte isolation. Cardiocytes were obtained from enzymatically digested hearts with modifications to methods previously described in detail (6, 7, 25). The original isolation protocol called for the heart to be perfused initially with a modified Krebs-Henseleit (KH) buffer containing 1.28 mM Ca²⁺ for 15 min, followed by perfusion with a nominal-Ca²⁺ KH buffer, and a solution containing 0.54 µM [Ca²⁺] and digestive enzymes. In the modified protocol, the first step was eliminated and the nominal-Ca²⁺ KH buffer was delivered first. Several additional modifications were made to the procedure for this study to improve aged cardiocyte survivability. First, the collagenase concentration used in the digestion solution was higher for the aged (419 U/ml) than the young (279 U/ml) animals. Second, the digestion solution perfusion pressure used for the aged hearts was higher than that used for the young hearts (46.5 vs. 32 mmHg). Cardiocytes were suspended in growth media, seeded onto laminin-coated glass coverslips, and incubated at 37°C in a 5% CO₂-21% O₂ (balance N₂) environment. All cardiocyte experiments were performed between 2 and 6 h after cardiocyte dispersion. All chemicals and reagents were acquired from Sigma Chemical (St. Louis, MO) unless otherwise noted.

Morphology experiment. At the time of death, adrenal gland and spleen weights and left tibial length were measured. Plantaris muscles were isolated and cross sections were homogenized and assayed for citrate synthase activity (36) to verify a peripheral training effect. Cardiocyte length, width, and area were measured after the cardiocytes were plated onto the glass coverslips and incubated for at least 30 min in 10 mM 2,3-butanedione monoxime (BDM) at 37°C. Cross-bridge formation is disrupted by BDM (11) and was used in these experiments to avoid potential between-group differences in cardiocyte morphology occurring as a result of differences in basal levels of contractile element activation. Western blots were conducted on LV tissue samples to determine the effect of training and advanced age on NCX1 expression with previously established procedures (50).

Cardiocyte pacing and caffeine contraction experiment. A Nikon Diaphot microscope fitted with a x40 oil-immersion objective was used for all experiments. [Ca²⁺]_i dynamics were measured with the method and fluor excitation filter pairing of Palmer et al. (32) and Palmer and Moore (31). Data were analyzed with a computer equipped with IonWizard 5.0 software (IonOptix, Milton, MA).

In our experiments, steady-state cardiocyte shortening dynamics and fluorescence ratio (R) data were recorded from cardiocytes isolated from 9 YS (39 cells), 11 AS (31 cells), and 11 AT (48 cells) rats after 7 min of electrical pacing. Only R data were recorded during caffeine-induced contractions in cardiocytes isolated from 9 YS (32 cells), 9 AS (26 cells), and 10 AT (39 cells) rats. We used a protocol previously established by our laboratory (29, 30), except that fura 5F (Molecular Probes, Eugene, OR) was used as the fluorescent Ca²⁺ indicator for its high selectivity for Ca²⁺ and wide dynamic range (K_d = 400 nM) (10, 14). An example of an R transient recorded during electrical stimulation and caffeine exposure and the R characteristics measured are illustrated in Fig. 2.

View larger version (30K): [in this window] [in a new window]   Fig. 2. An example of fluorescence ratio (R) transients recorded during electrically induced and caffeine-induced contractions. The R transients occurring in response to electrical pacing were recorded after a contractile steady state had been achieved. A caffeine contraction was initiated by the rapid application of a superfusate containing neither Na+ nor Ca2+, as indicated (between 6 and 11 s). A Na+-rich superfusate was then applied, and the rate of Na+-dependent Ca2+ efflux, presumed to be due to forward Na+/Ca2+ exchange, was characterized by the rate constant kefflux. Other characteristics of intracellular Ca2+ concentration ([Ca2+]i) dynamics that were analyzed include resting R during pacing [Rrest(p)], peak R value during pacing - Rrest(p) [Rdiff(p)], the exponential rate constant describing R transient return to Rrest(p) [kfall(p)], resting R immediately before caffeine exposure [Rrest(c)], peak R value elicited by caffeine exposure - Rrest(c) [Rdiff(c)], the rate constant describing the transition from the initial caffeine-induced R transient to Requil (kequil), and the plateau R value occurring just before exposure of the cell to extracellular Na+ (Requil).

Recorded R transients for both electrically paced and caffeine-exposed data were corrected for background fluorescence on a cell-by-cell basis and analyzed with IonOptix software as previously described (29, 32). It should be noted that the nomenclature for certain fluorescence metrics is similar among the pacing, caffeine contraction, and voltage-clamp protocols. Therefore, the subscript (p) is used to specify the measurements derived from paced cardiocytes, the subscript (c) for the caffeine contraction measurements, and the subscript (v) for the voltage-clamp measurements (see Voltage clamp experiment). The key R characteristics assessed during pacing were baseline (diastolic) R [R_rest(p)], time-to-peak R [TTP_(p)], R difference [peak R - R_rest(p), designated R_diff(p)], and the exponential rate constant, [k_fall(p)]. The R characteristics analyzed for the caffeine contraction experiment were baseline (diastolic) R immediately before caffeine exposure [R_rest(c)], R difference [peak R during caffeine exposure - R_rest(c), designated R_diff(c)], R plateau occurring during caffeine exposure before the reintroduction of extracellular Na⁺ (R_equil), and the exponential rate constants k_equil and k_efflux. All rate constants were determined by nonlinear, least-squares fitting of exponential functions to the recorded R transient (15, 29, 30).

Voltage-clamp experiment. Whole cell V_m was controlled with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and recorded onto a computer with pCLAMP 8.1 software (Axon Instruments). Cytosolic [Ca²⁺] ([Ca²⁺]_c) dynamics were measured as described in Cardiocyte pacing and caffeine contraction experiment, with the exception that fura 2 free acid (Molecular Probes) was used as the [Ca²⁺]_c indicator.

[Ca²⁺]_c data were recorded under voltage-clamp control in cardiocytes isolated from 11 YS (28 cells), 15 AS (25 cells), and 13 AT (21 cells) rats with a modification of the protocol established by Barcenas-Ruiz et al. (2). Na⁺/Ca²⁺ exchange was isolated from other cellular ion transport mechanisms by pharmacological blockade and chemical substitution with a bathing solution containing (in mM) 136.3 NaCl, 1 MgCl₂, 2.4 CaCl₂, 7.5 CsCl, 7.5 tetraethylammonium (TEA), 2.5 glucose, 10 HEPES, and 0.5 pyruvate and (in µM) 25 ryanodine, 3.75 thapsigargin, and 7.45 verapamil, pH 7.4 with NaOH. Verapamil was used to block L-type Ca²⁺ channel activity, thapsigargin to block sarcoplasmic reticular (SR) Ca²⁺ uptake, ryanodine to block SR Ca²⁺ release, and TEA and CsCl to block K⁺ currents. Cardiocytes were internally dialyzed for 5 min with a pipette (1.3-2.5 M) filling solution containing the free acid form of the calcium indicator fura 2 (70 µM) and (in mM) 5 NaCl, 130 CsCl, 2 MgCl₂, 10 HEPES, and 2 Na₂ATP, pH 7.2 with CsOH. Experiments were performed at 24 ± 1°C.

Our voltage-clamp protocol was designed to force Na⁺/Ca²⁺ exchange to work in both the forward (Na_in⁺/Ca_out²⁺ and reverse (Na_out⁺)/Ca_in²⁺) directions. Briefly, 2,500-ms voltage steps (to -40, 0, and +40 mV) were initiated from a holding potential of -80 mV (see Fig. 3). At the end of each voltage step, the V_m was returned to -80 mV and the next sweep was initiated after a 2-min interval. The three voltage steps (to -40, 0, and +40 mV) were selected to stimulate reverse Na⁺/Ca²⁺ exchange and intracellular Ca²⁺ loading. Repolarization to the holding potential (-80 mV) after each depolarizing step stimulated forward Na⁺/Ca²⁺ exchange and Ca²⁺ clearance from the cell, until resting [Ca²⁺]_c was achieved. The key measurements made during the voltage-clamp protocol were peak difference (peak [Ca²⁺]_c - baseline [Ca²⁺]_c) and k_rise and k_fall(v) (exponential constants characterizing the voltage-mediated rates of [Ca²⁺]_c rise and fall, respectively). All fluorescence ratiometric data were acquired from a discrete region of each cardiocyte with an adjustable iris (to exclude the recording pipette), and all R data were corrected for background fluorescence, which was determined by measuring the fluorescence of each cardiocyte before loading with fura 2.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Example of a cytosolic [Ca2+] ([Ca2+]c) transient measured during a 2,500-ms voltage step to +40 mV. A: representative [Ca2+]c transient in response to the voltage step protocol. Ratiometric fura 2 fluorescence (R) data, obtained with a 405 nm/365 nm excitation wavelength pairing, were transformed to [Ca2+]c as described in Palmer and Moore (31) with an assumed Kd of 145 nM. The dashed and solid lines through the [Ca2+]c transient data are representative exponential fits describing the rates of voltage-dependent rise and fall in [Ca2+]c, respectively. Peak difference, peak [Ca2+]c - baseline [Ca2+]c; krise, exponential rate constant describing the voltage-dependent rise in [Ca2+]c, assumed to occur via the actions of reverse Na+/Ca2+ exchange; CaPlat, plateau [Ca2+]c value derived from an average taken for 800 ms over the last third of the voltage step; kfall(v), exponential rate constant describing the voltage-dependent fall in [Ca2+]c, assumed to occur via the actions of forward Na+/Ca2+ exchange. Peak difference and krise were not different between groups (P > 0.05), suggesting that neither age nor run training influenced the initial rate of reverse Na+/Ca2+ exchange activity. B: voltage clamp protocol illustrating the 2,500-ms step from -80 to +40 mV. C: membrane current generated by the 120-mV voltage step.

Action potential experiment. Action potential recordings were made with fire-polished glass pipettes (3.0-5.0 M) containing an internal buffer composed of (in mM) 140 KCl, 10 HEPES, 4 MgCl₂, 5 Na₂ creatine phosphate, 5 EGTA, 3.1 Na₂ATP, and 0.06 CaCl₂, pH 7.1 with KOH. The external solution contained (in mM) 132 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.8 MgCl₂, 10 HEPES, 10 glucose, and 0.6 NaH₂PO₄, pH 7.4 with NaOH. Action potentials were elicited at a frequency of 0.2 Hz and a 5-ms maximally activating current pulse with an Axopatch 1D amplifier (Axon Instruments) in current-clamp mode. Steady-state action potentials were analyzed for resting V_m (V_rest), maximal (overshoot) V_m (V_max), and action potential amplitude (V_max - V_rest). Action potential duration was characterized by measuring the times required for membrane potential to return from V_max to 10%, 25%, 50%, 75%, and 90% of V_rest.

Data analysis. All statistical analyses were performed with SPSS 10.1 software (SPSS, Chicago, IL). To test age and training effects, a univariate ANOVA was performed on all electrophysiological and [Ca²⁺] dynamics data. In testing the response of each group to pacing and caffeine, and on all the myocyte shortening and morphology data, a one-way ANOVA was performed. Specific differences were identified and confirmed with a Bonferroni post hoc analysis of multiple comparisons. Post hoc comparisons were preceded by subjecting data to Levene's test of equality of variances. Our statistical significance reporting criteria for contrasts between data sets with equal and unequal variances were P < 0.05 and P < 0.10, respectively. The tests for significance across data sets with unequal variances are more conservative, and per the principles described by Williams et al. (46), the use of the latter significance minimizes the propensity to commit a type II interpretive error. All data in Figures 5, 6, and 8, and Tables 1, 2, 3, 4, 5 are presented as means ± SE.

View larger version (22K): [in this window] [in a new window]   Fig. 5. [Ca2+]c during the plateau phase (CaPlat) of the voltage step protocol. CaPlat (see Fig. 3) values for cardiocytes isolated from YS (filled bars), AS (open bars), and AT (gray bars) rats are shown. AS cardiocytes exhibited lower CaPlat than YS cardiocytes during depolarization steps (P < 0.05). Inset: the corresponding membrane currents (derived from an average taken for 800 ms over the last third of the voltage step), designated Iplat, were not different between groups.

View larger version (11K): [in this window] [in a new window]   Fig. 6. Effects of advanced age and endurance training on the rate of [Ca2+]c fall during the repolarizing phase of the voltage-clamp protocol. kfall(v) was significantly lower (P < 0.01) in YS cardiocytes () than in cardiocytes isolated from both aged groups: AS () and AT (). These data suggest that advanced age increased the rate of Na+/Ca2+ exchange-mediated [Ca2+]c removal from cardiocytes compared with YS cardiocytes (*P < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 8. Correlation between late AP recovery times and forward Na+/Ca2+ exchange-mediated [Ca2+]c removal rate [kfall(v)] after a +40-mV voltage step. Mean kfall(v) values for each experimental group were plotted against the mean times required for the AP to return to 75% (T75; filled symbols, solid regression line; r2 = 0.98, P < 0.05) and 90% (T90, open symbols, dotted regression line; r2 = 1.0, P < 0.05) of resting membrane potential. Circles, YS; triangles, AS; squares, AT.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Morphology, Western blot, and LV contractile function. Both age and training significantly increased cardiocyte length, width, and area (Table 1). Neither training nor age individually affected tibial length or adrenal weights. Body and spleen weights significantly increased with age, but training either prevented or reversed the age-related increases, respectively. Plantaris citrate synthase activity levels were significantly increased in AT compared with both AS and YS (P < 0.05), verifying peripheral training adaptations as previously reported (27).

To evaluate NCX1 expression, septal and LV free wall tissue samples from YS (n = 12), AS (n = 11), and AT (n = 8) hearts were homogenized and used for Western blot protein analysis with monoclonal antibody R3F1 that recognizes a single band (NCX1) at an apparent molecular mass of 160 kDa as shown in Fig. 4. Because previous work showed calsequestrin protein (CLSQ) levels to be uninfluenced by advanced age in the heart (48), CLSQ expression was used as an internal standard (48). After phosphorimager quantification, no between-group or regional differences were found in NCX1 and CLSQ expression or in NCX1-to-CLSQ ratios (P > 0.05).

View larger version (10K): [in this window] [in a new window]   Fig. 4. Western blot of cardiac Na+/Ca2+ exchanger protein (NCX1). Representative Western blots performed on homogenates of hearts from young sedentary (YS), aged sedentary (AS), and aged trained (AT) rats are shown. As shown by phosphorimager quantification of 12 YS, 11 AS, and 8 AT hearts, no between-group differences were found in NCX1 or calsequestrin protein (CLSQ) expression or NCX1-to-CLSQ ratios.

Isovolumic left heart experiment. Advanced age modestly impaired late LV relaxation in paced, isovolumic hearts (Table 2). The time required for hearts to achieve 75% (T₇₅) and 90% (T₉₀) maximal relaxation was slightly but significantly prolonged in hearts from AS rats compared with hearts from the YS animals (P < 0.05). Training improved late relaxation to the extent that T₉₀ was significantly shorter in AT than AS hearts (P < 0.05).

Cardiocyte pacing and caffeine contraction experiment. Neither age nor training significantly affected cardiocyte shortening characteristics (data not shown). Steady-state ratiometric fluorescence characteristics of the electrically paced cardiocytes are presented in Table 3. R_rest(p), R_diff(p), and k_fall(p) were not affected by advanced age or run training. TTP_(p) was slower in AT cardiocytes than in YS cardiocytes (P < 0.05). No other differences in fluorescence dynamics were observed between groups.

The R dynamics of caffeine-stimulated cardiocytes are shown in Table 4. Similar to results from the paced cardiocytes, R_rest(c) was not affected by advanced age or training. Values for R_diff(c), k_equil, k_efflux, and R_equil were also not influenced by age or training.

Voltage-clamp experiment. In these experiments, we examined V_m-dependent changes in [Ca²⁺]_c that were produced by forward and reverse Na⁺/Ca²⁺ exchange (see Fig. 3). Time-to-peak [Ca²⁺]_c (TTP_(v)), defined as the time elapsed between the onset of membrane depolarization and the apex of the [Ca²⁺]_c transient, was not influenced by advanced age or training (TTP_(v) values for the voltage step to +40 mV: YS = 688 ± 80 ms, AS = 731 ± 130 ms, and AT = 601 ± 60 ms; P > 0.05). It is relevant to note that the long TTP_(v) values observed in this study (ranging from 400 to 1,200 ms) provide evidence that SR Ca²⁺ release was successfully eliminated by pharmacological blockade (see METHODS) and that the voltage-dependent increase in [Ca²⁺]_c was due to reverse Na⁺/Ca²⁺ exchange. Neither the rate of [Ca²⁺]_c rise (k_rise; values expressed in s^-1 for the voltage step to +40 mV: YS = 5.2 ± 0.7, AS = 5.1 ± 0.9, and AT = 5.4 ± 0.6; P > 0.05) nor peak [Ca²⁺]_c difference (peak difference values expressed in nM for the voltage step to +40 mV: YS = 32.6 ± 5.3, AS = 24.9 ± 3.7, and AT = 27.7 ± 5.5; P > 0.05) were affected by age or training.

In each voltage-dependent [Ca²⁺]_c transient, a [Ca²⁺]_c plateau was evident during the last third of each voltage step (beginning 1.5 s into the voltage step), and this plateau value was designated CaPlat (see Fig. 3A). We found direct age-associated reductions in CaPlat, with the AS group exhibiting significantly lower CaPlat values than the YS group (P < 0.05; Fig. 5).

Advanced age was associated with an increase in the repolarization-induced rate of [Ca²⁺]_c fall [k_fall(v)] in patch-clamped cardiocytes compared with YS cardiocytes (P < 0.01; Fig. 6). This finding is indicative of an age-associated increase in forward Na⁺/Ca²⁺ exchange activity ([Ca²⁺]_c clearance).

Action potential experiment. V_rest was more positive in cardiocytes from both aged groups compared with YS cardiocytes (P < 0.05; Table 5). V_max and action potential amplitude were significantly greater in AS cardiocytes relative to YS cardiocytes (P < 0.05). Action potential duration was not different between groups until the later phase of recovery and then only in response to advanced age, not training. The time required to return to 75% and 90% of V_rest from V_max, T₇₅ and T₉₀, was significantly increased with age (P < 0.05) and can be seen in representative action potential tracings in Fig. 7. This age-associated prolongation was also evident in the AT cardiocytes, albeit to a lesser extent (P < 0.10).

View larger version (11K): [in this window] [in a new window]   Fig. 7. Effect of advanced age on the cardiac action potential in isolated rat cardiocytes. Representative examples of action potentials recorded from 2 myocytes isolated from YS and AS hearts are shown. The time required to return to 75% (T75; closed arrows) and 90% (T90; open arrows) of resting (Vrest) from maximal (Vmax) membrane potential was significantly (P < 0.05) increased with age (see Table 5). For purposes of temporal comparison, the amplitudes of AP data from both myocytes were sealed by expressing membrane voltages during the AP (V) as a fraction of AP amplitude (Vm).

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Slowed myocardial relaxation and action potential prolongation are commonly observed consequences of advanced aging (5, 16, 18, 23, 35, 37, 38, 44, 45). These two findings in the aging literature give rise to two mutually exclusive hypotheses for how advanced age might influence Na⁺/Ca²⁺ exchange function. First, because of its involvement in cardiocyte Ca²⁺ regulation, it is conceivable that decreases in Na⁺/Ca²⁺ exchange activity could contribute to age-related changes in myocardial relaxation. Alternatively, because of its electrogenic properties it is also possible that increases in Na⁺/Ca²⁺ exchange could be responsible for prolonged late action potential duration. Advanced age has been reported to decrease (13), increase (9), or not influence (1) Na⁺/Ca²⁺ exchange function in studies of isolated membrane vesicles and cardiac muscle strip preparations. Consequently, we directly examined the effects of aging and training on Na⁺/Ca²⁺ exchange function in intact ventricular cardiocytes and on NCX1 expression in ventricular myocardium.

We found no age-, training-, or region-related differences in NCX1 expression in our studies. At a fundamental level, these findings are significant in that they would indicate that neither advanced age (AS) nor training superimposed on advanced age (AT) elicits alterations in myocardial Na⁺/Ca²⁺ exchange activity via alterations in the amount of NCX1 expressed in the heart. On the other hand, because NCX1 activity can be modulated by a myriad of factors, our Western blot data cannot be interpreted to mean that myocardial Na⁺/Ca²⁺ exchange activity is uninfluenced by advanced age or training. Consequently, we used two different approaches to examine the operation of Na⁺/Ca²⁺ exchange in intact cardiocytes isolated from rat ventricular myocardium.

One approach involved the use of isolated cardiocytes that were electrically paced and then subjected to rapid solution changes that were designed to isolate and allow for the characterization of Na⁺/Ca²⁺ exchange-mediated Ca²⁺ clearance from the cell. Rapid solution switching strategies were used to isolate and characterize effects of advanced age and training on Na⁺-dependent Ca²⁺ removal from ventricular cardiocytes. We used this approach previously (29) to demonstrate that in mature adult rats a program of high-intensity endurance training elicits a reduction in the rate of cellular Ca²⁺ clearance via forward Na⁺/Ca²⁺ exchange, as reflected by the rate constant k_efflux, describing Na⁺/Ca²⁺ exchange-mediated Ca²⁺ clearance from the cell. In the present study, neither advanced age nor training influenced k_efflux. These negative results are subject to two different interpretations. First, if taken at face value, these findings indicate that in intact cardiocytes possessing a native intracellular milieu, neither advanced age nor training noticeably alters the contribution of Na⁺/Ca²⁺ exchange to sarcoplasmic Ca²⁺ clearance. The lack of influence of training on k_efflux in the present study may have been due to the fact that the aged animals were not able to train as intensely as the younger animals that were used in the earlier studies (29). Alternatively, it is possible that small age- or training-induced alterations in intrinsic Na⁺/Ca²⁺ exchange function may have been obscured in our experimental context. It is well known that Na⁺/Ca²⁺ exchange function is subject to control by V_m and the magnitude of transsarcolemmal [Na⁺] and [Ca²⁺] gradients. In our caffeine contraction experiments, we could tightly control the composition of the extracellular milieu, but we did not have control of or quantitative information about V_m and transsarcolemmal [Na⁺] and [Ca²⁺] gradients across our three experimental groups. Therefore, we conducted experiments to assess Na⁺/Ca²⁺ exchange function under conditions in which these key control parameters could be standardized.

Our whole cell voltage-clamp experiments allowed us to examine Na⁺/Ca²⁺ exchange-mediated changes in cardiocyte [Ca²⁺] under conditions in which V_m was carefully controlled, cells were internally dialyzed with solutions of known composition, and changes in [Ca²⁺]_c could be directly monitored. Under these tightly controlled conditions, the voltage-induced elevation in [Ca²⁺]_c was greater in YS than AS cardiocytes, perhaps because of age-induced diminution in reverse (Na_out⁺/Ca_in²⁺) Na⁺/Ca²⁺ exchange activity late in the voltage step (Fig. 5). It is generally thought that under most physiological conditions, the regulatory importance of cellular Ca²⁺ influx via reverse Na⁺/Ca²⁺ exchange is quite minor (4), and it is difficult to ascertain what the physiological significance of our finding might be.

More importantly, on hyperpolarization of Ca²⁺-loaded cardiocytes, we did find evidence of an age-induced increase in forward Na⁺/Ca²⁺ exchange activity that was not influenced by exercise training (see Fig. 6). This finding is significant for several reasons. Forward (Na_in⁺/Ca_out²⁺) Na⁺/Ca²⁺ exchange is central to overall cardiocyte Ca²⁺ homeostasis because it is the primary cellular Ca²⁺ efflux mechanism involved in quantitatively offsetting the Ca²⁺ influx that occurs during normal excitation-contraction coupling on a beat-to-beat basis. Efflux of Ca²⁺ via forward Na⁺/Ca²⁺ exchange has been implicated in late myocardial relaxation (3, 49), and, owing to the electrogenic nature of Na⁺/Ca²⁺ exchange, it has also been shown to influence late action potential repolarization (49). The age-associated acceleration in forward Na⁺/Ca²⁺ exchange-mediated Ca²⁺ clearance that we observed is not consistent with and cannot explain the age-associated prolongation in late LV relaxation, and subsequent normalization with training, that we reported in this study (Table 2). If late LV relaxation were significantly influenced by forward Na⁺/Ca²⁺ exchange, we would have expected to see an age-associated decrease, rather than an increase, in this type of exchange activity in isolated cardiocytes. Our forward Na⁺/Ca²⁺ exchange data are, however, sufficient to explain the late action potential prolongation that we observed in cardiocytes from aged rats.

Because forward Na⁺/Ca²⁺ exchange produces a net inward current (3Na_in⁺/1Ca_out²⁺), an increase in forward activity may influence action potential characteristics via membrane depolarization. In this study, we found that with advanced age, V_rest was slightly but significantly more positive, V_max was significantly more positive, and late action potential repolarization was slowed (Table 5). In different rat models of advanced aging, early and late action potential prolongation and pronounced V_max have also been observed (5, 12, 23, 44, 45), but none of these earlier studies addressed the ionic basis for late action potential prolongation (see below). We are aware of only one other study in which the effects of training on action potential characteristics were examined in myocardium isolated from aged rats. Whereas we found no evidence that training significantly altered any of the age-related changes in the cardiac action potential, Gwathmey et al. (12) reported that training tended to normalize the age-induced increase in V_max while further prolonging action potential duration. That our results do not exactly corroborate the findings of Gwathmey et al. (12) is not too surprising given the rat strain (FBN vs. Fischer 344) and training protocol (e.g., running speed, grade, and duration) differences between the two studies. What is intriguing in our study is that the age-related prolongations in late action potential duration that we observed were associated with the increases that we observed in forward Na⁺/Ca²⁺ exchange. Although the precise ionic events responsible for the age-dependent prolongation of the late repolarization phase of the rat action potential have not been previously identified, it has been suggested that a Ca²⁺-dependent inward current (24), perhaps a Na⁺/Ca²⁺ exchange current (44), may be involved.

The idea that Na⁺/Ca²⁺ exchange may contribute to prolonged action potential duration is supported by our work in that age-related increases in forward Na⁺/Ca²⁺ exchange were correlated with the changes in late action potential duration that we observed (see Fig. 8). Although the correlation certainly does not prove causality, it does provide a circumstantial case for the involvement of Na⁺/Ca²⁺ exchange in this age-dependent phenomenon. This is physiologically significant because it is known that hearts exhibiting prolonged action potential duration often display proarrhythmic phenomena (3).

In this study, the age-related increase in ventricular cardiocyte cell dimension and the slowing of LV relaxation corroborate the central cardiac effects of advanced aging described by others using different rat strains (8, 19, 23, 45). In addition, when endurance exercise training was superimposed on our model of aging, we observed further increases in cardiocyte dimension and an improvement in the lusitropic function of the heart isolated from aged animals. These types of observations have been described previously in the literature (21, 26, 39, 42, 47). However, we should point out that in our FBN rat model of advanced aging, the slowing of LV relaxation was quite subtle relative to that observed in other rat models of aging (19, 45). In addition, we found no evidence of age- or training-induced alterations in [Ca²⁺]_i dynamics during electrical pacing of single cardiocytes. The absence of gross age-associated alterations in [Ca²⁺]_i dynamics in paced cardiocytes is at odds with published work from other laboratories (16, 44). Several studies have demonstrated [Ca²⁺] transient prolongation with advanced aging in electrically paced papillary muscle and isolated cardiocyte preparations (28, 33). In general, it is thought that the prolongations of myocardial relaxation times that are typically observed in aged preparations (5, 39, 45) occur as a result of slowed [Ca²⁺] clearance from the sarcoplasm caused by a diminished expression and activity of the cardiac isoform of the sarco(endo)plasmic reticular Ca²⁺ ATPase (SERCA2) (40). It is relevant to note, however, that none of the studies that demonstrated large age-associated reductions in lusitropic function and SERCA2 activity, as well as [Ca²⁺] transient prolongation, used the FBN rat model of aging. Contrary to this previous body of work, Wahr et al. (43) concluded that SR function was maintained in advanced aging in intact FBN cardiocytes. The fact that we only observed a subtle diminution in LV lusitropic function and did not detect alterations in paced cardiocyte shortening or [Ca²⁺]_i dynamics is consistent with this point of view. The FBN rat strain exhibits less systemic and cardiovascular pathology with age and maintains normal cardiac function until later in life than other rat strains (43). Therefore, it seems reasonable that age-associated alterations in lusitropic function and Ca²⁺ regulation in the absence of overt animal pathology are less prominent or absent in FBN rats. We did, however, observe action potential prolongation in the aged FBN rat model, giving rise to the interesting possibility that action potential prolongation is a true age-related phenomenon, whereas reduced SR Ca²⁺ clearance occurs as a result of pathologies secondary to the aging process.

There are several other issues regarding the results of our studies warranting comment. First, although we did observe subtle lusitropic dysfunction in our isovolumic left heart preparation, we found no evidence for slowed Ca²⁺ clearance or mechanical relaxation in unloaded single myocytes. We believe that there is a simple explanation for this apparent paradox that centers on data variability inherent in different measurement techniques. The coefficients of variation of "relaxation" data derived from isovolumic heart pressure and fura 5F-reported [Ca²⁺] transient measurements were 3-6% and 30-50%, respectively. The small changes in late relaxation that were observed in the isovolumic heart studies would have been undetectable in our single-cell studies. Second, because we did observe an age-related increase (30-40%) in forward Na⁺/Ca²⁺ exchange-mediated Ca²⁺ clearance from myocytes studied under strict voltage control, the question arises as to why these changes were not manifest as alterations in the [Ca²⁺] decline phases in our electrically paced myocyte and caffeine contraction experiments. With regard to the latter, inspection of Table 4 reveals that the k_efflux values for the AS and AT cardiocytes were 10-20% higher than for YS cardiocytes, but owing to the high variability inherent in fura 5F data, we were not able to determine whether this apparent age-related k_efflux trend was reflective of pure chance or of a true increase in a Ca²⁺ clearance mechanism. As pointed out above, we believe that it was only under conditions with the strictest experimental control that age-related Na⁺/Ca²⁺ exchange-mediated Ca²⁺ clearance differences were statistically observable.

Finally, the question arises as to why age-related increases in Na⁺/Ca²⁺ exchange activity were not discernable in the shape of [Ca²⁺] transients recorded from electrically paced cells. In electrically paced cardiocytes, the rate constant of [Ca²⁺] decline occurs at 6.5 s^-1 (see Table 3), whereas Ca²⁺ clearance via Na⁺/Ca²⁺ exchange occurs at 1 s^-1 (see k_efflux data in Table 4 and Fig. 6); the rate of Ca²⁺ clearance via Na⁺/Ca²⁺ exchange is only 15% of that mediated by fast processes (i.e., the SR and intracellular buffers). In view of the very small contribution of Na⁺/Ca²⁺ exchange to Ca²⁺ clearance during a single contraction-relaxation cycle, it is unlikely that a modest (30-40%) alteration in Na⁺/Ca²⁺ exchange activity would be discernable in the [Ca²⁺] transient. This viewpoint is consistent with the observation that, in a murine model of NCX1 overexpression (49), an approximately threefold increase in Na⁺/Ca²⁺ exchange activity (i.e., 10x greater than that observed in this study) only produced a 30% acceleration in Ca²⁺ clearance during electrical pacing.

In summary, we found that in the FBN rat model of advanced age there was a very small but significant impairment in myocardial relaxation at the whole heart level that was ameliorated by a program of endurance treadmill running. This age-related impairment or its amelioration by training is not mediated by intrinsic alterations in Na⁺/Ca²⁺ exchange. In single LV cardiocytes studied under highly controlled conditions, we found evidence of an increase in forward Na⁺/Ca²⁺ exchange activity that was uninfluenced by exercise training. This occurred in the absence of age or training-induced changes in NCX1 expression in any region of the heart. The increases in Na⁺/Ca²⁺ exchange activity that we observed were associated with, and provide a possible explanation for, late phase action potential prolongation in advanced age.

	DISCLOSURES

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
This study was supported by National Institutes of Health Grants AG-13981 and HL-40306.

	ACKNOWLEDGMENTS

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
The authors are grateful for the expert technical assistance of Danielle Hodne, Katie Esquivel, Justin Myers, and Craig Emter.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. L. Moore, Dept. of Integrative Physiology, 354 UCB, Univ. of Colorado at Boulder, Boulder, CO 80309-0354.

Original submission in response to a call for papers on "Physiology of Aging."

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.

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TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 

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