Recent studies have suggested that treatment of skinned cardiac trabeculae with a histone deacetylase (HDAC) inhibitor increases calcium sensitivity, measured as the calcium required for half maximal force development (pCa50). The proposed mechanism is the acetylation of sarcomeric proteins by an endogenous histone acetyl transferase, p300/CREB binding protein associated factor (pCAF). As a test of this proposed mechanism, we treated skinned cardiac myocytes from 8 rat left ventricles (LV) with supra-physiological concentrations of pCAF, with Acetyl COA as an acetyl group donor, and supra-physiological concentrations of constitutively active forms of HDACs 3 and 4, and examined the force-calcium relation. Myocytes were prepared from frozen rat LV samples by gentle homogenization in rigor solution (mM: 50 Tris, 1 EGTA, 100 KCl, 2 MgCl2, 2 DTT and a protease inhibitor cocktail) containing 0.1% Triton X-100. The myocyte homogenates were washed and resuspended in relaxing solution (mM: 5 MgATP, 10 Creatine phosphate, 40 K-Propionate and 10-6 Ca-EGTA) divided into four 300 µl aliquots, One was an untreated control and three were treated with either i) HDAC3/Ncor or HDAC4 , ii) pCAF + 1mM Acetyl CoA, or ii) 1mM Acetyl CoA. Force-calcium relationships were determined in mixtures of relaxing and activating solution (mM: 5 MgATP, 10 Creatine phosphate, 0.5 K-Propionate, 10-1.5 Ca-EGTA) to give 6 calcium concentrations between 10-9 and 10-3.5 mM. While HDAC 3 or 4 treatment tended to decrease calcium sensitivity, and pCAF+Acetyl COA, and Acetyl COA alone, tended to increase calcium sensitivity compared to controls, we found no statistically significant effects of HDAC3 or 4, or pCAF on the calcium sensitivity, maximum developed force or co-operativity of the myocytes (ANOVA, n=8, P<0.05).
EWS-ATF-1 chimeric protein in soft tissue clear cell sarcoma associates with CREB-binding protein and interferes with p53-mediated trans-activation function
