The aim of this study was to test the hypothesis that persistent myocardial apoptosis contributes to progression of heart failure in a canine model of pacing-induced cardiomyopathy. Dogs were paced at 250 beats per minute for 1 week (n=9), 3 weeks (n=14) and 4 weeks (n=14) with normal dogs served as controls (n=12). Myocardial apoptosis was assessed by multiple methods including DNA fragmentation and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, bax and bcl-2 protein expression, and caspase activity. Pacing produced a progressive increase in left ventricular (LV) end diastolic pressure (LVEDP) and plasma norepinephrine levels with no significant increase in LV mass. The number of apoptotic cells was markedly increased after 1 week of pacing and remained increased at 4 weeks of pacing with characteristic DNA laddering. The increase in apoptosis was associated with bax protein expression and caspase activation while there was no detectable changes in bcl-2 protein expression. The estimated total number of apoptotic cells correlated with cardiac output and LVEDP (r=-0.69 and 0.59, respectively, P<0.001). Plasma norepinephrine and bax protein expression correlated significantly with the estimated total number of apoptotic myocytes (r=0.62 and 0.42, respectively, P<0.01). In conclusion, an early and persistent activation of myocardial apoptosis and pro-apoptotic factors is likely an important mechanism that contributes to the progression of heart failure in canine pacing-induced cardiomyopathy.