Effect of pH and temperature on age-associated alteration of actomyosin ATPase activity in human myocardium

The K⁺-EDTA-ATPase and Ca²⁺-ATPase activities of cardiac ventricular actomyosin prepared from autopsy samples from subjects of various ages were determined. We could not detect differences in the K⁺-EDTA-ATPase or Ca²⁺-ATPase activity of actomyosin prepared from the hearts of young (0–30 yrs.), middle-aged (31–60 yrs.) and old (61-yrs.) subjects at pH 7.0 and at 30°C conditions widely used in previous studies. However, there were significant decreases in both ATPase activities of actomyosin isolated from old and middle.aged subjects, compared with young subjects at pHs 6.0, 8.0 and 9.0 (at 30°C or at 37°C (at pH 7.0). The alkaline lability of the ATPase activities also increased significantly with age. There has been much debate as to whether or not there is a relationship between age and myocardial myosin ATPase activity. In many studies regarding myocardial myosin ATPase activity of hypertrophied or failed hearts, the age of the subjects was ignored. Our results draw attention to the effect of aging on the myocardial actomyosin ATPase activity and the importance of choosing appropriate assay conditions.     

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.     

Left ventricular hypertrophy and myocardial ischemia in systemic hypertension

Considerable attention has properly been focused in recent years on electrocardiographic abnormalities in patients with essential hypertension. It has been well established that both voltage evidence of left ventricular (LV) hypertrophy and LV hypertrophy and strain are ominous risk factors. A better understanding of the strain pattern in patients with LV hypertrophy has arisen from experimental animal studies showing how an increase in cardiac mass can lead to myocardial ischemia and from clinical studies showing that the patient with LV hypertrophy and strain is at risk from myocardial ischemia as a consequence of both associated coronary artery disease and increased LV mass. All the clinical syndromes associated with myocardial ischemia are increased in patients with LV hypertrophy and therefore earlier recognition of both cardiac involvement and myocardial ischemia is likely to improve survival in this particularly high-risk group of patients.     

Isoproterenol-induced cardiomegaly: assessment of myocardial protein content, actomyosin ATPase and heart rate

Rats with isoproterenol-induced cardiomegaly were evaluated for myocardial actomyosin and total protein content (mg/g tissue), hemodynamic changes and actomyosin adenosine triphosphatase (ATPase) activity.     

Age-related activity changes in actomyosin ATPase and arginine phosphokinase in male Drosophila melanogaster Meig

The specific activity of each of the two enzymes primarily concerned with the energy metabolism of flight muscle contraction was determined in male Drosophila melanogaster, from emergence through 5 weeks postemergence. Ca++-activated actomyosin ATPase activity rose rapidly from a minimum at emergence to a peak level at 4-5 days, falling gradually thereafter to a minimum level at 10-11 days postemergence, with no change thereafter. Arginine phosphokinase activity rose more gradually to a maximum at 11-12 days postemergence and then fell slowly to a minimum at 18-19 days. These data represent a sequential pattern of biochemical changes similar to those previously observed in other dipteran species, confirming the primary role of a genetic program for maturation and senescence of flight ability in holometabolous adult insects at the functional and underlying cellular, biochemical levels.     

Inhibition of actomyosin ATPase by vanadate.

Actin-myosin subfragment-1 (SF-1) or actin-heavy meromyosin is dissociated by the binding of ADP and vanadate (Vi) under conditions such that ADP alone does not dissociate the complex. The association constant of the stable complex M.ADP.Vi, in which M indicates myosin [Goodno, C. C. (1979) Proc. Natl. Acad. Sci. USA 76, 2620-2624] with actin is smaller than the average association constant of the intermediate states of the actin-SF-1 ATPase cycle. Actin-SF-1 ATPase activity is 90% inhibited by ADP plus vanadate. The reaction of actin with M.ADP.Vi produces a slow release of ADP and vanadate and quantitative recovery of ATPase activity. The rate of dissociation of ligands was almost linear in actin concentration; consequently, the rate constant of dissociation could only be roughly estimated as 0.5-1 sec-1. The rate of dissociation of ADP and vanadate is thus increased by a factor of 10(5) compared to M.ADP.Vi. The rate of release of ligands by regulated actin (actin-tropomyosin-troponin) was reduced to 1/10th to 1/20th by removal of calcium ion. Therefore the M.ADP.Vi complex has the properties of a more stable analogue of the myosin-ADP-phosphate complex that is generated in the normal ATPase cycle. The activation of ligand release (ratio of rate of dissociation of ADP and vanadate from actomyosin relative to myosin) is much larger than the activation of myosin ATPase by actin, whereas the actual rates of the reactions are much slower.     

Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution.

Crossbridge models of muscle contraction based on biochemical studies predict that there may be a relationship between the rate-limiting step in the actomyosin ATPase cycle in vitro and the rate of force development in vivo. In the present study, we measured the rate of force redevelopment in skinned rabbit muscle fibers following unloaded isotonic shortening and a rapid restretch. For comparison, ATPase activity was measured under identical conditions, using myosin subfragment-1 chemically crosslinked to actin. We found that the time course of force redevelopment is well fitted by a single exponential function, implying that force redevelopment is a first-order process, described by a single rate constant. The magnitude of this rate constant is in close agreement with the rate constant necessary to simulate the experimental force-velocity relation on the basis of a crossbridge model of the type proposed by A. F. Huxley in 1957. In addition, the observed close correlation between the rate constant for force redevelopment and the maximal actin-activated actomyosin ATPase rate under a variety of conditions suggests that the step that determines the rate of force generation in the crossbridge cycle may be the physiological equivalent of the rate-limiting step in the actomyosin ATPase cycle in solution.     

Hemodynamics and cardiac electrical activity in ventricular myocardial hypertrophy]

Comparison of the electrocardiographic characteristics of hypertrophy of the ventricular myocardium with the hemodynamic indices of systemic and pulmonary circulation and the indices of intracardiac hemodynamics provides testimony to the significance of the hemodynamic factor in the genesis of the electrophysiological changes. With due account for the role of the morphologic factor, these data allow the essence of myocardial hypertrophy to be placed in the electrophysiologic, hemodynamic, and morphologic category.     

Positron emission tomography in human myocardial ischemia

Positron emission tomography (PET) enables investigations of regional metabolic processes in myocardium on a noninvasive basis. This report deals with clinical studies employing C-11 palmitate and FDG (F-18-2 deoxyglucose). Experimental and clinical results have shown C-11 palmitate to be a well-suited marker for studies of myocardial fatty acid metabolism. Uptake and clearance of C-11 palmitate are proportional to cardiac work and oxygen consumption. In ten patients with coronary artery disease, at rest, there was no difference of C-11 palmitate uptake and clearance between "normal" and "ischemic" myocardium. In contrast, during atrial pacing, in normal myocardium there was a higher increase in C-11 palmitate uptake and more rapid clearance than in ischemic myocardial zones. In the presence of very compromised flow, however, due to diminished appearance of the marker, the activity curves cannot be reliably assessed. FDG is a useful marker for studies of glucose uptake and metabolic activity. In 13 of 15 patients studied two days to 13 weeks after myocardial infarction, in the hypoperfused myocardial zone there was increased FDG uptake. In studies in acute myocardial infarction (40 to 72 hours), zones devoid of FDG uptake subsequently were found to be irreversibly damaged while those with intact FDG uptake at the time of initial investigation were subsequently found to have reversible damage. In 17 patients with coronary artery disease and wall motion impairment, bypass surgery led to improved wall motion in 85% of zones with intact FDG uptake but only in 5% of zones with diminished FDG uptake. On comparison with thallium-201 scintigraphy during exercise, PET demonstrated viable myocardium in 58% or zones with fixed thallium defects while in only 42% was there agreement between the two methods with respect to necrosis. As compared with other diagnostic measures such as EKG, analysis of regional wall motion abnormalities and measurement of regional blood flow, the diagnostic accuracy of PET was clearly superior. New PET equipment in which substantial technological developments have been recently incorporated and new positron-emitting tracers (such as antimyosin antibodies or radioligands) will expand the possibilities for the study of regional myocardial tissue function in humans.     

Reduced myocardial actomyosin adenosine triphosphatase activity in the ageing male Fischer rat.

Actomyosin was extracted from myocardial homogenates from male rats of different ages of a long-inbred Fischer rat colony maintained under controlled conditions of temperature, humidity, and light. ATPase specific activity rose to a maximum at 2 months of age; this was followed by a progressive decline by about 25% at 16 months of age. However, the extractable actomyosin remained constant during this period. This loss in actomyosin ATPase specific activity is in good agreement with previously reported decrements in both stroke index and myocardial calcium content and an increase in myocardial contraction duration in aged rats.     

DNA stimulates ATP-dependent proteolysis and protein-dependent ATPase activity of protease La from Escherichia coli.

The product of the lon gene in Escherichia coli is an ATP-dependent protease, protease La, that also binds strongly to DNA. Addition of double-stranded or single-stranded DNA to the protease in the presence of ATP was found to stimulate the hydrolysis of casein or globin 2- to 7-fold, depending on the DNA concentration. Native DNA from several sources (plasmid pBR322, phage T7, or calf thymus) had similar effects, but after denaturation the DNA was 20-100% more effective than the native form. Although poly(rA), globin mRNA, and various tRNAs did not stimulate proteolysis, poly(rC) and poly(rU) were effective. Poly(dT) was stimulatory but (dT)10 was not. In the presence of DNA as in its absence, proteolysis required concomitant ATP hydrolysis, and the addition of DNA also enhance ATP hydrolysis by protease La 2-fold, but only in the presence of casein. At much higher concentrations, DNA inhibited proteolysis as well as ATP cleavage. Thus, association of this enzyme with DNA may regulate the degradation of cell proteins in vivo.     

Effect of inhibition of the mitochondrial ATPase on net myocardial ATP in total ischemia

The effect of inhibition of the mitochondrial ATPase with oligomycin on the rate of ATP depletion and anaerobic glycolysis was studied in the totally ischemic dog heart. An oxygenated, buffered crystalloidal solution containing 10 microM oligomycin and 12 mM glucose was delivered at 100 mmHg pressure to the circumflex bed of the excised cooled heart. Buffered solution without oligomycin was delivered simultaneously to the anterior descending bed of the same heart. Little metabolic evidence of ischemia developed until the heart was made totally ischemic by incubating it in a sealed plastic bag at 37 degrees C. Successful inhibition of the mitochondrial ATPase was confirmed by the absence of both mitochondrial ATPase activity and the loss of respiratory control in mitochondria isolated from treated tissue. ATP, glycolytic intermediates and catabolites of the adenine nucleotide pool were measured in the control and treated beds at various intervals during 120 min of ischemia. Inhibition of the ATPase resulted in slowing of the rates of ATP depletion and anaerobic glycolysis (estimated by lactate accumulation). Also, degradation of the adenine nucleotide pool occurred more slowly in the inhibited group. These data establish that about 35% of the ATP utilization observed during the first 90 min of total ischemia in the canine heart is due to mitochondrial ATPase activity.     

Endogenous nitric oxide prevents myocardial ischemia in patients with hypertension and left ventricular hypertrophy

Background Left ventricular hypertrophy (LVH) caused by chronic pressure overload is associated with increased risk of myocardial ischemia without epicardial coronary artery disease. We aimed to test the hypothesis that endogenous nitric oxide (NO) prevents myocardial ischemia in patients with LVH. Methods Epicardial coronary blood flow (Doppler wire and quantitative coronary arteriography) and myocardial lactate metabolism (paired arterial and coronary sinus blood sampling) were measured in 12 patients with hypertension, LVH, and angiographically normal epicardial coronary arteries and in 7 control subjects. Measurements were done under 3 pacing protocols: with no treatment (control), with intracoronary N^G-monomethyl-L-arginine (L-NMMA; NO synthesis inhibitor), and with intracoronary L-arginine (NO substrate). Results In control subjects the myocardial lactate extraction ratio was normal and stable during the 3 pacing protocols. In contrast, lactate uptake was significantly decreased from 0.21 ± 0.05 to 0.10 ± 0.06 (P <.05) during L-NMMA pacing in patients with LVH; in 6 of them, lactate production was demonstrated. After L-arginine administration, the lactate extraction ratio during pacing was normalized (0.18 ± 0.04) and lactate production was not observed in any patient. The level of myocardial lactate uptake at peak pacing after L-NMMA was correlated with that under untreated condition (P <.0001). Conclusions In patients with hypertension, LVH, and angiographically normal coronary arteries, inhibition of endogenous NO synthesis in the coronary circulation unmasked myocardial ischemia during tachycardia, and L-arginine reversed the adverse effects of L-NMMA. Although the precise mechanism remains to be determined, our results suggest that constitutive NO in the coronary circulation plays an anti-ischemic role in this population. (Am Heart J 2002;143:684-9.)     

Histoenzymological study of myocardial ATPase activity in experimental infarction in the rat

Summary Histoenzymological techniques were used to examine ATPase activity in rat heart muscle fibres after experimental infarction. 24 hours after coronary ligation, ATPase activity in all ventricular section fibres was high, homogeneous at pH 9.4 and inhibited by acid preincubation, and the same was observed for control heart sections. 48 hours after ligation, necrotic ventricular fibres appeared, leaving only a thin layer of fibres which had apparently preserved their myofibrillar ATPase. These results indicate that, unlike mitochondrial enzyme activity, myofibrillar ATPase activity is relatively resistant to ischaemia.     

老年急性心肌梗死患者红细胞形态与ATP酶活性

目的 探讨红细胞形态和ＡＴＰ酶活性及红细胞变形性对急性心肌梗死（ＡＭＩ）的影响。方法 选择正常对照组３０例,老年ＡＭＩ患者３３例,观察其红细胞几何形态,测定红细胞膜ＡＴＰ酶活性及红细胞变形性。结果 （１）ＡＭＩ组口形、嵴形、类球形等异常红细胞检出率明显高于正常对照组（Ｐ〈０．０１）;（２）ＡＭＩ最大变形指数（ＤＩｍａｘ）及综合变形指数（ＩＤＩ）均低于正常对照组（Ｐ〈０．０５）;（３）ＡＭＩ胞检出率     

On the mechanism of actomyosin ATPase from fast muscle.

The labeled inorganic phosphate formed by enzymatic hydrolysis of [gamma-18O]ATP in normal water was derivatized to trimethyl phosphate and analyzed for the proportions of [18O3]Pi, [18O2]Pi, [18O1]Pi, and [18O0]Pi. The proportions observed were correlated with the kinetics of intermediate exchange by using a kinetic relationship in which it is assumed that binding of ATP and subsequent release of products are irreversible. Actomyosin and acto-heavy meromyosin catalyze intermediate exchange at a mean rate that is more than 1 order of magnitude slower than that predicted by rapid kinetic studies or implied by the essentially complete intermediate exchange observed with myosin alone. The reason for the slow apparent exchange is that there are two ATPase activities with different exchange properties. The effect of varying heavy meromyosin concentrations at a constant actin concentration shows that the two activities are interrelated and suggests further that one is due to ATP hydrolysis by the undissociated actomyosin crossbridge.     

Abnormalities in myocardial perfusion during tachycardia in dogs with left ventricular hypertrophy: metabolic evidence for myocardial ischemia

This study tested the hypothesis that in the chronically hypertrophied left ventricle pacing stress may cause abnormalities of perfusion that result in myocardial ischemia. Left ventricular hypertrophy (LVH) was produced by banding the ascending aorta of 10 dogs at 6 weeks of age, and studies were carried out after the animals had reached adulthood and when mean left ventricular/body weight ratio was 74% greater than in eight control dogs. Myocardial blood flow was measured with microspheres during pacing at 100, 200, and 250 beats/min, while aortic and coronary sinus blood samples were obtained for determination of concentrations of lactate and the adenosine metabolites inosine and hypoxanthine. In the control dogs, increasing heart rates were associated with an increase in mean myocardial blood flow while subendocardial flow was maintained at a level equal to or greater than subepicardial flow. Myocardial lactate uptake ranged from +60% to -5%, and adenosine metabolites were not detected in coronary sinus blood (less than 0.5 microM/l). In four dogs that underwent aortic banding no production of lactate or adenosine metabolites was observed at any heart rate; in these animals subendocardial flow was maintained at a level equal to or greater than subepicardial flow at all pacing rates. The remaining six dogs with LVH demonstrated net lactate production significantly greater than control during pacing at 250 beats/min; five of these six animals also produced adenosine metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced membrane protein kinase C activity in myocardial ischemia

Summary The protein kinase activity in cytosol was similar in control, ischemic, and reperfused hearts; however, a 1.5-fold increase in membrane protein kinase activity was induced by ischemia and reperfusion. The H-7 inhibitable cytosolic protein kinase activity decreased by 40% with 30 min ischemia, while that of membrane fraction increased 1.8-fold. However, the CGS9343B inhibitable protein kinase activity in cytosolic fractions was unaffected by ischemia, while that of membrane increased by about 1.7-fold. These results suggest that myocardial ischemia is associated with enhanced protein kinase C and calmodulin-dependent kinase activities in membrane fraction. Furthermore, the results also suggest a translocation of protein kinase C activity from the cytosol to the membrane. Reperfusion of ischemic myocardium did not result in any further increase of protein kinase C and calmodulin-dependent kinase activities in the membrane. These enhanced protein kinase activities also resulted in an enhanced phosphorylation of endogenous membrane proteins. The creatine kinase released from the heart was increased by both ischemia and reperfusion. Therefore, these results suggest that biochemical cascades of reactions caused by enhanced membrane protein kinase C and calmodulin-dependent kinase activities may contribute to ischemic-reperfusion injury.