Cardiac CAMKIIδ Memory: How Post-Translational-Modifications Alter Calmodulin Affinity

Abstract

CaMKII is a central regulator of cardiac Ca handling, electrophysiology and transcription. Its persistent over-activation has been implicated in arrhythmias, heart failure, and other diseases. Typically, CaMKII activates upon Ca-CaM binding during elevated [Ca] free and inactivates upon Ca-CaM dissociation when [Ca] free drops. Several post-translational-modifications (PTMs) also trap CaMKII in an autonomously activated state even after [Ca] i declines. The PTMs include autophosphorylation, oxidation, S-nitrosylation, and O-GlcNAcylation. Exactly how these PTMs regulate CaM binding to CaM-KIId-the predominant cardiac isoform-is unknown. Schulman's group showed that autophosphorylation of CaMKIIa (neuronal isoform) increases its CaM affinity by 1,000-fold in lysates. CaMKII activity that persists during low [Ca] free creates a form of molecular memory. Here we perform novel tests of changes in CaM affinity (or trapping) for CaMKIId by known PTMs. Importantly, these are measured in adult rabbit ventricular myocytes, CaMKIId's native environment. Briefly, we express CaMKIId-GFP in isolated ventricular myocytes and load fluorescent-CaM after saponin-permeabilization of the myocytes. We use FRET as a readout for CaM dissociation upon the addition of unlabeled CaM or upon rapidly dropping [Ca] free with BAPTA. In myocytes, expressing the phosphomimetic CaMKIId (T287D) caused a dramatic slowing of CaM dissociation (>5 fold) upon abrupt reduction of [Ca] i (<50 nM) when compared to the non-phosphorylatable mutant (T287A). These differences provide a quantitative, mechanistic basis for PTM-dependent regulation of CaM-KIId in cardiac myocytes and its potential role in disease. We extended this analysis with CaMKIId-GFP mutants and corresponding agonists for other PTMs: e.g. 100uM H 2 O 2 -WT vs. M281/282V and analogous for the S-nitrosylation sites (C273S, C290A) and the O-GlcNAcylation site (S280A). This allows us to test which of these PTMs creates the longest CaM-binding state which will influence its relative impact on cardiac CaMKII memory and contributions to disease.