Abstract
Our previous study of homozygous mutants of the ventricular specific isoform of myosin light chain 2 (mlc-2v) demonstrated that mlc-2v plays an essential role in murine heart development (Chen, J., Kubalak, S. W., Minamisawa, S., Price, R. L., Becker, K. D., Hickey, R., Ross, J., Jr., and Chien, K. R. (1998) J. Biol. Chem. 273, 1252-1256). As gene dosage of some myofibrillar proteins can affect muscle function, we have analyzed heterozygous mutants in depth. Ventricles of heterozygous mutants displayed a 50% reduction in mlc-2v mRNA, yet expressed normal levels of protein both under basal conditions and following induction of cardiac hypertrophy by aortic constriction. Heterozygous mutants exhibited cardiac function comparable to that of wild-type littermate controls both prior to and following aortic constriction. There were no significant differences in contractility and responses to calcium between wild-type and heterozygous unloaded cardiomyocytes. We conclude that heterozygous mutants show neither a molecular nor a physiological cardiac phenotype either at base line or following hypertrophic stimuli. These results suggest that post-transcriptional compensatory mechanisms play a major role in maintaining the level of MLC-2v protein in murine hearts. In addition, as our mlc-2v knockout mutants were created by a knock-in of Cre recombinase into the endogenous mlc-2v locus, this study demonstrates that heterozygous mlc-2v cre knock-in mice are appropriate for ventricular specific gene targeting.
Highlights
Sarcomeres, the functional units of striated muscle, are composed of Ͼ20 known proteins, precisely organized in a nearly crystalline fashion
Heterozygous mutants can survive, but cannot fly due to dysfunctional flight muscles that display abnormal sarcomeric structure [14]. These heterozygous mutants display a 50% reduction in mlc-2 mRNA with a 25% reduction in Myosin light chain 2 (MLC-2) protein compared with wild-type litmyosin light chain 2; MLC-2v, ventricular MLC-2; MLC-2a, atrial MLC-2; TAC, transverse aortic constriction
We demonstrate that heterozygous mlc-2v cre knock-in mice are indistinguishable from wild-type mice in all aspects and display normal levels of MLC-2v protein despite a 50% reduction in mRNA
Summary
Sarcomeres, the functional units of striated muscle, are composed of Ͼ20 known proteins, precisely organized in a nearly crystalline fashion. Ultrastructural analysis revealed defects in sarcomeric assembly These data indicate that mlc-2v plays an important role in cardiac function and myofibrillogenesis during mammalian heart development. Heterozygous mutants can survive, but cannot fly due to dysfunctional flight muscles that display abnormal sarcomeric structure [14]. These heterozygous mutants display a 50% reduction in mlc-2 mRNA with a 25% reduction in MLC-2 protein compared with wild-type litmyosin light chain 2; MLC-2v, ventricular MLC-2; MLC-2a, atrial MLC-2; TAC, transverse aortic constriction. We have validated the potential utility of the mlc-2v cre knock-in system as a valuable approach for ventricular restricted gene targeting
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