The Ratio of RyR2 and CSQ2 in Calcium Handling

Abstract

Atrial fibrillation (AF) is the most common arrhythmia of the heart, affecting 2-3% of the population. AF decreases the quality of life and increases the risk of mortality in patients. Although many patients see improvement with available treatments, a significant number do not. This necessitates further study into mechanisms of AF to elucidate new therapeutic targets. Many arrhythmias arise from the dysfunction of calcium handling within cardiomyocytes. The contraction of cardiomyocytes relies on the regulated release of calcium from the sarcoplasmic reticulum (SR), via the ryanodine receptor (RyR2). In many arrhythmias, RyR2 becomes “leaky” and spontaneously releases calcium in a process called store overload-induced calcium release (SOICR). This can lead to the development of delayed afterdepolarisations and trigger arrhythmic contractions. The function of RyR2 can be modulated by many accessory proteins, including calsequestrin 2 (CSQ2). CSQ2 primarily buffers Ca2+ within the SR, but can also influence RyR2’s propensity for SOICR. Both human and animal studies indicate a relative increase of RyR2, and/or a relative decrease of CSQ2 (increased RyR2:CSQ2) can result in an arrhythmic phenotype.

My project aimed to investigate the functional effects of changing RyR2:CSQ2 on the propensity for SOICR by co-expressing RyR2 and CSQ2 within HEK293 cells, by using fluorescently tagged RyR2 and CSQ2. HEK293 cells were loaded with a Ca2+-dye and underwent single-cell calcium imaging with increasing [Ca2+]¬O (0.1-1 mM) to determine the effect of changing RyR2:CSQ2 on the propensity for SOICR. The results of this show a biphasic effect of decreasing RyR2:CSQ2 on the propensity for SOICR, where high RyR2:CSQ2 cells had a higher frequency of SOICR events than the CSQ2-null cells (p=0.0326), and the medium RyR2:CSQ2 cells (p=0.0008), and low RyR2:CSQ2 cells (p<0.0001). These findings indicate that a high RyR2:CSQ2 ratio increases RyR2’s propensity for SOICR more than a complete loss of CSQ2. Despite this, my finding of decreased RyR2:CSQ2 being protective against SOICR also provides hope for future novel therapeutics that can restore the balance of expression between RyR2 and CSQ2.