Comparison of ATP-dependent calcium transport and calcium-activated ATPase activities of cardiac sarcoplasmic reticulum and sarcolemma from rats of various ages

Mechanisms of Ageing and Development, 38 (1987) 127-143 Elsevier Scientific Publishers Ireland Ltd.

127

COMPARISON OF ATP-DEPENDENT CALCIUM TRANSPORT AND CALCIUMACTIVATED ATPase ACTIVITIES OF CARDIAC SARCOPLASMIC RETICULUM AND SARCOLEMMA FROM RATS OF VARIOUS AGES

NJANOOR NARAYANAN Department of Physiology, Health Sciences Centre, The University of Western Ontario, London N6A 5C1 (Canada) (Received September 5th, 1986) (Revision received January 3rd, 1987) SUMMARY Age-associated decline in the Ca2÷ pump function of cardiac sarcoplasmic reticulum (SR), and increase in the Ca 2+ pump activity of sarcolemma (SL) were suggested by my previous study which compared the ATP-energized in vitro Ca2÷ transport activities of these membranes from young (3--4-month-old) and aged (24-25-month-old) rat myocardium (Biochim. Biophys. Acta, 6 78 (1981) 442-459). In the present study, ATP.dependent Ca 2+ transport and Ca 2. sensitive ATPase activities of SR and SL derived from the myocardium of rats aged 3 (young), 6 (young adult), 12 (adult), 18 (aging) and 24 (aged) months were determined so as to further characterize age-related changes in the Ca 2÷ transport function of these membranes. The rates of ATP-dependent Ca 2÷ accumulation by SR from 3- and 6-month-old rats were virtuaUy similar whereas the rates of Ca2÷ accumulation by this membrane from 12-, 18- and 24-month-old rats were significantly lower when compared to 3- or 6-month-old rats; the magnitude of this age-related decline amounted to approx. 18, 45 and 50%, respectively, for SR from 12-, 18- and 24-month-old animals. In contrast to the above findings with SR, SL from 18- and 24-month-old rats displayed substantially higher rates (approx. 45 and 80% increase, respectively, at 18 and 24 months of age) of ATP-dependent Ca 2+ accumulation than SL preparations from 3-, 6- and 12-month-old rats; no significant age-related difference was evident between the latter three age groups. The divergent age-related changes in the Ca2÷ accumulating activities of SR and SL were seen at varying Ca2÷ concentrations (0.54-25.2 /aM). With either membrane, kinetic analysis showed that the velocity of Ca2* transport, but not the apparent affinity of the transport system for Ca2÷, underwent age-related changes. The Ca2+-stimulated ATPase activities of SR and SL were not altered significantly with increasing age from 3 to 24 months. Comparison of the 'combined Ca2÷ transport activity' of SR and SL from rats of various ages showed a significant overall age-related decline in the rates of Ca2÷ transport via the ATP-driven membrane Ca 2÷ pumps; this decrement in membrane function was moderate at 12 0047-6374/87/$03.50 Printed and Published in Ireland

© 1987 Elsevier Scientific Publishers Ireland Ltd.

128 months of age (approx. 16%) and became pronounced with advancing age thereafter (approx. 35 and 38%, respectively, at 18 and 24 months of age). Similar progressive agerelated decline was observed in the ATP-dependent Ca 2÷ sequestering activity of cardiac homogenates. No significant age-related difference was evident in the ATP- and respiration-supported Ca 2÷ uptake activity of mitochondria isolated from 6- and 24-month-old rats. The above findings suggest that, barring compensatory age-related increment in Ca 2÷ efflux v/a Na÷-Ca2÷ exchange across the SL, the overall ability of the myocardial cell to sequester Ca 2÷ from the sarcoplasm declines with aging, mainly due to deterioration in the Ca 2÷ pump activity of SR. This impairment in SR function is likely the major factor underlying the prolongation of cardiac relaxation seen with aging. The age-associated decline in the Ca 2÷ transport activity of SR may involve uncoupling of the energy transduction and ion translocation functions of this membrane Ca 2÷ pump.

K e y words: Aging heart;Ca2+transport;Ca2+-ATPase;Sarcoplasmic reticulum;Sarcolemma

INTRODUCTION Prolonged duration of contraction is one of the most striking and well documented effects of aging on the mechanical performance of cardiac muscle in a variety of species including rat [ 1 - 6 , and refs. therein], guinea pig [7], dog [8], rabbit [9] and man [10]. The prolongation of cardiac contraction seen with aging is recognized to be due to an increase in both time to peak force [1,6] and half relaxation time [2]. Conceivably, the duration of contraction may be determined to a large extent by the duration of excitation-induced elevation of Ca 2÷ concentration in the muscle sarcoplasm. In the heart, contraction- and relaxation-related Ca 2÷ translocation rests mainly on the sarcotubular and sarcolemmal membrane systems. Age-related changes in the Ca 2÷ translocation function of these membranes can, therefore, contribute to the prolonged contraction duration observed in the aging myocardium. Evidence supporting this possibility has been provided by previous studies in this and another laboratory. Thus, Froehlich et al. [6] observed decreased rates of ATP-supported Ca2÷ uptake by sarcoplasmic reticulum (SR)-enriched fraction from the myocardium of aged (24-26month-old) compared to young adult (6-8-month-old) rats. In a detailed comparative study using SR and sarcolemma (SL) from young (3-4-month-old) and aged (24-25month-old) rat hearts I have documented differential age-related alterations in the ATP-dependent Ca 2÷ transport activities of these two membrane systems [11]. It was observed that the ATP-dependent Ca 2+ transport rates and steady state Ca2+ accumulation by SR were reduced as much as 50% in the aged compared to young heart whereas these two parameters of Ca 2÷ transport were increased nearly twofold in SL of aged compared to young heart. The ATP-dependent in vitro Ca 2÷ uptake activity of SR and SL is thought to reflect the Ca 2÷ pump function of these membranes in vivo which

129 serves to promote muscle relaxation by sequestering Ca2+ from the sarcoplasm [ 1 2 15]. Therefore, the age-associated decline in the Ca2+ pump function of SR may be the major factor contributing to the increased relaxation time observed in the aging heart. The age-associated increase in the Ca2+ pump activity of SL may compensate, in part, for the diminution of SR Ca2÷ pump function in the senescent heart. Our findings [ 11 ] also suggested that Ca2÷ translocation but not energy transduction [Ca2*-activated ATP hydrolysis) by the membrane (SR and SL) Ca2* pumps undergo age-related changes. The present study was undertaken to determine: (a) the time of onset and progression of age-related changes in the ATP-energized Ca2+ pumps of cardiac SR and SL; (b) temporal relationship, if any, between the bidirectional age-related changes in the activity of the two membrane Ca2+ pumps; and (c) the net effect of age on the overall Ca 2÷ handling ability of the myocardial cell. To this end we have compared the ATPdependent Ca 2÷ transport and Ca2*-activated ATPase activities of SR and SL derived from the myocardium of rats aged 3, 6, 12, 18 and 24 months. The results are presented and discussed in this report. MATERIALS AND METHODS

Animals Virgin male Sprague-Dawley rats aged 3 months (young rats; 290-340 g body wt), 6 months (young adult rats; 440-480 g body wt), 12 months (adult rats; 580-630 g body wt), 18 months (aging rats; 600"-640 g body wt) and 24 months (aged rats; 5 9 0 630 g body wt) were obtained from Hadan Industries, Indianapolis, U.S.A. The animals were killed by a sharp blow to the base of the skull, the hearts were quickly removed and the ventricles were dissected free of atria and connective tissue, and were used for experiments. The weight of ventricles averaged 1.01 -+ 0.08 g, 1.26-+ 0.12 g, 1.55 -+ 0.14 g, 1.58-+0.16 g and 1.62-+0.18 g in the case of rats aged 3, 6, 12, 18 and 24 months, respectively. Chemicals 4SCaC12 (15.94 mCi/mg) and [~,-a2P]ATP (36 Ci/mmol) were purchased from New England Nuclear, Montreal, Canada. Reagents for electrophoresis were obtained from Bio-Rad Laboratories, Mississauga, Ontario, Canada. All other ~hemicals were of highest purity available from Sigma Chemical Co., St. Louis, MO, or Fisher Scientific Co., N J, U.S.A.

Isolation of membrane fractions enriched in SR and SL SR-enriched membrane fraction was prepared from rat heart ventricles according to the method of Harigaya and Schwartz [16] with minor modifications as described previously [17]. Following isolation, the membranes were suspended in 10 mM Trismaleate (pH 6.8) containing 100 mM KC1 to give a protein concentration of 2 mg/ml. Membrane fraction enriched in SLwas prepared from rat heart ventricles following the procedure of St. Louis and Sulakhe [18]. The isolated membranes were suspended in

130 10 mM Tris-HCl buffer (pH 7.5) containing 10% (W/V) sucrose/2 mM dithiothreitol to give a protein concentration of 2 mg/ml. For each experiment, SR and SL were isolated from single rat heart. All studies were carried out using freshly prepared membrane fractions. The SL preparations isolated from rat heart according to the procedure of St. Louis and Sulakhe [18] have been well characterized previously [ 11 ] ; these preparations were enriched (compared to homogenate activity) approx. 11-fold in the specific activity of ouabain-sensitive (Na++ K) ATPase and approx. 8-fold in the specific activity of adenylate cyclase. Also, as documented extensively in our previous study [11], the relative purity and stability of the membrane fractions (SR and SL) isolated from rat hearts of different ages were essentially similar (also see "Results and Discussion"). Preparation of heart homogenates and mitoehondria In addition to SR and SL, homogenates of ventricular myocardium from rats of different age groups were also used in some of the experiments. For this. the homogenates were prepared by homogenizing the ventricles in 10 vols. (based on tissue wt) of 10 mM Tris-maleate/100 mM KC1 buffer (pH 6.8) using a Polytron PT-10 homogenizer (three 10-s burst, with 30 s interval between bursts; setting 8; Brinkman Instruments, Westbury, NY). The mitochondrial fraction used in some experiments was obtained by differential centrifugation of heart homogenates prepared as above except that the homogenizing buffer consisted of 10 mM Tris-HC1/0.25 M sucrose/0.5 mM EDTA (pH 7.4). The homogenate was centrifuged at 1200 g for 10 rain and the resulting supernatant at 8000 g for 20 min. The pellet from the above step was washed twice by resuspension in buffer (10 mM Tris-HC1/0.25 M sucrose, pH 7.4) and subsequent centrifugation at 8000 g for 15 min. The final pellet was resuspended in 10 mM Tris-HCl/0.25 M sucrose (pH 7.4) and was used as the mitochondrial fraction. Determination of Ca2÷transport and Ca2÷A TPase activities ATP-dependent, oxalate-facilitated Ca 2+ accumulation by SR and SL was determined using a Millipore filtration technique as detailed elsewhere [11]. The standard incubation medium (total vol. 1 ml) contained 50 mM Tris-maleate (pH 6.8), 5 mM MgC12, 2.5 mM ATP, 120 mM KCI, 2.5 mM potassium oxalate, 5 mM NAN3, 0.1 mM EGTA, membrane or homogenate fraction (30 /2g protein in the case of SR: 80/ag protein in the case of SL and homogenate) and varying concentrations of 4SCaC12(6000-8000 cpm/ nmol). All assays were performed at 37°C; the Ca2+ transport reaction was initiated by the addition of membrane fraction following preincubation of the rest of the assay components for 3 rain. The Ca 2÷ transport reaction was carried out for I or 2 min during which the transport rates were linear. The free Ca 2÷ ion concentrations in the assay nledium were determined as described previously [11]. For a given experiment, the transport assays using membranes from 6-, 12- and 24-month-old rats were performed on

131 the same day; likewise, assays using membranes from 3- and 18-month-old rats were performed on the same day. The Ca2÷ uptake activity of mitochondrial fraction was determined under conditions similar to those described above except that NaN3 was excluded from the incubation medium and the assays were performed in the absence and presence of succinate (5 mM). The incubation medium used for the assay of Ca2+-ATPase was identical to that described for Ca 2÷ accumulation, except that [~,-32P]ATP was used instead of nonradioactive ATP and non-radioactive CaC12 was used instead of 4SCaC12. To determine the 'basal' ATPase (Mg:+-ATPase) activity, assays were also carried out in the absence of Ca 2÷ and in the presence of 0.2 mM EGTA. The incubations were carried out at 37°C for 3 min and the reaction was stopped by the addition of 1 ml 12% trichloroacetic acid/2 mM KH~PO4. Following this, 0.1 ml each of 25 mM ATP and 0.1% bovine serum albumin were added to the tubes. The tubes were centrifuged (3000 rev./min, 10 min) and the 32p released from [7-32P]ATP was extracted and quantitated as described by Sulakhe and Drummond [ 19]. The basal ATPase activity was substracted from the enzyme activity measured in the presence of Ca2+ to obtain the Ca2÷-ATPase activity.

SDS-polyacrylamide gel electrophoresis Protein composition of SR and SL prepared from rats of various ages was determined by SDS-polyacrylamide slab-gel electrophoresis as described previously [ 11 ].

Determination o f protein Protein was determined by the method of Lowry et al. [20] using defatted bovine serum albumin as standard.

Data analysis The results are presented as mean -+ S.D. Statistical significance was evaluated by Student's t-test; P < 0.05 was taken as the level of significance. RESULTS AND DISCUSSION The results presented in Fig. 1 compare the ATP-dependent Ca 2÷ accumulating activities of SR and SL derived from the myocardium of rats aged 3, 6, 12, 18 and 24 months. These results were obtained with a selected free Ca2÷ concentration of 11.9/aM in the assay medium which is sufficient for near maximal activation of these membrane Ca 2÷ pumps (see below, and ref. 11). It can be seen that the rates of Ca 2+ accumulation by SR from 3- and 6-month-old rats were virtually similar whereas the rates of Ca 2+ accumulation by this membrane from 12-, 18- and 24-month-old rats were significantly lower when compared to 3- or 6-month-old rats. The magnitude of the age-related decline in the Ca 2+ accumulating activity of SR amounted to approx. 18, 45 and 50%, respectively, for membranes from 12-, 18- and 24-month-old animals.

132

Age-associated decrease in the Ca 2÷ transport function of cardiac SR has been suggested by our previous study [11] and that by Froehlich et al. [6]; these studies, however, compared the in vitro Ca2÷ transport activity of cardiac SR from senescent (24-25month-old) rats with that of young ( 3 - 4 month old, ref. [ 1 1 ] ) o r young adult (6-8month-old) [6] rats, and hence, did not provide information about the time of onset and progression of age-related changes in the Ca 2÷ transport function of this membrane. Results of the present study, besides confirming the previously reported findings [6,11 ], show that, in rats, moderate yet significant age-associated decrease in the ATPenergized Ca 2÷ transport activity of cardiac SR becomes evident at the age of 12 months and marked progressive deterioration of this membrane function follows aging thereafter. In contrast to the results obtained with SR, SL from the myocardium of 18- and 24-month-old rats showed substantially higher (approx. 45 and 80% increase at 18 and 24 months of age, respectively) rates of ATP-dependent Ca2÷ accumulation than SL preparations from 3-, 6- and 12-month-old rats; no significant age-related difference was evident between the latter three age groups (Fig. 1). To my knowledge, the only published work describing the effects of age on ATP-energized Ca 2÷ transport activity of SL is that reported previously from this laboratory [11] which showed nearly twofold increase in the Ca 2+ transport activity of cardiac SL from senescent (24-25-month-old) compared to young (3-4-month-old) rats. The present results show that the ageassociated increase in Ca 2÷ transport activity of SL in rat myocardium is manifested late

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Fig. 1. Effect of age on ATP-dependent Ca 2÷ accumulation by rat cardiac sarcoplasmic reticulum (SR) and sarcolemma (SL). The Ca 2÷ accumulating activities of the m e m b r a n e s were determined as described under Materials and Methods using 11.9 #M free Ca 2÷. The results shown are m e a n _+ S.D. of 6 e x p e r i m e n t s in the case of SR and 5 e x p e r i m e n t s in the case of SL; each experiment was carried out using separate m e m b r a n e preparations. The Ca 2÷ accumulating activity of SR is significantly lower in 12- (P < 0.05), 18- (P < 0.001) and 24- (P < 0.001) m o n t h - o l d rats compared to 3- or 6-month-old rats. The Ca 2÷ accumulating activity o f SL is significantly higher in 18- (P < 0.01) and 24- (P < 0.001) m o n t h - o l d rats compared to 3-, 6- or 12-month-old rats.

133

during adult aging, subsequent to the occurrence of significant age-dependent diminution in the Ca2÷ transport function of SR. Figure 2 shows the effects of varying Ca 2+ concentrations on the ATP-dependent Ca 2÷ accumulating activities of SR (panel A) and SL (panel B) from 6 (young adult)-, 12 (adult)- and 24 (aged)-month-old rats. When the Ca2÷ concentration in the assay was varied from 0.54 to 11.9/aM SR from 24-month-old rats showed significantly lower rates (32-50%; P < 0 . 0 5 - 0 . 0 0 1 ) of Ca2÷ accumulation than the membranes from 6- or 12-month-old rats at all Ca2+ concentrations used. Also, at all but the lowest Ca2+ concentration used, the rates of Ca 2+ accumulation by SR from 12-month-old rats were significantly lower (17-23% ;P < 0.05 or 0.02) compared to SR from 6-month-old rats. Similar experiments using SL showed higher rates (approx. 45-100%; P < 0 . 0 1 or 0.001) of Ca 2÷ accumulation by this membrane from 24-month-old, compared to that from 6- or

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Free C a l c i u m ( u M ) Fig. 2. Effects of varying Ca 2+ concentrations on ATP-dependent Ca 2+ accumulation by cardiac sarcoplasmic reticulum (SR) and sarcolemma (SL) from 6,-12- and 24-month-old rats. The Ca 2÷ accumulating activity of the membranes were determined as described under Materials and Methods using varying concentrations of Ca 2÷ as indicated. Each data point represents mean -+ S.D. of 5 experiments using separate membrane preparations. In the case of SR, the differences between 6- and 12-monthold rats are significant (P < 0.05 or 0.02) at all but the lowest Ca 2÷ concentration used; the differences between 12- and 24-month-old rats are significant (P < 0.05 to 0.001) at all Ca 2÷ concentrations used. In the ease o f SL, the differences between 24- and 12- (or 6) month-old rats are significant (P < 0.01 or 0.001) at all Ca 2÷ concentrations used; the differences between 6- and 12-month-old rats are not significant.

134 12-month-old, rats at varying (0.54-25.2/aM) Ca2÷ concentrations (Fig. 2, panel B); no statistically significant difference was observed in the Ca2÷ accumulating activities of SL from 6- and 12-month-old rats. In the case of SR, double reciprocal transformation of the data resulted in curved plots concave upward (Fig. 3, panel A), a kinetic feature indicative of cooperative interaction between Ca2÷ and the transport system [6,12]. With SL, double reciprocal plots of the data were linear (Fig. 3, panel B). The kinetic parameters of Ca2÷ accumulation by SR (derived using the procedure described in ] 11 ] and SL (derived from double reciprocal plots) are summarized in Table I. These results, which conform to our previously reported data [ 11 ], indicate that divergent age-related changes occur in the Ca 2÷ transport activities of SR and SL without appreciable concurrent changes in the apparent affinities of the membrane transport systems for Ca2+. It is widely recognized that the membrane protein, Ca2+-ATPase, energizes the Ca 2÷ "pump" activities of both SR [12,14,15] and SL [13], and serves the dual functions of energy transduction (ATP hydrolysis) and Ca 2+ ion translocation. Age-related changes in the amount of this enzyme or its properties can lead to alterations in the Ca2÷ transport

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135 TABLE I EFFECT OF AGE ON THE KINETIC PARAMETERS OF ATP-DEPENDENT CALCIUM ACCUMULATION BY RAT CARDIAC SARCOPLASMIC RETICULUM (SR) AND SARCOLEMMA (SL) Kinetic parameters (values expressed as mean _+S.D.) were derived from the data on mean Ca2+ uptake velocities vs. free Ca2÷concentrations shown in Figs. 2 and 3. Age o f animals (months)

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Ca2÷concentration for half maximal velocity (Ko. s~ {tzM)

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21. +_0.40 2.2 _+0.35 2.5 +_0.40

a p < 0.01 compared to 6-month-old rats. bp < 0.01 compared to 6- or 12-month-old rats. c Not significantly different compared to 6-month-old rats. Ko. ~ values lbr Ca2÷ are not significantly different between age groups.

function of these membranes. However, as shown in Fig. 4, the Ca2+-stimulated ATPase activities of SR and SL did not differ significantly with age. This finding is in conformity with the results of our previous study [11] which compared Ca2+-ATPase activities of SL and SR from young (3-4-month-old) and aged (24-25-month-old) rats. Thus, the bidirectional age-related changes observed in the Ca 2* transport activities of SR and SL cannot be attributed directly to analogous age-associated changes in the ability of these membrane Ca 2÷ pumps to hydrolyze ATP. Using a variety of experimental approaches, previous study from this laboratory [ 11 ] has provided extensive data showing that the relative purity and stability of the membrane fractions (SR and SL) isolated from young (3-4-month-old) and aged ( 2 4 - 2 5 month-old) rat hearts (using procedures identical to that employed in the present study) are essentially similar. Therefore, it is most unlikely that the age-related changes in the Ca 2÷ transport activities of SR and SL observed here arise from differences in the purity and/or stability of cardiac membranes derived from rats of various age groups. In further support of this, the polypeptide composition of SR and SL (determined by SDS-polyacrylamide gel electrophoresis) derived from the myocardium of rats of various age groups used in the present study were found to be essentially similar (Fig. 5). Scanning and quantitation of the electropherograms did not reveal any significant age-related difference in the amount of protein in individual polypeptide bands including the 100 kDa band which most likely represents the monomeric Ca 2÷ATPase in the case of SR [12] and (Na ÷ + K ÷) ATPase, in addition to Ca2+-ATPase in the case of SL [21]. The latter observation conforms to the finding that Ca2÷-ATPase activities of these membranes did not differ with age.

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Fig. 4. Comparison of the Ca:+-stimulated ATPase activities of cardiac sarcoplasmic reticulum (SR) and sarcolemma (SL) from rats of various ages. The Ca2+-stimulated ATPase activity was determined as described under Materials and Methods using 11.9 uM free Ca ~+. The results represent mean ± S.D. of 6 experiments with SR and 5 experiments with SL; each experiment was carried out using separate membrane preparations. With either membrane, the differences between age groups are not significant.

Since the Ca 2÷ transport activities o f SR and SL showed divergent age-related changes additional experiments were performed to assess the net change, due to age, in the "combined Ca 2+ transport activity" o f the two membranes. In these experiments equal amounts of cardiac SR and SL from rats o f each age group were combined [the membranes were combined at a ratio of 1 : 1 as the yield of SR and SL from rat heart was found to be nearly similar (approx. 3 . 5 - 4 . 5 mg protein/g tissue, see ref. [ 11 ]); however, it is not certain whether the selected ratio is representative of the amounts of SR and SL in the intact heart] and then assayed for ATP-dependent Ca 2÷ accumulation in the presence of either 1.4 or 11.9 #M free Ca 2÷. The results are summarized in Table II. It can be seen that the combined Ca 2+ accumulating activity of membranes from 12-, 18- and 24-month-old rats was significantly lower than that observed with membranes from 6-month-old rats. At both Ca 2+ concentrations used, the magnitude of this age-related decrement was moderate (approx. 1 6 - 2 2 % ) in the case of membranes from 12-month-old rats but was more pronounced (approx. 3 2 - 3 8 % ) in the case of membranes from older animals. These results imply that the observed age-related increase in the Ca 2+ pump activity o f SL (Figs. 1 and 2) is inadequate to fully compensate for the decline in activity of the SR Ca 2+ pump (Figs. 1 and 2). Besides the ATP-energized SR and SL Ca 2+ pumps, two additional mechanisms known to participate in the sequestration of cytosolic Ca 2÷

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qg. 5. Comparison of the polypeptide composition of sarcoplasmic reticulum (SR) and sarcolemma (SL) isolated from the myocardium of rats of various ges. Lanes A, B, C, D and E represent cardiac membranes (SR or SL) from 3-, 6-, 12-, 18- and 24-months-old rats, respectively. Identical amounts of SR 35 tzg protein), or SL (45 t~g protein), derived from the myocardium of rats of various ages were fracti0nated by SDS-polyaerylamide slab gel (10%) ~lectrophoresis. The gels were stained with 0.025% coomassie blue and destained in 10% acetic acid. The procedures used for electrophoresis and determinaion of apparent molecular weights were as detailed by Narayanan [ 11 ]. Since SR and SL were fractionated on two separate gel slabs, the molecular weight ~eales (left for SR, right for SL) are not identical. Protein profiles similar to those shown here were obtained in two additional experiments using separate

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138 in heart muscle include Na÷-Ca 2÷ exchange across the SL [ 2 2 - 2 7 , and refs. therein] and ATP-supported Ca 2÷ uptake by mitochondria [ 2 8 - 3 0 , and refs. therein]. Although the Na+-Ca 2÷ exchanger, present in the SL, is thought to contribute significantly to the efflux o f Ca 2÷, it seems very likely that its direction varies during the heart contraction/relaxation cycle [22,25,27]. While the present study did not examine the effect of age on Na+-Ca 2÷ exchange across the SL, a recent report indicates that Na+-Ca2÷ exchange activity of isolated rat heart SL (both Ca2÷-influx into Na*-loaded SL vesicles as well as Na*-dependent Ca 2+ efflux from Ca2+-loaded SL vesicles) is lower in the case of aged ( 2 4 - 2 7 - m o n t h - o l d ) compared to young ( 3 - 4 - m o n t h - o l d ) or middle-aged ( 1 4 - 1 7 - m o n t h old) animals [31 ]. To assess the effect of age on the mitochondrial Ca 2÷ transport system, ATP-supported Ca 2÷ uptake by mitochondria isolated from 6- and 24-month-old rats was examined in the present study. As shown in Fig. 6, no significant age-related difference was observed in the rates of ATP-supported Ca 2÷ uptake by mitochondria when assays were performed in the absence or presence of an oxidizable substrate such as succinate; this finding is in conformity with previous observation that ATP- and respiration-supported Ca 2+ accumulation by mitochondria is not altered in the aged rat heart [11 ]. From the above findings it does not seem that age-related changes in the activities of Na÷-Ca 2÷ exchange system of SL or A T P - s u p p o r t e d Ca 2÷ transport system of mitochondria can compensate for the age-associated decline in the Ca 2÷ pump activity of SR. Thus, the data on the combined Ca 2÷ transport activities o f SR and SL (Table II) are consistent with the possibility that the overall ability of the myocardial cell to remove

TABLE I1 COMPARISON OF THE COMBINED CALCIUM TRANSPORT ACTIVITIES OF CARDIAC SARCOPLASMIC RETICULUM (SR) AND SARCOLEMMA (SL) FROM RATS OF VARIOUS AGES

Age of animals (months)

Sum o f Ca 2+accumulated by SR and SL (nmol Ca2+/mg protein/ min) leith 1.4 ~

6 12 18 24

40.6 31.7 27.8 25.4

Ca 2+

+-6.1 -+4.8 a (21.9) + 5.1b (31.5) + 4.6b (37.4)

leith 11.9 I~l Ca 2÷

122.0 102.1 78.3 75.4

+- 11.9 + 13.6 a (16.3) + 12.2 c (35.8) + 13.2 c (38.2)

Equal amounts of SR and SL derived from ventricles of each age group were combined and then assayed (20 /~g protein each of SR and SL per assay) for ATP-supported Ca~÷ accumulation in the presence of 1.4 or 11.9 /~M free Ca2÷. The data represent mean + S.D. of five experiments using separate membrane preparations. The numbers in parentheses represent percent decline in Ca 2+ accumulating activity of the membranes with increasing age when the corresponding acfivity of membranes from 6-month-old rats is taken as 100%. Significant differences from 6-month-old rats are ap < 0.05; bp < 0.01 ;cP < 0.001.

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Fig. 6. Time course of ATP-supported Ca 2+ accumulation by mitochondria from 6- and 24-month-old rats. Ca 2+ accumulation was determined as described under Materials and Methods using 11.9 taM free Ca 2+ in the absence of succinate (% o) and in the presence of 5 mM suecinate (o, =). The results shown are mean + S.E. of duplicate determinations using two separate preparations of mitochondria from each age group.

Ca 2* from the sarcoplasm (subsequent to activation of muscle contraction by excitationinduced elevation of intracellular Ca 2+) declines with aging. This possibility was further investigated by determining the rates of ATP-dependent Ca 2+ sequestration by homogenates of cardiac muscle from rats of various age groups. The results (see Fig. 7) showed significantly reduced rates of ATP-dependent Ca2+ sequestration by cardiac homogenates from 12-, 18- and 24-month-old rats when compared to those from 3- and 6-month-old rats; the Ca2+ sequestering activity of cardiac homogenates from the latter two age groups was essentially similar. It is worth noting that the magnitude of age-related decrement in ATP-dependent Ca2+ sequestration observed with cardiac homogenates from older rats (approx. 14, 33 and 34% respectively for rats aged 12, 18 and 24 months) is strikingly similar to that noted in experiments where the effect of age on the combined Ca2+ transport activity of cardiac membranes (SR and SL) was assessed (see Table lI). In conclusion, the present study has provided further characterization of the differential age-related alterations in the ATP-energized Ca2* transport activities of rat cardiac SR and SL reported previously from this laboratory [ 11 ]. Thus, the data presented here have shown that, in rat myocardium: (a) moderate yet significant age-related decline in the Ca2÷ pump activity of SR is manifested as early as 12 months of age and further progressive deterioration of this membrane function follows aging thereafter; (b) appreciable age-associated increase in the Ca 2÷ pump activity of SL becomes evident late during adult aging, subsequent to the occurrence of the diminution in SR Ca 2÷ pump activity;

140

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c

.c E

~

o

El

.; v_ u

9

3

6

12

18

24

Age (Months) Fig. 7. Effect o f age on ATP-dependent Ca 2+ accumulation by rat heart homogenates. The Ca 2+ accumulating activity was determined as described under Materials and Methods using 11.9 #M free Ca 2+. The results shown are mean ± S.D. o f 4 experiments using separate preparations o f homogenates. The decrement in Ca 2+ accumulating activity seen at 12, 18 and 24 months of age is significant (P < 0.05 or 0.001).

(c) the Ca2÷-stimulated ATPase activities of SR and SL are not altered significantly during the period of aging from 3 to 24 months; and (d) despite the age-related increment in the Ca2÷ pump activity of SL, the overall ability of the myocardial cell to sequester Ca 2+ from the sarcoplasm declines with aging, beginning at the age of 12 months. These findings are consistent with the possibility that age-associated deterioration in the Ca2÷ pump function of SR is a major factor underlying the prolongation of cardiac relaxation seen with aging [2,6]. In view of the lack of age-related changes in the Ca2÷-stimulated ATPase activities of SR and SL (also see ref. [11]), it is unlikely that the number of Ca2+ pump sites (i.e. the density of Ca2÷-ATPase) in these membranes is altered during aging. On the other hand, the observed age-related changes in the Ca2+ transport activities of the membranes may result from age-associated alterations in the efficacy of coupling ATP hydrolysis to Ca2÷ transport. A tight coupling between Ca2+-sensitive ATP hydrolysis and Ca 2+ transport, with a stoichiometry of 2 tool Ca2+ transported/tool Ca2+-stimulated ATP hydrolysis has been well documented in the case of SR from mammalian (generally rabbit) fast skeletal muscle (see ref. [12] for a review). Using SR from dog heart, some investigators have reported a similar stoichiometry between Ca 2+ transport and Ca2+stimulated ATP hydrolysis [32,33 ]. However, despite the cooperative dependence of Ca 2+ uptake seen with SR (Fig. 3A), the stoichiometry observed in the present study using rat heart membranes (SR or SL) was much lower (e.g. the estimated ratios of Ca2+

141 transport/Ca2+-stimulated ATP hydrolysis were 0.34 and 0.18, respectively, for SR from 6- and 24-month-old rats, and 0.09 and 0.17, respectively, for SL from 6- and 24-monthold rats). Several previously reported studies using cardiac SR from rats and other species have shown very low, and often variable, stoichiometry (ratio of Ca~÷ transported/ATP hydrolysis, approx. 0.2-0.7) of Ca2÷ transport and Ca2+-stimulated ATP hydrolysis [11, 3 4 - 4 0 ] although evidence for cooperative dependence on Ca2÷ uptake (Hill coefficients greater than 1 for Ca 2÷) could be observed [6,11,35]. Thus, it is possible that a substantial portion of Ca2+-dependent ATP hydrolysis measured under the experimental conditions employed in the present study (and other studies, refs. [11,34-40]) is not coupled to Ca2÷ translocation. While the exact reasons for this are not known our attempts to improve the coupling ratio of ATP hydrolysis and Ca 2÷ transport in rat heart SR by modifications in experimental conditions (which took into account a number of potential factors which might contribute to the poor stoichiometry) have met with only limited success (e.g., see ref. [35]). As discussed elsewhere [11], there also exists considerable evidence in the literature indicating that the intrinsic ATP hydrolyzing activity of the transport system may not be the sole determinant of ion transport function. In any case, as documented previously [11], the divergent age-related changes in the Ca2÷ transport activities of SR and SL, and the lack of significant age-related changes in the Ca2+-stimulated ATPase activities in these membranes cannot be accounted for by differences in the relative purity, stability, sidedness (i.e., the proportion of right-side-out and inside-out vesicles) or the permeability characteristics (leakiness) of the membrane vesicles isolated from rats of different age groups. Thus, the observed age-related alterations in Ca 2÷ transport but not Ca2÷-ATPase activities seem to imply age-associated changes in efficacy of energy utilization for Ca 2÷ transport in myocardial membranes. As suggested previously [11], the age-associated decline in the Ca 2÷ transport activity of SR may involve uncoupling of the energy transduction and ion translocation functions of this membrane Ca 2÷ pump. Studies aimed to characterize the molecular nature of the age-associated impairment in the Ca 2÷ transport function of SR are currently in progress in this laboratory. The age-associated enhancement in the Ca 2÷ pump activity of SL, observed late during adult aging, may be construed as an adaptive mechanism invoked by the myocardial cell to compensate (albeit partly) for the reduced rates of Ca2÷ sequestration by the SR Ca 2÷ pump. The mechanisms underlying this phenomenon remain to be investigated. ACKNOWLEDGEMENTS This work was supported by the Heart and Stroke Foundation of Ontario. The author is grateful to Ms. Nancy Wilson for secretarial assistance. REFERENCES 1 N.R. Alpert, H.H. Gale and N. Taylor, The effect of age on contractile protein ATPase activity and the velocity of shortening. In R.D. Tanz, K. Kavaler and J. Roberts (eds.), Factors Influencing Myocardial Contractility, Academic Press, New York, 1967, pp. 127-133.

142 2 E.G. Lakatta, G. Gerstenblith, C.S. Angell, N.W. Shock and M.L. WeisfeMt, Prolonged contraction duration in aged myocardium. J. Clin. lnvest., 55 (1975) 6 1 - 6 8 . 3 M.L. Weisfeldt, W.A. Loeven and N.W. Shock, Resting and active mechanical properties of trabeculae carneae from aged male rats. Am. J. Physiol., 220 (1971) 1921-1927. 4 E.G. Lakatta and F.C.P. Yin, Myocardial aging: functional alterations and related cellular mechanisms. Am. J. Physiol., 242 (1982) H927-H941. 5 J.Y. Wei, H.A. Spurgeon and E.G. Lakatta, Excitation-contraction in rat myocardium: alterations with adult aging. Am. J. Physiol., 246 (1984) H784-H791. 6 J.P. Froehlich, E.G. Lakatta, E. Beard, H.A. Spurgeon, M.L. Weisfeldt and G. Gerstenblith, Studies of sarcoplasmic retieulum function and contraction duration in young adult and aged rat myocardium.Z Mol. Cell Cardiol., 10 (1978) 4 2 7 - 4 3 8 . 7 E. Rumberger and J. Timmerman, Age-changes of the force-frequency relationship and duration of action potential in isolated papillary muscles of guinea pig. Eur. J. Appl. Physiol. Occup. Physiol., 34 (1976) 2 2 7 - 2 8 4 . 8 G.H. Templeton, J.T. Willerson, M.R. Platt and M. Weisfeldt, Contraction duration and diastolic stiffness in aged canine left ventricle. In T. Koybayashi, T. Sano and N.S. Dhalla (eds.), Recent Advances in Studies on Cardiac Structure and Metabolism - Heart Function and Metabolism, University Park, Baltimore, 1978, pp. 169-173. 9 V.V. Frolkis, V.V. Bezrukov and V.G. Shevtchuk, Hemodynamics and its regulation in old age. Exp. Gerontol., 10 (1975) 251-271. 10 T.R. Harrison, K. Dixon, R.O. Russel Jr., B.S. Bidwai and H.N. Coleman, The relation of age to the duration of contraction, ejection, and relaxation of the normal human heart. Am. Heart J., 67 (1964) 189-199. 11 N. Narayanan, Differential alterations in ATP-supported calcium transport activities of sarcoplasmic retieulum and sarcolemma of aging myocardium. Biochim. Biophys. Acta, 678 (1981) 4 4 2 459. 12 M. Tada, T. Yamamoto and Y. Tonomura, Molecular mechanism of active calcium transport by sarcoplasmic reticulum. Physiol. Rev., 58 (1978) 1-79. 13 P.V. Sulakhe and P.J. St. Louis, Passive and active calcium fluxes across plasma membranes, Prog. Biophys. Mol. Biol., 35 (1979) 135-195. 14 W. Hasselbach, Calcium-activated ATPase of the sarcoplasmic reticulum membranes. In S.L. Bonting and Y. Dupont (ed~), Membrane Transport, Elsevier Biomedical Press, Amsterdam, 1981, pp. 183-208. 15 G. lnesi, The sarcoplasmic reticulum of skeletal and cardiac muscle. In R.M. Dowben and J.W. Shay (eds.), Cell and Muscle Motility, Vol. I, Plenum Press, New York, 1981, pp. 6 3 - 9 7 . 16 S. Harigaya and A. Schwartz, Rate of calcium binding and uptake in normal animal and failing human cardiac muscle. Circ. Res., 25 (1969) 781-794. 17 P.V. Sulakhe and N. Narayanan, Microsomal and sarcolemmal adenylate cyclase in cardiac muscle: Differential effects of non-ionic detergents. Biochem. J., 1 75 (1978) 171-180. 18 P.J. St. Louis and P.V. Sulakhe, Isolation of sarcolemmal membranes from cardiac muscle. IntJ. Biochem., 7 (1976) 5 4 7 - 5 5 8 . 19 P.V. Sulakhe and G.I. Drummond, Protein kinase catalyzed phosphorylation of muscle sarcolemma. Arch. Biochem. Biophys., 161 ( 1 9 7 4 ) 4 4 8 - 4 5 5 . 20 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with phenol reagent. J. Biol. Chem., 193 (1951) 265-275. 21 P.J. St. Louis and P.V. Sulakhe, Protein analysis of cardiac sarcolemma: Effects of membrane perturbing agents on membrane proteins and calcium transport. Biochemistry, 1 7 (1978) 4540-4550. 22 H. Retuer, Na-Ca counter transport in cardiac muscle. In A. Martonosi (ed.), Membranes and Transport, Vol. 1, Plenum Press, New York, 1982, pp. 6 2 3 - 6 3 1 . 23 J.P. Reeves and J.L. Sutko, Sodium-calcium exchange in cardiac membrane vesicles. Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5 9 0 - 5 9 4 . 24 R.A. Chapman, Control of cardiac contractility at the cellular level. Am. J. Physiol., 245 (1983) H535-H552.

143 25 L.J. Mullins, The role of Na-Ca exchange in heart. In N. Sperelakis (ed.) Physiology and Pathophysiology o f the Heart, Martinus Nijhoff Publishing, Boston, 1984, pp. 199-214. 26 A. Fabiato and F. Fabiato, Calcium and excitation-contraction coupling. Annu. Rev. Physiol., 41 (1979) 4 7 3 - 4 8 4 . 27 J.H.B. Bridge, W.R. Cabeau, G.A. Langer and S. Reeder, Sodium efflux in rabbit myocardium: relationship to sodium-calcium exchange. J. Physiol., 316 (1981) 5 5 4 - 5 7 4 . 28 T. Kitazawa, Physiological significance of Ca uptake by mitochondrla in heart in comparison with that by cardiac sarcoplasmic retieulum. J. BiochertL, 80 (1976) 1129-1147. 29 D.G. Nicholls and M. Compton, Mitochondrial calcium transport. FEBS Lett., 111 (1980) 2 6 1 268. 30 E. Carafoli, M. Gavilanes, H. Affolter, M. Tunea de Gomez-Puyou and A. Gomez-Puyou, Regulation of ATP-supported Ca 2+ uptake by heart and liver mitochondria. Cell Calcium, 1 (1980) 255 -265. 31 C.E. Heyliger and J.H. McNeill, Alterations in membrane Na+-Ca2÷ exchange in the aging myocardium. J. Mol. Cell. Cardiol., 18, Suppl. 3 (1986) 34 (abstract). 32 M. Tada, M.A. Kirchberger, D.1. Repke and A.M. Katz, The stimulation of calcium transport in cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase. J. Biol. Chem., 249 (1974) 6174-6180. 33 M. Tada, M. Yamada, F. Ohmori, T. Kuzuya, M. Inui and H. Abe, Transient state kinetic studies of Ca2+-dependent ATPase and calcium transport by cardiac sarcoplasmic reticulum. J. Biol. Chem., 255 (1980) 1985-1992. 34 M.A. Kirchberger and T. Antonetz, Calmodulin-mediated regulation of calcium transport and (Ca2++ Mg2÷)-activated ATPase activity in isoalted cardiac sarcoplasmic reticulum. J. Biol. Chem., 257 (1982) 5685-5691. 35 N. Narayanan, Effects of adrenalectomy and in vivo administration of dexamethasone on ATPdependent calcium accumulation by sarcoplasmic reticulum from rat heart. J. Mol. Cell. Cardiol., 15 (1983) 7 - 1 5 . 36 A.M. Katz, D.I. Repke, J.E. Upshaw and M.A. Polascik, Characterization of dog cardiac microsomes. Use of zonal centrifugation to fractionate fragmented sarcoplasmic reticulum (Na + + K+) activated ATPase and mitochondrial fragments. Biochim. Biophys. Acta, 205 ( 1 9 7 0 ) 4 7 3 - 4 9 0 . 37 W.G. Nayler, 1. Dunnet and D. Berry, The calcium accumulating activity of subcellular fractions from rat and guinea-pig heart muscle. J. Mol. Cell. Cardiol., 7 (1975) 275-288. 38 J. Suko, The calcium pump of cardiac sarcoplasmic reticulum. Functional alterations at different level of thyroid states in rabbits. J. Physiol., 228 (1973) 5 6 3 - 5 8 2 . 39 J.M.J. Lamers and J.T. Stinis, Defective Ca 2÷ pump in sarcoplasmic reticulum of hyperthyroid rabbit heart. Life Sci., 24 (1979) 2313-2320. 40 J.J. Feher, F.N. Briggs and M.L. Hess, Characterization of cardiac sarcoplasmic reticulum from ischemic myocardium: comparison of isolated sarcoplasmic reticulum with unfractionated homogenates. J. Mol. Cell. Cardiol., 12 (1980) 4 2 7 - 4 3 2 .