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Effects of aging on phospholamban phosphorylation and calcium transport in rat cardiac sarcoplasmic reticulum
Mechanisms of Ageing and Development, 54 (1990)87--101 ElsevierScientificPublishersIrelandLtd.
87
EFFECTS OF AGING ON PHOSPHOLAMBAN PHOSPHORYLATION AND CALCIUM TRANSPORT IN RAT CARDIAC SARCOPLASMIC RETICULUM
MING-TAO JIANG and NJANOOR NARAYANAN* Department of Physiology, Health SciencesCenter, The Universityof WesternOntario, London, N6A 5C1 (Canada) (ReceivedOctober7th, 1989) SUMMARY Acceleration of cardiac relaxation upon beta adrenergic stimulation is due, in part, to enhancement in the rate of Ca 2*sequestration by the sarcoplasmic reticulum (SR) Ca 2* pump resulting from cAMP-mediated phosphorylation of the SR protein phospholamban. Our previous studies have shown that in rat myocardium, beta adrenergic activation of adenylate cyclase and the Ca 2* pump activity of SR decline with aging (Mech. Ageing Dev., 19 (1982) 127--139; 38 (1987) 127--143). In the present study, the effect of aging on phospholamban phosphorylation and consequent changes in SR Ca 2÷ pump activity were evaluated using cardiac SR from 6 (young adult), 12 (adult) and 28 (aged) months old rats. No age-related differences were observed in the rate or maximum level of phospholamban phosphorylation by exogenous cAMP-dependent protein kinase. The rates of ATP-dependent Ca 2÷ uptake by SR from young adult and aged rats were stimulated upon phospholamban phosphorylation, the percentage stimulation of Ca 2* uptake at varying Ca 2÷ concentrations (0.'24--11.9/~M) was not diminished with aging. However, the rates of Ca 2*uptake by phosphorylated and unphosphorylated SR were remarkably lower (35--50~70) in the aged. Regardless of the age of rats, the stimulatory effect of phosphorylation on Ca 2+uptake by SR was due to increase in Vm~ of Ca 2÷transport with no appreciable changes in Ko.5 for Ca 2÷. These findings imply that in spite of the ageassociated decline in SR Ca 2* pump activity, the ability of phospholamban to undergo cAMP-mediated phosphorylation and the relative responsiveness of the SR Ca 2÷ pump to phospholamban phosphorylation are not diminished in the aging heart.
Key words: Aging heart; Sarcoplasmic reticulum; Phosphorylation; Phospholamban; Ca 2÷transport "Towhomall correspondenceshouldbe addressed. 0047-6374/90/$03.50 Printedand Publishedin Ireland
© 1990ElsevierScientificPublishersIreland Ltd.
88 INTRODUCTION
The ability of the heart to respond to various forms of stress declines with aging in both humans and animals (for reviews see Refs. 1--3). Evidence from several studies suggests strongly that age-related diminution in the responsiveness of the heart to beta adrenergic stimulation is a key factor underlying this age-associated deficit in stress response. Thus, in normal aged humans, the increase in the heart rate response to various forms of stress that result in an increase in serum catecholamines (e.g., static exercise, hypoxia, hypercapnia) is diminished compared to that in the adult [4,5]. Also, in normal humans, beta adrenergic blockade reduces the age-related difference in cardiac output during dynamic exercise observed between adulthood and senescence [6]. The heart rate increase in response to infusion of the beta-receptor agonist, isoproterenol, is diminished in aged dogs [7], rats [8] and humans [9] when compared to their adult counterparts. Further, diminished contractile response to catecholamines in cardiac muscle isolated from the senescent compared to the adult rat has been demonstrated suggesting age-related deficit intrinsic to the myocardium [10,11]. The age-related alterations in postsynaptic mechanisms responsible for the diminished beta adrenergic responses of the aging heart are not yet clearly understood. In a study using interventricular septum from adult and aged rats, Guarnieri et al. [11] did not observe age-related differences in the density of beta receptors, isoproterenol-induced cyclic AMP accumulation or in the activity of cyclic AMP-dependent protein kinase, measured using an exogenous substrate (histone). They suggested that the age-related deficit limiting contractile response to beta adrenergic stimulation is at steps subsequent to protein kinase activation. While confirming the absence of age-related changes in the density of beta receptors, studies from this [12,13] and other [14--16] laboratories have shown that activation of adenylate cyclase by beta adrenergic agonists is significantly decreased in aging rat myocardium. Although the molecular nature of age-associated defect in the cardiac betareceptor cyclase system remains to be established it appears to involve alterations in furictional properties such as decrement in agonist binding affinity of the beta receptor, decreased ability of guanine necleotides to modulate receptor affinity for agonists and consequent impairement in the interactions between the three macromolecular components of the receptor cyclase system, viz. the beta adrenergic receptor, guanine nucleotide regulatory protein (N s) and the enzyme adenylate cyclase [12,151. Analysis of the effects of aging on cyclic AMP-mediated cellular processes (i.e., events distal to beta-receptor adenylate cyclase system) are also essential to gain further information about the mechanisms underlying the age-related decline in beta adrenergic responses of the heart. A major, well-characterized cellular process subject to regulation by cyclic AMP in cardiac muscle, is the active Ca 2÷ sequestration
89
function of the sarcoplasmic reticulum (SR) served by the ATP-energized Ca 2÷ pump, represented by the enzyme, Ca2+-ATPase. It is now well established that acceleration of cardiac relaxation upon beta adrenergic stimulation is due, in part, to enhancement in the rates of Ca 2÷sequestration by the SR Ca 2÷pump resulting fom cyclic AMP-mediated phosphorylation of the SR protein, phospholamban (see Refs. 17,18 for reviews). We [19,20] and others [21] have shown that the Ca 2÷pump activity of SR declines markedly with aging in rat myocardium, and this appears to be the biochemical basis of prolongation of cardiac contraction duration seen with aging. In this study, we have investigated the effects of aging on phospholamban phosphorylation in vitro by cyclic AMP-dependent protein kinase and the resultant changes in C a 2÷ pump activity of rat cardiac SR. The results demonstrate that, in isolated SR vesicles, the ability of phospholamban to undergo cyclic AMP-mediated phosphorylation, and the relative responsiveness of the SR Ca 2÷ pump to phospholamban phosphorylation, are not altered appreciably with aging. MATERIALS AND METHODS
Animals Male Fischer 344 rats aged 6 (young adult, 290--360 g body wt), 12 (adult, 340-410 g body wt), and 28 months (aged, 300--410 g body wt) were obtained from the aging Fischer rat colony of the National Institute on Aging (maintained by Harlan Industries, IN), U.S.A. Upon arrival, these animals were housed individually in cages with free access to food (Purina Chow containing 20070 protein) and water and were used for experiments within 2 weeks. Chemicals 45CaClz (15.96 mCi/mg) and [y-32p]ATP (36 Ci/mmol) were obtained from New England Nuclear, Montreal, Canada. Reagents for electrophoresis were from BioRad Laboratories, Mississauga, Ontario, Canada. Cyclic AMP-dependent protein kinase (catalytic subunit, from bovine heart) was purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. All other chemicals were of the highest purity available from Sigma Chemical Co. or BDH Chemicals, Toronto, Canada. Isolation o f SR-enriched membrane fraction SR-enriched membrane fraction was prepared from rat heart ventricles according to the method of Harigaya and Schwartz [22] with minor modifications as described previously [23]. Following isolation the membranes were suspended in 10 mM Tris-maleate (pH 6.8) containing 100 mM KCI to give a protein concentration of 3--4 mg/ml. Protein was determined by the method of Lowry et al. [24] using defatted bovine serum albumin as standard. All studies were carried out using freshly prepared membrane fractions.
90
SR phosphorylation Phosphorylation of SR membrane proteins by cyclic AMP-dependent protein kinase was carried out at 37°C for 0.5--3 rain. The standard incubation medium (total vol. 0.25 ml) contained 50 mM Tris--maleate (pH 6.8), 120 mM KCI, 2 mM MgCI2, 24 mM NaF, 1 mM [y-32p]ATP (10/aCi//amol) with or without 5/ag catalytic subunit of cyclic AMP-dependent protein kinase and SR membrane (400 /ag protein). The phosphorylation reaction was initiated by the addition of SR following preincubation of the rest of the assay components for 3 min. The reaction was terminated by adding 0.25 ml of electrophoresis sample buffer, which consisted of 0.125 M Tris--HC1 (pH 6.8), 4070 SDS, 20070 glycerol, 10070 beta-mercaptoethanol and 0.01 07o bromophenol blue. The samples were left at room temperature (23--24°C) overnight and aliquots of boiled (100°C for 5 min) and unboiled samples were fractionated by electrophoresis. Gel electrophoresis and autoradiography SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as detailed elsewhere [19] with two identical 8--18070 gradient acrylamide slab gels. Equal amount of membrane protein (between 80 and 130 big for different experiments) was applied to each lane and electrophoresis was conducted at 16 mA/gel through stacking gel and 25 mA/gel through separating gel. Following electrophoresis, the gels were fixed overnight in 25070 isopropyl alcohol/10070 acetic acid. One gel was stained with Coomassie Blue and the other, wrapped in plastic film, was used for autoradiography by placing in contact with Kodak X-Omat AR film in Kodak Lanex Regular Cassette (with intensifying screen) at - 8 0 ° C for 48--72 h. The radioactive bands corresponding to phopholamban were identified according to autoradiogram and then cut from the gel and counted in Beckman aqueous liquid scintillation cocktail (EP). Phosphate incorporated into phospholamban was quantified by dividing 32p incorporation by the specific radioactivity of [y-32p]ATP and expressed as pmo132pi p e r mg protein applied to each lane. Determination of calcium transport ATP-dependent, oxalate-facilitated C a 2÷ uptake by SR was determined using a Millipore filtration technique as described previously [19]. The standard incubation medium (total vol. 1 ml) contained 50 mM Tris--maleate (pH 6.8), 5 mM MgCl 2, 2.5 mM ATP, 120 mM KC1, 5 mM potassium oxalate, 5 mM NaN 3, 0.1 mM EGTA, SR membrane (30/ag protein) with or without 5/ag cyclic AMP-dependent protein kinase catalytic subunit and varying concentrations of 45CaCl2. All assays were performed at 37 °C; the Ca 2÷transport reaction was initiated by the addition of 45CaC12EGTA mixture to the rest of the assay components preincubated for 3 min. The free Ca 2÷ion concentration was determined as described previously [ 19].
91
Data analysis The results are presented as mean _+ S.E. Statistical significance was evaluated by Student's t-test; P < 0.05 was taken as the level of significance. RESULTS
Figure 1 compares protein phosphorylation in SR membranes derived from the myocardium of 6-, 12- and 28-month-old rats by exogenous cAMP-dependent protein kinase (cAMP-PK). In SR from all three age groups, cAMP-PK caused phosphorylation of two major peptides with apparent molecular weights of 24 kDa and
AGE (months) Pk
: ~
6 -
12 +
-
28 +
-
6 +
-
12 +
-
28 +
-
+ M.W,
~40k
PLM (H)
~24k
~l---15k ,~---12k
PLM(L)
UNBOILED
BOILED
Fig. 1. Autoradiogram depicting protein phosphorylation in cardiac SR isolated from rats aged 6, 12 and 28 months. The phosphorylation reaction was carried out for 1 min in the absence and presence of cAMP-dependent PK. Boiled and non-boiled samples of the phosphorylated membranes were subjected to SDS-PAGE and autoradiography (for details see Materials and Methods). PLM(H) and PLM(L) designate the high and low molecular weight forms of phospholamban, respectively. The results shown are typical of six experiments using separate SR preparations.
92 12 kDa (left panel). The electrophoretic mobility of the 24-kDa protein could be altered by conditions under which the membranes were solublized prior to electrophoresis. When the samples were boiled in SDS-sample buffer prior to electrophoresis, all of the radioactivity previously localized to 24-kDa band was recovered in the 12-kDa band (right panel). Such alteration in the electrophoretic mobility upon boiling in SDS is a characteristic property of phospholamban, and is attributed to dissociation of the polymeric high molecular weight form of the protein [25,26]. Therefore, the 24-kDa and 12-kDa phosphoproteins represent the high and low molecular weight forms of phospholamban. No significant age-related difference was evident in the extent of phospholamban phosphorylation caused by cAMP-PK. In addition to phospholamban, a protein of apparent molecular weight 15 kDa was also phosphorylated by cAMP-PK, albeit weakly, in SR preparations from all three age groups. This protein may correspond to the 15-kDa protein identified in sarcolemma, which is also a substrate for cAMP-PK [27,28]. This observation suggests slight degree of sarcolemmal contamination in the SR preparations. The degree of such contamination, however, is similar in cardiac SR preparations from all three age groups of rats as evidenced by the lack of age-related changes in the amount of this phosphoprotein. Further, as documented in our previous studies [19,20], the extent of sarcolemmal contamination in rat cardiac SR preparations obtained utilizing the procedure employed here is less than 12%, and more importantly, the relative purity of cardiac SR preparations from rats of different ages is very similar. SR incubated in the absence of cAMP-PK showed a phosphoprotein of molecular weight 40 kDa, apparently due to phosphorylation by endogenous protein kinase. The results shown in Fig. 1 were obtained when the phosphorylation was carried out for 1 min. In additional experiments the time course of phosphorylation was examined using cardiac SR preparations from 6- and 28-month-old rats. It was observed that phosphorylation of phospholamban by cAMP-PK was very fast and maximal phosphorylation occurred within 30 s of incubation (Fig. 2 and Table I). Quantitation of the amount of radioactive phosphate incorporated into phospholamban at various time-intervals showed no significant differences in the rate or maximum level of phospholamban phosphorylation. Although not readily evident from Fig. 2, cumulative data from several experiments showed slightly diminished levels of phospholamban phosphoprotein at 3 min compared to 0.5 or 1 min of incubation (Table 1), suggesting dephosphorylation by endogenous protein phosphatase known to be associated with the SR membrane [29,30]. Interestingly the level of phospholamban phosphoprotein at 3 min appeared to be lower, in the case of SR from 28-month-old compared to 6-month-old rats, suggesting slightly higher rates of dephosphorylation in the aged even though a phosphatase inhibitor (NaF) was included in the assay. The ATP-dependent Ca 2÷uptake activity of cardiac SR from 6 and 28-month-old rats were determined in the absence and presence of cAMP-PK so as to assess agerelated differences if any, in the ability of phosphorylated phospholamban to stimu-
93
AGE (Months)
6
Pk
28 +
6
28
28
6
- ÷
M.W.
4 - - 40k
PLM(H)
"~"-
24k
lSk 12k
PLM(L) I 2
TIME(rain) ~
3
4
5
6
0.5
7
8
9
I0
I
II
12
3
Fig. 2. Autoradiogram depicting time course of protein phosphorylation in cardiac SR isolated from rats aged 6 and 28 months. The phosphorylation reaction was carried out in the absence and presence of cAMP-dependent PK. Aliquots o f phosphorylated m e m b r a n e s (non-boiled) were subjected to SDSP A G E and autoradiography (for details see Materials and Methods). PLM(H) and PLM(L) designate the high and low molecular weight forms o f p h o s p h o l a m b a n , respectively. The results shown are typical of five experiments using separate SR preparations. TABLE I E F F E C T OF A G E ON P H O S P H O L A M B A N P H O S P H O R Y L A T I O N IN R A T C A R D I A C SR
Age o f animals (months)
6 28
Pmol J2P/mg protein 0.5 min
I min
3 min
114 ± 15.8 (n = 5) 108 ± 12.4" (n = 5)
101 ± 15.2 (n = 8) 100 ± 3.9" (n = 8)
93 (n 70 (n
± 9.1 c = 5) +_ 6.8 ",b = 5)
Phosphorylation reaction was carried out by incubating SR m e m b r a n e with c A M P - P K for various times as indicated (for details see Materials and Methods). Data represent means ± S.E. • Age-related difference not significant. b Significantly lower compared to the values obtained at 0.5 or 1 min, P < 0.05. Not significantly different compared to the values obtained at 0.5 or 1 min.
94
late SR Ca 2÷ p u m p activity. In the absence of c A M P - P K , the rates of Ca 2÷ uptake by SR f r o m 28-month-old rats were markedly lower (35--50°70) when compared to SR from 6-month-old rats (Fig. 3); this was true at low (0.92/~m) or high (5.9/am) concentrations of free Ca 2÷. These results confirm the striking age-related decline in Ca 2÷ p u m p activity o f SR in rat myocardium previously reported by us [19,20] and others [21]. In the presence of c A M P - P K , the rates of Ca 2+uptake by SR were increased (40 --50°70) in both age groups. In a preliminary report (abstract), K a d o m a et al. [31] also observed that c A M P - P K caused similar degree of stimulation (50070) of Ca 2÷ uptake by cardiac SR f r o m adult and aged rats. Figure 4 shows the effects of c A M P - P K on Ca 2÷ uptake by SR from 6- and 28month-old rats at varying free Ca 2* concentrations. The age-related difference in Ca 2÷ uptake (35--50070 lower in the aged, P < 0.01 or 0.001), and the stimulatory effects of c A M P - P K on Ca 2. uptake could be observed at the wide range o f free Ca 2. concentrations (0.24--11.9/~M) employed. It is noteworthy that the rates of Ca 2. uptake by SR f r o m aged rats measured in the presence of c A M P - P K were lower than the Ca 2÷ uptake rates of SR f r o m the young age group measured in the absence of
/
~"
J ± 28m, - P K • 28m, +PK 60 c~ 6 m , - P K • 6m, +PK
4s
I
120
//
?
90
Q. O~ E ~+ O o
E
~
60
ao
c ~.~
Q. .-,
ao
is
"6
E -!
® I
1
I
I
I
2 3 0 1 Incubation Time (rain)
I
I
2
3
o
Fig. 3. Time course of ATP-dependent Ca 2. uptake by cardiac sarcoplasmic reticulum from 6- and 28month-old rats in the absence and presence of cAMP-dependent PK. The Ca 2÷ uptake assays were performed as described under Materials and Methods with 0.92 ~M free Ca 2÷ (Panel A) or 5.9/~M free Ca 2÷ (Panel B) in the assay medium. Each data point represents mean _ S.E. of five experiments using separate membrane preparations. The differences between 6- and 28-month-old rats, in the presence and absence of PK, are significant ( P < 0.05 to 0.001).
95
60
E
~, 2 8 m , - P K • 28m,+PK o 6m,-PK • 6m,+PK 0.2
7 E"
45
t, --
o
30 0+1
K --'1
(..)
zx
15
0
® ~
I
+
4 6 Ca 2+, pM
II
12
i 1
[Ca 2+] '(tzM) '
Fig. 4. Effects of varying calcium concentration on ATP-dependent Ca2"uptake by cardiac sarcoplasmic reticulum from 6- and 28-month-old rats in the presence and absence of cAMP-dependent protein kinase (cAMP-PK). The Ca2" uptake assays were performed as described under Materials and Methods with varying free Ca2÷concentrations as indicated. In Panel A, mean Ca2+uptake velocities are plotted against free Ca2"concentrations in the assay; Panel B shows double reciprocal plots of the data. Each data point represent mean + S.E. of eight experiments using separate membrane preparations. The differences between 6- and 28-month-old rats, in the presence and absence of PK, are significant (P< 0.01 to 0.001). The stimulatory effect of PK is significant at all Ca z÷concentration employed (P < 0.01 to 0.001).
c A M P - P K . H o w e v e r , the percentage stimulation o f Ca 2÷ uptake due to c A M P - P K , tended to be slightly higher (12--25°70) in the aged at all but the lowest Ca 2+ concentration used (Fig. 5); this age-related difference was not statistically significant except at t w o Ca 2÷ concentrations (0.92 and 1.4 ~M, P < 0.05). The effects o f c A M P - P K on the kinetic parameters o f Ca 2+ uptake by SR, derived f r o m double reciprocal plots (Fig. 4B) o f the data on Ca 2÷ concentration-dependence, are s u m m a r i z e d in Table II. Regardless o f the age o f the rats, the stimulatory effect o f c A M P - P K on SR Ca 2+ uptake was due to increase in V o f Ca 2÷ transport; c A M P - P K did not s e e m to enhance appreciably the apparent affinity o f the transport system for Ca 2+ although c A M P - m e d i a t e d increase in the affinity o f Ca 2+A T P a s e for Ca 2 + has been reported for canine cardiac SR [32,33]. In agreement with our previous findings [19,20], the age-related decline in the SR Ca 2+ uptake activity was associated with selective decrement in Vm~ without appreciable alterations in the g0. 5 for Ca 2÷.
96
[] •
6m 28m
180
"6 ~
140
~
120
~
100
80
0.24
0.54
.
1.4
2.9
5.9
.
Ca 2 + ~M Fig. 5. Co mparison of the relative stimulatory effect of c A M P - P K on Ca 2* uptake by cardiac SR from 6and 28-month-old rats at varying Ca 2+ concentrations. The percentage Ca 2+ uptake values were derived from the data shown in Fig. 4 (Panel A). The age-related difference is significant only at two Ca 2÷concentrations, viz. 0.92 and 1.4/~M ( P < 0.05).
TABLE II EFFECTS OF A G E A N D P H O S P H O L A M B A N P H O S P H O R Y L A T I O N ON TH E KINETIC P A R A METERS OF A T P - D E P E N D E N T C A L C I U M U P T A K E BY RAT C A R D I A C SR
Age o f animals (months)
1 I (nmol Ca2*/mg protein per min) Control
6 28
53 - 2.8 26 _ 2.7 a
Phosphorylated
68 - 4.1 b 37 _ 2.6 a,h
Ca~÷concentration for half-maximal velocity k o5(laM) Control
Phosphorylated
2.39 _ 0.10 2.03 _+ 0.21
2.27 _+ 0.14 1.90 - 0.10
The kinetic parameters (values expressed as mean _+ S.E.) were derived from data on mean Ca 2÷ uptake velocities vs. free Ca 2÷concentrations shown in Fig. 4. a Age-related difference significant, P < 0.001. b Significantly higher compared to control, P < 0.001. k05 values for Ca 2÷ are not significantly different between age groups, or between control and phosphorylated.
97 DISCUSSION The results of the present study demonstrate that (a) in isolated rat cardiac SR vesicles, the ability of phospholamban to undergo phosphorylation by exogenously added cAMP-PK is not altered by aging and (b) the magnitude of the relative stimulatory effect of phosphorylated phospholamban on active Ca 2÷ sequestration by SR vesicles in vitro is not diminished by aging. On the other hand, the results presented here also confirm a striking age-related decline in the active C a 2÷ uptake activity of SR vesicles from rat myocardium previously reported by us [19,20] and others [21]. The interrelationship of these findings and their implications can be analysed by taking into consideration (a) available information about the mechanisms underlying the age-related decline in SR Ca 2÷ pump function and (b) the mechanisms postulated to underlie the regulation of SR Ca 2÷pump activity by phospholamban. With regard to (a) above, our previous studies have suggested that the age-related decline in the C a 2÷ pump activity of SR is due to impaired coupling between ATP hydrolysis and Ca 2÷translocation functions of the Ca 2÷pump since the latter but not the former decreases with aging [19,20]. Such an impairment, conceivably, can result from an age-related defect in the conformational response of the ATPase since ligand ( C a 2÷, ATP, MgE÷)-induced transitions in enzyme conformation are of prime importance in the ion translocation function [26,34]. The potential factors which might contribute to an age-related defect in the conformational response of the ATPase (see Ref. 19 for a discussion) include (i) an age-related molecular rigidity of the ATPase or a functional domain of the enzyme (e.g., Ca 2÷ transporting segment) and (ii) age-associated decrease in the fluidity of the membrane microenvironment (e.g., due to increase in cholesterol/phospholipid ratio) of the ATPase. With regard to (b) above, phospholamban is proposed to exert its actions by direct interaction with the Ca2*-ATPase, interaction of phosphorylated phospholamban producing enhancement in C a 2÷ pump activity through acceleration of the partial reactions of the CaE*-ATPase [17,18,26]. It has also been suggested that interaction of unphosphorylated phospholamban with the Ca2÷-ATPase may serve to suppress Ca 2÷pump activity [17,35]. Since no age-related decrease was observed in the degree of phospholamban phosphorylation and the magnitude of its stimulatory effect on the Ca 2÷pump, it can be concluded that age-related alterations in the physicochemical properties of the SR membrane, if they occur, do not occlude the phosphorylation site of phospholamban or hinder interactions between phospholamban and CaE÷ATPase. This does not seem surprising in view of recent information about the structural properties of phospholamban and its likely disposition in the SR. The phosphorylatable domain[containing the serine 16 (phosphoryiation site for cAMPPK) and threonine 17 (phosphorylation site for calmodulin-dependent PK) residues) located in the amino-terminal region of phospholamban molecule is hydrophilic and cytoplasmic, the carboxy-terminal being hydrophobic and membranous [26,36,37]. Further, there is good evidence that it is the cytoplasmic domain containing the
98 phosphorylation site that makes direct interaction with the Ca2+-ATPase[26,38]. Thus, any age-related structural changes in the hydrophobic core of the membrane would be expected to have only minimal direct impact on phospholamban phosphorylation and its interaction with the Ca2+-ATPase. Needless to add that the lack of age-related changes in phospholamban-CaE+-ATPase interactions, by no means rules out potential age-related alterations in the hydrophobic matrix of the SR membrane. Even though the magnitude of the relative stimulatory effect of phosphorylated phospholamban on SR Ca 2÷transport remained undiminished with aging, the actual C a 2÷ transport velocities were markedly lower in the aged heart SR even under conditions where phospholamban was maximally phosphorylated. Therefore, age-related enhancement in the postulated inhibitory action of unphosphorylated phospholamban [17,35] does not contribute to the age-related depression in SR Ca 2÷pump activity. Since the CaE÷-stimulated ATPase activity of cardiac SR is unaltered with aging [19,20], it is unlikely that age-related decrement in the density of Ca 2÷ pump units in the membrane or reduction in the turnover rates of the CaE+-ATPase contributes to the age-related decline in SR C a 2÷ pump activity. As noted earlier, a decrease in the efficiency of coupling A T P hydrolysis to Ca 2÷ transport appears to be the probable cause o f the age-related decline in SR Ca 2÷transport function. If so, one of two possible modes o f expression of impaired coupling can be envisioned: (a) All Ca 2÷pump units in the SR become partially uncoupled with aging, or (b) some of the Ca 2÷pump units in the SR membrane become totally uncoupled with aging while others remain well-coupled. If mode (a) is operative, fertile interactions (interaction resulting in stimulation of C a 2÷ transport) must occur between all of the phosphorylated phospholamban molecules and all of the Ca 2÷ pump units in SR [CaE+-ATPase and phospholamban are thought to exist in a 1: 1 stoichiometry in the SR [26]; we assume that this ratio is not altered with aging]. If mode (b) is operative, fertile interactions will occur only between the well-coupled C a 2÷ pump units and an equal number of phosphorylated phospholamban molecules, thus leaving a surplus of phosphorylated phospholamban molecules totally inert functionally. The lack of age-related changes in phospholamban phosphorylation, the undiminished relative stimulatory effect of phosphorylated phospholamban on SR Ca 2÷ transport with aging, and the agerelated decline in SR Ca 2÷ pump activity observed here are consistent with either modes of uncoupling envisioned above. No data are presently available to distinguish which of them is more probable. As noted in the introduction, cAMP-mediated phosphorylation of phospholamban and consequent stimulation o f SR C a 2÷ pump is the best known physiological mechanism underlying acceleration of cardiac muscle relaxation upon beta adrenergic stimulation. Interestingly, two reported studies have shown that the relaxant effect of beta adrenergic agonists is preserved in the aging heart whereas the inotropic effect is diminished [10,11]. Present data showing undiminished phosphorylation of phospholamban in v i t r o in cardiac SR of aged rats by cAMP-PK,
99
and the effect of phospholamban phosphorylation on Ca 2÷transport are consistent with the above observation. However, further studies using intact myocardium are necessary to determine whether age-related changes occur in the phosphorylation of phospholamban in vivo in response to beta adrenergic stimulation. Such studies are presently in progress in this laboratory. ACKNOWLEDGEMENTS
This work was supported by the Heart and Stroke Foundation of Ontario. The expert assistance of Mr. Emin Donat in some of the experiments is gratefully acknowledged. Dr. M.T. Jiang is a Research Trainee of the Canadian Heart Foundation. REFERENCES I
E.G. Lakatta, Age-related alterations in the cardiovascular response to adrenergic mediated stress. Fed. Pro(:., 39 (1980)3173--3177. 2 E.G. Lakatta, Heart and circulation.In C.E. Finch and E.L. Schneider (eds.),Handbook of the Biology of Aging, 2ndedn., N e w York, Van Nostrand Reinhold, 1985, pp. 377--409. 3 E.G. Lakatta, Cardiac muscle changes in senescence.Ann. Rev. PhysioL, 49 (1987) 519---531. 4 R.S. Kronenberg and C.W. Drage, Attenuation of the ventflatory and heart rate response to hypoxia and hypercapoia with aging in normal men. J. Clin.Invest.,25 (1973) 1812--1819. 5 J.S.Petrofsky and A.R. Lind, Isometric strength,endurance, and the blcod presure and heart rate responses during isometric exercisein healthy men and women, with special reference to age and body fat content. PluegersArch., 360 (1975) 49--6 I. 6 J.R. Conway and R. Sannerstedt, Sympathetic nervous activityduring exercisein relationto age. Cardiovasc. Res., 5 (1971) 577--581. 7 F.C.P. Yin, H.A. Spurgeon, H.L. Greene, E.G. Lakatta and M.L. Weisfeldt, Age-associated decrease in heart rateresponse to isoproterenolin dogs. Mech. Ageing Dev., I0 (1979) 17--25. 8 P.R. Le Blanc and K. Rakusan, Effects of age and isoproterenolon the cardiac output and regional blood flow in the rat.Can. J. CardioL, 3 (1987) 246--250. 9 F.C.P. Yin, H.A. Sporgeon, G.S. Ralzes, H.L. Greene, M.L. Weisfeldt and N.W. Shock, Age associated decrease in chronotropic response to isoproterenol (abstract). Circulation, 54 (Suppl.) II (1976) 167. I0 E.G. Lakatta, G. Gerstenblith,C.S. AngeIl, N.W. Shock and M.L. Weisfeldt,Diminished inotropic response of the aged myocardium to catecholamines. Circ.Res., 36 (1975) 262--269. I I T. Guarnieri, C.R. Filburn, G. Zitnik, G.S. Roth and E.G. Lakatta, Contractile and biochemical correlates of beta adrenergic stimulation of the aged heart. Am. J. Physiol., 239 (1980) H501-H508. 12 N. Narayanan and J.A. Derby, Alterationsin the propertiesof~-adrenergic receptorsof myocardial membranes in aging: Impairments in agonist-receptorinteractionsand guanine nucleotide regulation accompany diminished catacholamine-responsiveness of the adenylate cyclase. Mech. Ageing Dev., 19(1982) 127--139. 13 N. Narayanan and L. Tucker, Autonomic interactionssin the aging heart: Age-associated decrease in muscarinic cholinergic receptor mediated inhibitionof beta adrenergic activation of adenylate cyclase.Mech. Ageing Dev., 34 (I986) 249--259. 14 S.W. O'Connor, P.J. Sarpace and I.B. Arass, Age-associated decrease of adenylate cyclaseactivity in rat myocardium. Mech. Ageing Dev., 16 (198 I) 91--95. 15 P.J. Sarpace, Decreased beta adrenergic responsiveness during senescence. Fed. Proc., 45 (1986) 51--54.
lO0
16
T.M. Fan and S.P. Banerjee, Age-related reduction of beta adrenoceptor sensitivity in rat heart occurs by multiple mechanisms. Gerontology, 31 (1985) 373--380. 17 M. Tada and A.M. Katz, Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu. Rev. Physiol., 44 (1982) 401--423. 18 M. Tada, M. Shigekawa and Y. Nimura, Uptake of calcium by the sarcoplasmic reticulum and its regulation and functional consequences. In N. Sperelakis, II (ed.), Physiology and Pathophysiology o f the Heart, Martinus Nijhoff publishing, Boston, 1984, pp. 255--277. 19 N. Narayanan, Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium. Biochim. Biophys. Acta, 678 (1981) 442-459. 20 N. Narayanan, Comparison of ATP-dependent calcium transport and calcium activated ATPase activities of cardiac sarcoplasmic reticulum and sarcolemma from rats of various ages. Mech. Ageing Dev., 38 (1987) 127-- 143. 21 J.P. Froehlich, E.G. Lakatta, E. Beard, H.A. Spurgeon, M.L. Weisfeldt and G. Gerstenhlith, Studies of sarcoplasmic reticulum function and contraction duration in young and aged rat myocardium. J. Mol. Cell. Cardiol., 10 (1978)427--438. 22 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. 23 P.V. Sulakhe and N. Narayanan, Microsomal and sarcolemmal adenylate cyclase in cardiac muscle: Differential effects of non-ionic detergents. Biochem. J., 175 (1978) 171--180. 24 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randell, Protein measurement with phenol reagent. J. Biol. Chem., 193 (1951)265--275. 25 M.A. Kirchberger, T. Antonetz, Phospholamban: Dissociation of the 22,000 molecular weight protein of cardiac sarcoplasmic reticulum into 11,000 and 5,500 molecular weight forms. Biochem. Biophys. Res. Commun., 105 (1982) 152--156. 26 M. Tada, M. Kadoma, M. Inui and I.J. Fujii, Regulation of Ca2+-pump from cardiac sarcoplasmic reticulum. In S. Fleischer and B. Fleischer (eds.), Methods in Enzymology, Vol. 157, Academic Press, London, 1988, pp.107--154. 27 A.S. Manalan and L.R. Jones, Characterization of the intrinsic cAMP-dependent protein kinase activity and endogenous substrates in highly purified cardiac sarcolemmal vesicles. J. BioL Chem., 257(1982) 10052--10062. 28 C.F. Presti, L.R. Jones and J.P. Lindermann, lsoproterenol-induced phosphorylation of a 15-kilodalton sarcolemmal protein in intact myocardium. J. Biol. Chem., 260 (1985) 3860--3867. 29 P. LaRala and E. Morkin, Adenosine 3',5' monophosphate-dependentmembrane phosphorylation. A possible mechanism for the control of microsomal calcium transport in heart muscle. Circ. Res., 35 (1974) 298--306. 30 E.G. Kranias, Regulation of calcium transport by protein phosphatase activity associated with cardiac sarcoplasmic reticulum. J. Biol. Chem., 260 (1985) 11006--11010. 31 M. Kadoma, B. Sacktor and J.P. Froehlich, Stimulation by cAMP and protein kinase of calcium transport in sarcoplasmic reticulum from senescent rat myocardium (abstract). Fed. Proc. 39 (1980) 2040. 32 M. Tada, M. lnui, M. Yamada, M. Kadoma, T. Kuzuya, H. Abe and S. Kakiuchi, Effects of phospholamban phosphorylation catalyzed by adenosine 3' : 5'-monophosphate and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum. J. Mol. Cell. Cardiol., 15 (1983) 335--346. 33 M. Tada, M.A. Kirchberger, D.I. 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. 34 G. Inesi, Mechanism of calcium transport. Annu. Rev. Physiol., 47 (1985) 573--601. 35 M. Inui, B.K. Chamberlain, A. Sato, S. Fleischer, The nature of the modulation of Ca 2+transport as studied by reconstitution of cardiac sarcoplasmic reticulum. J. Biol. Chem., 261 0986) 1794-1800.
101 36
37
38
J. Fujii, A. Ueno, K. Kitano, S. Tanaka, M. Kadoma and M. Tada, Complete complementary DNA-derived amino acid sequence of canine cardiac phospholamban. J. Clin. Invest., 79 (1987) 301 --304. H.K.B. Simmerman, J.H. Collins, I.L. Thibert, A.D. Wegener and L.R. Jones, Sequence analysis of phospholamban: Identification of phosphorylation sites and two major structural domains. J. Biol. Chem., 261 (1986) 13333--13341. T. Suzuki and J.H. Wang, Stimulation of bovine cardiac sarcoplasmic reticulum Ca 2÷ pump and blocking of the phospholamban phosphorylation and dephosphorylation by a phospholamban monoclonal antibody. J. Biol. Chem., 261 (1986) 7018--7023.
87
EFFECTS OF AGING ON PHOSPHOLAMBAN PHOSPHORYLATION AND CALCIUM TRANSPORT IN RAT CARDIAC SARCOPLASMIC RETICULUM
MING-TAO JIANG and NJANOOR NARAYANAN* Department of Physiology, Health SciencesCenter, The Universityof WesternOntario, London, N6A 5C1 (Canada) (ReceivedOctober7th, 1989) SUMMARY Acceleration of cardiac relaxation upon beta adrenergic stimulation is due, in part, to enhancement in the rate of Ca 2*sequestration by the sarcoplasmic reticulum (SR) Ca 2* pump resulting from cAMP-mediated phosphorylation of the SR protein phospholamban. Our previous studies have shown that in rat myocardium, beta adrenergic activation of adenylate cyclase and the Ca 2* pump activity of SR decline with aging (Mech. Ageing Dev., 19 (1982) 127--139; 38 (1987) 127--143). In the present study, the effect of aging on phospholamban phosphorylation and consequent changes in SR Ca 2÷ pump activity were evaluated using cardiac SR from 6 (young adult), 12 (adult) and 28 (aged) months old rats. No age-related differences were observed in the rate or maximum level of phospholamban phosphorylation by exogenous cAMP-dependent protein kinase. The rates of ATP-dependent Ca 2÷ uptake by SR from young adult and aged rats were stimulated upon phospholamban phosphorylation, the percentage stimulation of Ca 2* uptake at varying Ca 2÷ concentrations (0.'24--11.9/~M) was not diminished with aging. However, the rates of Ca 2*uptake by phosphorylated and unphosphorylated SR were remarkably lower (35--50~70) in the aged. Regardless of the age of rats, the stimulatory effect of phosphorylation on Ca 2+uptake by SR was due to increase in Vm~ of Ca 2÷transport with no appreciable changes in Ko.5 for Ca 2÷. These findings imply that in spite of the ageassociated decline in SR Ca 2* pump activity, the ability of phospholamban to undergo cAMP-mediated phosphorylation and the relative responsiveness of the SR Ca 2÷ pump to phospholamban phosphorylation are not diminished in the aging heart.
Key words: Aging heart; Sarcoplasmic reticulum; Phosphorylation; Phospholamban; Ca 2÷transport "Towhomall correspondenceshouldbe addressed. 0047-6374/90/$03.50 Printedand Publishedin Ireland
© 1990ElsevierScientificPublishersIreland Ltd.
88 INTRODUCTION
The ability of the heart to respond to various forms of stress declines with aging in both humans and animals (for reviews see Refs. 1--3). Evidence from several studies suggests strongly that age-related diminution in the responsiveness of the heart to beta adrenergic stimulation is a key factor underlying this age-associated deficit in stress response. Thus, in normal aged humans, the increase in the heart rate response to various forms of stress that result in an increase in serum catecholamines (e.g., static exercise, hypoxia, hypercapnia) is diminished compared to that in the adult [4,5]. Also, in normal humans, beta adrenergic blockade reduces the age-related difference in cardiac output during dynamic exercise observed between adulthood and senescence [6]. The heart rate increase in response to infusion of the beta-receptor agonist, isoproterenol, is diminished in aged dogs [7], rats [8] and humans [9] when compared to their adult counterparts. Further, diminished contractile response to catecholamines in cardiac muscle isolated from the senescent compared to the adult rat has been demonstrated suggesting age-related deficit intrinsic to the myocardium [10,11]. The age-related alterations in postsynaptic mechanisms responsible for the diminished beta adrenergic responses of the aging heart are not yet clearly understood. In a study using interventricular septum from adult and aged rats, Guarnieri et al. [11] did not observe age-related differences in the density of beta receptors, isoproterenol-induced cyclic AMP accumulation or in the activity of cyclic AMP-dependent protein kinase, measured using an exogenous substrate (histone). They suggested that the age-related deficit limiting contractile response to beta adrenergic stimulation is at steps subsequent to protein kinase activation. While confirming the absence of age-related changes in the density of beta receptors, studies from this [12,13] and other [14--16] laboratories have shown that activation of adenylate cyclase by beta adrenergic agonists is significantly decreased in aging rat myocardium. Although the molecular nature of age-associated defect in the cardiac betareceptor cyclase system remains to be established it appears to involve alterations in furictional properties such as decrement in agonist binding affinity of the beta receptor, decreased ability of guanine necleotides to modulate receptor affinity for agonists and consequent impairement in the interactions between the three macromolecular components of the receptor cyclase system, viz. the beta adrenergic receptor, guanine nucleotide regulatory protein (N s) and the enzyme adenylate cyclase [12,151. Analysis of the effects of aging on cyclic AMP-mediated cellular processes (i.e., events distal to beta-receptor adenylate cyclase system) are also essential to gain further information about the mechanisms underlying the age-related decline in beta adrenergic responses of the heart. A major, well-characterized cellular process subject to regulation by cyclic AMP in cardiac muscle, is the active Ca 2÷ sequestration
89
function of the sarcoplasmic reticulum (SR) served by the ATP-energized Ca 2÷ pump, represented by the enzyme, Ca2+-ATPase. It is now well established that acceleration of cardiac relaxation upon beta adrenergic stimulation is due, in part, to enhancement in the rates of Ca 2÷sequestration by the SR Ca 2÷pump resulting fom cyclic AMP-mediated phosphorylation of the SR protein, phospholamban (see Refs. 17,18 for reviews). We [19,20] and others [21] have shown that the Ca 2÷pump activity of SR declines markedly with aging in rat myocardium, and this appears to be the biochemical basis of prolongation of cardiac contraction duration seen with aging. In this study, we have investigated the effects of aging on phospholamban phosphorylation in vitro by cyclic AMP-dependent protein kinase and the resultant changes in C a 2÷ pump activity of rat cardiac SR. The results demonstrate that, in isolated SR vesicles, the ability of phospholamban to undergo cyclic AMP-mediated phosphorylation, and the relative responsiveness of the SR Ca 2÷ pump to phospholamban phosphorylation, are not altered appreciably with aging. MATERIALS AND METHODS
Animals Male Fischer 344 rats aged 6 (young adult, 290--360 g body wt), 12 (adult, 340-410 g body wt), and 28 months (aged, 300--410 g body wt) were obtained from the aging Fischer rat colony of the National Institute on Aging (maintained by Harlan Industries, IN), U.S.A. Upon arrival, these animals were housed individually in cages with free access to food (Purina Chow containing 20070 protein) and water and were used for experiments within 2 weeks. Chemicals 45CaClz (15.96 mCi/mg) and [y-32p]ATP (36 Ci/mmol) were obtained from New England Nuclear, Montreal, Canada. Reagents for electrophoresis were from BioRad Laboratories, Mississauga, Ontario, Canada. Cyclic AMP-dependent protein kinase (catalytic subunit, from bovine heart) was purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. All other chemicals were of the highest purity available from Sigma Chemical Co. or BDH Chemicals, Toronto, Canada. Isolation o f SR-enriched membrane fraction SR-enriched membrane fraction was prepared from rat heart ventricles according to the method of Harigaya and Schwartz [22] with minor modifications as described previously [23]. Following isolation the membranes were suspended in 10 mM Tris-maleate (pH 6.8) containing 100 mM KCI to give a protein concentration of 3--4 mg/ml. Protein was determined by the method of Lowry et al. [24] using defatted bovine serum albumin as standard. All studies were carried out using freshly prepared membrane fractions.
90
SR phosphorylation Phosphorylation of SR membrane proteins by cyclic AMP-dependent protein kinase was carried out at 37°C for 0.5--3 rain. The standard incubation medium (total vol. 0.25 ml) contained 50 mM Tris--maleate (pH 6.8), 120 mM KCI, 2 mM MgCI2, 24 mM NaF, 1 mM [y-32p]ATP (10/aCi//amol) with or without 5/ag catalytic subunit of cyclic AMP-dependent protein kinase and SR membrane (400 /ag protein). The phosphorylation reaction was initiated by the addition of SR following preincubation of the rest of the assay components for 3 min. The reaction was terminated by adding 0.25 ml of electrophoresis sample buffer, which consisted of 0.125 M Tris--HC1 (pH 6.8), 4070 SDS, 20070 glycerol, 10070 beta-mercaptoethanol and 0.01 07o bromophenol blue. The samples were left at room temperature (23--24°C) overnight and aliquots of boiled (100°C for 5 min) and unboiled samples were fractionated by electrophoresis. Gel electrophoresis and autoradiography SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as detailed elsewhere [19] with two identical 8--18070 gradient acrylamide slab gels. Equal amount of membrane protein (between 80 and 130 big for different experiments) was applied to each lane and electrophoresis was conducted at 16 mA/gel through stacking gel and 25 mA/gel through separating gel. Following electrophoresis, the gels were fixed overnight in 25070 isopropyl alcohol/10070 acetic acid. One gel was stained with Coomassie Blue and the other, wrapped in plastic film, was used for autoradiography by placing in contact with Kodak X-Omat AR film in Kodak Lanex Regular Cassette (with intensifying screen) at - 8 0 ° C for 48--72 h. The radioactive bands corresponding to phopholamban were identified according to autoradiogram and then cut from the gel and counted in Beckman aqueous liquid scintillation cocktail (EP). Phosphate incorporated into phospholamban was quantified by dividing 32p incorporation by the specific radioactivity of [y-32p]ATP and expressed as pmo132pi p e r mg protein applied to each lane. Determination of calcium transport ATP-dependent, oxalate-facilitated C a 2÷ uptake by SR was determined using a Millipore filtration technique as described previously [19]. The standard incubation medium (total vol. 1 ml) contained 50 mM Tris--maleate (pH 6.8), 5 mM MgCl 2, 2.5 mM ATP, 120 mM KC1, 5 mM potassium oxalate, 5 mM NaN 3, 0.1 mM EGTA, SR membrane (30/ag protein) with or without 5/ag cyclic AMP-dependent protein kinase catalytic subunit and varying concentrations of 45CaCl2. All assays were performed at 37 °C; the Ca 2÷transport reaction was initiated by the addition of 45CaC12EGTA mixture to the rest of the assay components preincubated for 3 min. The free Ca 2÷ion concentration was determined as described previously [ 19].
91
Data analysis The results are presented as mean _+ S.E. Statistical significance was evaluated by Student's t-test; P < 0.05 was taken as the level of significance. RESULTS
Figure 1 compares protein phosphorylation in SR membranes derived from the myocardium of 6-, 12- and 28-month-old rats by exogenous cAMP-dependent protein kinase (cAMP-PK). In SR from all three age groups, cAMP-PK caused phosphorylation of two major peptides with apparent molecular weights of 24 kDa and
AGE (months) Pk
: ~
6 -
12 +
-
28 +
-
6 +
-
12 +
-
28 +
-
+ M.W,
~40k
PLM (H)
~24k
~l---15k ,~---12k
PLM(L)
UNBOILED
BOILED
Fig. 1. Autoradiogram depicting protein phosphorylation in cardiac SR isolated from rats aged 6, 12 and 28 months. The phosphorylation reaction was carried out for 1 min in the absence and presence of cAMP-dependent PK. Boiled and non-boiled samples of the phosphorylated membranes were subjected to SDS-PAGE and autoradiography (for details see Materials and Methods). PLM(H) and PLM(L) designate the high and low molecular weight forms of phospholamban, respectively. The results shown are typical of six experiments using separate SR preparations.
92 12 kDa (left panel). The electrophoretic mobility of the 24-kDa protein could be altered by conditions under which the membranes were solublized prior to electrophoresis. When the samples were boiled in SDS-sample buffer prior to electrophoresis, all of the radioactivity previously localized to 24-kDa band was recovered in the 12-kDa band (right panel). Such alteration in the electrophoretic mobility upon boiling in SDS is a characteristic property of phospholamban, and is attributed to dissociation of the polymeric high molecular weight form of the protein [25,26]. Therefore, the 24-kDa and 12-kDa phosphoproteins represent the high and low molecular weight forms of phospholamban. No significant age-related difference was evident in the extent of phospholamban phosphorylation caused by cAMP-PK. In addition to phospholamban, a protein of apparent molecular weight 15 kDa was also phosphorylated by cAMP-PK, albeit weakly, in SR preparations from all three age groups. This protein may correspond to the 15-kDa protein identified in sarcolemma, which is also a substrate for cAMP-PK [27,28]. This observation suggests slight degree of sarcolemmal contamination in the SR preparations. The degree of such contamination, however, is similar in cardiac SR preparations from all three age groups of rats as evidenced by the lack of age-related changes in the amount of this phosphoprotein. Further, as documented in our previous studies [19,20], the extent of sarcolemmal contamination in rat cardiac SR preparations obtained utilizing the procedure employed here is less than 12%, and more importantly, the relative purity of cardiac SR preparations from rats of different ages is very similar. SR incubated in the absence of cAMP-PK showed a phosphoprotein of molecular weight 40 kDa, apparently due to phosphorylation by endogenous protein kinase. The results shown in Fig. 1 were obtained when the phosphorylation was carried out for 1 min. In additional experiments the time course of phosphorylation was examined using cardiac SR preparations from 6- and 28-month-old rats. It was observed that phosphorylation of phospholamban by cAMP-PK was very fast and maximal phosphorylation occurred within 30 s of incubation (Fig. 2 and Table I). Quantitation of the amount of radioactive phosphate incorporated into phospholamban at various time-intervals showed no significant differences in the rate or maximum level of phospholamban phosphorylation. Although not readily evident from Fig. 2, cumulative data from several experiments showed slightly diminished levels of phospholamban phosphoprotein at 3 min compared to 0.5 or 1 min of incubation (Table 1), suggesting dephosphorylation by endogenous protein phosphatase known to be associated with the SR membrane [29,30]. Interestingly the level of phospholamban phosphoprotein at 3 min appeared to be lower, in the case of SR from 28-month-old compared to 6-month-old rats, suggesting slightly higher rates of dephosphorylation in the aged even though a phosphatase inhibitor (NaF) was included in the assay. The ATP-dependent Ca 2÷uptake activity of cardiac SR from 6 and 28-month-old rats were determined in the absence and presence of cAMP-PK so as to assess agerelated differences if any, in the ability of phosphorylated phospholamban to stimu-
93
AGE (Months)
6
Pk
28 +
6
28
28
6
- ÷
M.W.
4 - - 40k
PLM(H)
"~"-
24k
lSk 12k
PLM(L) I 2
TIME(rain) ~
3
4
5
6
0.5
7
8
9
I0
I
II
12
3
Fig. 2. Autoradiogram depicting time course of protein phosphorylation in cardiac SR isolated from rats aged 6 and 28 months. The phosphorylation reaction was carried out in the absence and presence of cAMP-dependent PK. Aliquots o f phosphorylated m e m b r a n e s (non-boiled) were subjected to SDSP A G E and autoradiography (for details see Materials and Methods). PLM(H) and PLM(L) designate the high and low molecular weight forms o f p h o s p h o l a m b a n , respectively. The results shown are typical of five experiments using separate SR preparations. TABLE I E F F E C T OF A G E ON P H O S P H O L A M B A N P H O S P H O R Y L A T I O N IN R A T C A R D I A C SR
Age o f animals (months)
6 28
Pmol J2P/mg protein 0.5 min
I min
3 min
114 ± 15.8 (n = 5) 108 ± 12.4" (n = 5)
101 ± 15.2 (n = 8) 100 ± 3.9" (n = 8)
93 (n 70 (n
± 9.1 c = 5) +_ 6.8 ",b = 5)
Phosphorylation reaction was carried out by incubating SR m e m b r a n e with c A M P - P K for various times as indicated (for details see Materials and Methods). Data represent means ± S.E. • Age-related difference not significant. b Significantly lower compared to the values obtained at 0.5 or 1 min, P < 0.05. Not significantly different compared to the values obtained at 0.5 or 1 min.
94
late SR Ca 2÷ p u m p activity. In the absence of c A M P - P K , the rates of Ca 2÷ uptake by SR f r o m 28-month-old rats were markedly lower (35--50°70) when compared to SR from 6-month-old rats (Fig. 3); this was true at low (0.92/~m) or high (5.9/am) concentrations of free Ca 2÷. These results confirm the striking age-related decline in Ca 2÷ p u m p activity o f SR in rat myocardium previously reported by us [19,20] and others [21]. In the presence of c A M P - P K , the rates of Ca 2+uptake by SR were increased (40 --50°70) in both age groups. In a preliminary report (abstract), K a d o m a et al. [31] also observed that c A M P - P K caused similar degree of stimulation (50070) of Ca 2÷ uptake by cardiac SR f r o m adult and aged rats. Figure 4 shows the effects of c A M P - P K on Ca 2÷ uptake by SR from 6- and 28month-old rats at varying free Ca 2* concentrations. The age-related difference in Ca 2÷ uptake (35--50070 lower in the aged, P < 0.01 or 0.001), and the stimulatory effects of c A M P - P K on Ca 2. uptake could be observed at the wide range o f free Ca 2. concentrations (0.24--11.9/~M) employed. It is noteworthy that the rates of Ca 2. uptake by SR f r o m aged rats measured in the presence of c A M P - P K were lower than the Ca 2÷ uptake rates of SR f r o m the young age group measured in the absence of
/
~"
J ± 28m, - P K • 28m, +PK 60 c~ 6 m , - P K • 6m, +PK
4s
I
120
//
?
90
Q. O~ E ~+ O o
E
~
60
ao
c ~.~
Q. .-,
ao
is
"6
E -!
® I
1
I
I
I
2 3 0 1 Incubation Time (rain)
I
I
2
3
o
Fig. 3. Time course of ATP-dependent Ca 2. uptake by cardiac sarcoplasmic reticulum from 6- and 28month-old rats in the absence and presence of cAMP-dependent PK. The Ca 2÷ uptake assays were performed as described under Materials and Methods with 0.92 ~M free Ca 2÷ (Panel A) or 5.9/~M free Ca 2÷ (Panel B) in the assay medium. Each data point represents mean _ S.E. of five experiments using separate membrane preparations. The differences between 6- and 28-month-old rats, in the presence and absence of PK, are significant ( P < 0.05 to 0.001).
95
60
E
~, 2 8 m , - P K • 28m,+PK o 6m,-PK • 6m,+PK 0.2
7 E"
45
t, --
o
30 0+1
K --'1
(..)
zx
15
0
® ~
I
+
4 6 Ca 2+, pM
II
12
i 1
[Ca 2+] '(tzM) '
Fig. 4. Effects of varying calcium concentration on ATP-dependent Ca2"uptake by cardiac sarcoplasmic reticulum from 6- and 28-month-old rats in the presence and absence of cAMP-dependent protein kinase (cAMP-PK). The Ca2" uptake assays were performed as described under Materials and Methods with varying free Ca2÷concentrations as indicated. In Panel A, mean Ca2+uptake velocities are plotted against free Ca2"concentrations in the assay; Panel B shows double reciprocal plots of the data. Each data point represent mean + S.E. of eight experiments using separate membrane preparations. The differences between 6- and 28-month-old rats, in the presence and absence of PK, are significant (P< 0.01 to 0.001). The stimulatory effect of PK is significant at all Ca z÷concentration employed (P < 0.01 to 0.001).
c A M P - P K . H o w e v e r , the percentage stimulation o f Ca 2÷ uptake due to c A M P - P K , tended to be slightly higher (12--25°70) in the aged at all but the lowest Ca 2+ concentration used (Fig. 5); this age-related difference was not statistically significant except at t w o Ca 2÷ concentrations (0.92 and 1.4 ~M, P < 0.05). The effects o f c A M P - P K on the kinetic parameters o f Ca 2+ uptake by SR, derived f r o m double reciprocal plots (Fig. 4B) o f the data on Ca 2÷ concentration-dependence, are s u m m a r i z e d in Table II. Regardless o f the age o f the rats, the stimulatory effect o f c A M P - P K on SR Ca 2+ uptake was due to increase in V o f Ca 2÷ transport; c A M P - P K did not s e e m to enhance appreciably the apparent affinity o f the transport system for Ca 2+ although c A M P - m e d i a t e d increase in the affinity o f Ca 2+A T P a s e for Ca 2 + has been reported for canine cardiac SR [32,33]. In agreement with our previous findings [19,20], the age-related decline in the SR Ca 2+ uptake activity was associated with selective decrement in Vm~ without appreciable alterations in the g0. 5 for Ca 2÷.
96
[] •
6m 28m
180
"6 ~
140
~
120
~
100
80
0.24
0.54
.
1.4
2.9
5.9
.
Ca 2 + ~M Fig. 5. Co mparison of the relative stimulatory effect of c A M P - P K on Ca 2* uptake by cardiac SR from 6and 28-month-old rats at varying Ca 2+ concentrations. The percentage Ca 2+ uptake values were derived from the data shown in Fig. 4 (Panel A). The age-related difference is significant only at two Ca 2÷concentrations, viz. 0.92 and 1.4/~M ( P < 0.05).
TABLE II EFFECTS OF A G E A N D P H O S P H O L A M B A N P H O S P H O R Y L A T I O N ON TH E KINETIC P A R A METERS OF A T P - D E P E N D E N T C A L C I U M U P T A K E BY RAT C A R D I A C SR
Age o f animals (months)
1 I (nmol Ca2*/mg protein per min) Control
6 28
53 - 2.8 26 _ 2.7 a
Phosphorylated
68 - 4.1 b 37 _ 2.6 a,h
Ca~÷concentration for half-maximal velocity k o5(laM) Control
Phosphorylated
2.39 _ 0.10 2.03 _+ 0.21
2.27 _+ 0.14 1.90 - 0.10
The kinetic parameters (values expressed as mean _+ S.E.) were derived from data on mean Ca 2÷ uptake velocities vs. free Ca 2÷concentrations shown in Fig. 4. a Age-related difference significant, P < 0.001. b Significantly higher compared to control, P < 0.001. k05 values for Ca 2÷ are not significantly different between age groups, or between control and phosphorylated.
97 DISCUSSION The results of the present study demonstrate that (a) in isolated rat cardiac SR vesicles, the ability of phospholamban to undergo phosphorylation by exogenously added cAMP-PK is not altered by aging and (b) the magnitude of the relative stimulatory effect of phosphorylated phospholamban on active Ca 2÷ sequestration by SR vesicles in vitro is not diminished by aging. On the other hand, the results presented here also confirm a striking age-related decline in the active C a 2÷ uptake activity of SR vesicles from rat myocardium previously reported by us [19,20] and others [21]. The interrelationship of these findings and their implications can be analysed by taking into consideration (a) available information about the mechanisms underlying the age-related decline in SR Ca 2÷ pump function and (b) the mechanisms postulated to underlie the regulation of SR Ca 2÷pump activity by phospholamban. With regard to (a) above, our previous studies have suggested that the age-related decline in the C a 2÷ pump activity of SR is due to impaired coupling between ATP hydrolysis and Ca 2÷translocation functions of the Ca 2÷pump since the latter but not the former decreases with aging [19,20]. Such an impairment, conceivably, can result from an age-related defect in the conformational response of the ATPase since ligand ( C a 2÷, ATP, MgE÷)-induced transitions in enzyme conformation are of prime importance in the ion translocation function [26,34]. The potential factors which might contribute to an age-related defect in the conformational response of the ATPase (see Ref. 19 for a discussion) include (i) an age-related molecular rigidity of the ATPase or a functional domain of the enzyme (e.g., Ca 2÷ transporting segment) and (ii) age-associated decrease in the fluidity of the membrane microenvironment (e.g., due to increase in cholesterol/phospholipid ratio) of the ATPase. With regard to (b) above, phospholamban is proposed to exert its actions by direct interaction with the Ca2*-ATPase, interaction of phosphorylated phospholamban producing enhancement in C a 2÷ pump activity through acceleration of the partial reactions of the CaE*-ATPase [17,18,26]. It has also been suggested that interaction of unphosphorylated phospholamban with the Ca2÷-ATPase may serve to suppress Ca 2÷pump activity [17,35]. Since no age-related decrease was observed in the degree of phospholamban phosphorylation and the magnitude of its stimulatory effect on the Ca 2÷pump, it can be concluded that age-related alterations in the physicochemical properties of the SR membrane, if they occur, do not occlude the phosphorylation site of phospholamban or hinder interactions between phospholamban and CaE÷ATPase. This does not seem surprising in view of recent information about the structural properties of phospholamban and its likely disposition in the SR. The phosphorylatable domain[containing the serine 16 (phosphoryiation site for cAMPPK) and threonine 17 (phosphorylation site for calmodulin-dependent PK) residues) located in the amino-terminal region of phospholamban molecule is hydrophilic and cytoplasmic, the carboxy-terminal being hydrophobic and membranous [26,36,37]. Further, there is good evidence that it is the cytoplasmic domain containing the
98 phosphorylation site that makes direct interaction with the Ca2+-ATPase[26,38]. Thus, any age-related structural changes in the hydrophobic core of the membrane would be expected to have only minimal direct impact on phospholamban phosphorylation and its interaction with the Ca2+-ATPase. Needless to add that the lack of age-related changes in phospholamban-CaE+-ATPase interactions, by no means rules out potential age-related alterations in the hydrophobic matrix of the SR membrane. Even though the magnitude of the relative stimulatory effect of phosphorylated phospholamban on SR Ca 2÷transport remained undiminished with aging, the actual C a 2÷ transport velocities were markedly lower in the aged heart SR even under conditions where phospholamban was maximally phosphorylated. Therefore, age-related enhancement in the postulated inhibitory action of unphosphorylated phospholamban [17,35] does not contribute to the age-related depression in SR Ca 2÷pump activity. Since the CaE÷-stimulated ATPase activity of cardiac SR is unaltered with aging [19,20], it is unlikely that age-related decrement in the density of Ca 2÷ pump units in the membrane or reduction in the turnover rates of the CaE+-ATPase contributes to the age-related decline in SR C a 2÷ pump activity. As noted earlier, a decrease in the efficiency of coupling A T P hydrolysis to Ca 2÷ transport appears to be the probable cause o f the age-related decline in SR Ca 2÷transport function. If so, one of two possible modes o f expression of impaired coupling can be envisioned: (a) All Ca 2÷pump units in the SR become partially uncoupled with aging, or (b) some of the Ca 2÷pump units in the SR membrane become totally uncoupled with aging while others remain well-coupled. If mode (a) is operative, fertile interactions (interaction resulting in stimulation of C a 2÷ transport) must occur between all of the phosphorylated phospholamban molecules and all of the Ca 2÷ pump units in SR [CaE+-ATPase and phospholamban are thought to exist in a 1: 1 stoichiometry in the SR [26]; we assume that this ratio is not altered with aging]. If mode (b) is operative, fertile interactions will occur only between the well-coupled C a 2÷ pump units and an equal number of phosphorylated phospholamban molecules, thus leaving a surplus of phosphorylated phospholamban molecules totally inert functionally. The lack of age-related changes in phospholamban phosphorylation, the undiminished relative stimulatory effect of phosphorylated phospholamban on SR Ca 2÷ transport with aging, and the agerelated decline in SR Ca 2÷ pump activity observed here are consistent with either modes of uncoupling envisioned above. No data are presently available to distinguish which of them is more probable. As noted in the introduction, cAMP-mediated phosphorylation of phospholamban and consequent stimulation o f SR C a 2÷ pump is the best known physiological mechanism underlying acceleration of cardiac muscle relaxation upon beta adrenergic stimulation. Interestingly, two reported studies have shown that the relaxant effect of beta adrenergic agonists is preserved in the aging heart whereas the inotropic effect is diminished [10,11]. Present data showing undiminished phosphorylation of phospholamban in v i t r o in cardiac SR of aged rats by cAMP-PK,
99
and the effect of phospholamban phosphorylation on Ca 2÷transport are consistent with the above observation. However, further studies using intact myocardium are necessary to determine whether age-related changes occur in the phosphorylation of phospholamban in vivo in response to beta adrenergic stimulation. Such studies are presently in progress in this laboratory. ACKNOWLEDGEMENTS
This work was supported by the Heart and Stroke Foundation of Ontario. The expert assistance of Mr. Emin Donat in some of the experiments is gratefully acknowledged. Dr. M.T. Jiang is a Research Trainee of the Canadian Heart Foundation. REFERENCES I
E.G. Lakatta, Age-related alterations in the cardiovascular response to adrenergic mediated stress. Fed. Pro(:., 39 (1980)3173--3177. 2 E.G. Lakatta, Heart and circulation.In C.E. Finch and E.L. Schneider (eds.),Handbook of the Biology of Aging, 2ndedn., N e w York, Van Nostrand Reinhold, 1985, pp. 377--409. 3 E.G. Lakatta, Cardiac muscle changes in senescence.Ann. Rev. PhysioL, 49 (1987) 519---531. 4 R.S. Kronenberg and C.W. Drage, Attenuation of the ventflatory and heart rate response to hypoxia and hypercapoia with aging in normal men. J. Clin.Invest.,25 (1973) 1812--1819. 5 J.S.Petrofsky and A.R. Lind, Isometric strength,endurance, and the blcod presure and heart rate responses during isometric exercisein healthy men and women, with special reference to age and body fat content. PluegersArch., 360 (1975) 49--6 I. 6 J.R. Conway and R. Sannerstedt, Sympathetic nervous activityduring exercisein relationto age. Cardiovasc. Res., 5 (1971) 577--581. 7 F.C.P. Yin, H.A. Spurgeon, H.L. Greene, E.G. Lakatta and M.L. Weisfeldt, Age-associated decrease in heart rateresponse to isoproterenolin dogs. Mech. Ageing Dev., I0 (1979) 17--25. 8 P.R. Le Blanc and K. Rakusan, Effects of age and isoproterenolon the cardiac output and regional blood flow in the rat.Can. J. CardioL, 3 (1987) 246--250. 9 F.C.P. Yin, H.A. Sporgeon, G.S. Ralzes, H.L. Greene, M.L. Weisfeldt and N.W. Shock, Age associated decrease in chronotropic response to isoproterenol (abstract). Circulation, 54 (Suppl.) II (1976) 167. I0 E.G. Lakatta, G. Gerstenblith,C.S. AngeIl, N.W. Shock and M.L. Weisfeldt,Diminished inotropic response of the aged myocardium to catecholamines. Circ.Res., 36 (1975) 262--269. I I T. Guarnieri, C.R. Filburn, G. Zitnik, G.S. Roth and E.G. Lakatta, Contractile and biochemical correlates of beta adrenergic stimulation of the aged heart. Am. J. Physiol., 239 (1980) H501-H508. 12 N. Narayanan and J.A. Derby, Alterationsin the propertiesof~-adrenergic receptorsof myocardial membranes in aging: Impairments in agonist-receptorinteractionsand guanine nucleotide regulation accompany diminished catacholamine-responsiveness of the adenylate cyclase. Mech. Ageing Dev., 19(1982) 127--139. 13 N. Narayanan and L. Tucker, Autonomic interactionssin the aging heart: Age-associated decrease in muscarinic cholinergic receptor mediated inhibitionof beta adrenergic activation of adenylate cyclase.Mech. Ageing Dev., 34 (I986) 249--259. 14 S.W. O'Connor, P.J. Sarpace and I.B. Arass, Age-associated decrease of adenylate cyclaseactivity in rat myocardium. Mech. Ageing Dev., 16 (198 I) 91--95. 15 P.J. Sarpace, Decreased beta adrenergic responsiveness during senescence. Fed. Proc., 45 (1986) 51--54.
lO0
16
T.M. Fan and S.P. Banerjee, Age-related reduction of beta adrenoceptor sensitivity in rat heart occurs by multiple mechanisms. Gerontology, 31 (1985) 373--380. 17 M. Tada and A.M. Katz, Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu. Rev. Physiol., 44 (1982) 401--423. 18 M. Tada, M. Shigekawa and Y. Nimura, Uptake of calcium by the sarcoplasmic reticulum and its regulation and functional consequences. In N. Sperelakis, II (ed.), Physiology and Pathophysiology o f the Heart, Martinus Nijhoff publishing, Boston, 1984, pp. 255--277. 19 N. Narayanan, Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium. Biochim. Biophys. Acta, 678 (1981) 442-459. 20 N. Narayanan, Comparison of ATP-dependent calcium transport and calcium activated ATPase activities of cardiac sarcoplasmic reticulum and sarcolemma from rats of various ages. Mech. Ageing Dev., 38 (1987) 127-- 143. 21 J.P. Froehlich, E.G. Lakatta, E. Beard, H.A. Spurgeon, M.L. Weisfeldt and G. Gerstenhlith, Studies of sarcoplasmic reticulum function and contraction duration in young and aged rat myocardium. J. Mol. Cell. Cardiol., 10 (1978)427--438. 22 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. 23 P.V. Sulakhe and N. Narayanan, Microsomal and sarcolemmal adenylate cyclase in cardiac muscle: Differential effects of non-ionic detergents. Biochem. J., 175 (1978) 171--180. 24 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randell, Protein measurement with phenol reagent. J. Biol. Chem., 193 (1951)265--275. 25 M.A. Kirchberger, T. Antonetz, Phospholamban: Dissociation of the 22,000 molecular weight protein of cardiac sarcoplasmic reticulum into 11,000 and 5,500 molecular weight forms. Biochem. Biophys. Res. Commun., 105 (1982) 152--156. 26 M. Tada, M. Kadoma, M. Inui and I.J. Fujii, Regulation of Ca2+-pump from cardiac sarcoplasmic reticulum. In S. Fleischer and B. Fleischer (eds.), Methods in Enzymology, Vol. 157, Academic Press, London, 1988, pp.107--154. 27 A.S. Manalan and L.R. Jones, Characterization of the intrinsic cAMP-dependent protein kinase activity and endogenous substrates in highly purified cardiac sarcolemmal vesicles. J. BioL Chem., 257(1982) 10052--10062. 28 C.F. Presti, L.R. Jones and J.P. Lindermann, lsoproterenol-induced phosphorylation of a 15-kilodalton sarcolemmal protein in intact myocardium. J. Biol. Chem., 260 (1985) 3860--3867. 29 P. LaRala and E. Morkin, Adenosine 3',5' monophosphate-dependentmembrane phosphorylation. A possible mechanism for the control of microsomal calcium transport in heart muscle. Circ. Res., 35 (1974) 298--306. 30 E.G. Kranias, Regulation of calcium transport by protein phosphatase activity associated with cardiac sarcoplasmic reticulum. J. Biol. Chem., 260 (1985) 11006--11010. 31 M. Kadoma, B. Sacktor and J.P. Froehlich, Stimulation by cAMP and protein kinase of calcium transport in sarcoplasmic reticulum from senescent rat myocardium (abstract). Fed. Proc. 39 (1980) 2040. 32 M. Tada, M. lnui, M. Yamada, M. Kadoma, T. Kuzuya, H. Abe and S. Kakiuchi, Effects of phospholamban phosphorylation catalyzed by adenosine 3' : 5'-monophosphate and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum. J. Mol. Cell. Cardiol., 15 (1983) 335--346. 33 M. Tada, M.A. Kirchberger, D.I. 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. 34 G. Inesi, Mechanism of calcium transport. Annu. Rev. Physiol., 47 (1985) 573--601. 35 M. Inui, B.K. Chamberlain, A. Sato, S. Fleischer, The nature of the modulation of Ca 2+transport as studied by reconstitution of cardiac sarcoplasmic reticulum. J. Biol. Chem., 261 0986) 1794-1800.
101 36
37
38
J. Fujii, A. Ueno, K. Kitano, S. Tanaka, M. Kadoma and M. Tada, Complete complementary DNA-derived amino acid sequence of canine cardiac phospholamban. J. Clin. Invest., 79 (1987) 301 --304. H.K.B. Simmerman, J.H. Collins, I.L. Thibert, A.D. Wegener and L.R. Jones, Sequence analysis of phospholamban: Identification of phosphorylation sites and two major structural domains. J. Biol. Chem., 261 (1986) 13333--13341. T. Suzuki and J.H. Wang, Stimulation of bovine cardiac sarcoplasmic reticulum Ca 2÷ pump and blocking of the phospholamban phosphorylation and dephosphorylation by a phospholamban monoclonal antibody. J. Biol. Chem., 261 (1986) 7018--7023.