Ryanodine receptor 2 (RyR2) is a major calcium release channel in the heart. Dysfunctional RyR2 is linked to cardiac arrhythmia and disease. Recent studies have identified a RyR2 splice variant encoding a RyR2 isoform with eight extra amino acids (RyR2-8aai) in cardiomyopathic human and mouse hearts with disruption of the muscle-specific splicing factor RNA binding motif 20 (RBM20). Given the limited research on alternative splicing of the RYR2 gene, our aim was to characterize the functional and biophysical properties of RyR2-8aai. We generated an antibody that selectively detects RyR2-8aai. Additionally, we generated HEK293 cell lines with inducible expression of recombinant WT or RyR2-8aai channels. Following induction, RyR2-8aai expression was significantly higher than WT. Inhibiting protein translation with cycloheximide uncovered a significantly prolonged half-life of RyR2-8aai channels, suggesting increased protein stability. 3 Hryanodine binding assays in recombinant protein, used to probe the RyR2 activity in response to Ca 2+ , revealed no differences in EC50 but showed a decrease in maximum binding capacity of RyR2-8aai. This suggests that RyR2-8aai channels may have cytosolic loss-of-function. We then exposed induced HEK293 cells to increasing extracellular Ca 2+ and used fluorescence imaging to detect cytosolic Ca 2+ oscillations resulting from spontaneous RyR2 openings, known as store overload-induced Ca 2+ release (SOICR). RyR2-8aai cells showed higher oscillation frequency and percentage of oscillating cells at all Ca 2+ studied. These results suggest that RyR2-8aai may display a luminal gain-of-function. Finally, western blot analysis showed that RyR2-8aai is expressed in hearts of mice with the mutation RBM20-S637A. This is the first demonstration that alternatively spliced RyR2 transcripts are translated into protein in RBM20-mutant hearts. Further investigation aims to resolve structural alterations induced in RyR2 by the eight amino acid insertion and the potential contribution of RyR2-8aai to RBM20 cardiomyopathy.
Estrada et al. (Sun,) studied this question.