Elevated local [Ca²⁺] and CaMKII promote spontaneous Ca²⁺ release in ankyrin-B-deficient hearts

Aims Loss-of-function mutations in the cytoskeletal protein ankyrin-B (AnkB) cause ventricular tachyarrhythmias in humans. Previously, we found that a larger fraction of the sarcoplasmic reticulum (SR) Ca²⁺ leak occurs through Ca²⁺ sparks in AnkB-deficient (AnkB^+/-) mice, which may contribute to arrhythmogenicity via Ca²⁺ waves. Here, we investigated the mechanisms responsible for increased Ca²⁺ spark frequency in AnkB^+/- hearts. Methods and results Using immunoblots and phospho-specific antibodies, we found that phosphorylation of ryanodine receptors (RyRs) by CaMKII is enhanced in AnkB^+/- hearts. In contrast, the PKA-mediated RyR phosphorylation was comparable in AnkB^+/- and wild-type (WT) mice. CaMKII inhibition greatly reduced Ca²⁺ spark frequency in myocytes from AnkB^+/- mice but had little effect in the WT. Global activities of the major phosphatases PP1 and PP2A were similar in AnkB^+/- and WT hearts, while CaMKII autophosphorylation, a marker of CaMKII activation, was increased in AnkB^+/- hearts. Thus, CaMKII-dependent RyR hyperphosphorylation in AnkB^+/- hearts is caused by augmented CaMKII activity. Intriguingly, CaMKII activation is limited to the sarcolemma-SR junctions since non-junctional CaMKII targets (phospholamban, HDAC4) are not hyperphosphorylated in AnkB^+/- myocytes. This local CaMKII activation may be the consequence of elevated [Ca²⁺] in the junctional cleft caused by reduced Na⁺/Ca²⁺ exchange activity. Indeed, using the RyR-targeted Ca²⁺ sensor GCaMP2.2-FBKP12.6, we found that local junctional [Ca²⁺] is significantly elevated in AnkB^+/- myocytes. Conclusions The increased incidence of pro-arrhythmogenic Ca²⁺ sparks and waves in AnkB^+/- hearts is due to enhanced CaMKII-mediated RyR phosphorylation, which is caused by higher junctional [Ca²⁺] and consequent local CaMKII activation.