Abstract 802: Dynamic FRET-Based Ca-Calmodulin Measurements in Intact Ventricular Myocytes Uncover Differential Signal Integration Due to Ca-Calmodulin Affinity

Abstract

Background: Myocyte [Ca] i varies beat-to-beat and calmodulin (CaM) transduces Ca signals to regulate many cardiac functions (e.g. via CaM-dependent kinase, CaMKII, and calcineurin, CaN). We hypothesize that the different Ca-CaM affinities of CaMKII (Kd ∼45 nM) and CaN (Kd ∼2 nM) will dictate their dynamic and tonic binding to Ca-CaM. Methods: Two adenoviral vectors encoding FRET (fluorescence resonance energy transfer) based biosensors with affinities similar to CaMKII and CaN (BsCaM-45 vs. BsCaM-2) were generated to monitor Ca-CaM dynamics in adult rabbit ventricular myocytes (with extra CaM expression via adenovirus). Results: Ca-CaM binding reduces FRET and increases the fluorescence emission ratio (F 480 /F 530 ). Figure shows the lower affinity sensor BsCaM-45 (like CaMKII) turns on and off completely with only slight diastolic increase at 1.0 Hz (5% ± 2.5%, vs. 0.1 Hz, n = 5). In contrast, the higher affinity sensor BsCaM-2 (like CaN) showed significant diastolic signal integration at 1.0 Hz (20% ± 3%, n = 5, p < 0.05). BsCaM-2 (n = 5) has slower kinetics vs. BsCaM-45 (n = 5): at 1.0 Hz, time to peak: 218 ± 11 vs. 244 ± 15 ms, ns; τ-decay: 647 ± 17 vs. 321 ± 25 ms, p < 0.05). Conclusion: CaM target proteins with different affinities (like CaMKII and CaN) respond very differently to the same Ca signal, (integrating vs. phasic), presumably tuned appropriately for their signaling pathway. This dynamic response shapes subsequent cellular processes in the heart. Indeed, altered diastolic [Ca] i or heart rate (as in pathophysiological states) can differentially influence downstream CaM-dependent pathways. Beat-to-beat FRET signal changes in Rabbit Ventricular Myocytes