Abstract 1382: The EF-hand Protein Calmyrin is a Novel Negative Regulator of Cardiac Myocyte Hypertrophy and Cell Death
Introduction: We identified the 22kDa protein calmyrin in a genetic screen for signaling molecules related to cardiomyocyte growth and death. Calmyrin contains an N-terminal myristoylation site and two C-terminal Ca++ binding EF-hand domains. This structure renders calmyrin a Ca++/myristoyl switch protein, which typically undergoes conformational changes upon Ca++ binding leading to membrane translocation and interaction with specific target proteins. Very little is known of calmyrin’s function, although it has been shown to affect cell death in Hela cells and spreading in platelets.
Results: Myocardial calmyrin expression was upregulated after 2 weeks of transverse aortic constriction in mice (TAC 190±20% vs. sham 100±15%, p<0.05). Immunohistochemical studies indicated that calmyrin translocates to the sarcolemma after TAC. Considering its increased expression during cardiac growth and hemodynamic stress, we analyzed the impact of calmyrin on cardiomyocyte hypertrophy and death in vitro. Overexpression of calmyrin with a recombinant adenovirus inhibited phenylephrine-induced increases in cell size (Ad.βgal 161±13% vs. Ad.calmyrin 113±12%, p<0.01), ANF expression (northern blot; Ad.βgal 1407±194% vs. Ad.calmyrin 862±170%, p<0.01) and sarcomere organization assessed by alpha-actinin staining. Calymrin overex-pression did not affect cardiomyocytes at baseline or reduce hypertrophy directed by insulin like growth factor-1. calmyrin overexpression also inhibited cardiomyocyte death following 96 h of serum deprivation as assessed by propidium-iodide exclusion (rate of cardiomyocyte death, Ad.βgal 22±2% vs. Ad.calmyrin 12±2%, p<0.05), Tunel staining (Ad.βgal 24±3% vs. Ad.calmyrin 9±1%, p<0.01) and caspase 3 activation (western-blot, Ad. βgal 100±17% vs. Ad.calmyrin 50±13%, p<0.05). Adenoviruses expressing calmyrin mutants defective in membrane localization or Ca++ binding did not inhibit cardiomyocyte growth or death, underscoring the importance of the intact Ca++/myristoyl switch for calmyrin’s function. Inducible calmyrin cardiac-specific transgenic mice have also been recently generated and will be presented.
Conclusion: Calmyrin inhibits cardiomyocyte growth and death.