Tetrahydrobiopterin improves diastolic dysfunction by reversing changes in myofilament properties.

Despite the increasing prevalence of heart failure with preserved left ventricular function, there are no specific treatments, partially because the mechanism of impaired relaxation is incompletely understood. Evidence indicates that cardiac relaxation may depend on nitric oxide (NO), generated by NO synthase (NOS) requiring the co-factor tetrahydrobiopterin (BH(4)). Recently, we reported that hypertension-induced diastolic dysfunction was accompanied by cardiac BH(4) depletion, NOS uncoupling, a depression in myofilament cross-bridge kinetics, and S-glutathionylation of myosin binding protein C (MyBP-C). We hypothesized that the mechanism by which BH(4) ameliorates diastolic dysfunction is by preventing glutathionylation of MyBP-C and thus reversing changes of myofilament properties that occur during diastolic dysfunction. We used the deoxycorticosterone acetate (DOCA)-salt mouse model, which demonstrates mild hypertension, myocardial oxidative stress, and diastolic dysfunction. Mice were divided into two groups that received control diet and two groups that received BH(4) supplement for 7days after developing diastolic dysfunction at post-operative day 11. Mice were assessed by echocardiography. Left ventricular papillary detergent-extracted fiber bundles were isolated for simultaneous determination of force and ATPase activity. Sarcomeric protein glutathionylation was assessed by immunoblotting. DOCA-salt mice exhibited diastolic dysfunction that was reversed after BH(4) treatment. Diastolic sarcomere length (DOCA-salt 1.70±0.01 vs. DOCA-salt+BH(4) 1.77±0.01µm, P<0.001) and relengthening (relaxation constant, τ, DOCA-salt 0.28±0.02 vs. DOCA-salt+BH(4) 0.08±0.01, P<0.001) were also restored to control by BH(4) treatment. pCa(50) for tension increased in DOCA-salt compared to sham but reverted to sham levels after BH(4) treatment. Maximum ATPase rate and tension cost (ΔATPase/ΔTension) decreased in DOCA-salt compared to sham, but increased after BH(4) treatment. Cardiac MyBP-C glutathionylation increased in DOCA-salt compared to sham, but decreased with BH(4) treatment. MyBP-C glutathionylation correlated with the presence of diastolic dysfunction. Our results suggest that by depressing S-glutathionylation of MyBP-C, BH(4) ameliorates diastolic dysfunction by reversing a decrease in cross-bridge turnover kinetics. These data provide evidence for modulation of cardiac relaxation by post-translational modification of myofilament proteins.

Hypersensitivity of myofilament response to Ca2+ in association with maladaptation of estrogen-deficient heart under diabetes complication.

The amelioration of cardioprotective effect of estrogen in diabetes suggests potential interactive action of estrogen and insulin on myofilament activation. We compared Ca2+-dependent Mg2+-ATPase activity of isolated myofibrillar preparations from hearts of sham and 10-wk ovariectomized rats with or without simultaneous 8 wk-induction of diabetes and from diabetic-ovariectomized rats with estrogen and/or insulin supplementation. Similar magnitude of suppressed maximum myofibrillar ATPase activity was demonstrated in ovariectomized, diabetic, and diabetic-ovariectomized rat hearts. Such suppressed activity and the relative suppression in alpha-myosin heavy chain level in ovariectomy combined with diabetes could be completely restored by estrogen and insulin supplementation. Conversely, the myofilament Ca2+ hypersensitivity detected only in the ovariectomized but not diabetic group was also observed in diabetic-ovariectomized rats, which was restored upon estrogen supplementation. Binding kinetics of beta1-adrenergic receptors and immunoblots of beta1-adrenoceptors as well as heat shock 72 (HSP72) were analyzed to determine the association of changes in receptors and HSP72 to that of the myofilament response to Ca2+. The amount of beta1-adrenoceptors significantly increased concomitant with Ca2+ hypersensitivity of the myofilament, without differences in the receptor binding affinity among the groups. In contrast, changes in HSP72 paralleled that of maximum myofibrillar ATPase activity. These results indicate that hypersensitivity of cardiac myofilament to Ca2+ is specifically induced in ovariectomized rats even under diabetes complication and that alterations in the expression of beta1-adrenoceptors may, in part, play a mechanistic role underlying the cardioprotective effects of estrogen that act together with Ca2+ hypersensitivity of the myofilament in determining the gender difference in cardiac activation.

Mg-ATPase and Ca+ activated myosin AtPase activity in ventricular myofibrils from non-failing and diseased human hearts--effects of calcium sensitizing agents MCI-154, DPI 201-106, and caffeine.

We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201-106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca2+ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca2+ sensitivity. Effects of DPI 201-106 were, however, different. Only at the 10(-6) M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201-106 caused a concentration-dependent increase in myofibrillar ATPase Ca2+ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201-106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca2+ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca2+ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.

Creatine transporter and mitochondrial creatine kinase protein content in myopathies.

Total creatine or phosphocreatine, or both, are reduced in the skeletal muscle of patients with inflammatory myopathy, mitochondrial myopathy, and muscular dystrophy/congenital myopathy. We used Western blotting techniques to measure skeletal muscle creatine transporter protein and sarcomeric mitochondrial creatine kinase (mtCK) protein content in patients with inflammatory myopathy (N = 8), mitochondrial myopathy (N = 5), muscular dystrophy (N = 7), and congenital myopathy (N = 3), as compared to a control group without a neuromuscular diagnosis (N = 8). Creatine transporter protein content was lower for all groups compared to control subjects (P < 0.05; P < 0.01 for congenital myopathy). Mitochondrial CK (mtCK) was lower for inflammatory myopathy (P < 0.05), higher for mitochondrial myopathy (P < 0.05), not different for muscular dystrophy, and markedly lower for the congenital myopathy group (P < 0.01), compared to control subjects. Together, these data suggest that the reduction in total creatine or phosphocreatine in patients with certain myopathies is correlated with creatine transporter and not mtCK protein content. This further supports the belief that creatine monohydrate supplementation may benefit patients with low muscle creatine stores, although the reduction in creatine transporter protein may have implications for dosing.

Effect of Arg145Gly mutation in human cardiac troponin I on the ATPase activity of cardiac myofibrils.

In order to determine the functional consequences of the Arg145Gly mutation in troponin I found in familial hypertrophic cardiomyopathy, human cardiac troponin I and its mutant were expressed in Escherichia coli and purified, and then their effects on the ATPase activity of porcine cardiac myofibrillar preparations from which both troponins C and I had been depleted were examined. Both the wild-type and mutant troponin Is suppressed the ATPase activity of the troponin C.I-depleted myofibrils, but the maximum inhibition caused by mutant troponin I was weaker than that by wild-type troponin I. In the Ca(2)(+)-activation profile of the myofibrillar ATPase activity after reconstitution with both troponins I and C, the Ca(2)(+)-sensitivity with mutant troponin I was higher than that with wild-type troponin I, whereas the maximum level of the ATPase activity with mutant troponin I was lower than that with wild-type troponin I. These findings strongly suggest that the Arg145Gly mutation in human cardiac troponin I modulates the Ca(2)(+)-regulation of contraction by impairing the interaction of troponin I with both actin-tropomyosin and troponin C.

Cellular and molecular remodeling in a heart failure model treated with the beta-blocker carteolol.

Broad-breasted white turkey poults fed furazolidone developed dilated cardiomyopathy (DCM) characterized by ventricular dilatation, decreased ejection fraction, beta1-receptor density, sarcoplasmic reticulum (SR) Ca2+-ATPase, myofibrillar ATPase activity, and reduced metabolism markers. We investigated the effects of carteolol, a beta-adrenergic blocking agent, by administrating two different dosages (0.01 and 10.0 mg/kg) twice a day for 4 wk to control and DCM turkey poults. At completion of the study there was 59% mortality in the nontreated DCM group, 55% mortality in the group treated with the low dose of carteolol, and 22% mortality in the group treated with the high dose of carteolol. Both treated groups showed a significant decrease in left ventricle size and significant restoration of ejection fraction and left ventricular peak systolic pressure. Carteolol treatment increased beta-adrenergic receptor density, and the high carteolol dose restored SR Ca2+-ATPase and myofibrillar ATPase activities, along with creatine kinase, lactate dehydrogenase, aspartate transaminase, and ATP synthase activities, to normal. These results show that beta-blockade with carteolol improves survival, reverses contractile abnormalities, and induces cellular remodeling in this model of heart failure.

Changes of enzyme activities in the rat myocardium caused by experimental hypoxia with and without ginkgo biloba extract EGb 761 pretreatment. A cytophotometrical study.

Using cytophotometry activity changes of succinate dehydrogenase, glycerol-3-phosphate dehydrogenase and myofibrillar adenosine triphosphatase, were measured in the rat myocardium under normal and different experimental conditions. After hypoxia all enzyme activities were significantly decreased in comparison to the normal situation, and the alterations differed in both ventricles. Ginkgo biloba extract treatment over three months before exposition to hypoxia resulted in a lower inhibition of succinate dehydrogenase, a higher inhibition of glycerol-3-phosphate dehydrogenase and an unchanged activity of adenosine triphosphatase after hypoxia of 20 min. These results were interpreted as a protective effect of the Ginkgo biloba extract on the hypoxic myocardium.

Dietary effects on cardiac metabolic properties in rodents.

Previous studies have shown that dietary provision of carbohydrate can alter cardiac isomyosin distribution in hormonally deficient rats. The main objective of this study was to determine if varying the heart's potential to utilize carbohydrate for energy provision can influence the cardiac isomyosin expression in normal weanling rats. Animals were assigned to one of five groups according to dietary and/or metabolic treatment: (1) mixed-control--(M); (2) high carbohydrate--(H); (3) low carbohydrate--(L); (4) mixed-diet supplemented with oxfenicine, a cardiospecific fatty acid oxidation inhibitor--(MO); and (5) high carbohydrate diet supplemented with oxfenicine--(HO). The results show that 4 weeks of dietary manipulations aimed to either increase or decrease carbohydrate supply to the heart, failed to induce any alterations in either cardiac myosin ATPase activity or isoenzyme pattern. However, extremes in carbohydrate provision altered the metabolic properties of both heart and skeletal muscle. A low carbohydrate diet increased 3-hydroxyacyl CoA dehydrogenase (P less than 0.05) and citrate synthase activities (P less than 0.05) and decreased glycogen content in both heart and soleus muscle; whereas, a high carbohydrate diet, in conjunction with oxfenicine, tended to increase hexokinase activity in these same tissues. These alterations provide indirect evidence that the contributions of both fat and carbohydrate to the energy balance of the heart and skeletal muscle were altered by the imposed dietary interventions. Collectively, these results suggest that although the substrate utilization patterns of the normal weanling heart can be modified via dietary manipulation, such shifts do not exert any regulatory influence on cardiac isomyosin expression.

Histochemical and biochemical characterization of two slow fiber types in decapod crustacean muscles.

Myofibrillar proteins in muscles of the claws and abdomen of lobster, Homarus americanus, and the claws of fiddler crab, Uca pugnax, and land crab, Gecarcinus lateralis, have been analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fibers contained numerous isoforms of structural and regulatory proteins in assemblages correlated with fiber type. One fast (F) and two slow (S1 and S2) fibers were identified. All F fibers possessed two isoforms of paramyosin (P1 and P2), while all slow fibers, with the exception of Uca major claw, contained only the P2 variant. S1 and S2 fibers were distinguished by the distribution of a large isoform of troponin-T (T1; Mr = 55,000); S2 fibers in all three species contained T1 in addition to one or two smaller-molecular-weight variants usually associated with S1 fibers. In order to determine whether the slow fibers differed in histochemical properties, land crab claw closer muscle was cryosectioned and stained for myofibrillar ATPase and NADH diaphorase activities. Most S2 fibers had lower ATPase and higher NADH diaphorase activities than S1 fibers, which indicated that S2 fibers had a lower rate of contraction and were more fatigue-resistant than S1 fibers. It is proposed that the S1 and S2 fibers defined by biochemical and histochemical criteria are identical to the slow-twitch and tonic fibers, respectively characterized physiologically.

Studies of myofibrillar ATPase in ragweed-sensitized canine pulmonary smooth muscle.

The maximal shortening velocities of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized dogs were significantly higher than those of muscles from their littermate controls. Myofibrils of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized and control dogs were obtained with use of Triton X-100 homogenizing solution. The myofibrillar adenosinetriphosphatase (ATPase) activities of the sensitized tissues were significantly higher (P less than 0.05) than those of their respective controls.

The effects of immobilization and of electrical stimulation on muscle glycogen and myofibrillar ATPase.

This study observed biochemical changes that occur when an athlete's limb is immobilized for six weeks, and determined if electrical stimulation could alter any of these changes. A control group (n = 3) and a stimulation group (n = 6) who had undergone major knee surgery, consented to a series of muscle biopsies of the vastus lateralis muscle. Both groups engaged in standard rehabilitative procedures following cast removal. The stimulation group's therapy also consisted of electrical stimulation (f = 2500 Hz.) of the quadricep group. The results indicate that ATPase activity decreased in the control group during the immobilization period (from 0.321 +/- 0.095 mumoles Pi/mg./min. to 0.205 +/- 0.032 mumoles Pi/mg./min.) but not in the stimulation group (from 0.268 +/- 0.022 mumoles Pi/mg./min. to 0.296 +/- 0.073 mumoles Pi/mg./min.), p less than 0.05. The stimulation did not appear to have any effect on glycogen concentrations. It is suggested that electrical stimulation retards the decrements in biochemical characteristics of human muscle due to immobilization.