Hormonal Control of Cardiac Myosin Adenosine Triphosphatase in the Rat

Transcription

1 Hrmnal Cntrl f Cardiac Mysin Adensine Triphsphatase in the Rat By Michael J. Rvett, Ake C. Hjalmarsn, Hward E. Mrgan, Michael J. Barrett, and Richard A. Gldstein ABSTRACT Cardiac functin was impaired in hearts frm hypphysectmized rats. Crrespnding t the decrease in perfrmance, mysin frm the hearts f these rats exhibited a decreased Ca 2 + -activated ATPase activity. Mysin ATPase activated by K + r NH + 4 was nt different in any f the preparatins studied. The decreased Ca 2 + -activated ATPase was bserved in cardiac mysin but nt in red r white skeletal muscle mysin. Treatment with thyrxine, but nt with grwth hrmne, restred cardiac mysin ATPase and perfrmance t nrmal. Hypthyridism prduced a decrease in cardiac mysin ATPase activity cmparable t that seen after hypphysectmy, suggesting that the absence f thyrid hrmne was respnsible fr the alteratins. Hwever, hyperthyridism did nt increase ATPase activity abve nrmal values. Adrenalectmy failed t alter mysin ATPase. Anin exchange chrmatgtaphy f the mysins and additin f varius cncentratins f Na +, Ca 2 +, and Mg 2+ did nt ablish the differences in mysin ATPase. The results strngly suggest that the lwer mysin ATPase activity in the absence f thyrid hrmne was the result f a change in the mysin mlecule per se and that the lwer enzymatic activity was invlved in the lwer cardiac perfrmance in these rats. KEY WORDS hyperthyridism cardiac functin thyrid hrmne hypthyridism adrenal insufficiency skeletal muscle mysin Several recent studies have shwn an alteratin in the enzymatic activity f mysin frm hearts f animals in abnrmal hrmnal states. A decrease was fund in ATPase activities f myfibrils, actmysin, and mysin frm adrenalectmized cats (1) that crrelated with cardiac functin (2). An increase in mysin ATPase activity was nted in hyperthyrid guinea pigs (3). In a preliminary reprt f the present wrk, we reprted that decreased ventricular functin and decreased ATPase activity f mysin were fund in hearts f hypphysectmized rats (4). The present wrk was undertaken (1) t further characterize the alteratin in cardiac This study was supprted in part by U. S. Public Health Service Grant HE frm the Natinal Heart and Lung Institute and by a grant frm the Pennsylvania Heart Assciatin. Dr. Rvett is a Pstdctral Fellw f the Natinal Institutes f Health. Received April 20, Accepted fr publicatin June 28, functin and mysin ATPase in hypphysectmized rats, (2) t identify the hrmne invlved in this abnrmal state, and (3) t try varius assay cnditins and mdificatins f mysin which might nrmalize the apparent lw specific enzymatic activity f cardiac mysin frm hypphysectmized animals. Methds ANIMAL TECHNIQUES Nrmal, hypphysectmized (hypx) r adrenalectmized (adrenex) male Sprague-Dawley rats were used. The adrenex rats were given 0.9* saline t drink but therwise nly rutine care and feeding was given t the rats. Thyrxine, grwth hrmne, and prpylthiuracil were administered subcutaneusly daily at the levels nted in the figures and tables. Cntrl animals received the slvent that was used fr each f the drugs. After 7-15 days f drug injectins r 7-15 days after peratin the animals were anesthetized with pentbarbitl sdium and the heart, gastrcnemius, sleus, and diaphragm muscles were quickly excised and placed in icecld 0.9% saline. The hearts were either perfused as wrking heart preparatins (5), hmgenized Circulatin Retmrcb, Vl. XXXI, Sipttmber

2 398 ROVETTO, HJALMARSON, MORGAN, BARRETT, GOLDSTEIN fr islatin f mysin, r frzen fr strage ver liquid nitrgen. The skeletal muscles were either hmgenized fr islatin f mysin r frzen fr later preparatin f the prtein. CARDIAC FUNCTION DETERMINATION The maximum rate f pressure develpment (dp/dt) and peak left ventricular pressure were measured at left atrial pressures frm 0 t 25 cm H. 2 O, at 5-cm steps during perfusin as wrking heart preparatins (5). Left atrial pressure was adjusted by varying the height f the left atrial bubble trap abve the heart. The pressure head against which the hearts pumped was maintained at 60 mm Hg. Left ventricular pressure and dp/dt were recrded via a Statham pressure transducer cnnected t a 20-gauge needle that was placed int the left ventricle thrugh the apex. Maximum rate f pressure develpment and peak left ventricular pressure were btained with Tektrnix type 3A8 peratinal and type 3A9 differential amplifiers, respectively. The utputs frm the amplifiers were recrded n a Tektrnix type RM564 strage scillscpe. Cardiac utput was calculated by summing the crnary flw and the vlume f fluid pumped by the heart against the hydrstatic pressure head f 60 mm Hg. MYOSIN ISOLATION Mysin was prepared by the differential centrifugatin methd f Weeds and Hartley (6). Frzen tissue that had been pulverized at liquid nitrgen temperatures in a percussin mrtar r fresh tissue that had been cut int small pieces was hmgenized in Dunce hmgenizers in 3 vlumes f a slutin cntaining 0.10M KH 2 PO 4, 0.05M K 2 HPO 4, 0.3M KC1, ph 6.5 at 0 C. Samples cntained 3 10 hearts, 3 5 gastrcnemius muscles r cmbined diaphragm and sleus muscles frm rats. N differences culd be detected in either yield r ATPase activity f mysin prepared frm fresh r frzen tissue. The remainder f the islatin prcedure was the same as that described by Weeds and Hartley (6) except that the step using batch treatment with DEAE Sephadex fr remval f nucleprcteins emplyed DEAE-A25 rather than DEAE- A50 Sephadex, and that the final precipitatin by dilutin emplyed lmm sdium-free ethylenediamintetraacetic acid (EDTA), ph 6.5, rather than disdium EDTA. The ph f all buffers was adjusted with either Trisbase r acetic acid, and the ATP that was used in the assays was the Tris salt. These steps minimized cntaminatin f samples with sdium. The final mysin slutin in 0.5M KC1 was diluted t abut 1 mg f prtein/ml befre assay f ATPase activity. Prtein cncentratin was determined by the absrbance at 280 and 260 m/i (7), the Lwry methd (8) using bvine serum albumin as a standard, r by absrbance in 0.5M NaOH at 291 and 350 m/j, (9). All three methds were standardized against micr-kjeldahl determinatins, assuming 16% nitrgen cntent. The absrbance at 280 and 260 mfi was rutinely used because f the rapidity with which determinatins culd be made. With this methd, the absrbance rati (280/260 m/i.) f mysin was between 1.15 and N significant differences in the rati were fund between mysins frm nrmal and experimental animals. DETERMINATION OF ATPASE ACTIVITY Assays were run fr 5 minutes at ph 7.5 and 25 C. The rate f ATP hydrlysis was fund t be linear ver this perid. The reactin was initiated by additin f mysin (0.2 ml) t a reactin mixture (1.8 ml) t give a final prtein cncentratin f 0.1 mg/ml. The reactin was stpped by adding 15% trichlracetic acid (1 ml), and the precipitated prtein was remved by centrifugatin. ATP hydrlysis was fllwed by measuring the appearance f inrganic phsphate in the supernatant fractin, using the prcedure f Harris and Ppat (10). The rutine reactin mixtures cntained 50 mm Tris and 5 mm Tris- ATP. In additin, Ca 2+ -activated ATPase was assayed in the presence f 0.1M KC1 and 10 mm CaCl 2. Reactin mixtures fr assay f mnvalent catin-activated ATPase cntained either 1M NH 4 C1 r 1M KC1 and 5 mm EDTA. Variatins in these assay mixtures are reprted with the table and figures. Fr simplicity in presentatin f results and discussin, ATPase activities measured in Ca 2 + -cntaining slutins r with EDTA in the presence f NH 4 + r K + will be referred t as Ca 2 + -ATPase, NH 4 +-ATPase, and K +-ATPase, respectively. COLUMN CHROMATOGRAPHY OF MYOSIN Cardiac mysin ( mg) islated as described abve was purified by chrmatgraphy n DEAE-A50 Sephadex using the methd f Richards et al. (11) r n a phsphcellulse clumn fllwed by a DEAE cellulse clumn (12). The clumns were 2.5 X cm. Mysin that was chrmatgraphed n DEAE-A50 Sephadex was dialyzed vernight against a phsphate buffer, ph 7.5 (0.15M K 2 HPO 4, 0.01M EDTA) and diluted t 2 3 mg prtein/ml. The mysin was applied t a clumn equilibrated with the same buffer. A linear gradient frm 0 t 0.5M KC1 was used t elute the mysin frm the clumn. The clumn effluent was cllected at 20-minute intervals (5 6 ml) and the 280-m/A absrbance was determined n each fractin. A majr peak beginning after 250 ml f eluent had passed thrugh the clumn cntained the mysin. This peak was pled and exhaustively dialyzed Circulatin Rtsurcb, Vl. XXXI, September 1972

3 MYOSIN ADENOSINE TRIPHOSPHATASE 399 against 0.04M KC1, 4 mm Tris, ph 6.5. The resulting precipitate was washed twice with fresh dialysis buffer t remve traces f phsphate. The precipitate was disslved in 0.5M KC1 and the ATPase activity was determined as abve. Abut 80S f the prtein placed n the clumn was recvered in the eluate, with 50 70* f the prtein in the mysin fractin. N significant differences in recvery f mysin frm hearts f nrmal and hypphysectmized rats was fund during the chrmatgraphic prcedure. Chrmatgraphy n phsphcellulse and DEAE cellulse was accmplished by precipitating mysin by dilutin t 0.04M KCl. The precipitate was disslved in an equal vlume f 0.8M KC1, 0.08M Tris, ph 7.8. The slutin was diluted t 5 8 mg prtein/ml by adding 0.4M KC1, 0.04M Tris, ph 7.8 and placed n a phsphcellulse clumn equilibrated with the same buffer. The majr peak f prtein eluted frm the clumn was immediately placed n a DEAE cellulse clumn equilibrated with the same buffer. The majr peak f prtein frm this clumn was pled, adjusted t ph 7.0, and fractinated with (NH 4 ) 2 SO 4. The fractin precipitating between 32 and 50% saturatin was redisslved and dialyzed against 0.04M KCl, 4 mm Tris, ph 6.5. The prtein was cllected by centrifugatin, washed twice with dialysis buffer, disslved in 0.5M KCl, and assayed as abve. The amunt f mysin recvered frm these clumns was less predictable, and the yield was lwer than frm the DEAE Sephadex clumns. Results CARDIAC PERFORMANCE IN ALTERED HORMONAL STATES The effects f hypphysectmy and f the treatment f hypphysectmized rats with grwth hrmne and thyrxine n perfrmance f the islated rat heart perfused with a left atrial filling pressure f 20 cm H 2 O are shwn in Table 1. Since hypphysectmy reduced heart size and prevented the animal frm grwing, cmparisns f perfrmance were made between nrmal and hypphysectmized rats matched bth fr bdy and heart weight. When cmparisns f cardiac functin were made between animals f the same bdy size (Table 1, Grup I nrmal and Grup I hypx and Grup II nrmal and Grup II hypx), lwer peak intraventricular pressure and dp/dt were fund in the hearts frm hypx animals. In additin, there was a larger cardiac utput in the nrmal II than in hypx II, which was prbably due t larger heart Circulatin Rtittrch, Vl. XXXI, Stpstmbtr 1972 ta &3 "3 F a, "3 S a ' a I 3 QO 00 to O CM -; t i d d> d d ^5 4J -H -H -H -a -a c~ c CO CO U H H -H H H g CM CD CO CO Ol CO OO OJ -H rvj CO. H i-h t>l -H -H -H -H -H -B c * «q q d c 6 c3; tm ' O -H -H H OS CD s CO CO CO CO CO CO ^ CO 08 3i q a CD lb CN OO -H -fi -H H in N Ol CM CO OO H Cq H OO ^ C3 CU O QJ a c c = c = c s "3 ~3 E O -H -H -H ^ CD t CO x x x x. a 6. c p, >> >> >i S KSSS a. Q. i <B V 1:.23 a. 3 I O. q d V a, X a. > CO «v H t f^ -H S 3 S. -i 2 > V S E 1 I > S 1* * - H^1 2 D. ca» 3

4 400 ROVETTO, HJALMARSON, MORGAN, BARRETT, GOLDSTEIN size in the nrmal animals. Cardiac atrphy ccurred in these hypx animals, as indicated by the lwer rati f heart t bdy weight. When cmparisns were made between hearts f the same size (Grup I nrmal and Grup II hypx), a decrease in peak pressure and dp/dt in the hypx preparatins was still bserved. Injectins f hypx animals with grwth hrmne (100 fig/day) r thyrxine (5 fi.g/day) fr 7 days maintained heart weight (Grup I hypx and Grup II nrmal). Althugh thyrxine (5 /^g/day) failed t maintain bdy weight in the hypx animals, it restred cardiac perfrmance t nrmal. On the ther hand, grwth hrmne failed t increase perfrmance t nrmal levels as judged frm develpment f peak ventricular pressure and dp/dt. Thus, dp/dt and develpment f peak pressure were decreased in hypx animals. Thyrxine, but nt grwth hrmne, was capable f maintaining these parameters at nrmal levels. Significant differences in develpment f peak ventricular pressure and dp/dt in hearts f hypx and nrmal rats als were bserved at 15 cm H^O left atrial filling pressure but nt at 10 cm H 2 O r belw. ATPase Aclivily f Cardiac Mysin Grup Treatment Nrmal Nne 3 GH, 100 Mg/day 3 T 4, 5 Mg/day 3 GH + T 4, 5 & 100 Mg/day 3 Hypx Nne 3 GH, 100 Mg/day 3 T 4, 5 Mg/day 3 T 4 + GH, 5 & 100 Mg/day 3 Nrmal Nne 24* Nrmal PTU, 20 mg/day 13 Nrmal PTU, 40 mg/day 8 Adrenex Nne 8 Nrmal T 4, 100 Mg/day 4 T,, 600 Mg/day 9 in Nrmal and CARDIAC MYOSIN ATPASE ACTIVITY IN ALTERED HORMONAL STATES The ATPase activity f cardiac mysin frm animals in varius states f hrmne balance is shwn in Table 2. The data in the first part f the table were btained frm experiments designed fr randmized blck data analysis. The values fr nrmal grups in the secnd part f the table have been cmbined int ne value fr cnvenience f reprting. Separate cntrl preparatins (8 each) were assayed with each f the experimental preparatins. When the NH 4 + -ATPase f the series given prpylthiuracil (20 mg/day) was cmpared with all cntrls, a significant difference was fund. Hwever, the NIL, + -ATPase f the grup f cntrls specifically prepared fr the prpylthiuracil series was 2.28 ± 0.12 yumles Pi/mg mirr 1, a value nt significantly different. The difference in NH4 + -ATPase values between the tw parts f this table reflected a lwering f this value ver the year in which the data were cllected. The nrmal and adrenex rats used in the studies in the lwer part f the table were males weighing g; thse in the upper part were females weighing g. Whether sex r age affects NH 4 + -ATPase has nt been systematically explred. TABLE 2 AUered Hrmnal Slates Myaln ATPase activity (jtsales Pi/mg mln-j) K + Ca> : = 0.06 = : 0.06 : 0.02 = f : f O.O65H J Values are means ="= BE. N = number f separate mysin preparatins assayed; Adrenex = adrenalectmy; PTU = prpylthiuracil, ther abbreviatins as in Table 1. * = Cmbined mean f cntrls fr all grups in the lwer part f the table; f =P< 0.05 vs. nrmal cntrl; tp < 0.01 vs. nrmal cntrl; P < vs. nrmal cntrl; \\P < vs. hypx + T 4 r hypx + T t and GH. Circulatin Rtsurcb, Vl. XXXI, Stptembtr 1972

5 MYOSIN ADENOSINE TRIPHOSPHATASE 401 The Ca 2 + -ATPase f mysin frm hearts f hypx rats was nly 50-70% f the activity f nrmal preparatins (Table 2). Neither the NH4 + -ATPase nr K + -ATPase exhibited any change in activity with hypphysectmy r in any f the ther hrmnal states shwn in this table. Grwth hrmne was ineffective in maintaining Ca 2 + -ATPase at nrmal levels, as it had been in maintaining nrmal cardiac perfrmance. In cntrast, thyrxine alne r with grwth hrmne cmpletely prevented the decrease in the Ca 2 + -ATPase f mysin when given t hypx animals. When thyrxine, grwth hrmne, r bth were given t nrmal animals, the activity f neither divalent nr mnvalent catin-activated ATPase was altered. Althugh there is a tendency fr the cmbinatin f thyrxine and grwth hrmne t increase the Ca 2 + -ATPase abve nrmal values, the significance f the change was questinable, since large dses f thyrxine failed t increase this activity (Table 2). T distinguish between the effects f deficiency f adrenal and thyrid hrmnes, cardiac mysin was islated frm adrenalectmized and prphylthiuracil-treated rats. Adrenal ectmy failed t alter either the mnvalent r divalent catin-activated ATPase. Prpylthiuracil treatment, n the ther hand, induced a dse-related decrease in the Ca 2 + -ATPase similar (40 mg/day) in magnitude t the reductin with hypphysectmy. Thus the lack f thyrxine, but nt f gluccrticids, appeared t be respnsible fr the change in enzymatic activity. Hwever, thyrxine at very high dses failed t enhance the activity f either the mnvalent r divalent catin-activated ATPase. In ther experiments, treatment with thyrxine (100 /ng/day) fr 30 days failed t alter the ATPase activity. Hwever, these large dses did induce cardiac hypertrphy. EFFECT OF CA24-, K +, AND NH.+ ON CARDIAC MYOSIN ATPASE The ATPase activity f mysin activated by a range f cncentratins f mnvalent and divalent catins is shwn in Figure 1. The Ca 2 + -ATPase activity f mysin frm hypx NORMAL. HYPOX Q-- HYPOX + T, m-- Effect f cncentratin f the activatr in n the Ca t +, K +, and NH^+ -activated cardiac mysin ATPase. Assay slutins cntained 50 min Tris, 5 m/tr Tris-ATP, and the cncentratin f activatr in shwn n the abscissa. The Ca* +-activated ATPase mixture als cntained 0.1M KCL. Each value is the mean f three mysin preparatins, and the vertical lines shw the SE. CircuUtin Resetrcb, Vl. XXXI, Stpttnher 1972

6 402 ROVETTO, HJALMARSON, MORGAN, BARRETT, GOLDSTEIN rats was significantly lwer than the mysin frm nrmal rats at all but the lwest Ca 2 + cncentratin (0.3 HIM CaCl 2 ). The Vmax and Km calculated frm these data were 1.10 and 0.80 fjimles Pi/mg min^1 and 1.82 and 2.02 mm fr mysins frm hearts f nrmal and hypx rats, respectively. Administratin f lw dses f thyrxine prevented the decrease in Ca 2 + -ATPase f mysin frm hypx rats, as indicated by an activatin curve that was similar t the nrmal. Althugh nt shwn here r in the next tw figures, the activatin curves fr mysin frm hypx rats treated with grwth hrmne were nt different frm untreated hypx animals. There was n difference in the relatin between cncentratin f activatr in and ATPase activity when cardiac mysin frm nrmal, hypx r thyrxine-treated hypx rats was assayed ver a range f K + and NH4 + cncentratins. Neither grwth hrmne nr thyrxine given t nrmal r t hypx animals altered these curves. The experiments described thus far have shwn that mechanical functin f the heart was impaired in hypphysectmized r hypthyrid rats. Impairment f cardiac functin was assciated with a reductin in Ca ATPase. The fllwing experiments were undertaken t establish whether the reductin in Ca ATPase was due t a change in the mysin mlecule r t the presence f cntaminating ins r prteins. EFFECT OF SODIUM AND MAGNESIUM IONS ON CARDIAC MYOSIN ATPASE Reductin f Ca 2 + -ATPase f mysin frm hypx hearts culd have resulted frm inhibitry ins that were adsrbed t the prtein. If this accunted fr the lwer Ca 2 + -ATPase, the difference between cardiac mysin f nrmal and hypx rats wuld be expected t disappear as increasing cncentratins f the inhibitry ins were added. As seen in Figure 2, hwever, the sensitivity f mysin frm nrmal, hypx, and thyrxine-treated hypx rats t inhibitin by sdium was similar ver a range f NaCl cncentratins frm 25 t NORMAL HYPOX HYPOX +T O $5 200 Na +, mm FIGURE 2 Effect f sdium cncentratin n the Ca t + - and K + -activated ATPase f cardiac mysin. The enzymatic activity teas determined as described in Methds. Sdium chlride was added at the cncentratin n the abscissa. CaCl, (10 THM) and KCl (1M) were added as the activating ins. Each value is the mean f three mysin preparatins and the vertical lines shw the SE. Circulatin Reittrcb, Vl. XXXI, Stpttmier 1972

7 MYOSIN ADENOSINE TRIPHOSPHATASE 403 mm. Althugh sdium inhibited bth Ca ATPase f cardiac mysin frm nrmal and hypx rats, the activity f the hypx rat mysin was significantly different at all sdium cncentratins. Inhibitin f K + -ATPase by sdium was mre marked than the inhibitin f Ca 2 + -ATPase, but n differences in the degree f inhibitin were fund with any f the mysins assayed. Sensitivity f mysin frm nrmal, hypx, r thyrxine-treated hypx rats t inhibitin by Mg 2+ was als similar (Fig. 3). Althugh Mg 2 + was a strng inhibitr f bth ATPases, it failed t ablish the differences in Ca ATPase between nrmal and hypx preparatins. The K + -ATPase was mre susceptible t Mg 2+ inhibitin than was Ca 2 + -ATPase. The half maximal inhibitins ccurred at abut 30 and 10 /XM Mg 2 + fr the Ca and K + - ATPases, respectively. EFFECT OF SULFHYDRYL REAGENTS ON CARDIAC MYOSIN ATPASE Preferential binding f heavy metal ins t the sulfhydryl grups f cardiac mysin f hypthyrid rats culd accunt fr the differences in Ca 2 + -ATPase. If this were the case, treatment f mysin with n-ethylmaleimide r dithithreitl wuld be expected t ablish the difference. Skeletal muscle mysin has been shwn t be activated by treatment with n-ethylmaleimide (13). This effect appeared t invlve mdificatin f sulfhydryl (SH) grups near the enzymatic site f mysin. In the present experiments, mysin was incubated fr 40 minutes at 0 C in the presence f varying cncentratins f n-ethylmaleimide. The activity f the enzyme thus treated was assayed at 37 in a reactin mixture that cntained 0.1M KC1, 10 mm CaCl 2, 5 mm Tris-ATP and 50 mm Tris, ph 7.5. There was n significant alteratin in Ca 2 + -ATPase when 1, 2, r 4 mles f n-ethylmaleimide per 10* g mysin were added. The largest increases were frm 1.08 t 1.11 and 0.72 t 0.75 ^mles P ( /mg min -1 fr mysin frm nrmal and hypx rats, respectively. Increasing the KC1 cncentratin in the assay mixture t 0.6M did nt mdify 0.1- NORMAL HYPOX D- HYPOX +T 4 m~ I J 0.3 I 3 10 Mg +? mm EDTA.mM Mg* E, um FIGURE 3 Effect f Mg 1+ cncentratin n Ca and K + -ATPase f cardiac mysin. The assay slutins fr Ca s + -ATPase were thse given in Methds plus the Mg* + cncentratin shwn n the abscissa. The slutins fr assay f K + -ATPase cntained 50 mm Tris, In KCl, 5 mm Tris-ATP, and EDTA r Mg* + cncentratin shwn n the abscissa. Each pint represents the mean f three mysin preparatins and the vertical lines shw the SE. Circulatin Rtiurcb, Vl. XXXI, Sepimbsr 1972 as K + ACTIVATION I \T T

8 404 ROVETTO, HJALMARSON, MORGAN, BARRETT, GOLDSTEIN the effect f n-ethylmaleimide; at 25 C, nly inhibitin ccurred with any cncentratin used. Additin f dithithreitl (5 mm) prduced nly a small activatin f Ca ATPase and did nt mdify the difference between cardiac mysins frm nrmal and hypx animals. COLUMN CHROMATOGRAPHY OF CARDIAC MYOSIN The reductin in Ca 2 + -ATPase f cardiac mysin frm hypx rats may have been caused by the presence f sme inhibitry prtein that cntaminated the mysin. T check this pssibility, mysin was islated in the usual manner and was then chrmatgraphed n tw systems. The first system emplyed a clumn f phsphcellulse fllwed by ne f DEAE cellulse. Since the recveries frm this system were variable, a secnd chrmatgraphy prcedure utilizing DEAE-A50 Sephadex als was emplyed. The results f the tw methds f purificatin were very similar (Table 3). With bth methds f purificatin, the difference between Ca 2 + -ATPase f cardiac mysin f nrmal and hypx rats still existed. There were n differences in either the K + - r NHL, + - ATPase. The enzymatic activities tended t increase fllwing chrmatgraphy, but Ca ATPase shwed the largest percent change. TABLE 3 Clumn Treatment f Mysin frm Nrmal and Hypphysectmized Rat Hearts Nrmal Befre After Hypx Befre After Nrmal Befre After Hypx Befre After (4) (3) (4) (4) (4) (4) (5) (5) NH.+ Phsphcellulse 2.62 ± ± =»= ± 0.06 The K + -ATPase f hypx mysin increased sufficiently t becme significantly higher than the nrmal value fllwing DEAE Sephadex chrmatgraphy. The significance f this is nt understd. The NH 4 + -ATPase f mysin prir t chrmatgraphy was significantly lwer in bth hypx grups as cmpared t nrmal values. This difference disappeared fllwing clumn fractinatin. Differences in NH 4 + -ATPase f nrmal and hypx mysin have nt been cnsistently bserved during the curse f these studies. These experiments indicated that the difference in Ca 2 + -ATPase between cardiac mysin f nrmal and hypx rats was an intrinsic prperty f the mysin mlecule and nt due t the presence f cntaminating ins r prteins. The final series f experiments was undertaken t determine whether a similar difference was present in skeletal muscle mysin. SKELETAL MUSCLE MYOSIN ATPASE Mysin islated frm skeletal muscle failed t exhibit any alteratins in either mnvalent r divalent catin-activated ATPase activity fllwing hypphysectmy. This was true at all cncentratins f activatr in (Fig. 4). The mnvalent and divalent catin ATPases were higher in the predminantly white K+ DEAE Cdlidse Chrmalgraphy 0.73 ± ± ± ± 0.08 DEAE A50 Sephadex Chrmalgraphy 2.64 ± ± ± ± ± ± ± 0.04* 1.16 ± 0.09* ± 0.04 ± 0.08 ± 0.02 ± O.OSf ± 0.03 ± 0.09 =t 0.02J ± 0.04J Values are means =±= SE. Number f mysin samples assayed is in parentheses. *P < 0.05; \P < 0.02; %P < 0.005; P < cmpared t cntrl values. CifcuUtin Rtnurcb, Vl. XXXI, Stpttmbtr 1972

9 MYOSIN ADENOSINE TRIPHOSPHATASE 405 (gastrcnemius) than in the mixture f tw predminantly red muscles (sleus + diaphragm). In cntrast t cardiac mysin, the K + -ATPase f gastrcnemius mysin was higher than Ca 2 + -ATPase. It is als interesting t nte that Ca 2 + -ATPase f cardiac mysin was higher at all Ca 2+ cncentratins than gastrcnemius mysin (Figs. 1 and 4). The reverse was true fr the K + - and NH ATPases. The degree f activatin by increasing cncentratins f activatr ins was cnsiderably different in skeletal muscle cmpared t cardiac muscle (Figs. 1 and 4). Increasing Ca 2+ cncentratin frm 0 t 10 mm nly slightly activated ATPase f skeletal muscle mysin, whereas a tenfld r greater activatin was achieved in cardiac mysin. The high cncentratins that gave maximal activatin in cardiac mysin inhibited bth types f skeletal muscle mysin. On the ther hand, the shape f K + -ATPase activatin curves was similar fr all three muscle types. K + -ATPase NH 4 + differed nly in the ttal activity at any given K + cncentratins. Discussin Decreased cardiac functin in hypphysectmized rats has been described in whle animals (14) and heart-lung preparatins (15). Als, decreased muscle perfrmance was evident in islated papillary muscle frm hypphysectmized rats, as shwn by decreased maximum tensin develpment and rate f tensin develpment (16). Treatment with thyrid hrmne, but nt with grwth hrmne, prevented these changes. The present wrk indicated that a psitive relatin existed between the reduced cardiac perfrmance and the Ca 2 + -ATPase f mysin. A similar crrelatin has been reprted in adrenalectmized cats (2). In thess later reprts (2, 15) and the present experiments, impairment f mechanical perfrmance was apparent nly at high left atrial filling pressures. Althugh Ca- + -ATPase and mechanical perfrmance were directly related in varius hrmnal deficiencies, it shuld be nted that actin is the physilgical activatr f mysin ATPase in intact muscle. Measurements f actmysin ATPase were nt per- mm NH + M K*, M I 0 I FIGURE 4 Effect f cncentratins f activating in n ATPase f skeletal muscle mysin. The assay cnditins were as described in Methds and Figure 1. Three mysin preparatins were assayed at each pint, and SE is indicated. Circulatin Reseercb, Vl. XXXI, Stpttmbtr 1972

10 406 ROVETTO, HJALMARSON, MORGAN, BARRETT, GOLDSTEIN frmed in the present experiments, but reductins in this activity were reprted earlier in adrenalectmized cats (1). The majr derangement in hypx rats that accunted fr decreased perfrmance appeared t invlve a reductin in thyrid functin. Thyrxine, but nt grwth hrmne, administered t these animals prevented bth the decline in cardiac functin and Ca ATPase f mysin. When thyrid functin was reduced by injectin f prpylthiuracil, Ca ATPase decreased t the same extent as with hypphysectmy. This result reinfrced the suggestin that the majr hrmnal deficiency respnsible fr changes in cardiac functin in hypx animals was in thyrid hrmne. The cntributin f crticsterids t this defect appeared t be f lesser imprtance as judged frm studies f cardiac mysin frm adrenalectmized rats. The failure t nte a change in Ca 2 + -ATPase in cardiac mysin f adrenalectmized rats was in cntrast t results btained with adrenalectmized cats (1, 2). The difference may reflect the well-knwn tlerance f saline-maintained rats t adrenalectmy. Injectin f large dses f thyrxine did nt increase the Ca 2 + -ATPase f cardiac mysin. These results are in cntrast t thse f Thyrum et al. (3), wh reprted that large dses f thyrxine (0.25 mg/kg day 1 ) increased Ca 2 + -ATPase f cardiac mysin in guinea pigs. Buccin et al. (17) nted an increased cntractility f papillary muscles frm hyperthyrid cats (1 mg thyrxine/kg day 1 ) and decreased cntractility f these muscles frm hypthyrid cats. In the present experiments, the lack f effect f txic dses f thyrxine (2 mg/kg day 1 ) n cardiac mysin suggested that thyrxine played a permissive rle in affecting activity f the prtein r, alternatively, that lw dses prduced the maximal change in activity. The cardiac hypertrphy that fllwed injectin f high dses f thyrxine indicated that it affected prtein turnver either directly r as a result f the increased cardiac wrk. Cardiac mysin btained frm many species has a lwer Ca 2 + -ATPase activity than des white skeletal muscle (18). Hwever, the present wrk demnstrated that the Ca ATPase f rat cardiac mysin was abut 1.3 times the activity f mysin prepared frm the predminantly white-fibered gastrcnemius. When mysin that was prepared frm predminantly white skeletal muscle f rabbits was assayed with the same reagents used fr assay f rat cardiac mysin, the rabbit skeletal muscle mysin had abut the same maximal activity f Ca 2 + -ATPase as mysin frm the gastrcnemius f the rat ( /i-mles Pi/mg min" 1 ). The relatin between ATPase activity and cncentratins f Ca 2 + suggested that the cncentratin f calcium required fr half maximal activatin f cardiac mysin frm hypx rats was unchanged but that the maximal Ca- + -ATPase was reduced. Similarly, Ca 2 + -ATPase was nt abnrmally sensitive t inhibitin by either Mg 2+ r Na +. Since the difference in Ca 2 + -ATPase between cardiac mysins f nrmal and hypx rats persisted ver a wide range f cncentratin f these inhibitry ins, the difference in Ca 2+ -ATPase wuld nt appear t have resulted frm the binding f greater amunts f these tw ins t the mysin islated frm hypphysectmized rats. Additinal evidence that Mg 2 + cntaminatin did nt accunt fr the difference in Ca 2 + -ATPase was the magnitude f activatin with mnvalent catins. Activatin by these ins was inhibited by Mg 2 +. This inhibitin culd be prevented by additin f EDTA (19, 20). If Mg 2+ had been present in greater amunts in the mysin preparatins frm hypx rats, additin f EDTA wuld have been expected t result in greater K + - ATPase. Hwever, the increase in activity resulting frm 1 and 5 mm EDTA was similar in mysin frm nrmal and hypx rats. Variatins in the cncentratins f Mg 2 +, Na +, K + +, r NH 4 failed t reveal any differences between the mnvalent catinactivated ATPase f cardiac mysin frm nrmal and hypphysectmized rats. These results suggested that the site n the enzyme respnsible fr calcium binding was distinct frm the binding site fr mnvalent catins. Circulatin Rtstarch, Vl. XXXI, StpUmbtr 1972

11 MYOSIN ADENOSINE TRIPHOSPHATASE 407 Only the site invlved in calcium binding appeared t be mdified in hrmnal deficiency. It has been suggested that the mnvalent catin and Ca 2 + -activated mysin ATPase invlved different sulfhydryl grups (13). If the SH grups invlved in the Ca 2 + -ATPase were mdified in hrmnal deficiency r during islatin f mysin t prduce the difference in Ca 2 + -ATPase between cardiac mysin f nrmal and hypphysectmized rats, reactin f these grups with sulfhydryl-blcking agents might be expected t remve any difference in Ca 2 + -ATPase. Hwever, n-ethylmaleimide failed t significantly enhance the Ca 2 + -ATPase f cardiac mysin frm either nrmal r hypx rats when assayed at 0.1M KC1. At 0.6M KC1, bth activities were slightly increased, but the difference in ATPase activity still persisted. These results suggest that mdificatin f SH grups was prbably nt invlved in the differences seen in hrmnal deficiency. These results als indicated that rat cardiac mysin was less sensitive t n-ethylmaleimide than rabbit skeletal muscle mysin (13, 21). The difference in Ca 2 + -ATPase f cardiac mysin frm nrmal and hypx rats might have resulted frm a prtein bund t the mysin. These cntaminants might inhibit the ATPase activity f hypx mysin mre strngly than nrmal mysin. Starr and Offer (22) have shwn that all but a small fractin f the cntaminating prteins culd be remved by chrmatgraphy n DEAE-Sephadex. The difference between the Ca 2 + -ATPase f cardiac mysin f nrmal r hypx rats was nt altered by chrmatgraphy n either DEAE- Sephadex r phsphcellulse and DEAEcellulse. These results further indicated that the difference in the ATPase activity f mysin frm hypx and nrmal rats culd nt be explained n the basis f selective inhibitin f mysin frm hypx rats by cntaminants in the preparatin but was due t a change in the mysin per se. The increase in Ca 2 + -, K + -, and NH 4 + -ATPase activities fllwing chrmatgraphy may have been due t remval either f inhibitry prteins r f CtrcaUUn Rutarcb, Vl. XXXI, Stpttmbn 1972 mysin that had been inactivated. Remval f inactive mysin wuld have been expected t increase the activity f bth mnvalent and divalent catin-activated ATPase t the same extent. The increase in Ca 2 + -ATPase was prprtinally greater, hwever, than either K + -r NH + 4 -ATPase. The altered ATPase activities f mysin f animals with heart failure (23 25), hrmnal deficiency, a perid f exercise (26), r crssinnervatin f skeletal muscle (27) may result frm similar changes in the mysin mlecule. Mysin cmpnents which might change under these circumstances include bth the light and heavy chains. Mysin light chains are distinctive fr different types f muscle (28-31) and may functin t mdify enzyme activity (32-34). It has recently been demnstrated that by exchanging light chains f red and white muscle mysin the specific activities f the recnstituted mysins were similar t the specific activity f the mysin frm which the subunits arse (35). In ther studies (36), the ATPase activity and the quantity f ne f the light chains were fund t be reduced in skeletal muscle frm vitamin E dystrphic rabbits. Heavy chains f mysin may be altered either by methylatin f lysine r histidine residues r by synthesis f new chains. Mysin frm white skeletal muscle has been fund t cntain 3-methylhistidine, but this amin acid was absent frm mysin f either red skeletal muscle r heart (37, 38). Hwever, cardiac muscle cntains bth mnand trimethyllysine (37). Althugh it has nt been demnstrated that methylatin f basic amin acids in the purified prtein can mdify mysin ATPase activity, the variatin in amunts f methylated amin acids between mysins f different muscle types suggests this pssibility. Since methylatin f amin acids appears t ccur fllwing peptide synthesis (39), cntent f methylated amin acids culd be due t a change in the rate f methylatin r demethylatin. Lbbey et al. (36) have fund that the amunt f 3- methylhistidine was decreased in mysin f skeletal muscle frm vitamin E dystrphic rabbits and resembled mysins frm adult red

12 408 ROVETTO, HJALMARSON, MORGAN, BARRETT, GOLDSTEIN muscle and nenatal skeletal muscle. The hrmnal effect n ATPase activity wuld nt appear t invlve synthesis f new heavy chains with altered prperties. This suggestin is based n the fact that the turnver time fr rat heart mysin is abut 21 days (40), whereas the hrmnal effect was well established after 6-7 days. Studies are presently in prgress t investigate pssible changes in light and heavy chains. In cntrast t the results with cardiac muscle, neither the mnvalent nr divalent catin-activated ATPase f skeletal muscle mysin was altered in hypphysectmized rats, suggesting that the hrmne effect may be specific fr the heart. The results are similar t thse fund in adrenalectmized cats in respect t the white-fibered gastrcnemius but are different frm thse reprted in the red-fibered sleus (2). A decrease in Ca 2 + -ATPase f mysin frm cat sleus was similar in magnitude t that fund in the heart. The apparent discrepancy between the present results in rats and the earlier studies in cats may be due t the species difference r t the use f a cmbinatin f diaphragm muscle and sleus t prepare the mysin frm redfibered muscle. A decrease in the Ca 2 + -ATPase f sleus mysin f hypphysectmized rats may have been bscured by the larger mass f diaphragm muscle. These muscles were cmbined in the present experiments t btain sufficient tissue fr the preparatin f mysin. References 1. ROVETTO, M.J., MURPHY, R.A., AND LEFER, A.M.: Cardiac impairment in adrenal insufficiency in the cat: Reduced adensinetriphsphatase activity f mycardial cntractile prteins. Circ Res 26:419-^28, ROVETTO, M.J., LEFEB, A.M., AND MURPHY, R.A.: Alteratins in mycardial cell functin in adrenal insufficiency. Pfluegers Arch 329:59-71, THYRUM, P.T., KRTTCHEH, E.M., AND Lucm, R.J.: Effect f Z-thyrxine n the primary structure f cardiac mysin. Bichim Biphys Acta 197: , HJALMAHSON, A.C., WHIT-FIELD, C.F., AND MORGAN, H.E.: Hrmnal cntrl f heart functin and mysin ATPase activity. Bichem Biphys Res Cmmun 41: , NEELY, J.R., LlEBEBMElSTEH, H., BATTEBSBY, E.J., AND MOHGAN, H.E.: Effect f pressure develpment n xygen cnsumptin by islated rat heart. Am J Physil 212: , WEEDS, A.G., AND HARTLEY, B.S.: Selective purificatin f the thil peptides f mysin. Bichem J 107: , LAYNE, E.: Spectrphtmetric and turbidimetric methds fr measuring prteins In Methds f Enzymlgy, vl. 3, edited by N. O. Kaplan and S. P. Clwick. New Yrk, Academic Press, 1957, pp LOWRY, O.H., ROSEBHOUGH, N.J., FARR, A.L., AND RANDALL, R.J.: Prtein measurement with the Flin phenl reagent. J Bil Chem 193: , LYMN, R.W., AND TAYLOR, E.W.: Transient state phsphate prductin in the hydrlysis f nucleside triphsphates by mysin. Bichemistry 9: , HARRIS, W.D., AND POP AT, P.: Determinatin f the phsphrus cntent f lipids. J Am Oil Chem Sc 31: , RICHARDS, E.G., CHUNC, C.-S., MENZEL, D.B., AND OLCOTT, H.S.: Chrmatgraphy f mysin n diethylaminethyl-sephadex A-50. Pichemistry 6: , HARMS, M., AND SUELTER, C.H.: Simple chrmatgraphic prcedure fr the preparatin f rabbit-muscle mysin A free frm AMP deaminase. Bichim Biphys Acta 133: , SEKINE, T., AND KIELLEY, W.W.: Enzymic prperties f n-ethylmaleimide mdified mysin. Bichim Biphys Acta 81: , BEZNAK, M.: Effect f grwth hrmne and thyrxine n cardivascular system f hypphysectmized rats. Am J Physil 204: , KOHECKY, B., BEZNAK, M., AND KORECKA, M.: Effect f hypphysectmy and hrmne treatment n the heart-lung preparatin f rats. Can J Physil Pharmacl 44:13-20, MINELLI, R., AND KORECKY, B.: Effect f grwth hrmne and thyrxine n ismetric cntractile mechanics f cardiac muscle. Can J Physil Pharmacl 47: , BUCCINO, R.A., SPANN, J.F., JR., SONNENBLICK, E.H., AND BRAUNWALD, E.: Effect f thyrid state n mycardial cntractility. Endcrinlgy 82: , KATZ, A.M.: Cntractile prteins f the heart Physil Rev 50:63-158, MUHLBAD, A., FABIAN, F., AND Bm, N.A.: On the activatin f mysin ATPase by EDTA. Bichim Biphys Acta 89: , CtrcuUtwn Ristmrcb, Vl. XXXI, Sspumber 1972

13 MYOSIN ADENOSINE TRIPHOSPHATASE OFFEH, G.W.: Antagnistic actin f magnesium ins and ethylenediaminetetraacetate n mysin A ATPase (ptassium activated). Bichim Biphys Acta 89: , SEIDEL, J.C.: Effect f salts f mnvalent ins n the adensine triphsphatase activities f mysin. J Bil Chem 244: , STARR, R., AND OFFER, G.: Plypeptide chaans f intermediate mlecular weight in mysin preparatins. FEBS Letters 15:40-^4, ALPERT, N.R., AND GORDON, M.S.: Myfibrillar adensine triphsphatase activity in cngestive heart failure. Am J Physil 202: , CHANDLER, B.M., SONNENBTJCK, E.H., SPANN, J.F., JR., AND POOL, P.E.: Assciatin f depressed myfibrillar adensinetriphsphatase and reduced cntractility in experimental heart failure. Circ Res 21: , GORDON, M.S., AND BHOWN, A.L., JR.: Myfibrillar adensinetriphsphatase activity f human heart tissue in cngestive failure: Effects f uabain and calcium. Circ Res 18: , WLLXERSON, J.E., AND EVONTUX, E.: Changes in cardiac and skeletal muscle mysin ATPase activities after exercise. J Appl Physil 30: , BuLLER, A.J., MOMMAERTS, W.F.H.M., AND SERAYDABIAN, K.: Enzymic prperties f mysin in fast and slw twitch muscles f the cat fllwing crss-innervatin. J Physil (Lnd) 205: , LOCKER, R.H., AND HACYARD, C.J.: Variatins in the small subunits f different mysins. Arch Bichem Biphys 122: , LOWEY, S., AND RISBY, D.: Light chains frm fast and slw muscle mysins. Nature (Lnd) 234:81-85, SARKAR, S., SHETER, F.A., AND GERGELY, J.: Light chains f mysins frm white, red, and cardiac muscles. Prc Natl Acad Sci USA 68: , WEEDS, A.G., AND POPE, B.: Chemical studies n light chains frm cardiac and skeletal muscle mysins. Nature (Lnd) 234:85-88, SAMAHA, F.J., GUTH, L., AND ALBERS, R.W.: Differences between slw and fast muscle mysin adensine triphsphatase activity and release f assciated prteins by p-chlrmercuriphenylsulfnate. J Bil Chem 245: , STRACHER, A.: Evidence fr the invlvement f light chains in the bilgical functining f mysin. Bichem Biphys Res Cmmun 35: , DHIEZEN, P., AND GERSHMAN, L.C.: Relatinship f structure t functin in mysin: II. Salt denaturatin and recmbinatin experiments. Bichemistry 9: , KIM, H.D., AND MOMMAERTS, W.F.H.M.: On the recnstitutin f rabbit mysin frm fast and slw muscle. Bichim Biphys Acta 245: , LOBLEY, G.E., PEBRY, S.V., AND STONE, O.: Structural changes in mysin induced by vitamin E dystrphy. Nature (Lnd) 231: , KUEHL, W.M., AND ADELSTEIN, R.S.: Absence f 3-methylhistidine in red, cardiac and fetal mysins. Bichem Biphys Res Cmmun 39: , KUEHL, W.M., AND ADELSTEIN, R.S.: Identificatin f n-mnmethyllysine and n-trimethyllysine in rabbit skeletal muscle. Bichem Biphys Res Cmmun 37:59-65, KIM, S., AND PAIX, W.K.: Purificatin and prperties f prtein methylase II. J Bil Chem 245: , MORCAN, H.E., EARL, D.C.N., BROADUS, A., WOLPERT, E.B., GlCER, K.E., AND JEFFERSON, L.S.: Regulatin f prtein synthesis in heart muscle: I. Effect f amin acid levels n prtein synthesis. J Bil Chem 246: , CircmUtun Rtiurcb, Vl. XXXI, Stpumbtr 1972