Transplantation of autologous skeletal myoblasts (SMBs) is a potential therapeutic approach for myocardial infarction. However, their clinical efficacy and safety is still controversial. Electrical coupling through gap junction between SMBs and host myocardium is essential for synchronized contraction and electrical stability. Here, we investigated the effect of heart beat-simulating environment, oscillating pressure, on the expression of connexin43 in two types of SMBs from rat and mouse. We found that connexin43 is markedly decreased under ischemia-mimicking conditions such as serum starvation and hypoxia (1% O(2)) in rat primary cultured SMBs and mouse C2C12 SMB cell line. Interestingly, the decrease of connexin43 expression under serum starvation was attenuated by oscillating pressure. Oscillating pressure treatment increased the expression of connexin43 twofold through AP-1 stimulation, which was blocked by PD98059, ERK inhibitor. In coculture of cardiomyocytes and C2C12, pressure-treated C2C12 and cardiomyocytes were able to form functional gap junction, which was demonstrated by both calcein-AM dye transfer assay and measurement of simultaneous contraction. In rat myocardial infarction model, transplantation of SMBs pretreated with oscillating pressure resulted in lesser ventricular dilatation and better systolic function than transplantation of untreated SMBs and control group. These results suggested that application of oscillating pressure on SMBs before transplantation may be useful to promote therapeutic efficacy for myocardial infarction by enhancing gap junction formation between transplanted and host cells.