ABSTRACT
Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to â¼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC.
Subject(s)
Actin Cytoskeleton/metabolism , Cardiomyopathy, Hypertrophic, Familial/metabolism , Mutation, Missense , Myocardial Contraction , Myosin Light Chains/metabolism , Ventricular Myosins/metabolism , Adenosine Triphosphate/metabolism , Animals , Cardiomyopathy, Hypertrophic, Familial/genetics , Cells, Cultured , Heart Ventricles/cytology , Heart Ventricles/metabolism , Mice , Myofibrils/metabolism , Myofibrils/physiology , Myosin Light Chains/chemistry , Myosin Light Chains/genetics , Protein Binding , Ventricular Myosins/geneticsABSTRACT
The contraction of the right ventricle (RV) expels blood into the pulmonary circulation, and the contraction of the left ventricle (LV) pumps blood into the systemic circulation through the aorta. The respective afterloads imposed on the LV and RV by aortic and pulmonary artery pressures create very different mechanical requirements for the two ventricles. Indeed, differences have been observed in the contractile performance between left and right ventricular myocytes in dilated cardiomyopathy, in congestive heart failure, and in energy usage and speed of contraction at light loads in healthy hearts. In spite of these functional differences, it is commonly believed that the right and left ventricular muscles are identical because there were no differences in stress development, twitch duration, work performance, or power among the RV and LV in dogs. This report shows that on a mesoscopic scale [when only a few molecules are studied (here three to six molecules of actin) in ex vivo ventricular myofibrils], the two ventricles in rigor differ in the degree of orientational disorder of actin within in filaments and during contraction in the kinetics of the cross-bridge cycle.
Subject(s)
Actins/chemistry , Actins/metabolism , Heart Ventricles/metabolism , Myosins/chemistry , Myosins/metabolism , Animals , Dogs , Female , Kinetics , Mice , Mice, Inbred C57BL , Models, Cardiovascular , Models, Molecular , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/metabolism , Myocardial Contraction , Myofibrils/chemistry , Myofibrils/metabolism , Tissue Distribution , Ventricular FunctionABSTRACT
A simple, precise, rapid, accurate and economic reverse phase high performance liquid chromatographic method has been developed for the estimation of prulifloxacin in tablet dosage form. The separation was achieved by using octadecylsilane column (C(18)) and KH(2)PO(4) buffer: acetonitrile adjusted to pH 7.3 with triethyl amine in proportion of 10:90 v/v as mobile phase, at a flow rate of 1.0 ml/min. The detection was carried out at 278 nm. The retention time of prulifloxacin was found to be 2.4 min. The limit of detection and limit of quantitation were found to be 0.14 µg/ml and 0.42 µg/ml respectively. The accuracy and reliability of the proposed method was ascertained by evaluating various validation parameters like linearity, precision, accuracy and specificity according to ICH guidelines. The proposed method provides an accurate and precise quality control tool for routine analysis of prulifloxacin in tablet dosage form.
