Abstract 1245: Protein Kinase A Induced Changes In Calcium Sensitivity Of Isolated Human Cardiac Myofibrils Do Not Depend Upon Changes In Total Phosphorylation Of Myofilament Proteins
Protein kinase A (PKA) treatment of skinned cardiac muscle preparations has been shown to decrease calcium sensitivity of force development. The change in calcium sensitivity is thought to be mediated mainly through phosphorylation of troponin I. However, other models of regulation of protein function by phosphorylation suggest that exchange of phosphate may also be an important regulatory process. We tested the effect of phosphate exchange versus changes in total phosphorylation on the calcium sensitivity of force in isolated human myofibrils. Preparations of cardiac myofibrils are often assumed to consist solely of sarcomeric proteins such as actin, myosin, the troponins, and tropomyosin, but regulatory signaling enzymes such as kinases and phosphatases are thought to be absent. We used PKA to phosphorylate several sarcomeric proteins (troponin I, troponin T, myosin binding protein C) in isolated myofibrillar preparations in the presence of 32P-ATP. We measured total phosphorylation using ProQ Diamond, a phosphoprotein specific gel stain and we also measured incorporation of 32P into the same target proteins. We also measured the force response to calcium in similarly treated isolated human cardiac myofibrils to assess changes in calcium sensitivity. Surprisingly, we found that PKA treatment of myofibrils did not significantly elevate the total phosphorylation of the target proteins despite producing the expected increase in pCa50 (ΔpCa50 = 0.11±0.03, n = 4, p = 0.006). However, autoradiograms of the same samples showed significant amounts of radioactive phosphate incorporation. These results show that phosphate incorporation can occur with little change in total protein phosphorylation. We propose that an endogenous phosphatase is present in myofibrillar preparations and dephosphorylates the target proteins allowing phosphate incorporation with no net change in total phosphorylation and it is this flux of phosphate that effects the change in pCa50 rather than sustained changes in total phosphorylation.