Abstract 203: Attenuation Of Myotrophin-induced Cardiac Hypertrophy By Mutation At Hairpin-like Tip
Myotrophin (myo), a 12-kD ankyrin repeat protein, stimulates protein synthesis and cardiomyocyte growth to initiate cardiac hypertrophy. It is also well established that myo activates NF-κB signaling cascade during cardiac hypertrophy and heart failure. However, it is not known what sites of myo are responsible for its stimulatory activity and for NF-κB activation. Based on the solution structure of myo, we identified potential functional sites in two hairpin-like tips that are responsible for these two activities. To explore the structure-function relationship of myo to its stimulatory activity, we generated mutant myo proteins and examined their stimulatory activity on rat cardiomyocytes. Mutation of glutamic acid 33 to alanine (E33A), located in the first hairpin-like tip, showed significant reduction in stimulatory activity. Using neonatal rat cardiomyocytes, we examined the localization and trafficking of Alexa-488-labeled wild-type myo and E33A after addition to external culture. We also evaluated NF-κB activation by immunolocalization of p65. Wild-type myo and E33A internalized into cells within 5 min after their addition into media. Within the next 20 – 60 min, wild-type myo translocated into the nucleus and activated the NF-κB pathway. In contrast, E33A did not migrate to the nucleus and failed to activate the NF-κB pathway; both E33A and p65 stayed in the cytoplasm. There was no appreciable increase in myocyte size (34190.3 μm2) after treatment with E33A in comparison with that of wild-type myo treatment (53698.8 μm2). We confirmed the loss of E33A activity by verifying inhibition of two markers for cardiac hypertrophy, atrial natriuretic factor and β-myosin heavy chain gene expression. Our study indicates that a change in a single amino acid (E33A) causes myo to lose its stimulatory activity with respect to cardiac hypertrophy. This observation will enable us to continue developing a therapeutic strategy against myotrophin-induced cardiac hypertrophy.