Abstract
Cardiovascular disease is one of the leading causes of mortality in New Zealand and is responsible for one third of all global deaths each year. Among cardiac diseases, arrhythmias are one of the most prevalent forms. A main mechanism of arrhythmia is the inappropriate release of Ca2+ (termed Ca2+ sparks) through ryanodine receptor 2 (RyR2), a sarcoplasmic reticulum-located channel that plays an essential role in cardiac excitation-contraction coupling, releasing the bulk Ca2+ required for contraction. This thesis aimed to determine the regulation of RyR2 by (casein kinase 2) CK2 phosphorylation in vivo. This was achieved using phospho-specific mutant mice, which expressed a variant of RyR2 unable to be phosphorylated by CK2 (RyR2-S2692/3AA).
To determine if loss of RyR2 phosphorylation by CK2 increases Ca2+ sparks, line-scan imaging was performed on isolated cardiomyocytes from RyR2-S2692/3AA and wildtype (WT) controls. Isolated cardiomyocytes from RyR2-S2692/3AA animals exhibited a significantly greater frequency of Ca2+ sparks combined with a shorter decay compared to WT animals. Next, to determine if this increase in Ca2+ spark frequency translated to an increased risk of arrhythmias, electrocardiograms were recorded before and after a pharmacological stress trigger, an intraperitoneal injection of caffeine (120 mg/kg) and epinephrine (1.6 mg/kg). In WT animals this procedure increased the heart rate but had little effect on arrhythmogenicity, with brief changes in sinus rhythm occurring in only 2 out of 9 animals. In contrast, RyR2-S2692/3AA, animals experienced a significant increase (7 out of 10 animals) in severe and prolonged non-sinus rhythm. Although RyR2-S2692/3AA mice showed altered rhythm under stress they displayed normal cardiac contractility and no hypertrophic remodelling compared to WT controls at rest.
This thesis also examined whether changes in CK2 phosphorylation resulted in other changes to RyR2 such as expression and phosphorylation by other kinases. Total protein expression and phosphorylation analysis of RyR2 indicated that the loss of phosphorylation of S2692 and S2693 causes an increase in phosphorylation of S2814 in young and S2808 in old animals.
Combined these data show that phosphorylation of RyR2 by CK2 is essential for normal channel function and Ca2+ release. It also proves that loss of CK2 phosphorylation increases Ca2+ leak and the susceptibility to arrhythmia. In addition to that, the phosphorylation of S2692 and S2693 also affects the phosphorylation level of other phosphorylation sites in RyR2. Clinically, identifying this pathway offers a new target for the treatment of cardiac arrhythmia.