CMYA5 establishes cardiac dyad architecture and positioning

Figures

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  Fig. 1. BioID identification of dyadic proteins. a Proximity proteomics strategy. BirA* and myc epitopes were fused to ASPH and TRDN, which interact with RYR2 in jSR of dyads. N-terminal fusion positions BirA* in the dyadic cleft. The fusion protein was expressed in vivo in cardiomyocytes using AAV9 and the cardiomyocyte-selective troponin T (Tnnt2) promoter. GFP was co-expressed using a P2A sequence. b Experimental timeline. Artwork from biorender.com. c Expression of BirA* dyadic biosensors in myocardium. Heart sections were stained for the myc epitope tag. Most cardiomyocytes were immunoreactive. Bar = 20 µm. Representative of 3 independent experiments. d Mature ventricular cardiomyocytes. In the left GFP + cell, myc immunoreactive signal co-localized with CAV3, a T-tubule marker. Bar = 10 µm. Representative of three independent experiments. e Protein lysates from mice expressing the indicated BirA*-fused biosensors or GFPs were analyzed by western blotting. AAV dose and treatment with exogenous biotin are indicated. M, middle dose (2 × 1010 vg/g). F, full dose (5.5 × 1010 vg/g). Ponceau S, total protein. SA-HRP labeled biotinylated proteins. Representative of two independent…

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  Fig. 2. Characterization of hearts lacking CMYA5. a Genomic structure of Cmya5 wild-type and Δ alleles. Cas9-mediated deletion of 9281 bp of exon 2 causes a frameshift (fs) after the 55th amino acid residue. qRTPCR and genotyping amplicons are indicated. b PCR genotyping using WT (1 + 2) and Δ (1 + 3) primers. Representative of five independent experiments. c Cmya5 cardiac mRNA levels. RT-qPCR amplicon 1 and 2 represent the deleted region and the 3’ end of the transcript, respectively. ANOVA with Dunnett’s multiple comparison test vs. WT of the same amplicon. n, number of hearts. Data are presented as mean ± SD. d Cardiac protein lysates were analyzed by western blotting. KO samples lacked CMYA5 immunoreactivity. Representative of five independent experiments. e Gross morphology of WT and KO hearts. Bar = 2.5 mm. Representative of three independent experiments. f Heart weight normalized to body weight, at the indicated ages. ANOVA with Dunnett’s multiple comparison test vs. WT at the same time point. n, number of hearts. g Echocardiographic measurement of systolic heart function. FS fractional shortening. n, sample size. Kruskal–Wallis with Dunn’s multiple comparison test vs. WT at the…

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  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…

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  Fig. 4. Altered dyadic Ca2+ release in CMYA5 KO cardiomyocytes. Ca2+ release in mature Cmya5 KO or WT ventricular cardiomyocytes was assessed using a dyad-localized Ca2+ nanosensor, GCaMP6f-Junctin (ASPH-G6f) and detected by confocal line scan imaging. a, b Surface plots of Ca2+ sparks in basal and isoproterenol (ISO)-stimulated (100 nM) cardiomyocytes. Quantification is shown in (b). Mann-Whitney test within basal or ISO conditions. c–f Ca2+ release during electrical pacing (red lines) under basal conditions or ISO stimulation. Dotted line, leading edge of the evoked Ca2+ transient. Arrowheads, aberrant Ca2+ release outside of the initial evoked Ca2+ transient, without (white) or with (red) wave-like propagation. d. Representative Ca2+ transients under basal (top) or ISO (bottom) conditions. e, f Quantification (mean ± SD) of Ca2+ transient amplitude and Ca2+ spark frequency. Mann-Whitney test within basal or ISO conditions. g–i Measurement of SR Ca2+ stores using low-affinity Ca2+-sensitive dye Fluo5N and AAV-mediated expression of SR-targeted esterase, which trapped Fluo5N in SR. g, Fluo5N distribution. h, i SR Ca2+ release induced by 10 mM caffeine. g–i are representative of three…

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  Fig. 5. CMYA5 protects the heart and dyads from the deleterious effects of pressure overload. Cmya5 KO and WT mice underwent baseline echocardiography (week 0) and then TAC or Sham surgery. After 4 weekly echocardiograms, necropsy was performed. a Systolic heart function. FS fractional shortening. Repeated-measures two-way ANOVA was performed for TAC cohort, with a comparison to WT-TAC (∧) at each time point. n, number of mice. b Gross cardiac morphology. Bar, 2.5 mm. c Heart weight normalized to body weight. Kruskal-Wallis with Dunn’s multiple comparison test vs.WT within Sham (*) or TAC (∧) cohorts. n number of mice. d–g Dyad architecture analyzed by transmission electron microscopy. Boxed regions are magnified in the bottom row. Bar, 500 nm. Quantification of T-tubule circularity, jSR-to-T-tubule coupling ratio, and Junction-Z-line distance. Kruskal–Wallis with Dunn’s multiple testing correction vs. WT Sham (*) or TAC (∧). n, number of dyads per group, from at least 10 cardiomyocytes from 3 different mice. h, i Cardiomyocytes were transduced with dyad-localized ASPH-G6f Ca2+ nanosensor. Images of nanosensor distribution indicate dyad disorganization induced by TAC and Cmya5 KO (h).…