Abstract 343: Histone Deacetylase 4 Binds to Muscle LIM Protein and Associates with the Z-Disc and I-Band of the Cardiac Muscle Sarcomeres
Histone deacetylase 4 (HDAC4), a member of class-II HDACs, is expressed both in the cytoplasm and nucleus of cardiac myocytes. In the nucleus, HDAC4 regulates expression of cardiac muscle specific genes; however its role in cytoplasm is not known. To examine the expression pattern of endogenous HDAC4 in cardiomyocytes, we double-stained neonatal mouse heart myocytes with HDAC4 and α-actinin specific antibodies and localized the protein expression using confocal microscopy. Alpha-actinin is a Z-disc associated protein and hence staining with actinin antibody revealed the expected striated staining pattern. When myocytes were examined for HDAC4 localization, we noticed a similar striated pattern. These results were intriguing because no HDAC isoform of any class has been shown to be associated with muscle sarcomere structures to date. The same experiment with adult mouse heart sections, stained for HDAC4, revealed nearly identical striated expression pattern that overlapped with anti-actinin staining, indicating that HDAC4 is likely to be a Z-disc associated deacetylase. For the negative control, we stained these heart sections with four other antibodies against SIRT1, HDAC1, H2b and HDAC6; none of them showed the striated staining pattern. We next performed immuno-electron microscopy to further confirm these results. In the heart sections, immunogold particles were found in the nucleus as well as on the sarcomeres. At higher magnification, we found that HDAC4 was mainly localized to the Z-disc, with a small portion of deacetylase also present at the I and A bands. However, no HDAC4 was found at M band and H zone of the sarcomere. Based on Far-Western, co-immunoprecipitation, and co-localization studies, we identified muscle LIM protein (MLP) as a sarcomeric binding partner of HDAC4. We also analyzed HDAC4 localization in mice hearts subjected to aortic banding-induced pressure overload hypertrophy. Results indicated that HDAC4 binding to sarcomeres of hypertrophied hearts was notably reduced (50%) as compared to control hearts. These results demonstrate that HDAC4 is a sarcomere associated deacetylase, with MLP as a binding partner. We speculate that HDAC4 may regulate myofilamental contractile function in patho-physiological conditions.