Calcineurin B homologous protein 3 (CHP3) negatively regulates cardiomyocyte hypertrophy in rat neonatal ventricular cardiomyocytes

Background: Pathological cardiac hypertrophy is the enlargement of cardiac muscle in response to various stimuli which increase in cardiac workload, and associated with a increase in the risk of cardiac death. Although many signaling factors are identified to regulate this process, the functions of some proteins are still unknown. Calcineurin B homologous protein 3 (CHP3) is an EF-hand calcium (Ca2+)-binding protein predominantly expressed in heart. However, the role is unclear. Methods: We used adenoviral-based RNA interference system to knockdown CHP3 expression in rat neonatal ventricular cardiomyocytes (NRVMs). The phosphorylation level of 27 kinds of kinases in CHP3 knockdown NRVM was analyzed by phosphokinase array. To examine the effect of Ca2+ on the function of CHP3, a Ca2+-binding-defective mutant of CHP3 (CHP3-D123A) was expressed in HEK293 cells, which do not express endogenous CHP3. The effect of CHP3 on phosphorylation of glycogen synthase kinase 3 (GSK3) was analyzed in vitro using phosphorylated (activated) protein kinase B (Akt) collected from insulin-treated HEK293 cells by immunoprecipitation (IP), GSK3 peptide and GST-CHP3. Protein-protein interaction was examined by Co-IP. Results: CHP3 knockdown resulted in significant enlargement of cardiomyocyte size and the increase in the protein expression level of atrial natriuretic peptide which is a pathological hypertrophy marker. In addition, phosphorylation level of GSK3 beta was dramatically elevated in CHP3 knockdown NRVMs. In contrast, the phosphorylation of GSK3 beta induced by insulin was inhibited in CHP3-overexpressed HEK293 cells. Interestingly, overexpression of CHP3-D123A also inhibited the phosphorylation. The phosphorylation level of GSK3 mediated by activated Akt was decreased in the presence of CHP3 in vitro. Co-IP experiments demonstrated the interaction of CHP3 with both Akt and GSK3 beta. Conclusions: These results suggest that CHP3 serves as a novel regulatory factor for cardiomyocyte hypertrophy by inhibiting Akt-mediated phosphorylation of GSK3 beta in a Ca2+-independent manner. Moreover, we will introduce the phenotype of CHP3 knockout mouse.