Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular pressure overload model (RVPO), we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24-48 h PO myocardium, and these changes returned to basal level by 1wk PO. Histochemical studies in 24 h PO myocardium revealed the presence of TUNEL-positive cardiomyocytes that exhibited enrichment of both calpain and gelsolin, and biochemical studies showed an increase in histone-H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, all indicating programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin by bolus intravenous injections twice (doses at 0.6 mg/kg), 15 min prior to and 6 h after the induction of PO for 24 h. Calpeptin administration blocked the following PO-induced changes: calpain enrichment, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone-H2B phosphorylation. Although similar administration of a caspase inhibitor, Z-VD-fmk, blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.