Abstract 450: Phosphorylation Sites of Cardiac Myosin Binding Protein-C are Required for Reduced Calcium Sensitivity of Myofilaments due to Protein Kinase-A
The cardiac isoform of myosin binding protein-C (cMyBP-C) possesses multiple phosphorylation sites (Ser-273, −282 and −302) near its N-terminus that are sensitive to protein kinase-A (PKA). However, the functional consequences of phosphorylating these sites by PKA are not known. We examined the effects of PKA exposure on myofilaments of chemically-skinned mouse myocardial strips that had or had not been genetically manipulated to contain: (i) wild-type cMyBP-C by non-transgenic methods (NTG, n=8), (ii) no cMyBP-C (t/t, n=8), (iii) wild-type cMyBP-C expressed on t/t background (WT, n=6), and (iv) wild-type cMyBP-C with Ser replaced by Ala expressed on t/t background (AllP-, n=8). Exposure of NTG myofilaments to PKA reduced half maximal calcium concentration (pCa50) of tension development significantly (5.75±0.03 vs 5.67±0.03, p<0.05). Conversely, PKA exposure in t/t enhanced calcium sensitivity of tension significantly (5.60±0.01 vs. 5.73±0.0.2, p<0.01). Furthermore, PKA exposure in the WT reduced pCa50 significantly (5.80±0.05 vs. 5.72±0.03, p<0.05) while PKA exposure in AllP- elevated pCa50 significantly (5.75±0.02 vs. 5.80±0.03, p<0.05). These results imply that the phosphorylation sites of cMyBP-C are specifically required for the reduction in myofilament calcium sensitivity normally induced by PKA. Interestingly, maximal tension was significantly (p<0.05) reduced in all populations by PKA: NTG 13.7±1.3 kN/m2 vs. 6.5±1.2 kN/m2; t/t 11.1±0.9 kN/m2 vs. 5.0±0.5 kN/m2; WT 16.0±1.2 kN/m2 vs. 6.7±0.7 kN/m2, and AllP- 15.2±1.4 kN/m2 vs. 7.7±0.7 kN/m2. Thus, reduction of maximal tension by PKA appears to be independent of cMyBP-C or its phosphorylation. We hypothesize that the structural and functional roles of the cMyBP-C N-terminus include a considerable influence on thin filament activation by calcium as part of contractile regulation in the heart.