Abstract 5408: PKA and PKC Phosphorylation of Troponin I Canonical Sites Regulate Calcium Sensitivity During Force Frequency Response
In failing hearts, the force-frequency response (FFR) is blunted, flat or negative. A positive FFR is crucial for healthy myocardium to respond to an increased working demand. There is no consensus in weather a positive FFR relies on myofilament Ca2+ sensitization or desensitization and weather this is modulated by cTnI phosphorylation. In the present work we aimed to address the FFR and Ca2+ cycling in intact mouse trabeculae loaded with Fura-2. To achieve this we used two transgenic models with pseudo phosphorylation mutants of troponin I (TnI), TnIDD22,23 mice, which mimic increased phosphorylation at PKA sites of TnI at Ser 22 and 23 and TnI PKA/PKC mice, which mimic dephosphorylation at same PKA sites and increased phosphorylation at PKC sites of TnI at Ser 42 and 44. We hypothesized that controlling for cTnI phosphorylation will clarify the contribution of cTnI to the differences in force and Ca2+ dynamics during FFR. When we examined the isometric contraction and Ca2+ dynamics in each of these lines (TnIDD22,23, n= 8; TnI PKA/PKC, n=6) and non transgenic controls (NTG, n=7) we found that all three groups showed a positive FFR, although peak Ca2+ increased with frequency rate in all three a less steep Ca2+ transient increase (myofilament Ca2+ sensitization) was observed in both transgenic lines compared to NTG (TnIDD22,23, p= 0.001; TnI PKA/PKC, p=0.03). Additionally, the peak force during the FFR was greater in the TnIDD22,23 mice compared to NTG (p < 0.0001), suggesting that TnIDD22,23 mice posses an enhanced frequency rate-related myofilament Ca2+ sensitivity. WB analysis of Ca2+ handling proteins including PLB, pPLB, SERCA2a and Ryanodine receptor normalized levels showed no major differences among all three groups, suggesting the differences observed in TnIDD22,23 mice were not due to altered Ca2+ handling but rather to myofilament Ca2+ sensitivity. We conclude that a positive systolic peak FFR is followed by increasing myofilament Ca2+ esensitization but mimicking increased phosphorylation at PKA sites of TnI Ser 22,23 enhances FFR and Ca2+ responsiveness. Overall, our results support the concept that myofilament alterations feedback onto Ca2+ handling mechanisms and these findings have important implications for human heart failure.