CaMKIIδ post-translational modifications increase affinity for calmodulin inside cardiac ventricular myocytes

Persistent over-activation of CaMKII (Calcium/Calmodulin-dependent protein Kinase II) in the heart is implicated in arrhythmias, heart failure, pathological remodeling, and other cardiovascular diseases. Several post-translational modifications (PTMs)—including autophosphorylation, oxidation, S-nitrosylation, and O-GlcNA-cylation—have been shown to trap CaMKII in an autonomously active state. The molecular mechanisms by which these PTMs regulate calmodulin (CaM) binding to CaMKIIδ—the primary cardiac isoform—has not been well-studied particularly in its native myocyte environment. Typically, CaMKII activates upon Ca-CaM binding during locally elevated [Ca](free) and deactivates upon Ca-CaM dissociation when [Ca](free) returns to basal levels. To assess the effects of CaMKIIδ PTMs on CaM binding, we developed a novel FRET (Forster resonance energy transfer) approach to directly measure CaM binding to and ¨ dissociation from CaMKIIδ in live cardiac myocytes. We demonstrate that autophosphorylation of CaMKIIδ increases affinity for CaM in its native environment and that this increase is dependent on [Ca](free). This leads to a 3-fold slowing of CaM dissociation from CaMKIIδ (time constant slows from ~0.5 to 1.5 s) when [Ca](free) is reduced with physiological kinetics. Moreover, oxidation further slows CaM dissociation from CaMKIIδ T287D (phosphomimetic) upon rapid [Ca](free) chelation and increases FRET between CaM and CaMKIIδ T287A (phosphoresistant). The CaM dissociation kinetics–measured here in myocytes–are similar to the interval between heartbeats, and integrative memory would be expected as a function of heart rate. Furthermore, the PTM-induced slowing of dissociation between beats would greatly promote persistent CaMKIIδ activity in the heart. Together, these findings suggest a significant role of PTM-induced changes in CaMKIIδ affinity for CaM and memory under physiological and pathophysiological processes in the heart.