Figure 5. RyR2^R420Q induces nanodomain structural alterations.

A. Deconvoluted super-resolution image of RyR2 in a cardiomyocyte from a WT mouse and a KI mouse. B. Cluster size relative frequency distribution in WT (open bars) and KI cells (red bars) (9516 clusters in 49 cells from 3 WT, 14165 clusters in 147 cells from 5 KI). C. Representative examples of Western Blot of RyR2 and GAPDH in WT and KI mice, as indicated. Right panel shows the quantification of the RyR2 bands, normalized to GAPDH in the two experimental groups (18 WT hearts, and 20 KI mice hearts). D. Relative frequency distribution of the distance of each cluster to the line connecting most, measured in WT cells (white wider bars), and KI cells (red narrower bars) from the same cell groups than in B. E. CRUs, specialized intracellular junctions between the jSR (labeled in yellow in a) and T-tubules (labelled in green in a). The cytoplasmic domains of RyR2, i.e. the feet, are visible as evenly spaced densities (pointed by a series of arrows in panel b) spanning the gap between jSR and T-tubule. The jSR is wrapped around the T-tubule to form a CRU (panel a), or associated to form a CRU with multiple (two or more) couplons (panel b). In WT, couplons are usually quite extended (panels a & b), while in KI cardiomyocytes we may find couplons, which appear fairly normal in length (panels c & e), and other ones in which the jSR is either shorter or apparently fragmented (panels d, f, g & h). The jSR contains a classic chain-like electron dense polymer (single arrows in panels b and d), representing CSQ2. In KI myocytes, the chain-like polymer of condensed CSQ2 may be sometimes missing in some portions of the jSR. F. Left: Western blot of immunoprecipitated proteins with JPH2 antibody from WT and KI hearts and then exposed successively (after stripping) to RyR2, Cav1.2 and JPH2 antibodies. Middle: Quantitative analysis of RyR2 co-IP with JPH2 in hearts from 5 WT mice and 12 KI mice. Right: Quantitative analysis of LTCC (Cav1.2) co-IP with the RyR2 in 5 WT and 8 KI hearts. G. Western blot of immunoprecipitated proteins with JPH2 antibody from control and RyR2^R420Q (Mu) h-iPS-CM and then exposed successively (after stripping) to RyR2, and JPH2 antibodies. On the right, quantitative analyses from 6 different differentiations each. H. Western Blot of JPH2 immunoprecipitated CSQ2 in 6 WT and 11 KI hearts. **p<0.05 and ***p<0.0005 Statistical tests and exact p values are provided in Online Table III.