Life Sciences, Vol . 24, pp . 2313-2320 Printed in the U .S .A .
Pergamon Press
DEFECTIVE CALCIUM PUMP IN THE SARCOPLASMIC RETICULUM OF THE HYPERTROPHIED RABBIT HEART J .M .J . Lamera and J .T . Stinis Department of Biochemistry I, Faculty of Medicine, Erasmus University Rotterdam P .O .Box 1738, 3000 DR Rotterdam, The Netherlands . (Received in final form April 23, 1979) Summary To evaluate Ca t+ -uptake in sarcoplasmic reticulum in the hypertrophied rabbit heart, microsomea were prepared from myocardium of rabbits with experimentally induced aortic atenosis . A significant reduction of microsomal Cat+-uptake was observed is hypertrophied left ventricle, 195+10 compared to 280+18 nmol/mg found ~n control animals . A similar pattern wasôbserved for the Ca +-stimulated ATPase (30+9 and 59+10 nmol/min/mg reap .) . A minimal activity difference of the microsomal marker enzyme rotenone-insensitive NADPH cyt . c reductase was found (7 .77+0 .05 and 8 .17+0 .11 nmol/min/mg reap .) . The specific activity of the latter enzyme was 5-6 fold increased in microsomea compared to homogenates in both animal groups, which excludes the possibility of increased amounts of contaminant or nonfunctional protein in sarcoplasmic reticulum prepared from hypertrophied myocardium . In addition the yield of microsomal protein did not differ significantly . Maximal phoaphorylation by exogenous cyclic AMP and protein kinase increased Cat+-uptake in both microsomal preparations (to 278+27 and 375+26 nmol/mg reap . for hypertrophied and control hearts), butCat+ -transport rate found in pathological hearts remained lower than is controls . These findings indicate that impairment of Ca t+ -metabolism in the hypertrophied heart is based on a defective Cat+-pump . Many investigations have been undertaken in order to correlate possible modifications of sarcolemma (SL) and sarcoplasmic reticulum (SR) function to contractile properties of hypertrophied and failing hearts (reviews : 1-4) . A reduced rate of Cat+-uptake, as has been found (5-7) in SR fra~tions from hypertrophied rabbit hearts, would lead to a reduced sarcotubular Ca +-pool, so that less Cat+ would be available for activation of the actomyoain ATPase . Changes in the SL function in hypertrophy have been described too (6,8-I1) and a possible cause-effect relationship of membrane changes may exist in the development of the hypertrophy (12) . Ito et al . (7) have demonstrated that ventricular hypertrophy is not necessarily accompanied by reduction of SR function . It may develop later as heart growth becomes more extensive . In the meantime it has been firmly established (13-15) that the Cat+-transport rate in SR in vitro can be regulated by cyclic AMP-dependent phosphorylation. Cyclic AMP levehn hypertrophied heart could be reduced, because the adenylate cyclase activity has been shown to be depressed (6,8,10), while phoaphodiesterase remained constant (8) . Already Limas and Cohn (16) demonstrated that the decreased Cat +0024-3205/79/252313-07$2 .00/0 Copyright (c) 1979 Pergamon Press Ltd
2314
Calcium Uptake in SR
Vol . 24, No . 25, 1979
accumulation in cardiac SR from spontaneous hypertensive rats might be explained by the reduced cyclic AMP-dependent protein kinase activity . t However, there is no information regarding the Ca +-transport in control and pathological SR membranes after phosphorylation by exogenous cyclic AMP and protein kinase . Consequently, we wished to study if functional changes in microsomes from hypertrophied heart originate from an altered degree of phosphorylation. Also it was of interest to determine whether besides the Ca t+ pump another microsomal function would be affected in hypertrophy . Therefore, we have examined activities of rotenone-insensitive NADPH cyt . ç reductase . This enzyme could also be used as a marker for the relative purity of SR membrane fractions . Methods Left ventricular hypertrophy was produced in New Zealand rabbits of either sex by constriction of the descending aorta as described in detail in an earlier report from our laboratory (10) . After one month the animals were killed by a blow on the head, the hearts quickly removed and dissected on ice to obtain atria, left and right ventricles . The apical part of the left ventricles (1 g muscle) was cut into small pieces, minced and homogenized in 5 ml medium (0 .25 M sucrose, 1 mM EGTA) with 10 strokes of a motor-driven Potter Elvehjem homogenizes . The sarcoplasmic reticulum was prepared from 3 .5 ml homogenate . Centrifugation was carried out at 600 (5 min), the gav pellet rehomogenized in 3 .5 ml medium (0 .25 M sucrose) and this procedure was repeated twice by centrifugation at 300 gav (5 min) . Thereafter the pellet was discarded and the combined supernatants were centrifuged at 39000 gav (10 min) to obtain a tighly packed mitochondrial fraction . A surface layer on top of the pellet was easily loosened by adding 2 ml medium and gentle rotation of the tube . This material was recombined with the supernatant, which was centrifuged again at 39000 gav (30 min) to yield the microsomal fraction, representing the sarcoplasmic reticulum. These vesicles were resuapended in 0 .4 ml 20 mM Tris-maleate (6 .8), containing 0 .1 M KC1 . Negligible activities of putative marker enzymes for mitochondria (auccinate dehydrogenase, determined as described in ref . 17) and for myofibrils (R+-stimulated ATPase, determined as described in ref . 18) were observed . The Cat+-uptake activity was determined by the Millipore filtration technique . The fractions were incubated in a medium containing 20 mM Tris-maleate (pH 6 .8), 100 mM KC1, 5 mM MgC12, 1 mt¢SATP, I mkf.PEP, 1 U/ml pyruvate kinase, 5 mM potassium-oxalate and 0 .05 mM CaC12 (14 dpm/umol, Ameraham, England) in a total volume of 1 ml . To increase the Cat+-transport rate 4l~y ~~clic AMPdependent phosphorylation, microsomes were preincubated in Ca -free transport assay medium to which 2 .6 mU/ml protein kinase (Type II from bovine heart) and 0 .01 mM cyclic AMP had been added . After 5 min the transport reaction was initiated by the addition of 0 .9 ml 45Ca2+ containing medium . Usually 20 ug membrane or 100 ug homogenate protein was present per ml assay medium and the reaction was stopped by Millipore filtration at various times (0 .45 mu filters were used) . Correction was made for the absorption of 45 Ca 2+ on the filter and 45 Ca 2+-uptake in the absence of ATP . Filters were solubilized in Instagel (Packard) for liquid scintillation counting . ATPase activity was determined in the same medium as used for Cat+-uptake. Cat+-independent ATPase was measured by inclusion of 1 mM EGTA in the Cat+-free medium . To initiate the reaction 10 ug of membrane protein was added to 1 ml of medium and incubation continued for 30 min at 25°C . The reaction was terminated by the addition of 0 .1 ml 41% trichloroacetic acid . The deproteinized extract was assayed for Pi as described earlier (19) . Rotenone-insensitive NADPH cytochrome c reductase was estimated at 30°C by thespectrophotometric procedure described by Scholte (18) . 5'-Nucleotidase was assayed essentially according to the method described by Avruch and Wallach (20), using ~U- 14 C~ AMP (0 .2
Vol . 24, No . 25, 1979
Calcium Uptake in SR
2315
uCi/lanol, Ameraham, England) as substrate . To estimate 32 P incorporation of sarcoplasmic reticulum,the reactionawere carried obtin a medium containing 50 mM potassiumphosphate (pH 6 .8), 10 mM MgC12, 0 .3 mM EGTA, 2 .5 mg/ml bovine serum albumin, 2 .5 mM theophyllin, 10 mM KF, 20 >lg membrane protein and 0 .05 mM ATP~Y-32 p~ labelled, 50-200 dpm/pmol, Amersham, England) in a volume of 0 .1 ml . When indicated, 2 .6 mU/ml protein kinase or 0 .01 mM cyclic AMP has been added (both sufficient to obtain maximal phoaphorylation, data not .shown) . The reaction was terminated after 5 min at 25°C with 0 .1 ml 10% trichloroacetic acid . After centrifugation the pellet was dissolved in 0 .5 N NaOH, the protein reprecipitated with 1 ml 10% trichloroacetic acid, filtrated on a glassfibre filter (Whatman CF/c), washed a few times with 12 ml 10% trichloroacetic acid and pellets were counted by liquid scintillation . Protein was determined by the Lowry procedure . Significant differences were calculated by Student's t-test . Results When the aorta in rabbits is clamped in vivo , left ventricular hypertrophy will develop after a few weeks . This can be seen from Table I . Similar results have been reported by us and others (5,7,9,10) . No evaluation of cardiovascular functional status of the rabbits is used in the present study, but pathological evidence of heart failure (pleural effusion and ascitea) could not be found . Moreover, the mean of right ventricular weights in the experimental group was increased, however, the difference with the control group appeared to be non-significant . TABLE I Tissue Weights in Control and Experimental Aortic Stenoeis in Rabbits Tissue weights in ~/kg body weight Control Aortic constriction (n=5b) (n=5) Total heart mass Left ventricle + septum Right ventricle Atria
1 .86+O .Ola 1 .29+0 .02 0 .37+0 .04 0 .20+0 .01
2 .91+0 .40 2 .05+0 .25 0 .53+0 .09 0 .31+0 .06
P
0 .025 X0 .01 NS
Pergamon Press
DEFECTIVE CALCIUM PUMP IN THE SARCOPLASMIC RETICULUM OF THE HYPERTROPHIED RABBIT HEART J .M .J . Lamera and J .T . Stinis Department of Biochemistry I, Faculty of Medicine, Erasmus University Rotterdam P .O .Box 1738, 3000 DR Rotterdam, The Netherlands . (Received in final form April 23, 1979) Summary To evaluate Ca t+ -uptake in sarcoplasmic reticulum in the hypertrophied rabbit heart, microsomea were prepared from myocardium of rabbits with experimentally induced aortic atenosis . A significant reduction of microsomal Cat+-uptake was observed is hypertrophied left ventricle, 195+10 compared to 280+18 nmol/mg found ~n control animals . A similar pattern wasôbserved for the Ca +-stimulated ATPase (30+9 and 59+10 nmol/min/mg reap .) . A minimal activity difference of the microsomal marker enzyme rotenone-insensitive NADPH cyt . c reductase was found (7 .77+0 .05 and 8 .17+0 .11 nmol/min/mg reap .) . The specific activity of the latter enzyme was 5-6 fold increased in microsomea compared to homogenates in both animal groups, which excludes the possibility of increased amounts of contaminant or nonfunctional protein in sarcoplasmic reticulum prepared from hypertrophied myocardium . In addition the yield of microsomal protein did not differ significantly . Maximal phoaphorylation by exogenous cyclic AMP and protein kinase increased Cat+-uptake in both microsomal preparations (to 278+27 and 375+26 nmol/mg reap . for hypertrophied and control hearts), butCat+ -transport rate found in pathological hearts remained lower than is controls . These findings indicate that impairment of Ca t+ -metabolism in the hypertrophied heart is based on a defective Cat+-pump . Many investigations have been undertaken in order to correlate possible modifications of sarcolemma (SL) and sarcoplasmic reticulum (SR) function to contractile properties of hypertrophied and failing hearts (reviews : 1-4) . A reduced rate of Cat+-uptake, as has been found (5-7) in SR fra~tions from hypertrophied rabbit hearts, would lead to a reduced sarcotubular Ca +-pool, so that less Cat+ would be available for activation of the actomyoain ATPase . Changes in the SL function in hypertrophy have been described too (6,8-I1) and a possible cause-effect relationship of membrane changes may exist in the development of the hypertrophy (12) . Ito et al . (7) have demonstrated that ventricular hypertrophy is not necessarily accompanied by reduction of SR function . It may develop later as heart growth becomes more extensive . In the meantime it has been firmly established (13-15) that the Cat+-transport rate in SR in vitro can be regulated by cyclic AMP-dependent phosphorylation. Cyclic AMP levehn hypertrophied heart could be reduced, because the adenylate cyclase activity has been shown to be depressed (6,8,10), while phoaphodiesterase remained constant (8) . Already Limas and Cohn (16) demonstrated that the decreased Cat +0024-3205/79/252313-07$2 .00/0 Copyright (c) 1979 Pergamon Press Ltd
2314
Calcium Uptake in SR
Vol . 24, No . 25, 1979
accumulation in cardiac SR from spontaneous hypertensive rats might be explained by the reduced cyclic AMP-dependent protein kinase activity . t However, there is no information regarding the Ca +-transport in control and pathological SR membranes after phosphorylation by exogenous cyclic AMP and protein kinase . Consequently, we wished to study if functional changes in microsomes from hypertrophied heart originate from an altered degree of phosphorylation. Also it was of interest to determine whether besides the Ca t+ pump another microsomal function would be affected in hypertrophy . Therefore, we have examined activities of rotenone-insensitive NADPH cyt . ç reductase . This enzyme could also be used as a marker for the relative purity of SR membrane fractions . Methods Left ventricular hypertrophy was produced in New Zealand rabbits of either sex by constriction of the descending aorta as described in detail in an earlier report from our laboratory (10) . After one month the animals were killed by a blow on the head, the hearts quickly removed and dissected on ice to obtain atria, left and right ventricles . The apical part of the left ventricles (1 g muscle) was cut into small pieces, minced and homogenized in 5 ml medium (0 .25 M sucrose, 1 mM EGTA) with 10 strokes of a motor-driven Potter Elvehjem homogenizes . The sarcoplasmic reticulum was prepared from 3 .5 ml homogenate . Centrifugation was carried out at 600 (5 min), the gav pellet rehomogenized in 3 .5 ml medium (0 .25 M sucrose) and this procedure was repeated twice by centrifugation at 300 gav (5 min) . Thereafter the pellet was discarded and the combined supernatants were centrifuged at 39000 gav (10 min) to obtain a tighly packed mitochondrial fraction . A surface layer on top of the pellet was easily loosened by adding 2 ml medium and gentle rotation of the tube . This material was recombined with the supernatant, which was centrifuged again at 39000 gav (30 min) to yield the microsomal fraction, representing the sarcoplasmic reticulum. These vesicles were resuapended in 0 .4 ml 20 mM Tris-maleate (6 .8), containing 0 .1 M KC1 . Negligible activities of putative marker enzymes for mitochondria (auccinate dehydrogenase, determined as described in ref . 17) and for myofibrils (R+-stimulated ATPase, determined as described in ref . 18) were observed . The Cat+-uptake activity was determined by the Millipore filtration technique . The fractions were incubated in a medium containing 20 mM Tris-maleate (pH 6 .8), 100 mM KC1, 5 mM MgC12, 1 mt¢SATP, I mkf.PEP, 1 U/ml pyruvate kinase, 5 mM potassium-oxalate and 0 .05 mM CaC12 (14 dpm/umol, Ameraham, England) in a total volume of 1 ml . To increase the Cat+-transport rate 4l~y ~~clic AMPdependent phosphorylation, microsomes were preincubated in Ca -free transport assay medium to which 2 .6 mU/ml protein kinase (Type II from bovine heart) and 0 .01 mM cyclic AMP had been added . After 5 min the transport reaction was initiated by the addition of 0 .9 ml 45Ca2+ containing medium . Usually 20 ug membrane or 100 ug homogenate protein was present per ml assay medium and the reaction was stopped by Millipore filtration at various times (0 .45 mu filters were used) . Correction was made for the absorption of 45 Ca 2+ on the filter and 45 Ca 2+-uptake in the absence of ATP . Filters were solubilized in Instagel (Packard) for liquid scintillation counting . ATPase activity was determined in the same medium as used for Cat+-uptake. Cat+-independent ATPase was measured by inclusion of 1 mM EGTA in the Cat+-free medium . To initiate the reaction 10 ug of membrane protein was added to 1 ml of medium and incubation continued for 30 min at 25°C . The reaction was terminated by the addition of 0 .1 ml 41% trichloroacetic acid . The deproteinized extract was assayed for Pi as described earlier (19) . Rotenone-insensitive NADPH cytochrome c reductase was estimated at 30°C by thespectrophotometric procedure described by Scholte (18) . 5'-Nucleotidase was assayed essentially according to the method described by Avruch and Wallach (20), using ~U- 14 C~ AMP (0 .2
Vol . 24, No . 25, 1979
Calcium Uptake in SR
2315
uCi/lanol, Ameraham, England) as substrate . To estimate 32 P incorporation of sarcoplasmic reticulum,the reactionawere carried obtin a medium containing 50 mM potassiumphosphate (pH 6 .8), 10 mM MgC12, 0 .3 mM EGTA, 2 .5 mg/ml bovine serum albumin, 2 .5 mM theophyllin, 10 mM KF, 20 >lg membrane protein and 0 .05 mM ATP~Y-32 p~ labelled, 50-200 dpm/pmol, Amersham, England) in a volume of 0 .1 ml . When indicated, 2 .6 mU/ml protein kinase or 0 .01 mM cyclic AMP has been added (both sufficient to obtain maximal phoaphorylation, data not .shown) . The reaction was terminated after 5 min at 25°C with 0 .1 ml 10% trichloroacetic acid . After centrifugation the pellet was dissolved in 0 .5 N NaOH, the protein reprecipitated with 1 ml 10% trichloroacetic acid, filtrated on a glassfibre filter (Whatman CF/c), washed a few times with 12 ml 10% trichloroacetic acid and pellets were counted by liquid scintillation . Protein was determined by the Lowry procedure . Significant differences were calculated by Student's t-test . Results When the aorta in rabbits is clamped in vivo , left ventricular hypertrophy will develop after a few weeks . This can be seen from Table I . Similar results have been reported by us and others (5,7,9,10) . No evaluation of cardiovascular functional status of the rabbits is used in the present study, but pathological evidence of heart failure (pleural effusion and ascitea) could not be found . Moreover, the mean of right ventricular weights in the experimental group was increased, however, the difference with the control group appeared to be non-significant . TABLE I Tissue Weights in Control and Experimental Aortic Stenoeis in Rabbits Tissue weights in ~/kg body weight Control Aortic constriction (n=5b) (n=5) Total heart mass Left ventricle + septum Right ventricle Atria
1 .86+O .Ola 1 .29+0 .02 0 .37+0 .04 0 .20+0 .01
2 .91+0 .40 2 .05+0 .25 0 .53+0 .09 0 .31+0 .06
P
0 .025 X0 .01 NS
