Overexpression of HAX-1 protects cardiac myocytes from apoptosis through caspase-9 inhibition.

Caspase-9 is a critical regulator of mitochondria-mediated apoptosis. We found that adult cardiac myocytes, but not nonmyocytes, have high caspase-9 expression, and exhibit relative resistance to caspase-9-induced cell death. Thus, we hypothesized that cardiac myocytes possess factors that resist apoptosis. Through a yeast two-hybrid screening of adult human heart cDNA library, we identified HS-1 associated protein-1 (HAX-1), a 35-kD BH-domain containing protein localized to the mitochondria as one of the molecules that interacts with caspase-9. Recombinant HAX-1 protein inhibited caspase-9 processing in a dose-dependent manner in a cell-free caspase activation assay. Overexpression of HAX-1 in adult cardiac myocytes conferred 30% protection from apoptosis as compared with the control. Suppression of HAX-1 expression using siRNA-HAX-1 resulted in significant cell death in adult cardiac myocytes, suggesting the importance of HAX-1 in cardiac myocyte resistance to apoptotic stimulation. On apoptotic stimulation, some caspase-9 translocated to the mitochondria and co-localized with HAX-1, confirming the spatial proximity of caspase-9 and HAX-1. In summary, HAX-1 is a newly identified anti-apoptotic factor and its mechanism of action is through caspase-9 inhibition.