Abstract 5408: PKA and PKC Phosphorylation of Troponin I Canonical Sites Regulate Calcium Sensitivity During Force Frequency Response

Abstract

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 Ca 2+ sensitization or desensitization and weather this is modulated by cTnI phosphorylation. In the present work we aimed to address the FFR and Ca 2+ 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), TnIDD 22,23 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 Ca 2+ dynamics during FFR. When we examined the isometric contraction and Ca 2+ dynamics in each of these lines (TnIDD 22,23 , 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 Ca 2+ increased with frequency rate in all three a less steep Ca 2+ transient increase (myofilament Ca 2+ sensitization) was observed in both transgenic lines compared to NTG (TnIDD 22,23 , p= 0.001; TnI PKA/PKC, p=0.03). Additionally, the peak force during the FFR was greater in the TnIDD 22,23 mice compared to NTG (p < 0.0001), suggesting that TnIDD 22,23 mice posses an enhanced frequency rate-related myofilament Ca 2+ sensitivity. WB analysis of Ca 2+ handling proteins including PLB, pPLB, SERCA2a and Ryanodine receptor normalized levels showed no major differences among all three groups, suggesting the differences observed in TnIDD 22,23 mice were not due to altered Ca 2+ handling but rather to myofilament Ca 2+ sensitivity. We conclude that a positive systolic peak FFR is followed by increasing myofilament Ca 2+ esensitization but mimicking increased phosphorylation at PKA sites of TnI Ser 22,23 enhances FFR and Ca 2+ responsiveness. Overall, our results support the concept that myofilament alterations feedback onto Ca 2+ handling mechanisms and these findings have important implications for human heart failure.