Abstract 1065: Class-II Histone Deacetylases Regulate Myofilament Calcium Sensitivity in the Mouse Heart

Abstract

The role of phosphorylation of myofilaments in regulating cardiac muscle calcium sensitivity and force production is well established. Recently it was shown that trichostatin A (TSA), a class-I and II histone deacetylase (HDAC) inhibitor, improved cardiac contractile function in vivo . Because class-II HDACs are able to translocate to the cytoplasm from the nucleus, it is postulated that some HDACs may interact with contractile proteins and thereby regulate myofilament calcium sensitivity and/or force production. Papillary muscle strips from mouse hearts were skinned for 4 hours and then treated overnight with two structurally dissimilar HDAC inhibitors, TSA and MS275. Both HDAC inhibitors significantly increased myofilament calcium sensitivity as quantified by pCa 50 [Baseline (n=9): 5.75±0.04; TSA (n=6): 5.84±0.02; MS275 (n=7): 5.87±0.03; P <0.05 vs . Baseline], without any significant changes in maximally activated force. Similar increase in calcium sensitivity was also observed when skinned fibers were incubated overnight in a buffer containing acetyl-CoA [acetyl-CoA (n=6): 5.84±0.04; P <0.05 vs . Baseline]. These observations suggest that acetylation of myofilament proteins can regulate myofilament calcium sensitivity. Western blot analyses of skinned fibers revealed that HDAC4 and HDAC5 were specifically associated with myofilament proteins, and acetylation of several myofilament proteins was increased following TSA treatment. One of these acetylated proteins was identified as muscle LIM protein (MLP). Protein-protein interaction analyses and co-localization studies showed that MLP specifically binds to HDAC4. Functional studies with skinned fibers isolated from MLP knock out [MLP −/− ] mice (C57BL6 strain) indicated that TSA-induced increase in calcium sensitivity was completely prevented [MLP −/− -Baseline (n=6): 5.60±0.03; MLP −/− -TSA (n=6): 5.61±0.02; P <NS] compared to wild-type (WT) mice of the same strain [WT-Baseline (n=6): 5.59±0.02; WT-TSA (n=6): 5.70±0.02; P <0.05]. These data demonstrate, for the first time, that class-II HDACs bind to cardiac myofilament proteins, with MLP being one of the binding partners, and play a role in regulating myofilament contractile function.