Abstract 3209: MYFbx, a Novel Cardiac-specific E3-ligase, is a Component of the Sarcomeric Z-disc and Mediates Degradation of α-Actinin and Filamin C
The emerging role of the ubiquitin-proteasome system in cardiomyocyte function and homeostasis implies the necessity of a tightly controlled mechanism by which substrate-specific ubiquitination and degradation is achieved. Yet, little is known about cardiac-specific components of this machinery. Performing an “in silico”-screen of the EST database for cardiac-enriched F-box proteins/E3-ligases we identified MYFbx (Myocardial F-box protein). Cardiac-specific expression was confirmed by multiple tissue Northern and Western Blot analyses as well as qRT-PCR on a human cDNA-library. Immunocolocalization with Calsarcin-1 in neonatal and adult rat ventricular myocytes as well as murine heart tissue located MYFbx to the sarcomeric Z-disc. To detect cardiac interaction partners we performed a yeast two-hybrid screen employing MYFbx as a bait. Coimmunoprecipitation confirmed the identified interactions of MYFbx with Skp1 as well as Cullin1, two critical components of SCF (Skp1/Cul1/F-Box) E3-ligases. Moreover, we identified several potential substrates, including the z-disc proteins α-actinin and filamin C. Consistently, in vitro overexpression of MYFbx mediated degradation of both substrates in a dose-dependent fashion while simultaneous proteasome inhibition with MG-132 markedly attenuated degradation. As ubiquitination is known to regulate cardiac hypertrophy we tested for altered MYFbx-levels in hypertrophy models. Interestingly, we observed a significant downregulation of MYFbx in phenylephrine (PE)-treated cardiomyocytes (−79%, p<0.001) as well as in hearts subjected to aortic banding (−42%, p<0.05). Conversely, adenovirally mediated overexpression of MYFbx attenuated PE-mediated induction of the hypertrophic gene program with downregulation of ANF (−35%, p<0.05) and (BNP −58%, p<0.001). Furthermore, cell size analyses revealed a significant inhibition of hypertropy (cell size -23%, p<0.001). In summary, we identified a cardiac-specific F-box protein termed MYFbx that mediates degradation of α-actinin and filamin C. MYFbx inhibits cardiomyocyte growth as well as ANF and BNP expression and may thus represent a novel component of hypertrophic signaling pathways.