Abstract 14349: CaMKII-Dependent Troponin-I Phosphorylation Contributes to the Frequency-Dependent Acceleration of Relaxation in Ventricular Myocytes
Background: Frequency-dependent acceleration of relaxation (FDAR) is an intrinsic mechanism in ventricular myocytes allowing a faster ventricular relaxation (and diastolic filling) at fast heart rates. Previous studies suggest that CaMKII activity is required for FDAR but the molecular targets remain elusive.
Objective: We propose that CaMKII regulates FDAR by a mechanism that involves CaMKII-dependent alteration of myofilament sensitivity to Ca2+.
Methods: [Ca2+]i and sarcomere length were measured by IonOptix Ca2+ image system. Myofilament sensitivity to Ca2+ was assessed by measuring the gradient of cell length-fura2 trajectory during contraction and late relaxation. Western blot was used to detect the target proteins.
Results: Increasing pacing rate from 0.5Hz to 4Hz in left ventricular (LV) myocytes accelerated Ca2+ decline and sarcomere relaxation time constants (from 152±13ms to 60±5ms and from 36±2ms to 18±1ms, respectively, p<0.05, n=27) and increased the length-fura2 trajectory gradient and shifted the trajectory loop to the right (ECa50 increased from 1.62±0.06 to 1.84±0.06, p<0.05), indicating a consistency of FDAR with the reduction of myofilament sensitivity to Ca2+. Inhibition of PKA (H89, 1υM) or PKC (CHE, 1υM) had no effect on myofilament Ca2+ desensitization and FDAR, whereas CaMKII inhibitor KN93 (1υM) abolished frequency-dependent myofilament desensitization to Ca2+ and FDAR. Because cardiac troponin I (Tn-I) is the major regulator for myofilament sensitivity to Ca2+ and both PKA and PKC share the same phosphorylation sites Ser23/24, we determined the Ser23/24 phosphorylation in ventricular myocytes and found that Ser23/24 phosphorylation was largely reduced by PKA and PKC inhibitors but not by CaMKII inhibition. However, a phospho-Ser-antibody showed that CaMKII inhibitor KN93 significantly reduced Tn-I phosphorylation in the Tn-I immunoprecipitates, indicating that CaMKII phosphorylates Tn-I at sites different from the PKA and PKC sites. Indeed, a co-immunoprecipitation of CaMKII and Tn-I has been detected.
Conclusion: Our results suggest that FDAR is regulated by a frequency-dependent desensitization of myofilament sensitivity to Ca2+, in which CaMKII-dependent Tn-I phosphorylation plays a major role.
- © 2011 by American Heart Association, Inc.