Elevated local [Ca ²⁺ ] and CaMKII promote spontaneous Ca ²⁺ release in ankyrin-B-deficient hearts

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) Ca ²⁺ leak occurs through Ca ²⁺ sparks in AnkB-deficient (AnkB ^+/− ) mice, which may contribute to arrhythmogenicity via Ca ²⁺ waves. Here, we investigated the mechanisms responsible for increased Ca ²⁺ spark frequency in AnkB ^+/− hearts.

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 Ca ²⁺ 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 [Ca ²⁺ ] in the junctional cleft caused by reduced Na ⁺ /Ca ²⁺ exchange activity. Indeed, using the RyR-targeted Ca ²⁺ sensor GCaMP2.2-FBKP12.6, we found that local junctional [Ca ²⁺ ] is significantly elevated in AnkB ^+/− myocytes.

The increased incidence of pro-arrhythmogenic Ca ²⁺ sparks and waves in AnkB ^+/− hearts is due to enhanced CaMKII-mediated RyR phosphorylation, which is caused by higher junctional [Ca ²⁺ ] and consequent local CaMKII activation.