Time dependent response of cardiac myofibrillar ATPase activity to exercise

1. The purpose of the present study was to investigate the time course of run training effects on the Ca2+ kinetics of the cardiac myofibrillar ATPase activity in female Sprague-Dawley rats. 2. The cardiac myofibrillar ATPase activity was measured at varying Ca2+ levels, and the Hill-n and pCa50 were measured in the hearts of rats after 3, 6 and 9 weeks of running training with a training program that began with an initially high intensity (HINT) and a training program with a more progressive increase in intensity (PROG). 3. After 3 and 6 weeks of training cardiac myofibrillar ATPase activity in the hearts of the trained rats in both training programs was elevated by 28-40% over the control group (P less than 0.05) at a pCa5 but was not different from the control groups after 9 weeks of training (P greater than or equal to 0.05). 4. Also the Ca2+ co-operativity as measured by the Hill-n was elevated in the hearts of the trained rats after 6 and 9 weeks of training when compared to control groups suggesting changes in the regulatory proteins of the myofibrils of hearts from trained rats. 5. The elevations in cardiac myofibrillar ATPase activity suggest that the myocardium responded to the training stimulus in a phasic manner. 6. The regression of cardiac myofibrillar ATPase in the late weeks of training might be related to a reduction or a loss of a specific training stimulus for the myocardium.     

Lack of effect of prostaglandins on rat heart myofibrillar ATPase activity

Although various prostaglandins have been shown to elicit an isotropic response in the rat heart, the subcellular basis responsible for this effect is unknown. The purpose of this study was to examine the influence of three prostaglandins with varying inotropic potencies on myofibrillar ATPase activity in the rat heart. PGF2α, PGI2 and PGE2 were found to have no influence on basal or Ca-stimulated myofibrillar ATPase activity. In addition, no influence ²⁺ s observed on the sensitivity of myofibrillar ATPase activity to Ca . Alternative mechanisms to explain the isotropic effect are discussed.     

Formation of superoxide radicals in isolated cardiac mitochondria: effect of adriamycin

The effect of adriamycin (doxorubicin) on superoxide radical formation in isolated rat heart mitochondria was studied by the spin trapping technique. The samples were placed into the cavity of EPR spectrometer in thin - wall gas - permeable capillary tubes, which allowed keeping the mitochondria of suspension in aerobic conditions. TIRON was used as a spin trap. We demonstrated that the rate of superoxide generation by isolated mitochondria depended radically on the presence of 1-150 microM adriamycin in incubation medium and was considerably higher than in control. The effect of adriamycin could be observed in the presence of both complex I (succinate) or complex II (glutamate and malate) substrates. The results obtained let to conclude that isolated cardiac mitochondria modified by adriamycin have a higher rate of production of superoxide radicals, which can react with spin traps not penetrating through the internal membrane.     

Assay of sulfhydryl groups in cardiac myofibrillar proteins: effect of oxygen radicals in vitro

Myofibrils from rat hearts were prepared in conditions maintaining their redox state, and their sulfhydryl groups were measured using a solution of urea and sodium dodecyl sulfate (SDS) as denaturant. The sulfhydryl content was 92 n mol/mg of protein, indicating that cysteins are in reduced form. In the presence of superoxide radicals generated in vitro with purine and xanthine oxidase, the myofibrillar sulfhydryl groups were oxidized.     

Phosphorylated cardiac myofibrils and their effect on ATPase activity.

Control guinea pig cardiac myofibrils were isolated in the presence of Triton X-100. Experimental myofibrils, prepared in the presence of Triton X-100, NaF, cyclic AMP and ATP, possessed a reduced myofibrillar ATPase activity. When myofibrils isolated under control conditions were incubated for two hours at 25°C with NaF, ATP and cyclic AMP, the ATPase activity was also decreased; however, the ATPase activity was not reduced as much as that of myofibrils isolated under experimental conditions. Incubation of myofibrils with $\text{E. coli}$ aklaline phosphatase and guinea pig heart phosphoprotein phosphatase resulted in an increase in ATPase activity and a decrease in phosphoprotein phosphate. Thus there appeared to be an inverse relationship between myofibrillar ATPase activity and phosphoprotein phosphate content. The results indicated that a protein kinase is associated with the Triton X-100 purified myofibrils and supports the notion that intact myofibrils can exist in at least two catalytic forms.     

Effect of endurance swimming on rat cardiac myofibrillar ATPase with experimental diabetes

Diabetes is characterized by depressed cardiac functional properties attributed to Ca2+-activated ATPase activity. In contrast, endurance swimming enhances the cardiac functional properties and Ca2+-activated myofibril ATPase. Thus, the purpose of this study was to observe if the changes associated with experimental diabetes can be ameliorated with training. Diabetes was induced with a single i.v. injection of streptozotocin (60 mg/kg). Blood and urine glucose concentrations were 802 +/- 44 and 6965 +/- 617 mg/dL, respectively. The training control and training diabetic animals were made to swim (+/- 2% body weight) 4 days/week for 8 weeks. Cardiac myofibril, at 10 microM free Ca2+ concentration was reduced by 54% in the sedentary diabetics compared with sedentary control animals (p less than 0.05). Swim training enhanced the Ca2+-activated myofibril ATPase activities for the normal animals. The diabetic animals, which swam for 8 weeks, had further reduced their Ca2+-activated myofibril ATPase activity when compared with sedentary diabetics (p less than 0.05). Similarly, the Mg2+-stimulated myofibril ATPase activity was depressed by 31% in diabetics following endurance swimming. It is concluded that the depressed Ca2+-activated myofibril ATPase activity of diabetic hearts is not reversible with endurance swimming.     

Paradoxical effects of endurance training and chronic hypoxia on myofibrillar ATPase activity

This study aimed to determine the changes in soleus myofibrillar ATPase (m-ATPase) activity and myosin heavy chain (MHC) isoform expression after endurance training and/or chronic hypoxic exposure. Dark Agouti rats were randomly divided into four groups: control, normoxic sedentary (N; n = 14), normoxic endurance trained (NT; n = 14), hypoxic sedentary (H; n = 10), and hypoxic endurance trained (HT; n = 14). Rats lived and trained in normoxia at 760 mmHg (N and NT) or hypobaric hypoxia at 550 mmHg (approximately 2,800 m) (H and HT). m-ATPase activity was measured by rapid flow quench technique; myosin subunits were analyzed with mono- and two-dimensional gel electrophoresis. Endurance training significantly increased m-ATPase (P < 0.01), although an increase in MHC-I content occurred (P < 0.01). In spite of slow-to-fast transitions in MHC isoform distribution in chronic hypoxia (P < 0.05) no increase in m-ATPase was observed. The rate constants of m-ATPase were 0.0350 +/- 0.0023 s(-1) and 0.047 +/- 0.0050 s(-1) for N and NT and 0.033 +/- 0.0021 s(-1) and 0.038 +/- 0.0032 s(-1) for H and HT. Thus, dissociation between variations in m-ATPase and changes in MHC isoform expression was observed. Changes in fraction of active myosin heads, in myosin light chain isoform (MLC) distribution or in MLC phosphorylation, could not explain the variations in m-ATPase. Myosin posttranslational modifications or changes in other myofibrillar proteins may therefore be responsible for the observed variations in m-ATPase activity.     

Specific enhancement of the cardiac myofibrillar ATPase by bound creatine kinase.

The kinetic influence of bound creatine kinase (CK) on the Ca(2+)-activated myosin ATPase was evaluated. ATPase rates were measured from 0.8 microM to 3.2 mM MgATP. Under control conditions, the apparent KmATP was 79.9 +/- 13.3 microM. In contrast, the addition of 12.2 mM phosphocreatine (PCr) decreased the apparent KmATP to a value of 13.6 +/- 1.4 microM. To determine if this reduction was merely the result of an ATP maintenance system, ATP was regenerated using either phosphoenolpyruvate and pyruvate kinase (PEP-PK), or PCr and soluble bovine cardiac CK. Data obtained with PEP + PK indicated an apparent KmATP of 65.5 +/- 7.3 microM. To study the effects of exogenous CK, the endogenous CK was irreversibly inhibited with 1 mM iodoacetamide. The kinetics of the ATPase were then examined by adding soluble CK to the incubation medium. Under these conditions, the KmATP was 56.4 +/- 0.86 microM. Therefore, these two ATP regeneration systems could not duplicate the effects of endogenous CK. The reduction of the apparent KmATP by endogenous CK was not the result of an altered inhibition by MgADP. MgADP inhibition was determined to be non-competitive, with a Ki of 5.0 +/- 0.1 mM. These data suggest that the observed kinetic effects reflect the proximity of the enzymes in the myofibrillar bundle, thus emphasizing the importance of bound CK for the localized regeneration of MgATP utilized by the myosin ATPase.     

Phosphorylation of guinea pig cardiac natural actomyosin and its effect on ATPase activity.

Guinea pig cardiac natural actomyosin incubated with commercial protein kinase, Mg²⁺-ATP, and cyclic AMP produced little or no change in actomyosin ATPase activity. However, addition of sodium fluoride, a known phosphatase inhibitor resulted in a decreased actomyosin ATPase at all measured calcium concentrations. The presence of phosphatase activity in actomyosin and protein kinase was confirmed with ?-nitrophenyl phosphate. These results indicate the importance of inhibiting phosphatase activity, particularly when measuring biological or enzymatic activity as a function of phosphorylation.     

Effects of isoproterenol treatment of isolated perfused rat hearts on myofibrillar phosphorylation and ATPase activity

Perfusion of isolated rat hearts with isoproterenol resulted in increases in the level of protein-bound phosphate of the myofibrils. After perfusion of the hearts with 32P, followed by SDS-polyacrylamide gel electrophoresis of the purified myofibrils, four major 32P-containing protein bands were identified. Most of the increased 32P incorporation produced by isoproterenol was localized on the troponin I and myosin light chain bands, and, to lesser extent, on the M-protein band. ATPase activity was tested in the purified myofibrils. No changes in Ca2+ requirement for activation were found after isoproterenol perfusion. However, maximal ATPase activity was markedly reduced in the myofibrils obtained from isoproterenol-treated hearts. It would appear that the myofibrillar protein phosphorylation induced by isoproterenol perfusion results in a decrease in actomyosin ATPase activity.     

Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni

Malolactic fermentation (MLF) is carried out by Oenococcus oeni under very harsh conditions. This paper shows that stress compounds in wine such as SO(2), fatty acids and copper have an inhibitory effect on cell growth and MLF duration, and relates this effect to an inhibition of ATPase activity. Of the stress compounds, SO(2) and dodecanoic acid had the strongest effect, decreasing the ATPase specific activity to 37% and 58%, respectively. It can be concluded that ATPase is a good indicator of the physiological state of the cells and their ability to lead MLF.