Abstract 13519: ZFYVE7 Promotes Autophagy in Cardiomyocytes
Using a bioinformatic approach for screening of EST libraries we aimed to identify novel genes that are predicted to be specifically expressed in heart and skeletal muscle. In this screen we identified a previously little characterized protein, termed “ZFYVE7”. A highly striated muscle-specific expression pattern could be confirmed by multiple tissue real time PCR and Northern blot. To assess the cellular function of ZFYVE7 we performed a yeast two-hybrid screen that revealed LC3 as an interacting protein. LC3 is an essential protein in autophagy, a lysosome-dependent degradation mechanism. Overexpression of ZFYVE7 in neonatal rat cardiomyocytes (NRCM) by adenoviral gene transfer resulted in increased LC3-II levels (3.6-fold, p<0.05), an acivated form of LC3. As a control we used adenoviruses encoding for b-galactosidase. Under starvation conditions of glucose deprivation for 24h, which strongly induces autophagy, we found a 3.7-fold upregulation of LC3-II in control NRCMs. This effect was significantly exaggerated by overexpression of ZFYVE7 (10.3-fold). Overexpressed and endogenous ZFYVE7 localized to the cytosol in a punctuated pattern and co-localized with LC3. Next, we generated adenoviruses encoding for synthetic miRNAs targeting ZFYVE7. The most promising miRNA resulted in a knockdown of ZFYVE7 mRNA by 93%. After 72h we detected a complete protein knockdown by Western blot. NRCMs were then treated with adenoviruses carrying either a miRNA that is predicted not to have any target (control) or a synthetic miRNA against ZFYVE7. Using a reporter adenovirus encoding for GFP-tagged LC3, we detected autophagosomes in control NRCMs after 24h of glucose deprivation by immunocytochemistry. In contrast, ZFYVE7 knockdown led to a marked reduction in cells showing vesicular staining. In order to examine the cellular function of ZFYVE7 in vivo, we generated a constitutive knockout mouse model. Homozygous mice were born in the expected mendelian ratios. Body and heart weight as well as cardiac function did not differ from wildtype littermates. Since autophagy is induced under stress conditions we are currently performing aortic banding experiments in order to assess a potential differential response of ZFYVE7-deficient to mice to pressure overload.
- © 2011 by American Heart Association, Inc.