Differential integration of Ca²⁺-calmodulin signal in intact ventricular myocytes at low and high affinity Ca²⁺-calmodulin targets

Cardiac myocyte intracellular calcium varies beat-to-beat and calmodulin (CaM) transduces Ca²⁺ signals to regulate many cellular processes (e.g. via CaM targets such as CaM-dependent kinase and calcineurin). However, little is known about the dynamics of how CaM targets process the Ca ²⁺ signals to generate appropriate biological responses in the heart. We hypothesized that the different affinities of CaM targets for the Ca ²⁺-bound CaM (Ca²⁺-CaM) shape their actions through dynamic and tonic interactions in response to the repetitive Ca²⁺ signals in myocytes. To test our hypothesis, we used two fluorescence resonance energy transfer-based biosensors, BsCaM-45 (K_d = ∼45 nM) and BsCaM-2 (K_d = ∼2 nM), to monitor the real time Ca ²⁺-CaM dynamics at low and high affinity CaM targets in paced adult ventricular myocytes. Compared with BsCaM-2, BsCaM-45 tracks the beat-to-beat Ca²⁺-CaM alterations more closely following the Ca²⁺ oscillations at each myocyte contraction. When pacing frequency is raised from 0.1 to 1.0 Hz, the higher affinity BsCaM-2 demonstrates significant elevation of diastolic Ca²⁺-CaM binding compared with the lower affinity BsCaM-45. Biochemically detailed computational models of Ca²⁺-CaM biosensors in beating cardiac myocytes revealed that the different Ca ²⁺-CaM binding affinities of BsCaM-2 and BsCaM-45 are sufficient to predict their differing kinetics and diastolic integration. Thus, data from both experiments and computational modeling suggest that CaM targets with low versus high Ca²⁺-CaM affinities (like CaM-dependent kinase versus calcineurin) respond differentially to the same Ca²⁺ signal (phasic versus integrating), presumably tuned appropriately for their respective and distinct Ca²⁺ signaling pathways.