Fig. 3. CMYA5 positions jSR adjacent to Z-lines.

a P7 ventricular cardiomyocyte co-immunostained for CMYA5 and RYR2. Bar, 10 µm. Bottom, spatial profile plot demonstrates co-localization of CMYA5 and RYR2 in a striated pattern. b Localization of FSD2 in P7 ventricular cardiomyocytes. FSD2 adopted a striated pattern. Bar, 10 µm. Representative of two independent experiments. c jSR (RYR2) and T-tubule (CAV3) organization in WT and KO P7 ventricular cardiomyocytes. CMYA5 ablation caused loss of jSR organization. At this stage, T-tubules were not yet present in either genotype. Bar, 10 µm. Representative of three independent experiments. d RYR2, CMYA5, and ACTN2 localization in WT E15.5 ventricular myocardium. Assembling sarcomere Z-lines (ACTN2) co-localized with CMYA5 alone (red arrowheads), CMYA5 and RYR2 (yellow arrowheads), or neither (white arrowheads). Bar, 5 µm. Representative of five independent experiments. e, f Effect of CASAVV-mediated ablation of RYR2 on CMYA5 and FSD2 localization. CASAAV somatic mutagenesis was used to deplete RYR2 in a subset of cardiomyocytes. CMYA5 and FSD2 localization was evaluated in RYR2-deficient (GFP+) and control (GFP−) cardiomyocytes. Spatial profiles of boxed areas in f, plotted at right, demonstrate that RYR2 ablation did not impact CMYA5 or FSD2 localization. Bar = 10 µm (e), 5 µm (f). Representative of three independent experiments. g, h Effect of CASAAV-mediated ablation of MYH6 on CMYA5 and ACTN2 localization. CASAAV was used to deplete MYH6 in a subset of cardiomyocytes. MYH6-deficient (GFP+) had impaired sarcomerogenesis and disorganization of Z-line marker ACTN2, and CMYA5 organization was correspondingly deranged. Signal intensities in boxed areas in h are plotted to the right. Bar = 10 µm (g), 5 µm (h). Representative of three independent experiments.