BPS2026 – Temporal dynamics of calcium release-dependent CaMKIIδ activation in live cardiac myocytes

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key regulator of numerous functions in cardiac myocytes including calcium (Ca) regulation and contraction. Its activation is dependent on binding calcified calmodulin (Ca/CaM), but its deactivation can be complicated by post-translational modifications (PTMs), like autophosphorylation at Thr287, that can delay the dissociation of CaM and promote persistent CaMKII activity implicated in cardiac disease. While it is well known that a regulatory feedback interaction exists between Ca and CaMKII (via CaM), how CaMKII activation varies in real time relative to dynamic Ca release events in live cardiac myocytes has not been thoroughly studied. Here, we investigate how the timing and kinetics of CaMKII conformational state varies during Ca transients using our FRET-based CaMKIIδ conformational state reporter, Camui (RFP-CaMKIIδ-GFP), and its phosphomimetic (T287D) and phosphoresistant (T287A) mutant variants, expressed via adenoviral infection in intact cardiac myocytes. We found that the rates of rise and fall of Camui transients—the real-time conformation-dependent FRET signal response—for WT and phosphoresistent T287A were similar to the rates of Ca release and reuptake, respectively, of field-stimulation-evoked Ca transients. The whole-cell average state of activation gradually increased until reaching steady-state plateau during pacing but returned rapidly to the deactivated state upon pacing cessation. Phosphomimetic T287D Camui exhibited longer transient durations, paralleling its altered Ca transient kinetics regulated by increased CaMKII activity. While the whole-cell average state of activation was rapid during pacing, its return to the deactivated state upon pacing cessation was considerably slower. These findings suggest that while the timing and kinetics of Camui conformation state change parallel the kinetics of Ca transients on each beat, autophosphorylation of CaMKII confers a substantial Ca-independent effect.