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Effects of severe hemodynamic pressure overload on the properties of canine left ventricular myosin: Mechanism by which myosin ATPase activity is lowered during chronic increased hemodynamic stress
Journal
of Molecular
and Cellular
Cardiolo~g~ (1976)
8, 263-270
Effects of Severe Hemodynamic Pressure Overload on the Properties of Canine Left Ventricular Myosin: Mechanism By Which Myosin ATPase Activity is Lowered During Chronic Increased Hemodynamic Stress JOAN WIKMAN-COFFELT, TEIKO KAMIYAMA, Sec.tion of Cardiovascular
(&eked
ROBERT WALSH, ANTONE SALEL AND
Medicine, Department Medicine, Davis, 6 A%g
1975,
CLAUDIA FENNER, DEAN T. MASON
of Medicine, liniversity of California, CaliSornia, 95616, iiS.A.
accepted in revised
form
16 June
Davis
School
of
1975)
J. WIKMAN-COFFELT, R. M'ALSH, C. FEWER, T. KAMIYAMA, A. SALEL AND D. T. MASON. Effects of Severe Hemodynamic Pressure Overload on the Properties of Canine Left Ventricular Myosin: Mechanism By Which Myosin ATPase Activity is Lowered During Chronic Increased Hemodynamic Stress. Journal of Noleculnr and Cellular Cardioloa (1976) 8, 263-270. Left ventricular myosin ATPase activity. expressed as enzymatic b’,,,, values, was analyzed in dogs subjected to severe left ventricular pressure overload (aortic stenosis). K- and Ca27 activated myosin ATPase activities in the left ventricle (LV) were significantly depressed (P < .Ol) in the experimental animals. For normai Ii+ activated myosin the l,,, values in micromoles of Pi per mg per min were: right ventricle 2.10; left ventricle, 2.84. For Ca’* activated myosin thr r,max values were: right ventricle. 0.77; left ventricle 0.97, when assayed at 37°C. Myosin enzymatic activity in the left ventricle progressively declined following se\-ere aortic banding, reaching a value similar to that observed for normal right ventricular myosin; NH4 - activated left ventricular myosin ATPase activity remained unchanged (7.20 + 0.4 pm01 POd/mg.min). Lrft ventricular myosin from the hearts subject to severe stress simulated normal right vcntricular myosin in ATPase activity, chain proportions and degree of calcium binding, Sormal left ventricular myosin contained approximately lOq6 of the myosin protein concentration in the light chains: myosin from the lrft ventricles of the hemodynamically ovrrloaded hearts contained 20’1; of the myosin protein concentration in the light chains ‘P < ,001). With only one of the myosin light chains binding calcium left ventricular In>-osin from the stressed hypertrophied tissue bound approximately 2 mol Caz.‘- mol-1 In)-osin similar to myosin of thr normal right vmtric Ic: normal Irft ventricular myosin holmd approxirnatclv 1 mol of Ca”’ molk’ myosin. KEY JSORM: Cardiac myosin: ( ‘nl(~ilm~ binding in myosin.
XIyosin
ATPase
nctkity:
Lc.ft ventricle
pressure;
Overload;
1. Introduction In the normal canine heart, the K+ and Ca 2+ activated myosin ATPase values of the right ventricle have been shown to be approximately 25% lower than those of the left ventricle [25]. Further, in these normal animals, myosin of the right ventricle has been found to contain more light chains, to have a higher molecular weight [IS]. and a greater degree of calcium binding [IO] than left ventricular myosin. It
264
J. WIKMAN-COFFELT
ETA.
has also been demonstrated recently that canine right ventricular myosin activities (K~c and Gas+ systems) become elevated during early stages of mild pulmonic stenosis; these activities progressively declined at longer postoperative intervals [26, 271. In contrast, when severe pulmonic stenosis was produced in dogs, K+ and Gas+ activated ATPase levels in the markedly pressure overloaded right ventricles were never elevated, but decreased initially and remained depressed with prolonged duration of stress [23]. During the early periods of mild pulmonic stenosis when K+ and Casf activities of right ventricular myosin were elevated, the enzymatic I’,,, values and myosin chain proportions were similar to those of normal left ventricles [27]. A low myofibrillar ATPase activity has been reported in several models of cardiac dysfunction [I, 2, 14, 151, including aortic regurgitation [19, 201; the biochemical events leading to such an enzymatic alteration have not been defined. From the studies reported here on chronic hemodynamic overloading of the canine left ventricle, a mechanism is formulated by which left ventricular myosin decreases in activity and becomes like that of the right ventricle relative to enzymatic activity, chain proportions and calcium binding during severe cardiac dysfunction induced by left ventricular systolic pressure overload. 2. Materials
and Methods
Surgical intervention and hemodynamic measurements Severe systolic pressure overloading of the left ventricle was caused in 10 dogs by banding of the ascending aorta affecting a transaortic peak systolic pressure gradient of approximately 50 mm Hg. Hemodynamic measurements were made prior to banding and immediately before sacrifice under light pentobarbital anesthesia as described earlier [30]. Myosin pur&ation Hearts were frozen in acetone and dry ice and stored (-4°C). The weights of the free walls of the ventricles were obtained prior to myosin purification. Procedures for purification of myosin have been described earlier [9, 21-241, as well as criteria of purity [12]. In addition to the hemodynamically overloaded hearts, myosin was obtained from left ventricles of normal dogs for control values. Analyses of myosin Enzymatic analyses of K+, Casf and NH4f activated myosin have been described recently [7], as well as calcium binding methods [IO] and procedures for gel electrophoresis [S]. Quantification of protein in myosin chains have also been delineated [8].
SEVERE
HEMODYNAMIC
PRESSURE
OVERLOAD
ON
VENTRICULAR
MYOSIN
265
3. Results In the dogs with severe aortic stenosis, left ventricular systolic and end-diastolic pressures were increased as documented by the measurements attained at 5 and 16 weeks after aortic banding. With 5 weeks of severe aortic stenosis the left ventricular end-diastolic pressure in mm Hg was 6.2 & 1.8 (P < .05) and by 16 weeks of banding rose to 11.4 h 2.1 (P < .Ol j. (Normal values were 3.1 & 1.7.) The left ventricle peak systolic pressure was 167 & 8 mm Hg (P < .Ol) with 5 weeks of severe aortic banding, and 164 rt 7 mm EIg with 16 weeks of severe aortic peak systolic pressure was stenosis (P < .Ol). The normal value for left ventricular 1 l!) j, 7 mm Hg. A comparison of both left and right ventricle weights (dry weight of the free wall) normalized for body weights were studied in both normal and experimental dogs. For the left ventricles with 5 weeks of severe aortic stenosis the value in grams x 10-3 was 2.20 f 0.10 (P < .Ol), and with 16 weeks of severe aortic banding the value was 1.80 & 0.12 (P e< .05). The value for the normal left ventricle was 1.50 & 0.05. The weights of the free wall of the right ventricles relative to body weights were not significantly different in the experimental zx normal dogs ; the normal value in grams x 10-3 was 0.71 & 0.03. Aunt Weeks
sknosis postoprratlve
15 16 16
myosin FIGURE 1. Average enzymatic Vmax values for K+ and Ca 3+ activated left ventricular ATPase. Incubation mixture contained 0.1 M Tris. HCl, pH 7.5, 0.001 M EDTA, 0.65 M KC1 and varying concentrations of ATP for K+ activated myosin and 0.2 M Tris maleate, pH 6.5, 0.01 M Ca Cl2 and varying concentrations of ATP were used for analyzing Ca2f activated myosin. Conditions wrre as those described earlier except myosin was assayed at 37°C. a higher temperature than that used previously [25, 261. The rXax values were obtained from double reciprocal plots of rate ~8s substrate concentration. Mean values of percent decreases in V,,, (& S.E.M.) are shown at 5 and 16 weeks after aortic banding. Normal values of left ventricular myosin V,,, values in pmol PO4 mg.min.-1 relative to the activation cation used were: K * = 2.84 & 0.22; Cat+ = 0.97 & 0.14: NHJ+ = 7.20 $ 0.40. Statistical comparisons relate postoperative to normal values (for all analysrq I’ < .Ol).
266
J.
WIKhlAN-COFFELT
El- /IL.
Canine left ventricular myosin from normal hearts, hearts after 5 weeks of severe aortic stenosis, and hearts after 16 weeks of severe aortic banding were electrophoretically pure without hydrolytic breakdown, and had identical electrophoretic mobility on dodecylsulfate gels; gel patterns were similar to those shown earlier [2.5, 271. The enzymatic V,,, values of left ventricular myosin progressively declined following severe aortic stenosis (Figure 1) with K+ or Ca”‘- used as the activator cation. With NHd+ as the activator cation left ventricular myosin activity was not altered in the hemodynamically overloaded heart.
0
Heavy
chams
Light
chaws
FIGURE 2. For a comparative analysis of protein present in the chains of myosin, left ventricular myosin was analyzed on dodecylsulfate gels as described earlier [IZ] and protein concentration determined by quantification of eluted dye [8]. Chain proportions were assessed in the myosin analyzed for ATPase activity. N = normal left Lentricle; PO = left ventricular pressure overload (aortic stenosis); Wk : postoperative weeks. Statistical values compare PO to K.
Accompanying the decline in K+ and Gas+ activated myosin ATPase values in the canine left ventricles subjected to chronic left ventricular pressure overload there was an increase in the proportion of light to heavy chains in the myosins (Figure 2). Thus, the left ventricular myosin in the hemodynamically overloaded animals became similar to that previously observed in normal right ventricular myosin, both in regard to enzymatic activity and chain proportions [25]. With an increase in proportion of light chains in myosin from relatively hypertrophied left ventricular tissue, there was also an increase in the number of calcium binding sites (Figure 3), again simulating the number of calcium binding sites present in normal right ventricular myosin [IO]. The K+ and Cast activated myosin ATPase V mx values for right ventricular myosin were elevated 20% with 16 weeks of severe aortic stenosis. Right ventricular myosin ATPase values were never depressed as observed in these studies but were significantly elevated (P < .O 1).
SEVERE
HEMODYNAMIC
PRESSURE
OVERLOAD
ON
VENTRICULAR
MYOSIN
267
FIGURE 3. Representative Scatchard plots for the binding of Ca” + by (a! normal left ventricular myosin, (b) left ventricular myosin 16 weeks after pressure overload. Binding parameters were calculated from least squares regression lines for Scatchard plots [17]. The molecular weight for left ventricular myosin was established by sedimentation equilibrium studies [18]. The number of calcium binding sites for normal left ventricular myosin was 1.30 + 0.05: for left ventricular myosin subjected to severe pressure overload it was 2.35 +. 0.09 (P < .Ol I : (r, -~ 31.
4. Discussion In the present study, analyses of enzymatic V msx values of myosin of the left ventricle were carried out at a stage in dogs with relative hypertrophy of this chamber, but in which congestive heart failure had not developed. Thus, the myosin was not cleaved; subluxation and cleavage of myosin have been observed by Bing et al. [4, 241 in humans with heart failure. Unlike mild pressure overload of the left ventricle, induction of severe pressure overload of this chamber caused decreases in both K+ and Gas+ activated myosin ATPase activities. Severe aortic stenosis in the present study was defined as an aortic-systemic arterial peak systolic pressure gradient of approximately 50 mm Hg at time of sacrifice, as compared to mild left ventricular pressure overload with a pressure gradient of 20 mm Hg. The latter was defined as mild aortic stenosis [29]. With mild aortic stenosis myosin ATPase
J. WIKMAN-COFFELT
268
E7AL.
activity was elevated. Unlike Hoar et al. [II] who found no difference in K+ activated myosin ATPase but a significant difference in Caz+ activated myosin ATPase in cats, we noted in dogs a significant decrease in both K+ and Ca2+ myosin ATPase activities in the left ventricle but not the right ventricle in animals subjected to severe aortic stenosis. The initial observations at 5 weeks following the onset of severe left ventricular systolic pressure overloading indicated a decline in K+ and Caz+ activated myosin ATPase activities; earlier deviations from normal values were not found. By 16 weeks of severe aortic stenosis left ventricular K+ and Ca2f myosin enzymatic activities were diminished further to 25% below those of normal values. This decrease in myosin ATPase Vmax values is consistent with the diminution in contractility observed by other investigators [5, 16, 211. In previous reports from our laboratory on the properties of right and left ventricular myosins in normal dogs, it was shown that there was a greater concentration of myosin light chains relative to heavy chains, less myosin activity and an increased number of calcium binding sites in myosin of the right ventricle as compared to the left ventricle [25]. In the study presented here on the properties of myosin in the left ventricle in hemodynamic overloading, there was a decrease in left ventricular myosin ATPase activity with an increase in proportion of myosin light chains present and an increase in the number of calcium binding sites present in myosin. Thus, the myosin in the left ventricle of these hemodynamically overloaded animals became similar in its characteristics to that of normal right ventricular myosin. It was proposed by Draper et al. [6] that the depression in actomyosin ATPase following severe aortic stenosis in guinea pigs may be caused by a decreased Ca2+ sensitivity or an alteration in myosin Caaf binding sites. A greater number of light chains, and an increase in the number of calcium binding sites appear to be associated with a decrease in both K+ and Ca2+ activated myosin ATPase V,,, values.
Acknowledgements This study was supported by Research Program Project Grant HL-14780 and AM-NS- 167 16-01 AMP, from the National Institutes of Health, and the American Heart Association (Golden Empire Chapter) and a GRS Grant to the University of California, Davis.
REFERENCES 1. ALPERT, N. R., relates of cardiac 2. ALPERT, N. R. congestive heart
B. B. & HALPERN, W. Mechanical and biochemical corhypertrophy. Circulation Research 34 and 35, II-7 1, II-8 1 (1974). & GORDON, M. S. Myofibrillar adenosine triphosphatase activity in failure. American Journal of Physiology 202, 940-946 (1962). HAMRELL,
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l%rk
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attractions
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ions.
:&tnnZJ
of
the
51. 660-670 (1949).
D. T. & WIKMAN-COFFELT, J. Molecular weight studies of canine FEBS Lettfr.r 43, 104-106 (1974 I. SWYNGHEDAUW, B., BOUVERET~ P. & PIGUET. \-. 1.. Le doasage du phosphore inorganique en presence d’adenosine tri-phosphate. .4nnnlac de Liologie clinique 28, 235-247 ,197O). SWYNGHEDAUW, B.. KLOTZ, C., LEGER, J. J., PRETESCILLE, LI., CHATEAU, F., DRONNE, ;CI. & COUTALON, A. Heart myosin adenosine triphosphatase and light subunits in experimental chronic aortic insufficiency in the rabbit. Journal of ,%folecnlar and Cellular cnrddog’ 5, 501-514 (1973). SMITH,
cardiac
19.
PRESSURE
BERSON, G. & SWYNGHEDAUW, B. Cardiac myofibrillar ATPase and electrophoretic pattern in experimental heart failure produced by a two-step mechanical overloading in the rat. Cardiovascular Research 7, 464-469 (1973). BING, R. J. & KAKO, K. Contractile proteins of heart muscle in man. Circulation 24, 483-490 (1961). CHANDLER, B. M.. SOKNENBLICK, E. H., SPANN, J. F. tit POOL, P. E. Association of depressed myolibrillar adenosine triphosphate and reduced contractility in experimental heart failure. Circulation Research 21, 717-725 (1967). DRAPER, M., TAYLOR, N. & ALPERT, R. N. Alteration in the contractile protein in hypertrophied guinea pig hearts in Cardiac Hypertrophy. N. A. Alpert, Ed. pp. 3 15-33 1 New York: Academic Press (1971). FENNER, C., MASON, D. T., ZEIJS, R. & WIKMAN-COFFELT, J. Regulatory properties of myocardiac myosin. Proceedin,<.c of the National Acrrdeny qf Sciences CL%4 70, 3205-3209 (1973). FENXER. CL, TRAUT, R. R.. MASON, D. T. & WIKMAN-COFFEL.~, J, Quantification of coomassie blue stained protein in polyacrylamide gels based on analyses of eluted dye. Analytical Biochemistry 63, 595-602 (1975). FENNER, C., VALENTINE, R., MASON, D. T. (Ir W’IKMAN-COFFELT, J. Purification and characterization of cardiac tropomyosins. Preparative Biochemistry 5, 189-197 (1975). FENNER, C., ANDREASEN, ‘1‘., MASON, D. T. & WIKMAN-COFFELT, J. Variances in calcium binding of canine right and left ventricular myosins. Biochemical and Biophysical Research Communications (Submitted). HOAR, F. P., SHIVERICK. ‘I. K., HAMRELL. B. B. & ALPERT, R. iX. The ATPase activity and Gas’ affinity of myosin B from hypertrophied nonfailing cat hearts. In Cardiac Hypertrophy. N. A. Alpert. Ed. pp. 315-331. New York: Academic Press (1971). A\ICPHERSON, J., TRAUT, R. R., MASON, D. ‘I’.. ZELIS. R. & WIKMAN-COFFELT, J. Resolution of myocardiac myosin light chains by two-dimensional gel electrophoresis, Journal of Biological Chemistry 249, 994996 (1974). MEERSON, F. Z. Dynamics of nucleic acid and protein synthesis of myocardium in hyperfunction and hypertrophy. Circulation Research 25 Suppl. 2, 82-155 (1969). NEBEL, M. L. & BING, R. J. Contractile proteins of normal and failing human heart. .IrchiveJ oj’lnternal .Ifedicine 111, 190-195 (1963). OC;ANESSY.I\N, S., ZAMINIAN, T., BAY, N., PETROSIAN, v., KOSCH~AR~AN, A., MARTIROSIAN, I. (3r ELOYAN, M. Structural and functional chauges of the contractile proteins in experimental-induced cardiac hypertrophy in animals. and heart failure in man. Journal of .Ifolecular and Cellular CardioloD 5, 1-9-k i 1973). OSCAR, H. L., BIN<:, S. 51.. B.~RRY, L. F. & HERBERT. J. L., Mechanical properties of rat cardiac muscle during experimental hypertrophl,. Circulation Re.rearrh 28, 234-245 [1971).
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MASON,
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VITALI-MAZZA, L., ~~VERSA, l’.. 'I‘EDESCHI, F., .UASTANDREA, K.. U~VILLA. \'. & VIsmLI, 0. Ultrastructural basis of acute left ventricular failure from severe acute aortic stenosis in the rabbit. j’ownal qf Molecular and Cellular Cardiolo~gy 4, 661-671 (1972). WIKMAN-COFFELT, J.. ZELIX. K., FENNE.R, C. &Z MASOK. 1). ‘I‘. Comparative purification of myocardial myosin and anti,qcllic ~prcificil? of the Iwo liqhl chains. Preparative Biochemistry 3, -139-W? ( 1973). WIKMAN-COFFELT. .J., ZELIS. K., FENNER, C. 8r MASON, D. T. Myosin chains ofmyocardial tissue 1. Purifcation and immunological properties of myosin heavy chains. Biochemical and Biopt~~iicai Research Communications 51, 1097--l 105. WIKMAN-COFFELT. J.. ZELIS, R., FENNER. C. & MASON. D. ‘r. Studies on the synthesis and degradation of light and hcav\chains of cardiac myosin. Journal of Biological Chemistry 248, 5206-5210 (1973). ~VIKMAN-COFFELT. J., FEWER. C.. SMITH, A. & MASON, D. ‘1’. Comparative analyses of kinetics and subunits of myosins from canine skeletal ~~lusclc and cardiac tissue. Journal of Biological Chcmihtry 250, 1257-1262 (1975). WIKMAN-COFFELT. J., FENNER? C.. COFFELT, R. J.- SALEL, h., E;AMIYAMA, 'r. & MASON, D. ‘I‘. Chronological effects of mild pressure overload on myosin ATPase activity in the canine right ventricle. Journal of Molecular and Ceklar Gwdiologv 7, 2 1% 22-2 (1975). WIKMAN-COFFELT, J.. FENNER, C.. ~ICPHERSON, J.. ZELIS, R. & MASON. D. T. Alterations of subunits composition and ATPasr activity of myosin in early hypertrophied right ventricles of dogs with mild experimental pulmonic stenosis. Journal of Molecular and Cellular Cardiology 7, 513-522. WIKMAN-COFFELT, J.? FENNER, C., SALEL, A., ~~AMIYAMA, 'I'. & MASON, 1). T. Comparison of mild us severe pressure overload on the enzymatic activity of myosin in the canine ventricles. Bioch.emical Aledicine (In Press). WIKMAN-COFFELT, J., MASON, D. T. & LAKS, l&I. Effects of mild aortic stenosis on myosin ATPase activity, Circulation Research (Submitted). ZELIS, R., WIKMAN-COFFELT, J., KAMIYAMA, T., PENG, C. L., SALEL, i\., AMSTERDAM, E. & MASON, D. T. Acute right ventricular stress as a stimulus for left ventricular RNA and protein synthesis. In Myocardiology. Bajusz, E., Ed. 3> 625-635. Baltimore: TJniversit? Park Press (1973).
of Molecular
and Cellular
Cardiolo~g~ (1976)
8, 263-270
Effects of Severe Hemodynamic Pressure Overload on the Properties of Canine Left Ventricular Myosin: Mechanism By Which Myosin ATPase Activity is Lowered During Chronic Increased Hemodynamic Stress JOAN WIKMAN-COFFELT, TEIKO KAMIYAMA, Sec.tion of Cardiovascular
(&eked
ROBERT WALSH, ANTONE SALEL AND
Medicine, Department Medicine, Davis, 6 A%g
1975,
CLAUDIA FENNER, DEAN T. MASON
of Medicine, liniversity of California, CaliSornia, 95616, iiS.A.
accepted in revised
form
16 June
Davis
School
of
1975)
J. WIKMAN-COFFELT, R. M'ALSH, C. FEWER, T. KAMIYAMA, A. SALEL AND D. T. MASON. Effects of Severe Hemodynamic Pressure Overload on the Properties of Canine Left Ventricular Myosin: Mechanism By Which Myosin ATPase Activity is Lowered During Chronic Increased Hemodynamic Stress. Journal of Noleculnr and Cellular Cardioloa (1976) 8, 263-270. Left ventricular myosin ATPase activity. expressed as enzymatic b’,,,, values, was analyzed in dogs subjected to severe left ventricular pressure overload (aortic stenosis). K- and Ca27 activated myosin ATPase activities in the left ventricle (LV) were significantly depressed (P < .Ol) in the experimental animals. For normai Ii+ activated myosin the l,,, values in micromoles of Pi per mg per min were: right ventricle 2.10; left ventricle, 2.84. For Ca’* activated myosin thr r,max values were: right ventricle. 0.77; left ventricle 0.97, when assayed at 37°C. Myosin enzymatic activity in the left ventricle progressively declined following se\-ere aortic banding, reaching a value similar to that observed for normal right ventricular myosin; NH4 - activated left ventricular myosin ATPase activity remained unchanged (7.20 + 0.4 pm01 POd/mg.min). Lrft ventricular myosin from the hearts subject to severe stress simulated normal right vcntricular myosin in ATPase activity, chain proportions and degree of calcium binding, Sormal left ventricular myosin contained approximately lOq6 of the myosin protein concentration in the light chains: myosin from the lrft ventricles of the hemodynamically ovrrloaded hearts contained 20’1; of the myosin protein concentration in the light chains ‘P < ,001). With only one of the myosin light chains binding calcium left ventricular In>-osin from the stressed hypertrophied tissue bound approximately 2 mol Caz.‘- mol-1 In)-osin similar to myosin of thr normal right vmtric Ic: normal Irft ventricular myosin holmd approxirnatclv 1 mol of Ca”’ molk’ myosin. KEY JSORM: Cardiac myosin: ( ‘nl(~ilm~ binding in myosin.
XIyosin
ATPase
nctkity:
Lc.ft ventricle
pressure;
Overload;
1. Introduction In the normal canine heart, the K+ and Ca 2+ activated myosin ATPase values of the right ventricle have been shown to be approximately 25% lower than those of the left ventricle [25]. Further, in these normal animals, myosin of the right ventricle has been found to contain more light chains, to have a higher molecular weight [IS]. and a greater degree of calcium binding [IO] than left ventricular myosin. It
264
J. WIKMAN-COFFELT
ETA.
has also been demonstrated recently that canine right ventricular myosin activities (K~c and Gas+ systems) become elevated during early stages of mild pulmonic stenosis; these activities progressively declined at longer postoperative intervals [26, 271. In contrast, when severe pulmonic stenosis was produced in dogs, K+ and Gas+ activated ATPase levels in the markedly pressure overloaded right ventricles were never elevated, but decreased initially and remained depressed with prolonged duration of stress [23]. During the early periods of mild pulmonic stenosis when K+ and Casf activities of right ventricular myosin were elevated, the enzymatic I’,,, values and myosin chain proportions were similar to those of normal left ventricles [27]. A low myofibrillar ATPase activity has been reported in several models of cardiac dysfunction [I, 2, 14, 151, including aortic regurgitation [19, 201; the biochemical events leading to such an enzymatic alteration have not been defined. From the studies reported here on chronic hemodynamic overloading of the canine left ventricle, a mechanism is formulated by which left ventricular myosin decreases in activity and becomes like that of the right ventricle relative to enzymatic activity, chain proportions and calcium binding during severe cardiac dysfunction induced by left ventricular systolic pressure overload. 2. Materials
and Methods
Surgical intervention and hemodynamic measurements Severe systolic pressure overloading of the left ventricle was caused in 10 dogs by banding of the ascending aorta affecting a transaortic peak systolic pressure gradient of approximately 50 mm Hg. Hemodynamic measurements were made prior to banding and immediately before sacrifice under light pentobarbital anesthesia as described earlier [30]. Myosin pur&ation Hearts were frozen in acetone and dry ice and stored (-4°C). The weights of the free walls of the ventricles were obtained prior to myosin purification. Procedures for purification of myosin have been described earlier [9, 21-241, as well as criteria of purity [12]. In addition to the hemodynamically overloaded hearts, myosin was obtained from left ventricles of normal dogs for control values. Analyses of myosin Enzymatic analyses of K+, Casf and NH4f activated myosin have been described recently [7], as well as calcium binding methods [IO] and procedures for gel electrophoresis [S]. Quantification of protein in myosin chains have also been delineated [8].
SEVERE
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PRESSURE
OVERLOAD
ON
VENTRICULAR
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265
3. Results In the dogs with severe aortic stenosis, left ventricular systolic and end-diastolic pressures were increased as documented by the measurements attained at 5 and 16 weeks after aortic banding. With 5 weeks of severe aortic stenosis the left ventricular end-diastolic pressure in mm Hg was 6.2 & 1.8 (P < .05) and by 16 weeks of banding rose to 11.4 h 2.1 (P < .Ol j. (Normal values were 3.1 & 1.7.) The left ventricle peak systolic pressure was 167 & 8 mm Hg (P < .Ol) with 5 weeks of severe aortic banding, and 164 rt 7 mm EIg with 16 weeks of severe aortic peak systolic pressure was stenosis (P < .Ol). The normal value for left ventricular 1 l!) j, 7 mm Hg. A comparison of both left and right ventricle weights (dry weight of the free wall) normalized for body weights were studied in both normal and experimental dogs. For the left ventricles with 5 weeks of severe aortic stenosis the value in grams x 10-3 was 2.20 f 0.10 (P < .Ol), and with 16 weeks of severe aortic banding the value was 1.80 & 0.12 (P e< .05). The value for the normal left ventricle was 1.50 & 0.05. The weights of the free wall of the right ventricles relative to body weights were not significantly different in the experimental zx normal dogs ; the normal value in grams x 10-3 was 0.71 & 0.03. Aunt Weeks
sknosis postoprratlve
15 16 16
myosin FIGURE 1. Average enzymatic Vmax values for K+ and Ca 3+ activated left ventricular ATPase. Incubation mixture contained 0.1 M Tris. HCl, pH 7.5, 0.001 M EDTA, 0.65 M KC1 and varying concentrations of ATP for K+ activated myosin and 0.2 M Tris maleate, pH 6.5, 0.01 M Ca Cl2 and varying concentrations of ATP were used for analyzing Ca2f activated myosin. Conditions wrre as those described earlier except myosin was assayed at 37°C. a higher temperature than that used previously [25, 261. The rXax values were obtained from double reciprocal plots of rate ~8s substrate concentration. Mean values of percent decreases in V,,, (& S.E.M.) are shown at 5 and 16 weeks after aortic banding. Normal values of left ventricular myosin V,,, values in pmol PO4 mg.min.-1 relative to the activation cation used were: K * = 2.84 & 0.22; Cat+ = 0.97 & 0.14: NHJ+ = 7.20 $ 0.40. Statistical comparisons relate postoperative to normal values (for all analysrq I’ < .Ol).
266
J.
WIKhlAN-COFFELT
El- /IL.
Canine left ventricular myosin from normal hearts, hearts after 5 weeks of severe aortic stenosis, and hearts after 16 weeks of severe aortic banding were electrophoretically pure without hydrolytic breakdown, and had identical electrophoretic mobility on dodecylsulfate gels; gel patterns were similar to those shown earlier [2.5, 271. The enzymatic V,,, values of left ventricular myosin progressively declined following severe aortic stenosis (Figure 1) with K+ or Ca”‘- used as the activator cation. With NHd+ as the activator cation left ventricular myosin activity was not altered in the hemodynamically overloaded heart.
0
Heavy
chams
Light
chaws
FIGURE 2. For a comparative analysis of protein present in the chains of myosin, left ventricular myosin was analyzed on dodecylsulfate gels as described earlier [IZ] and protein concentration determined by quantification of eluted dye [8]. Chain proportions were assessed in the myosin analyzed for ATPase activity. N = normal left Lentricle; PO = left ventricular pressure overload (aortic stenosis); Wk : postoperative weeks. Statistical values compare PO to K.
Accompanying the decline in K+ and Gas+ activated myosin ATPase values in the canine left ventricles subjected to chronic left ventricular pressure overload there was an increase in the proportion of light to heavy chains in the myosins (Figure 2). Thus, the left ventricular myosin in the hemodynamically overloaded animals became similar to that previously observed in normal right ventricular myosin, both in regard to enzymatic activity and chain proportions [25]. With an increase in proportion of light chains in myosin from relatively hypertrophied left ventricular tissue, there was also an increase in the number of calcium binding sites (Figure 3), again simulating the number of calcium binding sites present in normal right ventricular myosin [IO]. The K+ and Cast activated myosin ATPase V mx values for right ventricular myosin were elevated 20% with 16 weeks of severe aortic stenosis. Right ventricular myosin ATPase values were never depressed as observed in these studies but were significantly elevated (P < .O 1).
SEVERE
HEMODYNAMIC
PRESSURE
OVERLOAD
ON
VENTRICULAR
MYOSIN
267
FIGURE 3. Representative Scatchard plots for the binding of Ca” + by (a! normal left ventricular myosin, (b) left ventricular myosin 16 weeks after pressure overload. Binding parameters were calculated from least squares regression lines for Scatchard plots [17]. The molecular weight for left ventricular myosin was established by sedimentation equilibrium studies [18]. The number of calcium binding sites for normal left ventricular myosin was 1.30 + 0.05: for left ventricular myosin subjected to severe pressure overload it was 2.35 +. 0.09 (P < .Ol I : (r, -~ 31.
4. Discussion In the present study, analyses of enzymatic V msx values of myosin of the left ventricle were carried out at a stage in dogs with relative hypertrophy of this chamber, but in which congestive heart failure had not developed. Thus, the myosin was not cleaved; subluxation and cleavage of myosin have been observed by Bing et al. [4, 241 in humans with heart failure. Unlike mild pressure overload of the left ventricle, induction of severe pressure overload of this chamber caused decreases in both K+ and Gas+ activated myosin ATPase activities. Severe aortic stenosis in the present study was defined as an aortic-systemic arterial peak systolic pressure gradient of approximately 50 mm Hg at time of sacrifice, as compared to mild left ventricular pressure overload with a pressure gradient of 20 mm Hg. The latter was defined as mild aortic stenosis [29]. With mild aortic stenosis myosin ATPase
J. WIKMAN-COFFELT
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activity was elevated. Unlike Hoar et al. [II] who found no difference in K+ activated myosin ATPase but a significant difference in Caz+ activated myosin ATPase in cats, we noted in dogs a significant decrease in both K+ and Ca2+ myosin ATPase activities in the left ventricle but not the right ventricle in animals subjected to severe aortic stenosis. The initial observations at 5 weeks following the onset of severe left ventricular systolic pressure overloading indicated a decline in K+ and Caz+ activated myosin ATPase activities; earlier deviations from normal values were not found. By 16 weeks of severe aortic stenosis left ventricular K+ and Ca2f myosin enzymatic activities were diminished further to 25% below those of normal values. This decrease in myosin ATPase Vmax values is consistent with the diminution in contractility observed by other investigators [5, 16, 211. In previous reports from our laboratory on the properties of right and left ventricular myosins in normal dogs, it was shown that there was a greater concentration of myosin light chains relative to heavy chains, less myosin activity and an increased number of calcium binding sites in myosin of the right ventricle as compared to the left ventricle [25]. In the study presented here on the properties of myosin in the left ventricle in hemodynamic overloading, there was a decrease in left ventricular myosin ATPase activity with an increase in proportion of myosin light chains present and an increase in the number of calcium binding sites present in myosin. Thus, the myosin in the left ventricle of these hemodynamically overloaded animals became similar in its characteristics to that of normal right ventricular myosin. It was proposed by Draper et al. [6] that the depression in actomyosin ATPase following severe aortic stenosis in guinea pigs may be caused by a decreased Ca2+ sensitivity or an alteration in myosin Caaf binding sites. A greater number of light chains, and an increase in the number of calcium binding sites appear to be associated with a decrease in both K+ and Ca2+ activated myosin ATPase V,,, values.
Acknowledgements This study was supported by Research Program Project Grant HL-14780 and AM-NS- 167 16-01 AMP, from the National Institutes of Health, and the American Heart Association (Golden Empire Chapter) and a GRS Grant to the University of California, Davis.
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