INTRODUCTION

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IN SEVERAL MAMMALIAN SPECIES, including humans, postmaturational aging is accompanied by a prolongation of cardiac muscle contraction duration (22, 23). The prolonged contraction duration in the senescent heart is caused by an age-associated increase in time to peak tension and a slower rate of relaxation (12, 17, 22, 23). The cellular and molecular mechanisms underlying these age-associated changes have only been partially defined to date. Evidence from a number of studies using the rat model suggests that an age-associated decline in the Ca²⁺-sequestering activity of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase is a major factor contributing to the slowing of cardiac muscle relaxation seen with aging (12, 30, 31, 43). An age-related decrease in the Ca²⁺-ATPase content of rat cardiac SR has been reported in some studies (41) but not others (17, 30, 31), and the mechanistic basis for the diminished Ca²⁺-sequestering activity of the SR in the aging heart remains to be resolved.

Ca²⁺-cycling proteins in the SR, in addition to Ca²⁺-ATPase, include the intraluminal Ca²⁺-storage protein calsequestrin (28), the ryanodine receptor-Ca²⁺-release channel (RyR-CRC) (5), and the Ca²⁺-ATPase-regulatory protein phospholamban (28, 40). Age-related changes in the content of these SR Ca²⁺-cycling proteins and/or their functional properties can, conceivably, influence the time course of contraction and relaxation as well as contractile force development. However, very little is known about the impact of aging on these SR Ca²⁺-cycling proteins in cardiac muscle. Studies using rat cardiac SR indicated that the ability of phospholamban to undergo phosphorylation by exogenous cAMP-dependent protein kinase (PKA) in vitro and the stimulatory effect of phospholamban phosphorylation on Ca²⁺ uptake are not altered by aging (18). On the other hand, in studies using isolated perfused beating rat hearts, -adrenergic-mediated cAMP accumulation and phospholamban phosphorylation were found to be significantly reduced with aging (17). Also, age-associated decrements in the inotropic and lusitropic responses to -adrenergic stimulation have been observed in studies using intact hearts and cardiac tissue preparations (8, 13, 17). Besides PKA, Ca²⁺/calmodulin-dependent protein kinase (CaM kinase), associated with cardiac SR (and present in the cytosol), is implicated in the modulation of the Ca²⁺-uptake and -release functions of the SR through phosphorylation of phospholamban (7, 20, 35, 39), RyR-CRC (14, 42, 45), and Ca²⁺-ATPase (15, 44, 47, 48). To our knowledge, the effect of aging on CaM kinase-mediated phosphorylation of the SR Ca²⁺-cycling proteins has not been reported. The present study was designed to investigate the influence of aging on the contents of major SR Ca²⁺-cycling proteins and their phosphorylation by the SR-associated CaM kinase in rat myocardium.

	METHODS

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Animals. Male virgin Fischer 344 rats, ages 6-8 mo (adult animals, 340-375 g body wt) and 26-28 mo (aged animals, 330-385 g body wt), were obtained from the aging Fischer rat colony of the National Institute of Aging (maintained by Harlan Industries). On arrival, these animals were housed individually in plastic cages in the Health Sciences Center animal care facility of this institution at 23°C on a 12:12-h light-dark cycle. The animals had free access to food (Purina Chow containing 20% protein) and water and were used for experiments within 2 wk.

Chemicals. Reagents for electrophoresis were obtained from Bio-Rad Laboratories (Mississauga, ON, Canada), [-³²P]ATP was purchased from Amersham (Oakville, ON, Canada), and ⁴⁵CaCl₂ was from NEN (Mississauga, ON, Canada). Monoclonal antibodies against ryanodine receptor (1), SR Ca²⁺-ATPase (19), and calsequestrin (21) were purchased from Affinity BioReagents (Golden, CO). Anti-phospholamban monoclonal antibody (38) was obtained from Upstate Biotechnology (Lake Placid, NY). Anti--CaM kinase II polyclonal antibody was a generous gift from Dr. H. A. Singer (Weis Center for Research, Danville, PA). All other chemicals were from Sigma Chemical (St. Louis, MO) or BDH Chemicals (Toronto, ON, Canada).

Isolation of SR vesicles. SR-enriched membrane vesicles were prepared from the ventricular myocardium of adult and aged rats as described previously (37). The wet weight of the ventricles averaged 0.82 ± 0.06 and 0.92 ± 0.05 g, respectively, in the case of adult and aged rats. After isolation, the SR vesicles were suspended in 10 mM Tris-maleate (pH 6.8) containing 100 mM KCl and stored at 80°C after quick-freezing in liquid N₂. Protein was determined by the method of Lowry et al. (27) using bovine serum albumin as standard. The yield of SR membranes from adult and aged hearts was similar (~2 mg protein/g wet tissue). Enzyme markers were assessed as described previously (30). Mitochondrial contamination was ~12% as assessed from azide-sensitive Ca²⁺-Mg²⁺-ATPase activity measured in the presence of 8.2 µM free Ca²⁺, 5 mM MgCl₂, 5 mM ATP, and 5 mM NaN₃. Sarcolemmal contamination was ~10% as assessed from ouabain-sensitive Na⁺-K⁺-ATPase activity measured in a reaction medium composed of 50 mM imidazole-HCl (pH 7.5), 100 mM NaCl, 10 mM KCl, 5 mM MgCl₂, 5 mM ATP, 1 mM ouabain, and 125 µg SDS/mg SR protein (30). The Ca²⁺-stimulated ATPase activity of SR membranes (measured in the presence of 8.2 µM free Ca²⁺, 5 mM MgCl₂, and 5 mM ATP) amounted to 191 ± 20 nmol P_i · mg protein¹ · min¹. No age-related difference was observed in these enzyme markers. Furthermore, as documented extensively in our previous studies (17, 30, 31), the polypeptide composition and relative purity of SR membranes isolated from adult and aged rat hearts were essentially similar (also see RESULTS).

Preparation of muscle homogenates. In addition to SR, homogenates from adult and aged hearts were used in some experiments. The homogenates were prepared by homogenizing the ventricular tissue in 10 volumes (based on tissue wt) of 10 mM Tris-maleate-100 mM KCl buffer (pH 6.8) using a Polytron PT-10 homogenizer (three 10-s bursts with 30-s interval between bursts; setting 8; Brinkman Instruments, Westbury, NY). The homogenates were filtered through four layers of cheesecloth and used for experiments.

Immunoblotting of SR Ca²⁺-cycling proteins. Western immunoblotting techniques were used for the detection and estimation of the relative amounts of SR Ca²⁺-cycling proteins in adult and aged rat hearts. For immunoassay of RyR-CRC, Ca²⁺-ATPase, phospholamban, calsequestrin, and CaM kinase, homogenate (25 µg protein/lane) or SR (25 µg protein/lane) samples were first subjected to SDS-PAGE in 6% (for RyR-CRC), 10% (for Ca²⁺-ATPase, calsequestrin and CaM kinase), or 15% (for phospholamban) gels. The protein samples separated by gel electrophoresis were then electroblotted to nitrocellulose membranes. The membranes were probed with antibodies specific for cardiac RyR-CRC [monoclonal (1), dilution 1:2,500], cardiac sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2) [monoclonal (19), dilution 1:3,000], phospholamban [monoclonal (38), 0.5 µg/ml], calsequestrin [monoclonal (21), dilution 1:1,000], or CaM kinase II (polyclonal, dilution 1:1,000). A peroxidase-linked anti-mouse (for RyR-CRC, Ca²⁺-ATPase, phospholamban, and calsequestrin) or anti-rabbit (for CaM kinase) IgG at a dilution of 1:5,000 was used as the secondary antibody. Protein bands reactive with antibodies were visualized using the enhanced chemiluminescence detection system from Amersham and quantified using laser-scanning densitometry of the Western immunoblots. The Western blotting detection system was determined to be linear with respect to the amount of SR/homogenate protein in the range of 10 to 40 µg using the laser-scanning densitometry method.

Phosphorylation assay. The standard incubation medium (total volume 50 µl) for phosphorylation by endogenous CaM kinase contained 50 mM HEPES (pH 7.4), 10 mM MgCl₂, 200 µM CaCl₂, 200 µM EGTA, 1 µM calmodulin, 0.8 mM [-³²P]ATP (specific activity 250-350 counts · min¹ · pmol¹), and SR (30 µg protein). Identical amounts of SR protein from adult and aged hearts were used in each individual experiment. The initial free Ca²⁺ concentration, determined using the computer program of Fabiato (11), was 5.4 µM. The phosphorylation reaction was initiated by the addition of [-³²P]ATP after preincubation of the rest of the assay components for 3 min at 37°C. The reaction was terminated after 2 min by the addition of 15 µl of SDS-sample buffer, and the samples were analyzed in 4-18% SDS-polyacrylamide gels. The gels were stained with Coomassie brilliant blue, dried, and autoradiographed (18). Quantification of phosphorylation was carried out by liquid scintillation counting after excision of the radioactive bands from the gels (18). The Ca²⁺- and calmodulin-dependence of phosphorylation was monitored in parallel assays in which Ca²⁺ (1 mM EGTA present) and/or calmodulin was lacking in the assay medium.

Determination of Ca²⁺ uptake. ATP-dependent, oxalate-facilitated Ca²⁺ uptake was determined in cardiac SR using the Millipore filtration technique as detailed elsewhere (30). The standard incubation medium (total volume 250 µl) contained 50 mM HEPES (pH 7.2), 120 mM KCl, 5 mM NaN₃, 5 mM ATP, 6 mM MgCl₂, 0.1 mM EGTA, 25 µM ruthenium red, 5 mM potassium oxalate, SR membranes (7.5 µg protein), and differing amounts of ⁴⁵CaCl₂ to yield the desired free Ca²⁺ concentration. The initial concentration of free Ca²⁺ in the assay medium was determined using the computer program of Fabiato (11). To evaluate the effect of endogenous CaM kinase-mediated phosphorylation on Ca²⁺ uptake, the assays were performed in the absence of calmodulin and in the presence of 1 µM calmodulin in the incubation medium. The Ca²⁺-uptake reaction was initiated by the addition of SR to the rest of the assay components preincubated for 3 min at 37°C and was allowed to proceed for 2 min, during which the Ca²⁺-uptake rates were found to be linear. In separate experiments (using [-³²P]ATP and nonradioactive CaCl₂), it was established that the Ca²⁺-uptake assay conditions used permitted maximal phosphorylation of SR proteins within the first 45 s after incubation in the presence of calmodulin.

Treatment of SR with anti-phospholamban monoclonal antibody. In some experiments, the SR membranes were treated with anti-phospholamban monoclonal antibody (38) before the effects of Ca²⁺/calmodulin-dependent phosphorylation on Ca²⁺ uptake were determined. The SR membranes (60 µg protein) were incubated in a medium (total volume 110 µl) containing 10 mM Tris-maleate (pH 6.8), 120 mM KCl, and 40 µg anti-phospholamban monoclonal antibody for 10 min at 24°C and for an additional 20 min at 4°C. Subsequently, the SR membranes were recovered by centrifugation (at 15,000 rpm for 40 min in a microcentrifuge) and used for phosphorylation and Ca²⁺-uptake assays. SR membranes subjected to the same experimental protocol in the absence of anti-phospholamban monoclonal antibody in the incubation medium served as the control for these experiments.

Data analysis. Results are presented as means ± SE. Statistical significance was evaluated with the Student's t-test; P < 0.05 was taken as the level of significance.

	RESULTS

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SR Ca²⁺-cycling proteins in adult and aged hearts. Major Ca²⁺-cycling proteins in the SR include the RyR-CRC, through which Ca²⁺ is released into the cytosol on myocyte excitation to induce muscle contraction (5); Ca²⁺-ATPase, which actively sequesters Ca²⁺ back into the SR lumen to promote muscle relaxation (28); calsequestrin, which serves to bind and store Ca²⁺ within the SR lumen (28); and phospholamban, which serves to regulate Ca²⁺-ATPase function (7, 28, 40). In the present study, we utilized monoclonal antibodies specific for each of these SR Ca²⁺-cycling proteins to perform Western immunoblotting analysis of their relative amounts in cardiac SR isolated from adult and aged rats. The results from these experiments are summarized in Fig. 1. No significant age-related difference was evident in the relative amounts of RyR-CRC, Ca²⁺-ATPase, calsequestrin, or phospholamban in cardiac SR. Similar findings were also obtained in experiments in which Western immunoblotting analysis of these proteins was performed using unfractionated cardiac muscle homogenates from adult and aged rats (results not shown). Analysis of the phospholamban-to-Ca²⁺-ATPase ratio in individual experiments showed variation in the range of 3 to 25% (data pooled from 10 separate experiments yielded an average value of 14 ± 3%); this variation was found to be independent of age.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Detection and estimation of relative amounts of Ca2+-cycling proteins in cardiac sarcoplasmic reticulum (SR) isolated from adult (open bars) and aged (filled bars) rats by Western blotting. Identical amounts (25 µg protein) of cardiac SR from adult and aged rats were subjected to Western immunoblotting analysis of ryanodine receptor-Ca2+-release channel (RyR-CRC), Ca2+-ATPase, calsequestrin, and phospholamban as described in METHODS. Immunoblots (bottom) were obtained with 5 separate SR preparations from adult and aged rats. The content of each Ca2+-cycling protein in several SR preparations was determined by laser-scanning densitometry of immunoblots, and results are presented as means ± SE (n = 12, 16, 6, and 11, respectively, for RyR-CRC, Ca2+-ATPase, calsequestrin, and phospholamban) in bar graphs. Immunoblots for phospholamban show high (H)- and low-molecular-weight (L) forms of protein. Age-related difference is not significant for any of the Ca2+-cycling proteins.

CaM kinase and SR protein phosphorylation in adult and aged hearts. CaM kinase II, associated with the SR (and present in the cytosol), is implicated in the regulation of the Ca²⁺-uptake and -release functions of cardiac SR through phosphorylation of phospholamban (7, 20, 35, 40), RyR-CRC (14, 42, 45), and Ca²⁺-ATPase (15, 44, 47, 48). In the present study, we utilized a CaM kinase II polyclonal antibody (specific for the -isoform predominantly expressed in the heart) to perform Western blotting analysis of this enzyme in adult and aged rat hearts. The relative amount of CaM kinase II in cardiac muscle homogenates and SR membranes was found to be significantly lower (~50%) in the aged compared with adult rat heart (Fig. 2).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Detection and estimation of relative amounts of Ca2+/calmodulin-dependent protein kinase (CaM kinase II) in cardiac muscle homogenates and cardiac SR preparations from adult (open bars) and aged (filled bars) rats by Western blotting. Identical amounts of cardiac homogenates and SR membranes (25 µg protein) from adult and aged rats were subjected to Western immunoblotting analysis of CaM kinase II (-isoform) as described in METHODS. Immunoblots (bottom) were obtained with 3 separate homogenate and SR preparations from adult and aged rats. CaM kinase II content was quantified by laser-scanning densitometry of immunoblots obtained using 6 preparations each of homogenate and SR from adult and aged rats, and results are presented as means ± SE in bar graph. * P < 0.0001 vs. adult.

View larger version (92K): [in this window] [in a new window]   Fig. 3.   Comparison of endogenous CaM kinase-mediated protein phosphorylation in cardiac SR isolated from adult and aged rats. Phosphorylation reaction was carried out under standard assay conditions in presence of Ca2+ and calmodulin as described in METHODS. Left: Coomassie blue-stained SDS-PAGE gel depicting protein profiles of 3 separate cardiac SR preparations each from adult and aged rats. Molecular mass markers are in kDa. Right: autoradiogram of same gel depicting protein phosphorylation. Ca2+-cycling proteins undergoing phosphorylation included RyR-CRC, Ca2+-ATPase, and phospholamban (PLB; H and L forms). Phosphorylation of these substrates did not occur in absence of Ca2+ or calmodulin in assay medium (not shown). Note diminished phosphorylation of SR Ca2+-cycling proteins in aged compared with adult heart. Quantitative data on substrate phosphorylation from several experiments are summarized in Fig. 4.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Endogenous CaM kinase-mediated phosphorylation of RyR-CRC, Ca2+-ATPase, and phospholamban in cardiac SR isolated from adult (open bars) and aged (filled bars) rats. After phosphorylation under standard assay conditions in presence of Ca2+ and calmodulin, SR proteins were fractionated by SDS-PAGE and 32P incorporation into peptide bands representing RyR-CRC, Ca2+-ATPase, and phospholamban was determined by liquid scintillation counting as described in METHODS. Data represent means ± SE (n = 12, 15, and 15, respectively for RyR-CRC, Ca2+-ATPase, and phospholamban). * P < 0.01-0.001 vs. adult.

Effect of CaM kinase-mediated phosphorylation on Ca²⁺ uptake by SR. To assess the effect of endogenous CaM kinase-mediated phosphorylation on Ca²⁺ uptake, ATP-dependent, oxalate-facilitated Ca²⁺ uptake by SR was determined in the absence and presence of calmodulin in the assay medium (see METHODS). Under the experimental conditions employed, the addition of calmodulin to the Ca²⁺-uptake assay medium promotes phosphorylation of CaM kinase substrates in the SR (RyR-CRC, Ca²⁺-ATPase, and phospholamban) through activation of the endogenous enzyme (15, 47). As shown in Fig. 5, the presence of calmodulin (1 µM) in the assay medium resulted in stimulation of Ca²⁺ uptake by SR from adult and aged hearts. This stimulatory effect of calmodulin was observed when assays were performed with a subsaturating (0.6 µM) or saturating (8.2 µM) concentration of free Ca²⁺. The Ca²⁺-uptake rates measured in the absence and presence of calmodulin were significantly lower in the aged (30-40%) compared with adult heart. The calmodulin-induced stimulation of Ca²⁺ uptake by SR was ~36-38% in the aged compared with ~60-65% in the adult heart.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of activation of endogenous CaM kinase by calmodulin (CaM) on Ca2+-uptake activity of cardiac SR isolated from adult and aged rats. Ca2+-uptake reaction was carried out under standard assay conditions at a subsaturating (0.6 µM) or saturating (8.2 µM) free Ca2+ concentration, in absence of CaM (CaM) and in presence of 1 µM CaM (+CaM), in incubation medium as described in METHODS. Ca2+-uptake data from experiments using 6 SR preparations each from adult and aged hearts are presented as means ± SE. # P < 0.01 vs. CaM. * P < 0.05 vs. adult.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of activation of endogenous CaM kinase by CaM on Ca2+-uptake activity of anti-phospholamban antibody (PLBab)-treated cardiac SR from adult and aged rats. Control SR and PLBab SR were prepared as described in METHODS. Ca2+-uptake reaction was carried out under standard assay conditions at a subsaturating (0.6 µM) or saturating (8.2 µM) free Ca2+ concentration in absence of CaM or in presence of CaM (1 µM). Ca2+-uptake data from experiments using 3 SR preparations each from adult and aged hearts are presented as means ± SE. * P < 0.05 vs. adult. # P < 0.05 vs. control.  P < 0.05-0.01 vs. PLBab.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Autoradiogram showing effect of PLBab treatment of cardiac SR from adult and aged rats on endogenous CaM kinase-mediated protein phosphorylation. Control SR and SR treated with PLBab were prepared, and phosphorylation reaction was carried out, under standard assay conditions as described in METHODS. Results shown are typical of 3 experiments using separate cardiac SR preparations from adult and aged rats. Note that phosphorylation of phospholamban (PLB; H and L forms) is abolished in PLBab SR.

	DISCUSSION

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The results presented here demonstrate that the contents of the SR Ca²⁺-cycling proteins RyR-CRC, Ca²⁺-ATPase, calsequestrin, and phospholamban are not altered significantly during postmaturational aging in the rat heart. On the other hand, it is shown that significant age-associated decrements occur in 1) the amount of -CaM kinase II in the rat heart, 2) the endogenous CaM kinase-mediated phosphorylation of SR Ca²⁺-cycling proteins, and 3) the phosphorylation-dependent stimulation of SR Ca²⁺ sequestration. These findings are of considerable significance in the context of analyzing the mechanisms associated with the well-documented age-related alterations in intrinsic contractile properties of the heart and the physiological regulation of heart function.

In the rat, altered contractile properties of cardiac muscle with aging include prolonged contraction duration caused by increases in time to peak tension and relaxation time (12, 17, 22) and attenuation of developed peak tension at high-frequency (>120 beats/min) stimulation (17) but not at low-frequency (<100 beats/min) stimulation (3, 13, 22). An age-associated decrease in the speed of Ca²⁺ cycling between the myofilaments and the Ca²⁺ reservoirs (SR compartment and extracellular fluid) is a likely factor underlying the altered contractile properties of the senescent myocardium (22, 23). Our finding that the content of RyR-CRC remains unaltered with aging in the rat heart suggests that the age-related increase in time to peak tension and decrease in developed peak tension cannot be attributed to a decrease in the density of RyR-CRC in the SR. At present, it is not known whether aging alters the functional properties of RyR-CRC in cardiac muscle. A recent study (6) has reported twitch prolongation and altered Ca²⁺ and caffeine sensitivity of RyR-CRC, but not RyR-CRC content, with aging in fast-twitch tibialis anterior muscle of the rat.

Consistent with previous reports from this (18, 30, 31) and other laboratories (12, 43), the ATP-dependent Ca²⁺-uptake activity of SR was found to be significantly lower in the aged heart. The observed age-related decline in the Ca²⁺-uptake activity of SR is likely a major factor contributing to the slow rate of cardiac muscle relaxation seen with aging. A previous study attributed the age-associated decline in Ca²⁺-uptake activity of rat cardiac SR to an age-related decrease in the SR Ca²⁺-ATPase content (41). We did not observe any significant age-related difference in the immunoreactive SR Ca²⁺-ATPase content in the rat heart in studies using SERCA2-specific monoclonal (this report) or polyclonal (17) antibodies. Our earlier studies have shown an age-related decline in Ca²⁺ uptake but not Ca²⁺-stimulated ATP hydrolysis by the SR Ca²⁺-ATPase in the rat heart (30, 31) and slow-twitch soleus muscle (33). These observations support the view that age-associated changes might occur in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport in the SR, resulting in reduced Ca²⁺ sequestration. Apparent uncoupling of ATP hydrolysis from Ca²⁺ transport may be observed because of an age-related increase in Ca²⁺ efflux through RyR-CRC, "downhill" movement of Ca²⁺ mediated by the Ca²⁺-ATPase (29), and/or an age-related decrease in the Ca²⁺ storage capacity of the SR. A significant age-related increase in Ca²⁺ efflux from the SR caused by any of these factors, however, seems unlikely in view of the lack of significant age-related changes in the contents of RyR-CRC, Ca²⁺-ATPase, and calsequestrin reported here.

To our knowledge, this report is the first to describe the effects of aging on Ca²⁺/calmodulin-dependent SR protein phosphorylation in the myocardium. Our results have revealed a significant age-related decrease in the phosphorylation of RyR-CRC, Ca²⁺-ATPase, and phospholamban by the endogenous SR-associated CaM kinase. Because aging did not significantly alter the levels of CaM kinase substrates, this decrease in phosphorylation may be attributed to the observed age-related decrease in the amount of -CaM kinase II, which is the predominant CaM kinase II isoform present in cardiac cytosol (10, 36) and SR (2, 48). The functional consequence of cardiac RyR-CRC phosphorylation has not yet been clearly established. In reconstituted lipid vesicles, phosphorylation of RyR-CRC by CaM kinase II has been shown to either increase or decrease channel openings (14, 26, 45). Because CaM kinase II has been found to phosphorylate a single serine residue (Ser²⁸⁰⁹) in the cardiac RyR-CRC (45), it is unclear how such divergent effects on Ca²⁺ release would ensue on phosphorylation of the same site. Recently, CaM kinase II inhibitors as well as protein phosphatases have been found to reduce SR Ca²⁺-release channel activity in intact cardiomyocytes (9, 25). These findings are consistent with an increased SR Ca²⁺-release channel activity on RyR-CRC phosphorylation by CaM kinase II. In any case, the observed age-related decline in RyR-CRC phosphorylation can be expected to impact on the modulation of SR Ca²⁺ release and, therefore, myofilament activation.

Phosphorylation of phospholamban by PKA (at Ser¹⁶) and CaM kinase (at Thr¹⁷) is well known to stimulate Ca²⁺ uptake by SR, apparently by relieving an inhibitory effect exerted by dephosphorylated phospholamban on the Ca²⁺-ATPase (4, 7, 28, 39, 40). Recently, Ser³⁸ phosphorylation of the cardiac SR Ca²⁺-ATPase by CaM kinase also has been shown to result in stimulation of ATP hydrolysis and Ca²⁺ transport (15, 44, 47). Some studies have, however, questioned the physiological role of Ca²⁺-ATPase phosphorylation. Thus a study by Odermatt et al. (34) showed CaM kinase-mediated phosphorylation of Ca²⁺-ATPase in native rabbit cardiac SR as well as of the cardiac isoform of Ca²⁺-ATPase (SERCA2a) expressed in HEK-293 cells but failed to observe a significant stimulatory effect on Ca²⁺-ATPase function. Another study, by Reddy et al. (35), reported the failure to observe phosphorylation of the Ca²⁺-ATPase in canine cardiac SR or of purified Ca²⁺-ATPase reconstituted in lipid vesicles. These studies have attributed the stimulatory effect of CaM kinase on SR Ca²⁺ uptake [i.e., an increase in Ca²⁺ affinity but not maximum velocity of Ca²⁺ transport (V_max)] solely to the phosphorylation of phospholamban. We have, however, observed stimulation of Ca²⁺ uptake by SR (increase in V_max) under experimental conditions in which the Ca²⁺-ATPase, but not phospholamban, underwent selective phosphorylation by CaM kinase (32). The present observations showing age-related decrements in CaM kinase-mediated phosphorylation of phospholamban and Ca²⁺-ATPase as well as in the stimulatory effect of phosphorylation on Ca²⁺ uptake suggest attenuation of this regulatory pathway in the aging heart. This might impact adversely on the speed of muscle relaxation in aging. Whether the age-associated decline in the phosphorylation of CaM kinase substrates observed in the isolated SR membranes in vitro also holds true under in vivo conditions, however, remains to be established. Furthermore, age-associated decrements in CaM kinase-mediated phosphorylation of Ca²⁺-ATPase and calmodulin-stimulated Ca²⁺ uptake were observed under conditions in which the inhibitory interaction of phospholamban with Ca²⁺-ATPase was completely disrupted and phospholamban phosphorylation was abolished (Figs. 6 and 7). These findings imply age-associated diminution in the regulation of SR Ca²⁺-pump function through CaM kinase-mediated phosphorylation of the Ca²⁺-ATPase. In this regard, it is noteworthy that evidence from recent studies has revealed acceleration of cardiac muscle relaxation caused by a CaM kinase-mediated increase in SR Ca²⁺-pump activity, independent of phospholamban phosphorylation (16, 24).

Besides its influence on SR Ca²⁺ cycling, CaM kinase likely plays a critical role in modulating Ca²⁺ influx though L-type Ca²⁺ channels in the sarcolemma during membrane depolarization (25, 46). Therefore, it is conceivable that the observed age-related decrease in CaM kinase content in the cardiomyocyte may also adversely impact on this crucial excitatory event, contributing to some of the alterations in the excitation-contraction coupling process seen in the aging heart (23).