Effect of sulfhydryl group modification on age-associated alteration of actomyosin ATPase activity in human myocardium

Summary Cardiac ventricular actomyosin was prepared from autopsy samples from humans ranging in age from one to 83 years, and its Ca²⁺-ATPase and K⁺-EDTA-ATPase activities were determined in the presence or absence of a sulfhydryl reagnet, N-ethylmaleimide (NEM). The Ca²⁺-ATPase activity increased in the presence of appropriate concentrations of NEM. The extent of the stimulation of Ca²⁺-ATPase activity by NEM decreased significantly with age. The ratio of K⁺-EDTA-ATPase activity to Ca²⁺-ATPase activity also decreased with age. This suggests that there is an age-related modification of sulfhydryl groups in the myosin molecule.     

Increased cardiac myosin ATPase activity as a biochemical adaptation to running training: enhanced response to catecholamines and a role for myosin phosphorylation

The effects were studied of prior running training on protein phosphorylation and adenosine triphosphatase (ATPase) activities of natural actomyosin isolated from perfused rat hearts. Myosin Ca²⁺-ATPase activities were significantly higher in running-trained hearts than in controls, whereas the Ca²⁺-stimulated, Mg²⁺-dependent ATPase activities of natural actomyosin were not changed. After treatment of isolated perfused hearts with the β-agonist isoproterenol, both troponin-I and myosin P light chains became phosphorylated. Troponin-I phosphorylation (1 mol/mol) was the same in both sets of hearts and was accompanied by similar changes in cardiac cyclic AMP contents. The V_max values for myosin Ca²⁺-ATPase activity were increased after isoproterenol treatment in all the perfused hearts, but to a significantly greater extent in the hearts of running trained animals; this was correlated with enhancement of both the rate and extent of myosin P light chain phosphorylation. Enhanced Ca²⁺-dependent myosin P light chain phosphorylation, further enhanced by β-adrenergic stimulation, represents, at the molecular level, a biochemical response to running training.     

Effects of extracellular calcium concentrations on myosin P light chain phosphorylation in hearts from running-trained rats

Certain biochemical responses to clevated perfusate calcium concentrations were studied in the isolated hearts of sedentary and running-trained rats from which natural actomyosin was prepared. Both the V_max and Ca²⁺ sensitivity of the Ca²⁺-stimulated, Mg²⁺-dependent actomyosin adenosine triphosphatase (ATPase) were similar in the hearts of trained and sedentary animals, and were not altered when the concentration of extracellular Ca²⁺ was increased. Enhanced myosin Ca²⁺-ATPase activities and phosphate contents of myosin P light chains were found in the hearts of trained animals compared with controls, and these differences were still maintained when perfusate Ca²⁺ concentrations were increased. Although treatment of the perfused hearts with isoproterenol also increased both parameters in the trained and sedentary series, the V_max for myosin Ca²⁺-ATPase and the alkali-labile phosphate contents of myosin P light chains remained, throughout, significantly greater in the hearts of trained rats than in their sedentary counterparts. These differences were not eliminated by the combined use of isoproterenol and high perfusate Ca²⁺. The results suggest that an enhanced capacity for trans-sarcolemmal Ca²⁺ flux in the hearts of trained animals may be responsible for enhanced Ca²⁺-dependent phosphorylation of myosin P light chains, and thus improving cardiac function.     

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.

Left ventricular myosin ATPase activity, expressed as enzymatic V_max values, was analyzed in dogs subjected to severe left ventricular pressure overload (aortic stenosis). K⁺ and Ca²⁺ activated myosin ATPase activities in the left ventricle (LV) were significantly depressed (P < .01) in the experimental animals. For normal K⁺ activated myosin the V_max values in micromoles of Pi per mg per min were: right ventricle 2.10; left ventricle, 2.84. For Ca²⁺ activated myosin the V_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 severe 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 μmol PO₄/mg.min). Left ventricular myosin from the hearts subject to severe stress simulated normal right ventricular myosin in ATPase activity, chain proportions and degree of calcium binding, Normal left ventricular myosin contained approximately 10% of the myosin protein concentration in the light chains; myosin from the left ventricles of the hemodynamically overloaded hearts contained 20% of the myosin protein concentration in the light chains (P < .001). With only one of the myosin light chains binding calcium left ventricular myosin from the stressed hypertrophied tissue bound approximately 2 mol Ca²⁺ mol^?1 myosin similar to myosin of the normal right ventricle; normal left ventricular myosin bound approximately 1 mol of Ca²⁺ mol^?1 myosin.     

Comparison of ATPase activity and light subunits in myosins from left and right ventricles and atria in seven mammalian species.

Comparison of myosins from left and right ventricles and atria of rat, cat, hare, rabbit, dog, pig and cow resulted in four observations: (i) the Ca²⁺- and K⁺-ATPase activities and electrophoretic patterns of both light subunits were identical in left and right ventricle myosins, whatever the species (respective mol. wts of subunits: 26 000 and 18 000). (ii) There were no inter-species differences between left and right atrial myosins from hare, dog, cow and pig, but Ca²⁺- and K⁺-ATPase activity was usually higher in atrial than ventricular myosin, in the same species. In addition, the mol. wts of the two light atrial myosin subunits (24 000 and 20 000 respectively) differed from the mol. wts of the ventricular subunits. As with fast skeletal myosin, dithio-bis-nitrobenzoic acid eliminated one of the light subunits (mol. wt 20 000) from atrial but not from ventricular myosin. (iii) The Ca²⁺-ATPase activity of ventricular and atrial myosins was higher in the hearts of small animals like the rat than in the hearts of larger animals. There were no differences between species with regard to the mol. wts of light subunits. (iv) Ca²⁺-ATPase in ventricular myosin was more active in the hare than the domestic rabbit, even though the rabbit's heart is half the size of the hare's, which suggests that myosin ATPase activity is essentially adapted to the requirements of the animal's predominant physical activity.     

The ATPase activity of cardiac myosin from failing and hypertropied hearts.

We have measured the EDTA and Ca²⁺ activated ATPase activity of myosin from hearts of normal dogs, dogs in gross heart failure and dogs with cardiac hypertrophy without failure. Heart failure was caused by systolic and/or diastolic hemodynamic loads. Under one or more assay conditions, depressed ATPase activity was consistently found for myosin from failing hearts. In contrast to myosin from hearts with diastolic loads, myosin from failing hearts with pulmonic stenosis in the presence of Ca²⁺ and 0.1 m KCl showed normal mean ATPase activity with several preparations having elevated activity. Elevated ATPase activities were obtained for myosin from the non-failing, but hypertrophied hearts of dogs with aortic stenosis. The data suggest that the interaction of ionic strength and pH with the ATPase active site of suggest that the interaction of ionic strength and pH with the ATPase active site of myosin from failing hearts is altered, thus modifying the ATPase activity in the presence of Ca²⁺ or EDTA. The altered interaction may be modified by the presence of hypertrophy, resulting in normal or increased activity in the presence of Ca²⁺ and decreased activity in the presence of EDTA.     

Activities of Transport Adenosine Triphosphatases in Erythrocyte Membranes of Healthy and Hypertensive Subjects

The Na⁺, K⁺-ATPase activity in the erythrocyte membrane was measured in 25 untreated essential hypertensive patients and 25 age-matched healthy normotensive subjects. In addition, the Ca²⁺, Mg²⁺-ATPase activity was measured in 20 hypertensive and 25 age-matched normotensive subjects. The Na⁺, K⁺-ATPase activity of healthy Chinese measured in this study was similar to the data reported in a Dutch study. We therefore could not support a theory which speculated an ethnic influence on Na⁺, K⁺-ATPase activity. Both Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase activities were slightly lower in hypertensive patients as compared with those in normotensive subjects, but the differences were not statistically significant. We concluded that the transport ATPase activities were not impaired in the erythrocyte membranes of hypertensive patients.     

A biphasic change in contractile proteins during the development of cardiac hypertrophy in pigs

Summary A non-failing hypertrophy of the left ventricle was produced in the pig heart by supravalvular banding of aorta for 4, 8 and 12 weeks and the myosin and myofibrillar adenosine triphosphatase activities were measured. A significant increase in myosin Ca²⁺-ATPase activity was seen at 4 weeks of hypertrophy, but at 8 and 12 weeks this activity was significantly decreased compared to sham control. Similar changes were also seen in actin-activated myosin ATPase activities at 4, 8 and 12 weeks of hypertrophy. There were no changes in the K⁺- and NH₄ ⁺-EDTA-stimulated ATPase activities of myosin. Basal ATPase activities of myofibrils were decreased at 4 and 8 weeks of hypertrophy and there was no change in this activity at 12 weeks of hypertrophy. Ca²⁺ stimulated ATPase activity of myofibrils was significantly increased at 4 weeks, normal at 8 weeks and significantly reduced at 12 weeks of hypertrophy. The changes in ATPase activities were not due to any alterations of proteins by high concentrations of salts during the purification of myosin. The non-hypertrophied right ventricle from the banded animals did not show any change in the basal or Ca²⁺ stimulated myofibrillar ATPase activities. It is suggested that hypertrophy of the myocardium is accompanied by specific changes in the enzyme activities of the contractile proteins and the biphasic responses may correlate with the functional state of the myocardium subjected to a chronic increase in pressure.This investigation was supported by a grant from the Medical Research Council of Canada. Dr. Panagia is a Manitoba Heart Foundation Scholar and Dr. Singal is a Canadian Heart Foundation Scholar.     

The effect of streptozotocin-induced diabetes in rats on cardiac contractile proteins

In order to determine whether diabetic cardiomyopathy in rats is associated with altered contractile proteins, male and female rats were made diabetic with intravenous streptozotocin (STZ). Calcium ATPase activity of cardiac actomyosin was significantly decreased after 1 week of diabetes and was depressed by 60% by 2 weeks. Rats pretreated with 3-O-methyl glucose to prevent the hyperglycemia caused by STZ had normal Ca2+-actomyosin ATPase activities, and non-diabetic rats whose food was restricted to keep their body and heart weights similar to those found in diabetic animals had only a slight fall in actomyosin ATPase activity. Ca2+-ATPase and actin-activated ATPase activities of pure myosin were similarly depressed in preparations from hearts of diabetic animals. Sodium dodecylsulfate gel electrophoresis and isoelectric focusing failed to reveal differences in the patterns of contractile proteins or light subunits between diabetics and controls, but pyrophosphate gels showed a shift in the myosin pattern. Because of depressed circulating thyroid hormone levels in diabetic animals, cardiac contractile proteins were also studied in preparations from thyroidectomized rats. Calcium activities of actomyosin and myosin ATPase were lower than values found in hearts of diabetic rats. When diabetic animals were kept euthyroid with thyroid replacement, actomyosin ATPase activity was still depressed. Thus STZ diabetes causes a significant decrease in cardiac contractile protein ATPase activity. This may be related to altered proportions of myosin isoenzymes.     

Heart myosin adenosine triphosphatase and light subunits in experimental chronic aortic insufficiency in the rabbit

Cardiac myofibrillar and myosin ATPases were studied in experimentally induced aortic insufficiency in the rabbit, in order to elucidate the pathogenesis of the defect in myofibrillar ATPase shown in chronically hypertrophied and/or failing hearts. The rabbits were killed 345 ± 32 days after the operation; 24% of them had clinical or anatomical signs of failure. They all had hypertrophic hearts (mean degree of hypertrophy, 64%) with dilatation.Heart myofibrillar Ca²⁺ ATPase was found to be lowered in aortic insufficiency. Three different preparations of myosin were also studied. The purity of these preparations was assessed by urea or SDS polyacrylamide gel electrophoresis or by measuring Mg²⁺-ATPase in a medium of low ionic strength. The first preparation (homogenized muscle) was highly contaminated by thin-filament proteins; the second (minced muscle) was rather pure but still contaminated by a nucleoprotein; the latter was separated by chromatography in the third preparation. Myosin Ca²⁺-ATPase measured in a medium of low or high ionic strength at pH 7.0 was decreased in aortic insufficiency when all three preparations were tested.On electrophoresis myosin light subunits were normal in aortic insufficiency. The search for an inhibitor was unsuccessful: the two myosin ATPase activities were additive and ribonuclease treatment did not normalize hypertrophied heart myosin. The “nucleoprotein” peak separated from myosin during chromatography had an inhibitory effect on myosin ATPase but was present in controls as well as in aortic insufficiency.This work suggests that myosin itself is abnormal in chronic aortic insufficiency, but an abnormality residing in a very closely associated factor could not definitely be excluded. Because myosin subunits were only studied by electrophoresis, their structures and relative amounts would need more detailed study before final conclusions could be reached.     

ATPase activity and proteolysis of human myocardial actomyosin in ischemia and hypertrophy

Recently we have shown that actomyosin ATPase activity decreases with age when measured under appropriate conditions (Yoshida K et al, Age 12: 97-102, 1989). Many previous studies, which examined changes in ATPase activity in myosin, actomyosin, or myofibrils under pathological states, ignored the age-related changes. In this study actomyosin was isolated from myocardia of middle-aged subjects (37-49 years old) and examined for ATPase activity under various conditions and protein composition. Proteolysis of myosin and troponin was more frequently observed in ischemic heart disease (IHD) subjects than in non-IHD subjects. The proteolysis was associated with a decrease in Ca2+ sensitivity of Mg2(+)-ATPase activity and enhanced stimulation of Ca2(+)-ATPase activity with a sulfhydryl reagent, N-ethylmaleimide. Hypertrophy appeared not to significantly affect ATPase activity.     

Altered distribution of myosin isoenzymes in the cardiomyopathic Syrian hamster (BIO 8.262)

Summary Myosin of the ventricular myocardium of the cardiomyopathic Syrian hamster and of control animals was analysed using non-dissociating pyrophosphate electrophoresis. Three different myosin isoenzymes exhibiting different Ca²⁺ activated ATPase activities were demonstrated in the ventricular myocardium of the Syrian hamster. As shown by peptide mapping, ventricular myosin isoenzymes differ in their heavy chain composition. In the cardiomyopathic hamster a shift to myosins of lower Ca²⁺-activated ATPase activities occurs in the stage of insufficiency (age 220 days), whereas no different isoenzyme pattern could be found at the age of 65 days compared to control animals. We conclude that this redistribution of myosin isoenzymes is the basis of reduced myosin ATPase activity in the ventricular myocardium of the cardiomyopathic Syrian hamster during the development of myocardial insufficiency.Supported by the SFB 89, Göttingen     

Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na⁺, K⁺-and Ca²⁺-ATPase in crude homogenates of rat ventricular myocardium by determination of K⁺-and Ca²⁺-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K⁺-and Ca²⁺-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min^–1×g^–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na⁺, K⁺-and Ca²⁺-ATPase concentrations were estimated to 2 and 10 nmol×g^–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca²⁺-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K⁺-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K⁺-and Ca²⁺-dependentpNPPase activity gradually increased to a plateau. The present assay for Na⁺, K⁺-ATPase quantification has the advantage over [³H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K⁺-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca²⁺-ATPase on crude myocardial homogenates. Age-dependent changes in K⁺-and Ca²⁺-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na⁺, K⁺-and Ca²⁺-ATPase in excitable tissues.Abbreviations Na⁺, K⁺-ATPase sodium, potassium-dependent ATPase - Ca²⁺-ATPase caldium-dependent ATPase - pNP p-nitrophenyl - pNPP p-nitrophenyl phosphate - 3-0-MFP 3-0 methylfluorescein phosphate - DOC sodium deoxycholate     

The effect of hyperthyroidism on the sarcoplasmic reticulum and myosin ATPase of dog hearts.

Large doses of desiccated thyroid were administered to dogs for from 55 to 176 days (mean 118 days) to evaluate the effect of long-term administration upon the myocardium. An increased pulmonary vascular bed and cardiac enlargement were found in most animals. Calcium-activated myosin adenosine-triphosphatase (ATPase) activity was significantly elevated at pH values of 7.0 and below. The ATPase activity of sulfhydryl-modified myosin was also altered. However, in the presence of K⁺ and EDTA, the myosin ATPase activity was similar to that of myosin from normal dog hearts. The calcium uptake by the cardiac sarcoplasmic reticulum (CSR) (in the presence of oxalate) was significantly less than normal for the thyroid-treated dogs. The calcium content of the CSR was also less than normal for the treated dogs. The norepinephrine content of the myocardium from the thyroid-treated dogs was less than normal, but when corrected for hypertrophy was within normal limits.     

Dephosphorylation of myofibrillar proteins in actomyosin preparations and in isolated perfused rat hearts after β-agonist withdrawal

The in vitro dephosphorylation of myosin P light chains and troponin-I was studied in preparations of natural actomyosin isolated after isoproterenol treatment of perfused rat hearts. These preparations contained endogenous phosphatase(s) which on incubation removed incorporated ²²P from myosin P light chains more rapidly than from troponin-I. The in situ effects of isoproterenol pulse-treatment were also studied in perfused rat hearts, with respect to myofibrillar protein phosphorylation and the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ and Ca²⁺-ATPase activities of natural actomyosin. Abrupt removal of the β-adrenergic stimulus from perfused rat hearts caused the (incomplete) dephosphorylation of both myosin P light chains and troponin-I; the former was dephosphorylated in apparent synchrony with the slowing of the beating rates and falling cyclic AMP contents (definitely decreased after 1 min), whereas troponin-I dephosphorylation only began after 2 min and was correlated with changes in force development. The dephosphorylation of P light chains was accompanied by a decrease in the V_max |ATP| of the myosin Ca²⁺-ATPase activities, but the latter reached pre-stimulation values before basal phosphorylation levels of the protein were attained. An increase in the V_max |Ca²⁺| of $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ activities and an increase in their sensitivity to Ca²⁺ was correlatable with the removal of phosphate from troponin-I. The results suggest that the phosphorylations of troponin-I and myosin light chains in adrenergically stimulated hearts serve separate functions in the context of positive inotropism.     

Heavy long-term ethanol consumption induces an alpha- to beta-myosin heavy chain isoform transition in rat

Alcoholic heart muscle disease is characterized by structural changes which include chamber dilation, ventricular hypertrophy, and myocyte damage. These effects often lead to contractile dysfunction and ultimately to heart failure if alcohol consumption is not terminated. In rat models for heart failure in which heart failure is induced by pressure or volume overload, there is a shift in the myosin heavy chain (MHC) isoforms, from α to β. As a result of this MHC transition, there is typically a decrease in myosin ATPase activity. We utilized a rat model of chronic alcohol consumption in order to determine if alcohol causes a similar shift in MHC isoforms and changes in myosin ATPase activity. A liquid diet containing 9% ethanol (46% of daily calories; 11.8 g/kg/day) was administered to adult rats for a period of 60 or 90 days. This heavy consumption of ethanol resulted in an average blood ethanol content of 150 mg%. The relative abundance of β-MHC isoform protein increased from a control level of 9.7% to 35.1% in hearts of ethanol-fed rats, following 90 days of ethanol consumption. In a separate set of experiments, the levels of α-MHC and β-MHC mRNA were demonstrated to increase by 150% and 230%, respectively. Following a 60 day treatment, there was a significant reduction in the actomyosin Mg²⁺-ATPase activity in the myofibrillar preparations from hearts of ethanol-fed rats compared to hearts from control-fed rats. In addition, the myosin Ca²⁺-ATPase activity was decreased 17% and 30% after 60 and 90 days of ethanol consumption, respectively. The present study demonstrates that chronic ethanol consumption induces an increase in the proportion of the total MHC content composed of the β-isoform. This isoform transition is accompanied by an accumulation of β-MHC mRNA, suggesting that the switch is organized pretranslationally. A functional consequence of this transition in MHC phenotype is demonstrated by significant decreases in the myofibrillar and myosin ATPase activities. Received: 17 November 1998, Returned for 1. revision: 4 January 1999, 1. Revision received: 22 March 1999, Returned for 2. Revision: 12 April 1999, 2. Revision received: 31 May, Accepted: 31 May 1999     

Cardiac contractile proteins in hypertrophied and failing guinea pig heart.

OBJECTIVE: The aim was to study changes in contractile proteins which accompany marked hypertrophy and heart failure in mammalian hearts initially containing predominantly V3 isomyosin. METHODS: Left ventricular myosin and myofibrillar ATPase activity and right ventricular actomyosin ATPase activity were measured in normal guinea pig hearts, in hearts which were hypertrophied as a result of progressive left ventricular systolic overload following ascending aortic banding, and in hypertrophied hearts from animals which showed signs of overt congestive heart failure. Male guinea pigs weighing 225-275 g at the time of aortic banding were used for the studies. RESULTS: Left ventricular myosin and myofibrillar ATPase activity and right ventricular actomyosin ATPase activity were correlated with body weight, left and right ventricular weight, and left ventricular peak systolic pressure during aortic occlusion. Left ventricular myosin ATPase activity and right ventricular actomyosin ATPase activity were markedly depressed in hypertrophied ventricles compared to control ventricles. Cardiac myofibrillar ATPase activity was lower in hypertrophied failing hearts than in control hearts over a wide range of calcium concentrations. In control animals and in those without heart failure, there was a nearly identical inverse relationship between left ventricular mass up to 1600 mg and myosin ATPase activity. Hypertrophied failing hearts were larger but showed little further reduction in cardiac myosin ATPase activity. Representative gel scans of non-dissociating pyrophosphate gels of left ventricular myosin from an 8 d postoperative aortic constricted animal and from its age and weight matched control showed predominantly V3 isomyosin with small amounts of V1 isoenzyme. However, preparations taken from guinea pigs 16 d after aortic constriction showed only the V3 isoform, whereas the V1 isoform was still apparent in control. Hypertrophied failing left ventricles developed less pressure per unit mass during brief aortic occlusion than non-failing left ventricles with comparable myosin ATPase activities. CONCLUSIONS: These observations raise important questions as to the distribution of myosin isoforms in the normal adult guinea pig, and the possibility that myosin ATPase activity might be altered by post-translational modification. Although cardiac myosin ATPase activity correlates with left ventricular performance, it cannot fully explain the depressed performance of failing hearts in this model. Additional immunological studies of cardiac contractile proteins are required as well as studies designed to explore the implications of altered myosin ATPase activity for both contractile function and overall cellular homeostasis.     

Changes in rat cardiac myosin during development and in culture

Developmental changes in the subunit composition and ATPase activity of myosin isolated from rat ventricular myocardium and 5-day-old myocardial tissue cultures were examined. Electrophoretic analysis of cardiac myosin from 12-week-old adults, 9-, and 16-day neonates, and 5-day tissue cultures demonstrated two light chains (Mol. wt 25 500 and 20 000) with a molar ratio of 1: 1. In 21-day fetal myosin, three light chains were observed (Mol. wt: 25 500; 24 500; 20 000), with a molar ratio of 0.82:0.15: 1.0. Analysis of ATPase activity in the presence of three activators (Ca²⁺, K⁺, and F-actin) showed significant differences between these myosin preparations, though the time course of the change was activator specific. The change in K⁺-activated ATPase activity occurred soon after birth and correlated with the disappearance of the third light chain (Mol. wt 24 500) and a partial isozymic shift from V₃ to V₁ myosin. The Ca²⁺- and actin-activated ATPase activities increased more slowly and were accompanied by continuation of the V₃ to V₁ myosin shift. Thus, it appears that V₃ myosin is heterogeneous. Moreover, kinetic analysis of tissue culture myosin is consistent with the predominance of V₃ myosin with low K⁺-activated ATPase activity.     

Effects of aging on atrial and ventricular human myosin

Enzymatic and structural studies of human cardiac myosin from young and old subjects have been investigated to determine possible changes in myosin properties in aging hearts. Human ventricular myosin from old subjects (47-70 years old) has lower actin-activated ATPase activity than and increased alkaline sensitivity as compared to myosin from young subjects (1-132 months old). Ca2+-and K+(EDTA)-ATPase activities, pyrophosphate gel patterns and one-dimensional peptide mapping of heavy chains of ventricular myosin from old subjects are similar to those observed for myosin from young subjects. Atrial myosin from human hearts differs significantly from ventricular myosin in that the Ca2+-, Mg2+- and actin-activated myosin Mg2+-ATPase activities of atrial myosin are significantly higher than those of ventricular myosin. Pyrophosphate gel electrophoresis patterns and peptide mapping of heavy chains of atrial myosin are also different from those of ventricular myosin. Unlike ventricular myosin, atrial myosin from young hearts is similar to that of atrial myosin from old hearts in its enzymatic and structural properties.     

Phospholipase A activity of highly enriched preparations of cardiac sarcolemma from hamster and dog.

Cardiac sarcolemma was isolated from hamster and dog and was examined for phospholipase A activity using 1-acyl [2-¹⁴C]-linoleoyl3-glycerophosphorylethanolamine as substrate. Hamster sarcolemmal preparations contained phospholipases A₁ which had optimal activity at pH 6.0 and pH 9.0 in the presence of 5 mm Ca²⁺; EDTA was a potent inhibitor of phospholipase activity at both pHs. The specific activities of the hamster sarcolemmal phospholipases A₁ were increased 5.9-fold (pH 6.0) and 8.4-fold (pH 9.0) over the homogenate, while the increase in specific activity of the sarcolemmal marker enzyme, ouabain-sensitive (Na⁺-K⁺)-Mg⁺-ATPase, was 7.5-fold; thus both the (Na⁺+K⁺)-Mg⁺-ATPase and phospholipase A₁ activities were associated with enriched cardiac sarcolemmal membranes. Canine myocardial sarcolemmal preparations also contained a phospholipase A that had optimal activity at pH 7.0 in the presence of 5 mm Ca²⁺ and the enzyme exhibited apparent specificity for the 2-position. The phospholipase A and (Na⁺+K⁺)-Mg²⁺-ATPase activities were similarly enriched in the canine sarcolemmal preparation. These endogenous, calcium-stimulated phospholipase A activities may be important constituents of the myocardial sarcolemmal membrane; they may modulate the lipid environment of the sarcolemma and may regulate the activity of lipid-dependent enzymes as well as alter membrane permeability.