Abstract
Background: Nearly 18 million people die from cardiovascular disease (CVD) each year according to the World Health Organisation. One of the outcomes of CVD is the development of cardiac arrhythmias. A mechanism through which arrhythmias occur is due to abnormal calcium (Ca2+) signalling via the Ryanodine Receptor Type II (RyR2). Several kinases are known to alter the activity of RyR2, with the consensus being that hyperphosphorylation leads to abnormal Ca2+ release and arrhythmia. Within the Jones’ lab, a novel kinase, CK2, has been shown to alter RyR2 activity. In contrast to most kinases, removing RyR2 phosphorylation by CK2 results in an increased ‘leak’ of Ca2+ from RyR2, proving pro-arrhythmogenic. However, whether CK2-mediated hyperphosphorylation of RyR2 is protective against Ca2+ leak and arrhythmia has yet to be investigated.
Hypothesis: We hypothesized that CK2-mediated hyperphosphorylation of RyR2 will reduce Ca2+ leak.
Methods: Cardiomyocytes were isolated via collagenase digestion from mice with phosphomimetic mutations at the CK2 phosphorylation sites Serine-2692 and Serine-2963. These mutations mimic complete hyperphosphorylation of RyR2. Ca2+ leak was determined using line scanning confocal microscopy of Fluo-4-AM-loaded cardiomyocytes. ImageJ SparkMaster analysis was used to determine the amount of Ca2+ leak occurring. The cardiomyocytes were imaged under baseline conditions and under isoproterenol exposure to stimulate the β-adrenergic stress response. Normal Ca2+ release was also assessed using electrical pacing to determine if changes also occurred in normal Ca2+ handling of the mutant cardiomyocytes.
Results: In cardiomyocytes from wildtype animals, the application of isoproterenol resulted in a significant increase (p = 0.0019) in Ca2+, compared to baseline. In contrast, cardiomyocytes from both the heterozygous and homozygous mutant mice exhibited no change in Ca2+ leak in response to β-adrenergic stimulation (p = 0.7044 and 0.1550, respectively). There were no differences to normal Ca2+ handling (Ca2+ release amplitude, duration and Tau Decay) between genotypes.
Conclusions: The results suggest that stress-induced Ca2+ leak is attenuated in the mutant cardiomyocytes compared to wildtype. This indicates hyperphosphorylation of RyR2 by CK2 has a protective effect against the increased Ca2+ leak. Therefore, this supports the working hypothesis and suggests that CK2-mediated hyperphosphorylation may also reduce cardiac arrhythmias. Ultimately, this mechanism has the potential to be a novel therapeutic target for preventing cardiac arrhythmias.