Given the essential role played by gap junctions in the coordination of cardiac muscle contraction, it is plausible that down-regulation of gap junctional conduction is in part responsible for the contractile dysfunction observed in hypertrophied and failing hearts. In the present study, we analyzed the expression and function of the gap junction protein, connexin43, in the ventricular myocardium of hereditary cardiomyopathic, Syrian BIO 14.6 hamsters. Immunoprecipitation and immunoblot analyses revealed that levels of tyrosine phosphorylated connexin43 were increased in BIO 14.6 hamsters at the late stage of congestive heart failure. Furthermore, the increased tyrosine phosphorylation was correlated with increased c-Src activity. The functional consequences of tyrosine phosphorylation of connexin43 in gap junction were assessed using transfected cells expressing constitutively active c-Src. It was found that constitutively active c-Src diminished propagation of Ca(2+) waves in HEK293 cells and reduced gap junctional conductance between pairs of cardiac myocytes. We, therefore, conclude that during the progression of cardiac dysfunction in the cardiomyopathic heart, gap junctional communication is reduced via c-Src-mediated tyrosine phosphorylation of connexin43.