Mechanism of Doxorubicin-Induced Suppression of Cardiac Ankyrin Repeat Protein: Implications for Anthracycline Cardiomyopathy

Doxorubicin (adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin, however, the mechanism(s) of doxorubicin-induced CARP suppression and its specific role in cardiomyocyte biology remains to be elucidated. In this study, we report that treatment of cardiomyocytes with doxorubicin resulted in complete suppression of CARP promoter activity, decreased CARP protein levels, and marked sarcomere disarray. Transfection of CARP siRNA in cardiomyocytes resulted in a complete depletion of CARP and significant disruption of sarcomere ultrastructure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. GATA4 has previously been shown to regulate CARP, thus we examined the role of GATA4 in doxorubicin-induced CARP depletion. Cardiomyocytes treated with doxorubicin show a concomitant depletion of CARP and GATA4 protein levels. GATA4 siRNA inhibits while GATA4 overexpression enhances CARP promoter activity in cardiomyocytes. Both GATA4 and CARP siRNA significantly repressed titin and actin promoter activity. These data show that in cardiomyocytes transcription factor GATA4 is upstream of CARP and that doxorubicin induces a rapid down-regulation of GATA4 resulting in inhibition of CARP transcription. Our data further support a role for a GATA4/CARP signaling axis in sarcomere maintenance and that suppression of this pathway contributes, in part, to the overall pathophysiology of doxorubicin cardiomyopathy.