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Decrease in rat cardiac myosin ATPase with aortic constriction: Prevention by thyroxine treatment
Pergamon Preas
Life Sciences, Vol . 25, pp . 353-364 Printed in the U .S .A .
DECREASE IN RAT CARDIAC MYOSIN ATPase WITH AORTIC CONSTRICTION : PREVENTION BY THYROXINE TREATMENT 1 John J . Afflitto 2 and Mario A . Inchiosa, Jr .3 Department of Pharmacology, New York Medical College, ~b Ihalla, New York 10595 (Received in final form June 11, 1979) Summary Severe aortic constriction in rats produced cardiac hypertrophy and a chronic decrease in cardiac actomyosin ATPase activity during a six week postoperative period . Two weeks following aortic constriction, Cat+ stimulated cardiac myosin ATPase activity was also depressed; the Km and Vn,~ were decreased by 86 .2% (p X0 .0025) and 84 .4% (p<0 .0025), respectively, when compared to sham operated controls . Administration of thyroxine (100 Ng/kg/day for 14 days), which was initiated on the same day as aortic constriction, prevented, to a large extent, the decrease in cardiac myosin ATPase activity . The Kn, and Vmax of myosin from animals with aortic constriction showed substantially smaller decreases as a result of concomitant thyroxine administration (p<0 .0025 for the chmge from aortic constriction without thyroxine treatment) . Thyroxine treatment in rats with aortic stenosis resulted in m additional increment of cardiomegaly when compared to animals with aortic constriction alone . The results of this study indicate that thyroxine, which normally has no effect on Ca 2 +activated cardiac myosin ATPase in the rat, can prevent the decrease in myosin ATPase activity which results from severe aortic stenosis . Depressed cardiac myosin ATPase activity has been reported following chronic experimental pressure overload of the heart in a number of different animal species (1-b) . In some studies, an increase in cardiac myosin and actomyosin ATPase activity has been ob served following constriction of the pulmonary artery (7) and the aorta (8,9). However, these findings appear to be associated with a mild cardiac pressure overload md,/or a relatively early stage in the development of cardiomegaly . In contrast to the depressed cardiac myosin ATPase usually observed following pressure induced hypertrophy, administration of thyroxine results in substantial cardiac hypertrophy accompanied by an increased Ca t+ stimulated myosin ATPase activity in the rabbit (1014), guinea pig (15,16) and dog (9), but not in the rat or mouse (13,17) . Rat and mouse iThédataprésentedin~paper are tan from a thesis presented by John J . Afflitto in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacology, New York Medical College, 1978 . 2 Present address: Dept . of FNiarmacology, Colgate-Palmolive Co ., Piscataway, N .J . 3 Correspondence 0024-3205/79/040353-12$02 .00/0 Copyright (c) 1979 Pergamon Presa Ltd
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cardiac myosin ATPase activities, which are comparatively high, ap°ear to be maximally activated and cannot be further stimulated by thyroxine administration or by titration of the most sensitive class of myosin sulfhydryl groups ($H1) with thiol reagents (13, 17) . Previous studies from this laboratory (10,18) and by others (12), have demonstrated that the enhanced cardiac myosin ATPase activity observed subsequent to chronic thyroxine treatment in rabbits is characterized by an increase in both Vm~ and Kn., when compared to placebo injected animals . Thyroxine treatment also produced substantial increases in actomyosin content (14,19) . The extent to which these effects of thyroxine are responsible for the increased contractile performmce of thyrotoxic cardiac muscle has not been established . The present studies concern the effects of thyroxine treatment on cardiac myosin ATPase activity in animals with aortic constriction . The "chronic" animal model used for these investigations is also characterized . Methods Aortic constriction : The abdominal aorta was constricted immediately below the diophrogm in ma e, Prague-Dawley rats under ether anesthesia . A metal rod of specific diameter was placed next to the aorta, a ligature was tied around the rod and the aorta, completely occluding the vessel, and the rod was then withdrawn . The same procedure was followed for sham operated animals with the exception that the ligature was used only to temporarily occlude the aorta and was then removed with the metal rod . Preparation of cardiac actom sin : Cardiac tissue was obtained and prepared for myosin an actomyosin extraction according to methods previously described (20) . The animals were sacrificed by decapitation and the beating heart was rapidly excised and placed in cold distilled water . All subsequent procedures were carried out at 4°C . A finely minced homogeneous mixture of right and left ventricular tissue was used for either myosin or actomyosin extraction . Quantitative extraction and purification of cardiac actomyosin was performed according to a described method (20), which is summarized as follows: Approximately 0 .5 grams of minced ventricular tissue was homogenized For b minutes with 5 volumes of extraction solution (0 .3 M KCI, 0 .15 M Kg HP04 , 0 .15 M KH 2 P04 and 0 .8 mM ATP 2; final pH, 6 .5) . Actomyosin was extracted overnight (16-20 hr) with gentle stirring . The homogentle was centrifuged at 18,500 g for 15 minutes and the clear supernatant extract was saved . The sediment was resuspended in 0.6 M KCI and centrifuged at 18,500 g for 10 minutes. The supematmts were combined and diluted with water to ionic strength 0.065 . The actomyosin was allowed to precipitate for two hours and was then collected by centrifugation at 1600 g . The precipitate was then washed with 0 .065 M KCI . A portion of the actomyosin was resuspended in water and assayed for protein nitrogen by a modified biuret procedure (21) . A similar portion of actomyosin was dissolved by the addition of solid KCI (final concentration, 0 .4 M) and stored overnight at 5°C . Actomyo sin suspensions were assayed for ATPase activity on the following day. 1 antes ua omogen zer, matching surfaces ground glass . 2 Crystalline di-Na ATP (Sigma) adjusted with KOH to pH 7 .4 .
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Preparation of cardiac myosin : Complete details for the preparation of cardiac myosin have en previous y scri e ) . Essentially, the procedure is a modification of techniques described by Brahms and Kay (23) . Parts of other methods are incorporated, including the three successive precipitation-dissolution cycles outlined by Shiverick et al . (24) and a final overnight dialysis of the concentrated myosin solution employed by Mueller et al . (25) . Myosin prepared by this procedure showed absorbante ratios for 280 nrq/260 nm ranging from 1 .47 to 1 .58. Shiverick et al . (7, 24) demonstrated that myosin with an A280 nrrJA260 nm above 1 .40 contains less thin 1% nucleic acid contamination . These investigators have noted the importmce of A28p nrrJA260 nm ratios as a criterion for documenting the purity of myosin preparations . They have also demonstrated that cardiac myosin preparations showing high A280 nm/A26p nn~, ratios contain no detectable contamination by actin, troponin or tropomyosin as determined by SDS polyacrylomide gel electrophoresis (7, 24) . ATPase assay: The components of reaction mixtures are described in the individual assays . ATPase activity was determined by measurement of the rate of inorganic phosphate liberation . Protein was added to the reaction mixtures last, at 5°C . The temperature of the assay tubes was then equilibrated to 25oC . Reactions were initiated by the addition of ATP and terminated by deproteinizotion with 4 volumes of cold, 4% trichloroacetic acid The hydrolytic rates were linear within the assay periods which were used (26) . Inorgmic phosphate was determined by the method of Fiske and SubbaRow (27) . Results In m initial study of the effects of aortic constriction on heart weight and mortality in the rat, it was determined that a rat in the body weight range of 220 to 280 grams with an internal aortic diameter of 0.77 mm following aortic constriction would serve as an optimal experimental model (2) . This degree of aortic constriction produced the greatest cardiac hypertrophy and a 46% mortality . When aortic constriction was more severe, the degree of cardiomegaly was not increased and mortality rose substmtially . Death uwally occurred 1 to 3 days following surgery and in most instances was apparently due to acute pulmonary edema. An aortic stenosis less severe than 0 .77 mm in 250 gram rats produced wb-optimal amounts of cardiac hypertrophy . Changes in body weight, ventricular dry weight, cardiac actomyosin content and ATPase activity were studied at 1, 2, 3 and b weeks following aortic constriction to deter mine a postoperative period during which the effects of thyroxine administration could be most advmtageously studied (Figs. 1 - 4) . Rats with aortic constriction lost body weight during the first week following wrgery but thereafter gained weight rapidly. By the sixth postoperative week, the mean body weights of sham operated and constricted animals were essentially the same (Fig . 1) . The mean dry weight of the combined right and left ventricles of constricted mimals rapidly increased during the first week following surgery (Fig . 2) . Increases in ventricular dry weight of constricted mimals ringed from 3996 at one week, to 51% at b weeks, when compared to sham operated mimals . Ventricular actomyosin content (total mg of ventricular actomyosin as a percentage of dry ventricular weight) tended to be lower in constricted animals than sham operated animals throughout the b week postoperative period (Fig . 3) . However, none of these differ entes were statistically significant .
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FIG . 1 Chrnges in the body weight of rats at intervals of time following aortic constriction (broken line) or sham operation (solid line) . In this figure and in subsequent figures and tables the number of animals in each group is indicated in parentheses . Also, all data are presented as the mean t $ .E .M . Cardiac actomyosin suspensions from constricted and sham operated animals were assayed for ATPase activity in the presence of Cat+ and Mgt+ at pH 6 .8 (Fig . 4) . Actomyosin from constricted animals exhibited statistically significant decreases in ATPase activity at l, 2, 3 and 6 weeks postoperatively, the average decreases being 24 .6, 30 .4, 23 .1 and 25 .6%, respectively . The effects of chronic thyroxine administration were superimposed upon the chronic animal model of aortic constriction described above . Purified cardiac myosin was prepared for these studies . The kinetic parameters of rat cardiac myosin ATPase activity were de termined 2 weeks following sham operation, aortic constriction alone and aortic constriction plus thyroxine administration (100 Na/kg/day for 14 days) . Thyroxine administration was initiated on the day of surgery . The results presented in Fig . 5 show the calculated least squares curves for each assay presented as a double reciprocal plot of enzyme activity vs substrate concentration . Cardiac myosin ATPase activity of mimals with aortic stenosis was significantly depressed at all substrate concentrations . Kinetic analysis revealed an average, 86 .2% decrease in maximal velocity and an average, 84 .4% decrease in Km when compared to sham operated animals (Table 1) . Administration of thyroxine to animals with aortic stenosis prevented, to a great extent, the decrease in enzymatic activity and resulted in a significant increase in both the Vm~ (+355%) and the Km (+277%) of myosin in rats with aortic constriction (Table 1) .
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A comparison of fresh heart weight for sham operated and experimental mimals (Table 2) demonstrated that thyroxine treatment of animals with aortic stenosis resulted in an additional increment of cardiac hypertrophy when compared to animals with aortic stenosis alone . Aortic constriction produced a 29 .2% (p<0 .0005) increase in fresh heart weight while aortic constriction plus thyroxine produced a 51 .3% (p<0 .0005) increase in fresh heart weight (comparisons with sham operated animals) . Thyroxine treatment increased fresh heart weight by 14 .6% (p~0 .01) when compared to animals with aortic constriction alone. Thyroxine administration reduced survival rate from 53 .4%, observed in rnimals with aortic stenosis alone, to 36 .0% in the group of animals with the combined treatment of aortic constriction plus thyroxine (Table 2) . The mean increase in body weight during a two week postoperative period was significmtly greater in the thyroxine treated group than the group of animals with only aortic constriction (Table 2) . Both experimental groups gained less body weight than the sham operated animals . Di scussi on The chronic study of ventricular dry weight, body weight and actomyosin ATPaseactivity following aortic constriction was useful for characterization of a model of compensotory cardiac hypertrophy in the rat (Figs . 1-4) . The degree of aortic constriction was severe and the mortality rate was high (46%) . However, some animals survived by meets of a rapid compensatory cardiac hypertrophy, most of which occurred during the first postoperative week (Fig . 2) . Beznak has demonstrated that the cardiac hypertrophy seen following aortic constriction, in the rat, is essentially complete by the seventh postoperative day, a time when cardiac output and cardiac reserve force have returned to near normal levels (29) . The rapid recovery of normal body weight in young rats with aortic stenosis (Fig . 1), which begins after the first postoperative week, appears to be related to the cardiac compensation which has occurred . Actomyosin content as a percentage of dry ventricle weight tended to be lower in rats with aortic stenosis than in sham operated mimals . These changes were not statistically significant at any of the individual time periods of measurement (Fig . 3) . A decreased contractile protein content during the early stages of experimental cardiac hypertrophy has been reported (30-33) and appears to be related to m increased proliferation of mhochrondria at this time (30, 34-36) . Although the rats with aortic constriction appeared to have compensated completely by the sixth week following surgery, cardiac actomyosin ATPase activity remained depressed to approximately 75% of that seen in sham operated rats (Fig . 4) . Depressed cardiac myosin and actomyosin ATPase activities have frequently been reported in states of severe work induced hypertrophy and also in cardiac failure in animals and man . Whether depressed myosin ATPase activity is a defect of the overstressed myocardium, or is part of a compensatory mechanism to reduce energy utilization, is unknown . There is evidence to suggest that myosin, which appears to be an iscenzyme system, demonstrates chmges in substructure and ATPase activity in response to altered functional states (4, 6, 12, 14, 18, 37) . Calcium stimulated cardiac myosin ATPase activity, measured 2 weeks following aortic constriction, was depressed throughout the range of substrate concentrations (Fig . 5). The decreases in Km and Vm~ of myosin of constricted animals were similar to the findings of Shiverick et al . for pulmonary artery constriction in the rabbit (7) . Using SDS poly-
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acrylamide gel electrophoresis, these investigators also demonstrated that there were no chmges in myosin light chains in hypertrophied rabbit myocardium (7) . They concluded that the decreases in Km and Vm~ were not attributable to changes in the composition of the major myosin subunits but more probably involved subtle modifications which influenced the active site . Katagiri and Morkin (38) have likewise found no evidence for chmges in the content or composition of calf myosin subunits in left ventricular hypertrophy produced by aorti c coarctati on .
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FIG . 4 Changes in ventricular actomyosin ATPase activity at various time intervals following aortic constriction . The broken line represents values for constricted rats and the solid line represents values for sham operated animals . Assay conditions : 0 .08 M KCI ; 0 .1 mMCaCl2; 0.3 mM MgCl 2; 20 mM histidine buffer (pH 6 .8); 5 mM ATP; 100 Ng of actomyosin N/ml . Assayed 10 min at 25oC . Cat + stimulated cardiac myosin ATPase activity is known to be related to the level of thyroid activity (17, 39) . Thyroxine administration to euthyroid rabbits results in increased Ca t+ activated cardiac myosin ATPase activity (9, 10, 12, 37, 39, 40) . Flink and Morkin (41) have demonstrated the presence of a new myosin isozyme in the thyrotoxic rabbit . Rat and mouse cardiac myosin ATPase activity, which is high in comparison to other species, is not increased by thyroxine administration (17, 39). In the present study, concomitant administration of thyroxine (100 Na/kg/day for 14 days) to constricted animals resulted in cardiac hypertrophy which was opparently additive to that caused by aortic Stenosis alone (Table 2) . However, the most important result of these studies was the observation that thyroxine administration prevented, to a great extent,
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FIG . 5 Lineweaver-Burk plots of rat cardiac myosin ATPase activity : ~---+sham operation; x---~c aortic constriction ; o---o aortic constriction plus thyroxine treatment . Reaction mixtures : 0.6 M KCI; 0 .1 M Tris-HCI, pH 9 .0 ; CaCl2 in concentrations which were 0 .5 mM greater than the substrate, ATP; 25 Ng myosin N~ml . Assayed 5 min at 25°C . The curves were tested for linearity by the t test (28) . Each plot represented a statistically signifiant regression line; p values ranged from <0 .025 to <0 .0001 .
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the decrease in the Km and maximal velocity of myosin ATPase in rats with aortic stenosis (Fig . 5, Table 1) . Although the dosage of thyroxine did not completely restore the Vm~ and Km of myosin from constricted animals to normal levels, it did increase the Vm~ac by 335% (p<0 .0025) and the Km by 227% (p<0 .0025) when compared to animals with aortic constriction alone . It is of interest that thyroxine, which does not affect C~ + activated ATPase in the normal rat (17, 39), effectively increased the depressed Ca t+ activated cardiac myosin ATPase activity in rats with aortic constriction (Fig . 5 Table 1) . TABLE 2 Effect of Aortic Constriction Plus Thyroxine Treatment on Body Weight, Fresh Heart Weight and Survival in the Rat No .
% Survival
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These findings might have been mticipated since thyroxine has been shown to cause changes in cardiac myosin in normal rabbits (i .e ., increased Vm~ and Km; refs . 10, 12) which are directly opposite to those associated with severe pulmonic stenosis (24) . The apparent decrease in ATP-myosin affinity (md increased ATPase) with thyroxine treatment in normal rabbits has been demonstrated to rewlt from a modification of SH1 thiol groups associated with the active site (9, 12, 18, 39). The observation of an increased Vm~ associated with a decreased affinity is in accord with the reaction mechanism of myosin ATPase proposed by Lymn and Taylor whose kinetic findings indicated that release of the enzyme bound products is the rate limiting step of the reaction (42) . The apparent increased affinity of ATP for myosin in rats with aortic stenosis (Fig . 5, Table 1) might be expected to result, therefore, from molecular modifications which increase SH1 thiol group affinity or availability . This interpretation is supported by the findings of Shiverick et al . (7), who studied the effects of SH1 group modification with p-chloromercuribenzoate on C°2+ _pendent myosin ATPase in rabbits with pulmonary artery constriction . They also concluded that the SH1 groups of myosin from hypertrophied hearts were function-
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ally altered when compared with normal myosin . It is concluded that thyroxine prevented, to a great extent, the decrease in myosin ATPase with aortic stenosis by virtue of its apparently opposite effects upon the myosin molecule . References l. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . Z0 . 21 . 22 . 23 . 24 . 25 . 26 .
G . CONWAY, R. A . HEAZLITT, R.A . FOWLER, M . GABEL, and S . GREEN, J . Mol . 8 Cell . Cardiol . 8 39-51 (1976) . FLITT , odors dissertation, New York Medical College (1978) . M . DRAPER, N . TAYLOR, and N .R . ALPERT, in Cardiac Hypertrophy , pp . 315-331, Academic Press, New York (1971) . J . WIKMAN COFFELT, R . WALSH, C . FENNER, T . KAMIYAMA, A . SALEL, and D .T . MASON, J . Mol . 8. Cell . Cardiol . 8 263-270 (1976) . NB I K, J.F . SPANN, and P .E . POOL, B.M CHANDLER, E .H . Circulation Res . 21 717-725 (1967) . J . IK OFFELT, C . FENNER, R. J . COFFELT, A . SALEL, and D .T . MASON, J . Mol . 8 Cell . Cardiol . 7 219-224 (1975) . K. . , BBB . HA7~ARELL, and N .R . ALPERT, J . Mol . & Cell . Cardio l . 8 837-851 (1976) . ~. OGANESSYAN, T . ZAMINIAN, N . BAY, V. PETROSIAN, A . KOSCHARIAN, I . MARTIROSIAN, and M . ELOYAN, J . Mol . 8 Cell . Cardiol . 5 1-24 (1973) . . . fFIGLY, J . Mol . 8~ G . CONWAY, R.A . HEAZLITT, J . , an Cell . Cardiol . 7 817-826 (1975) . S .F . KU Z NSKI and M .A . INCHIOSA, Jr ., Abstracts of Papers, 164th National Meeting, A.C .S ., Division of Biol . Chem . 128 T . KATAGIRI, A .S . FREEDBERG, and E . MORKIN, Life Sci . 16 1079-1087'1975) . S .K . BANERJEE, I .L . FLINK, and E . MORKIN, CircuTati x._39 319-326 (1976) . 1 7 Y . YAZAKI and M.S . RABEN, Circulation Res . 35 15M .A . INCHIOSA, Jr ., in Biomo ecu ar Structuré and Function, pp . 455-461, Academic Press, New York 19 M.J . GOODKIND, G .F . DAMBACH, and R.J . LUCHI, J . clin . Invest . _48 30A (1969) . S . GUDBJARNASON, M . TELERMAN, C .CHIBA, P .L . WOLF, and R .J . BING, J . Lab.clin . Med . 63 245-253 (1964) . . . , ~C . HJALMARSON, H .E . MORGAN, M .J . BARBETT, and R.A . GOLDSTEIN, Circulation Res . 31 397-409 (1972) . S . F . KUCZYNSKI, Doctors isserta iôn, New York Medical College (1974) . M .A . INCHIOSA, Jr . and A .S . FREEDBERG, in Current Topics in Thyroid Research , pp . 394-402, Academic Press, New York (1965) . M.A . INCHIOSA, Jr ., Am . J . Ph siol . 206 541-546 (1964) . M.A . INCHIOSA, Jr ., J . La . c in . srb3 319-324 (1964) . V . T . PAGANO and M. . I , r . ~es . Commun . Chem . Pathol .& Pharmacol . 23 37-47 (1979) . . J ~AAFufS and C .M . KAY, J . molec . Biol . 5 132-137 (1962) . K . T . SHIVERICK, L .L . THO , m . .ÂLRERT, Biochim . biophys. Acts 393 124-133 (1975) . IT . MUELLER, J . FRANZEN, R .V . RICE, and R .E . OLSON, J . biol . Chem . _239 1447-1456 (1964) . M .A . INCHIOSA, Jr ., Biochem . Pharmac. 16 329-344 (1967) .
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27 . C, H . FISKE and Y . SUBBAROW, J . biol . Chem, 66 375-400 (1925) . 28 . G .W, SNEDECOR and W .G, COCHR , tatistica Methods, 6th Ed ., pp, 135171, Iowa State Univ . Press, Ames (1967) . 29 . M, BEZNAK, Circulation Res . 6 207-212 (1958) . AYEVA, S .S . LUGACHEV, and M .G . PSKNNIKOVA, 30 . F .A . MEERSO , . . E~ I . Cell . Res. 36 568-578 (1964) . 31 . . . , . K~BATA, and W .V . WHITEHORN, Circulation Res . 12 118-124 (1963) . 32 . E. PAGE and L,P . McCALLISTER, Am . J . Cardio l . 31 172-181 (1973) . 33 . A .L . GORDON, M .A . INCHIOSA, Jr ., and D . LET-IR, J . Mol . 8 Cell . Cardio l . 4 543-557 (1972) . 34 . F. Y . HATT, G, BERJAL, J . MORAVEC, and B . SWYGHENDAUW, J . Mol, & Cell . Cardiol . 1 235-247 (1970) . 35 . 1uT .lE~II~OWITZ, V . ASCHENBRENNER, R . ALBIN, N .J . GROSS, R. ZAK, and K .G . NAIR, in Cardiac H ertrophy, pp . 283-299, Academic Press, New York (1971), 36 . M . RABINOWIT , m . . ar io . 31 202-210 (1973) . , iôchim, biophys . Acta 481 68(1`688 (1977) . 37 . L .L . THOMAS and 38 . T . KATAGIRI and E . MORKIN, Bioc im, iop s. cta ßf62-274 (1974) . 39 . Y . YAZAKI and M .S . RABEN, Circu ation Res . 3 208-215(1975) . 40 . M .J . GOODKIND, G .E . DA , P . . H17f~UM, and R .J . LUCHI, Am . J . Physiol . 226 66-72 (1974) . 41 . FLINKand E . MORKIN, Febs Letters 81 391-394 (1477) . 42 . R,W . LYMN and E .W . TAYLOR~iôc,~istry 9 2975-2983 (1970) .
Life Sciences, Vol . 25, pp . 353-364 Printed in the U .S .A .
DECREASE IN RAT CARDIAC MYOSIN ATPase WITH AORTIC CONSTRICTION : PREVENTION BY THYROXINE TREATMENT 1 John J . Afflitto 2 and Mario A . Inchiosa, Jr .3 Department of Pharmacology, New York Medical College, ~b Ihalla, New York 10595 (Received in final form June 11, 1979) Summary Severe aortic constriction in rats produced cardiac hypertrophy and a chronic decrease in cardiac actomyosin ATPase activity during a six week postoperative period . Two weeks following aortic constriction, Cat+ stimulated cardiac myosin ATPase activity was also depressed; the Km and Vn,~ were decreased by 86 .2% (p X0 .0025) and 84 .4% (p<0 .0025), respectively, when compared to sham operated controls . Administration of thyroxine (100 Ng/kg/day for 14 days), which was initiated on the same day as aortic constriction, prevented, to a large extent, the decrease in cardiac myosin ATPase activity . The Kn, and Vmax of myosin from animals with aortic constriction showed substantially smaller decreases as a result of concomitant thyroxine administration (p<0 .0025 for the chmge from aortic constriction without thyroxine treatment) . Thyroxine treatment in rats with aortic stenosis resulted in m additional increment of cardiomegaly when compared to animals with aortic constriction alone . The results of this study indicate that thyroxine, which normally has no effect on Ca 2 +activated cardiac myosin ATPase in the rat, can prevent the decrease in myosin ATPase activity which results from severe aortic stenosis . Depressed cardiac myosin ATPase activity has been reported following chronic experimental pressure overload of the heart in a number of different animal species (1-b) . In some studies, an increase in cardiac myosin and actomyosin ATPase activity has been ob served following constriction of the pulmonary artery (7) and the aorta (8,9). However, these findings appear to be associated with a mild cardiac pressure overload md,/or a relatively early stage in the development of cardiomegaly . In contrast to the depressed cardiac myosin ATPase usually observed following pressure induced hypertrophy, administration of thyroxine results in substantial cardiac hypertrophy accompanied by an increased Ca t+ stimulated myosin ATPase activity in the rabbit (1014), guinea pig (15,16) and dog (9), but not in the rat or mouse (13,17) . Rat and mouse iThédataprésentedin~paper are tan from a thesis presented by John J . Afflitto in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacology, New York Medical College, 1978 . 2 Present address: Dept . of FNiarmacology, Colgate-Palmolive Co ., Piscataway, N .J . 3 Correspondence 0024-3205/79/040353-12$02 .00/0 Copyright (c) 1979 Pergamon Presa Ltd
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cardiac myosin ATPase activities, which are comparatively high, ap°ear to be maximally activated and cannot be further stimulated by thyroxine administration or by titration of the most sensitive class of myosin sulfhydryl groups ($H1) with thiol reagents (13, 17) . Previous studies from this laboratory (10,18) and by others (12), have demonstrated that the enhanced cardiac myosin ATPase activity observed subsequent to chronic thyroxine treatment in rabbits is characterized by an increase in both Vm~ and Kn., when compared to placebo injected animals . Thyroxine treatment also produced substantial increases in actomyosin content (14,19) . The extent to which these effects of thyroxine are responsible for the increased contractile performmce of thyrotoxic cardiac muscle has not been established . The present studies concern the effects of thyroxine treatment on cardiac myosin ATPase activity in animals with aortic constriction . The "chronic" animal model used for these investigations is also characterized . Methods Aortic constriction : The abdominal aorta was constricted immediately below the diophrogm in ma e, Prague-Dawley rats under ether anesthesia . A metal rod of specific diameter was placed next to the aorta, a ligature was tied around the rod and the aorta, completely occluding the vessel, and the rod was then withdrawn . The same procedure was followed for sham operated animals with the exception that the ligature was used only to temporarily occlude the aorta and was then removed with the metal rod . Preparation of cardiac actom sin : Cardiac tissue was obtained and prepared for myosin an actomyosin extraction according to methods previously described (20) . The animals were sacrificed by decapitation and the beating heart was rapidly excised and placed in cold distilled water . All subsequent procedures were carried out at 4°C . A finely minced homogeneous mixture of right and left ventricular tissue was used for either myosin or actomyosin extraction . Quantitative extraction and purification of cardiac actomyosin was performed according to a described method (20), which is summarized as follows: Approximately 0 .5 grams of minced ventricular tissue was homogenized For b minutes with 5 volumes of extraction solution (0 .3 M KCI, 0 .15 M Kg HP04 , 0 .15 M KH 2 P04 and 0 .8 mM ATP 2; final pH, 6 .5) . Actomyosin was extracted overnight (16-20 hr) with gentle stirring . The homogentle was centrifuged at 18,500 g for 15 minutes and the clear supernatant extract was saved . The sediment was resuspended in 0.6 M KCI and centrifuged at 18,500 g for 10 minutes. The supematmts were combined and diluted with water to ionic strength 0.065 . The actomyosin was allowed to precipitate for two hours and was then collected by centrifugation at 1600 g . The precipitate was then washed with 0 .065 M KCI . A portion of the actomyosin was resuspended in water and assayed for protein nitrogen by a modified biuret procedure (21) . A similar portion of actomyosin was dissolved by the addition of solid KCI (final concentration, 0 .4 M) and stored overnight at 5°C . Actomyo sin suspensions were assayed for ATPase activity on the following day. 1 antes ua omogen zer, matching surfaces ground glass . 2 Crystalline di-Na ATP (Sigma) adjusted with KOH to pH 7 .4 .
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Preparation of cardiac myosin : Complete details for the preparation of cardiac myosin have en previous y scri e ) . Essentially, the procedure is a modification of techniques described by Brahms and Kay (23) . Parts of other methods are incorporated, including the three successive precipitation-dissolution cycles outlined by Shiverick et al . (24) and a final overnight dialysis of the concentrated myosin solution employed by Mueller et al . (25) . Myosin prepared by this procedure showed absorbante ratios for 280 nrq/260 nm ranging from 1 .47 to 1 .58. Shiverick et al . (7, 24) demonstrated that myosin with an A280 nrrJA260 nm above 1 .40 contains less thin 1% nucleic acid contamination . These investigators have noted the importmce of A28p nrrJA260 nm ratios as a criterion for documenting the purity of myosin preparations . They have also demonstrated that cardiac myosin preparations showing high A280 nm/A26p nn~, ratios contain no detectable contamination by actin, troponin or tropomyosin as determined by SDS polyacrylomide gel electrophoresis (7, 24) . ATPase assay: The components of reaction mixtures are described in the individual assays . ATPase activity was determined by measurement of the rate of inorganic phosphate liberation . Protein was added to the reaction mixtures last, at 5°C . The temperature of the assay tubes was then equilibrated to 25oC . Reactions were initiated by the addition of ATP and terminated by deproteinizotion with 4 volumes of cold, 4% trichloroacetic acid The hydrolytic rates were linear within the assay periods which were used (26) . Inorgmic phosphate was determined by the method of Fiske and SubbaRow (27) . Results In m initial study of the effects of aortic constriction on heart weight and mortality in the rat, it was determined that a rat in the body weight range of 220 to 280 grams with an internal aortic diameter of 0.77 mm following aortic constriction would serve as an optimal experimental model (2) . This degree of aortic constriction produced the greatest cardiac hypertrophy and a 46% mortality . When aortic constriction was more severe, the degree of cardiomegaly was not increased and mortality rose substmtially . Death uwally occurred 1 to 3 days following surgery and in most instances was apparently due to acute pulmonary edema. An aortic stenosis less severe than 0 .77 mm in 250 gram rats produced wb-optimal amounts of cardiac hypertrophy . Changes in body weight, ventricular dry weight, cardiac actomyosin content and ATPase activity were studied at 1, 2, 3 and b weeks following aortic constriction to deter mine a postoperative period during which the effects of thyroxine administration could be most advmtageously studied (Figs. 1 - 4) . Rats with aortic constriction lost body weight during the first week following wrgery but thereafter gained weight rapidly. By the sixth postoperative week, the mean body weights of sham operated and constricted animals were essentially the same (Fig . 1) . The mean dry weight of the combined right and left ventricles of constricted mimals rapidly increased during the first week following surgery (Fig . 2) . Increases in ventricular dry weight of constricted mimals ringed from 3996 at one week, to 51% at b weeks, when compared to sham operated mimals . Ventricular actomyosin content (total mg of ventricular actomyosin as a percentage of dry ventricular weight) tended to be lower in constricted animals than sham operated animals throughout the b week postoperative period (Fig . 3) . However, none of these differ entes were statistically significant .
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FIG . 1 Chrnges in the body weight of rats at intervals of time following aortic constriction (broken line) or sham operation (solid line) . In this figure and in subsequent figures and tables the number of animals in each group is indicated in parentheses . Also, all data are presented as the mean t $ .E .M . Cardiac actomyosin suspensions from constricted and sham operated animals were assayed for ATPase activity in the presence of Cat+ and Mgt+ at pH 6 .8 (Fig . 4) . Actomyosin from constricted animals exhibited statistically significant decreases in ATPase activity at l, 2, 3 and 6 weeks postoperatively, the average decreases being 24 .6, 30 .4, 23 .1 and 25 .6%, respectively . The effects of chronic thyroxine administration were superimposed upon the chronic animal model of aortic constriction described above . Purified cardiac myosin was prepared for these studies . The kinetic parameters of rat cardiac myosin ATPase activity were de termined 2 weeks following sham operation, aortic constriction alone and aortic constriction plus thyroxine administration (100 Na/kg/day for 14 days) . Thyroxine administration was initiated on the day of surgery . The results presented in Fig . 5 show the calculated least squares curves for each assay presented as a double reciprocal plot of enzyme activity vs substrate concentration . Cardiac myosin ATPase activity of mimals with aortic stenosis was significantly depressed at all substrate concentrations . Kinetic analysis revealed an average, 86 .2% decrease in maximal velocity and an average, 84 .4% decrease in Km when compared to sham operated animals (Table 1) . Administration of thyroxine to animals with aortic stenosis prevented, to a great extent, the decrease in enzymatic activity and resulted in a significant increase in both the Vm~ (+355%) and the Km (+277%) of myosin in rats with aortic constriction (Table 1) .
Vol . 25, No . 4, 1979
Myosin ATPase - Aortic Stenoeis and Ty
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A comparison of fresh heart weight for sham operated and experimental mimals (Table 2) demonstrated that thyroxine treatment of animals with aortic stenosis resulted in an additional increment of cardiac hypertrophy when compared to animals with aortic stenosis alone . Aortic constriction produced a 29 .2% (p<0 .0005) increase in fresh heart weight while aortic constriction plus thyroxine produced a 51 .3% (p<0 .0005) increase in fresh heart weight (comparisons with sham operated animals) . Thyroxine treatment increased fresh heart weight by 14 .6% (p~0 .01) when compared to animals with aortic constriction alone. Thyroxine administration reduced survival rate from 53 .4%, observed in rnimals with aortic stenosis alone, to 36 .0% in the group of animals with the combined treatment of aortic constriction plus thyroxine (Table 2) . The mean increase in body weight during a two week postoperative period was significmtly greater in the thyroxine treated group than the group of animals with only aortic constriction (Table 2) . Both experimental groups gained less body weight than the sham operated animals . Di scussi on The chronic study of ventricular dry weight, body weight and actomyosin ATPaseactivity following aortic constriction was useful for characterization of a model of compensotory cardiac hypertrophy in the rat (Figs . 1-4) . The degree of aortic constriction was severe and the mortality rate was high (46%) . However, some animals survived by meets of a rapid compensatory cardiac hypertrophy, most of which occurred during the first postoperative week (Fig . 2) . Beznak has demonstrated that the cardiac hypertrophy seen following aortic constriction, in the rat, is essentially complete by the seventh postoperative day, a time when cardiac output and cardiac reserve force have returned to near normal levels (29) . The rapid recovery of normal body weight in young rats with aortic stenosis (Fig . 1), which begins after the first postoperative week, appears to be related to the cardiac compensation which has occurred . Actomyosin content as a percentage of dry ventricle weight tended to be lower in rats with aortic stenosis than in sham operated mimals . These changes were not statistically significant at any of the individual time periods of measurement (Fig . 3) . A decreased contractile protein content during the early stages of experimental cardiac hypertrophy has been reported (30-33) and appears to be related to m increased proliferation of mhochrondria at this time (30, 34-36) . Although the rats with aortic constriction appeared to have compensated completely by the sixth week following surgery, cardiac actomyosin ATPase activity remained depressed to approximately 75% of that seen in sham operated rats (Fig . 4) . Depressed cardiac myosin and actomyosin ATPase activities have frequently been reported in states of severe work induced hypertrophy and also in cardiac failure in animals and man . Whether depressed myosin ATPase activity is a defect of the overstressed myocardium, or is part of a compensatory mechanism to reduce energy utilization, is unknown . There is evidence to suggest that myosin, which appears to be an iscenzyme system, demonstrates chmges in substructure and ATPase activity in response to altered functional states (4, 6, 12, 14, 18, 37) . Calcium stimulated cardiac myosin ATPase activity, measured 2 weeks following aortic constriction, was depressed throughout the range of substrate concentrations (Fig . 5). The decreases in Km and Vm~ of myosin of constricted animals were similar to the findings of Shiverick et al . for pulmonary artery constriction in the rabbit (7) . Using SDS poly-
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acrylamide gel electrophoresis, these investigators also demonstrated that there were no chmges in myosin light chains in hypertrophied rabbit myocardium (7) . They concluded that the decreases in Km and Vm~ were not attributable to changes in the composition of the major myosin subunits but more probably involved subtle modifications which influenced the active site . Katagiri and Morkin (38) have likewise found no evidence for chmges in the content or composition of calf myosin subunits in left ventricular hypertrophy produced by aorti c coarctati on .
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Vol . 25, No, 4, 1979
the decrease in the Km and maximal velocity of myosin ATPase in rats with aortic stenosis (Fig . 5, Table 1) . Although the dosage of thyroxine did not completely restore the Vm~ and Km of myosin from constricted animals to normal levels, it did increase the Vm~ac by 335% (p<0 .0025) and the Km by 227% (p<0 .0025) when compared to animals with aortic constriction alone . It is of interest that thyroxine, which does not affect C~ + activated ATPase in the normal rat (17, 39), effectively increased the depressed Ca t+ activated cardiac myosin ATPase activity in rats with aortic constriction (Fig . 5 Table 1) . TABLE 2 Effect of Aortic Constriction Plus Thyroxine Treatment on Body Weight, Fresh Heart Weight and Survival in the Rat No .
% Survival
Chmge in body weight 2 weeks postoperative (grams) +105 .0 t 6 .0
Fresh heart weight (grams) 0 .996 t 0 .018
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27
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° Compared w i th sham operation (p <0.001) b
Compared with sortic constriction (p <0.005 ) Compared with aortic constriction (p~0 .01)
These findings might have been mticipated since thyroxine has been shown to cause changes in cardiac myosin in normal rabbits (i .e ., increased Vm~ and Km; refs . 10, 12) which are directly opposite to those associated with severe pulmonic stenosis (24) . The apparent decrease in ATP-myosin affinity (md increased ATPase) with thyroxine treatment in normal rabbits has been demonstrated to rewlt from a modification of SH1 thiol groups associated with the active site (9, 12, 18, 39). The observation of an increased Vm~ associated with a decreased affinity is in accord with the reaction mechanism of myosin ATPase proposed by Lymn and Taylor whose kinetic findings indicated that release of the enzyme bound products is the rate limiting step of the reaction (42) . The apparent increased affinity of ATP for myosin in rats with aortic stenosis (Fig . 5, Table 1) might be expected to result, therefore, from molecular modifications which increase SH1 thiol group affinity or availability . This interpretation is supported by the findings of Shiverick et al . (7), who studied the effects of SH1 group modification with p-chloromercuribenzoate on C°2+ _pendent myosin ATPase in rabbits with pulmonary artery constriction . They also concluded that the SH1 groups of myosin from hypertrophied hearts were function-
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ally altered when compared with normal myosin . It is concluded that thyroxine prevented, to a great extent, the decrease in myosin ATPase with aortic stenosis by virtue of its apparently opposite effects upon the myosin molecule . References l. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . Z0 . 21 . 22 . 23 . 24 . 25 . 26 .
G . CONWAY, R. A . HEAZLITT, R.A . FOWLER, M . GABEL, and S . GREEN, J . Mol . 8 Cell . Cardiol . 8 39-51 (1976) . FLITT , odors dissertation, New York Medical College (1978) . M . DRAPER, N . TAYLOR, and N .R . ALPERT, in Cardiac Hypertrophy , pp . 315-331, Academic Press, New York (1971) . J . WIKMAN COFFELT, R . WALSH, C . FENNER, T . KAMIYAMA, A . SALEL, and D .T . MASON, J . Mol . 8. Cell . Cardiol . 8 263-270 (1976) . NB I K, J.F . SPANN, and P .E . POOL, B.M CHANDLER, E .H . Circulation Res . 21 717-725 (1967) . J . IK OFFELT, C . FENNER, R. J . COFFELT, A . SALEL, and D .T . MASON, J . Mol . 8 Cell . Cardiol . 7 219-224 (1975) . K. . , BBB . HA7~ARELL, and N .R . ALPERT, J . Mol . & Cell . Cardio l . 8 837-851 (1976) . ~. OGANESSYAN, T . ZAMINIAN, N . BAY, V. PETROSIAN, A . KOSCHARIAN, I . MARTIROSIAN, and M . ELOYAN, J . Mol . 8 Cell . Cardiol . 5 1-24 (1973) . . . fFIGLY, J . Mol . 8~ G . CONWAY, R.A . HEAZLITT, J . , an Cell . Cardiol . 7 817-826 (1975) . S .F . KU Z NSKI and M .A . INCHIOSA, Jr ., Abstracts of Papers, 164th National Meeting, A.C .S ., Division of Biol . Chem . 128 T . KATAGIRI, A .S . FREEDBERG, and E . MORKIN, Life Sci . 16 1079-1087'1975) . S .K . BANERJEE, I .L . FLINK, and E . MORKIN, CircuTati x._39 319-326 (1976) . 1 7 Y . YAZAKI and M.S . RABEN, Circulation Res . 35 15M .A . INCHIOSA, Jr ., in Biomo ecu ar Structuré and Function, pp . 455-461, Academic Press, New York 19 M.J . GOODKIND, G .F . DAMBACH, and R.J . LUCHI, J . clin . Invest . _48 30A (1969) . S . GUDBJARNASON, M . TELERMAN, C .CHIBA, P .L . WOLF, and R .J . BING, J . Lab.clin . Med . 63 245-253 (1964) . . . , ~C . HJALMARSON, H .E . MORGAN, M .J . BARBETT, and R.A . GOLDSTEIN, Circulation Res . 31 397-409 (1972) . S . F . KUCZYNSKI, Doctors isserta iôn, New York Medical College (1974) . M .A . INCHIOSA, Jr . and A .S . FREEDBERG, in Current Topics in Thyroid Research , pp . 394-402, Academic Press, New York (1965) . M.A . INCHIOSA, Jr ., Am . J . Ph siol . 206 541-546 (1964) . M.A . INCHIOSA, Jr ., J . La . c in . srb3 319-324 (1964) . V . T . PAGANO and M. . I , r . ~es . Commun . Chem . Pathol .& Pharmacol . 23 37-47 (1979) . . J ~AAFufS and C .M . KAY, J . molec . Biol . 5 132-137 (1962) . K . T . SHIVERICK, L .L . THO , m . .ÂLRERT, Biochim . biophys. Acts 393 124-133 (1975) . IT . MUELLER, J . FRANZEN, R .V . RICE, and R .E . OLSON, J . biol . Chem . _239 1447-1456 (1964) . M .A . INCHIOSA, Jr ., Biochem . Pharmac. 16 329-344 (1967) .
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27 . C, H . FISKE and Y . SUBBAROW, J . biol . Chem, 66 375-400 (1925) . 28 . G .W, SNEDECOR and W .G, COCHR , tatistica Methods, 6th Ed ., pp, 135171, Iowa State Univ . Press, Ames (1967) . 29 . M, BEZNAK, Circulation Res . 6 207-212 (1958) . AYEVA, S .S . LUGACHEV, and M .G . PSKNNIKOVA, 30 . F .A . MEERSO , . . E~ I . Cell . Res. 36 568-578 (1964) . 31 . . . , . K~BATA, and W .V . WHITEHORN, Circulation Res . 12 118-124 (1963) . 32 . E. PAGE and L,P . McCALLISTER, Am . J . Cardio l . 31 172-181 (1973) . 33 . A .L . GORDON, M .A . INCHIOSA, Jr ., and D . LET-IR, J . Mol . 8 Cell . Cardio l . 4 543-557 (1972) . 34 . F. Y . HATT, G, BERJAL, J . MORAVEC, and B . SWYGHENDAUW, J . Mol, & Cell . Cardiol . 1 235-247 (1970) . 35 . 1uT .lE~II~OWITZ, V . ASCHENBRENNER, R . ALBIN, N .J . GROSS, R. ZAK, and K .G . NAIR, in Cardiac H ertrophy, pp . 283-299, Academic Press, New York (1971), 36 . M . RABINOWIT , m . . ar io . 31 202-210 (1973) . , iôchim, biophys . Acta 481 68(1`688 (1977) . 37 . L .L . THOMAS and 38 . T . KATAGIRI and E . MORKIN, Bioc im, iop s. cta ßf62-274 (1974) . 39 . Y . YAZAKI and M .S . RABEN, Circu ation Res . 3 208-215(1975) . 40 . M .J . GOODKIND, G .E . DA , P . . H17f~UM, and R .J . LUCHI, Am . J . Physiol . 226 66-72 (1974) . 41 . FLINKand E . MORKIN, Febs Letters 81 391-394 (1477) . 42 . R,W . LYMN and E .W . TAYLOR~iôc,~istry 9 2975-2983 (1970) .