Fig 2: Effect of Tdn and Jct on e-c coupling and ECCE.A) Representative traces of K+ -dose responses of primary cultured myotubes from wild type (WT), triadin-null (Tdn), junctin-null (Jct) and triadin/juctin double-null (Tdn/Jct) mouse muscles. B) Average peak fluorescent amplitude of depolarization-induced Ca2+ release response of WT (black, n = 114 cells), Jct-null (green, n = 83 cells), Tdn-null (blue, n = 57 cells) and Tdn-/Jct null (red, n = 68 cells) myotubes. Myotubes were loaded with 5 µM Fura-4F and exposed to increased concentrations of K+ for 5 s. C) Average rate of decrease in Fura-2 signal by Mn2+ quench during depolarization with 80 mM KCl. Numbers in the bars indicate the number of cells analyzed per condition. The data are from 3–4 experiments and are presented as mean ± SEM (***p<0.001 (ANOVA, One-way analysis of variance, Tukey's multiple comparison test)).

Fig 3: A model proposing the contribution of Tdn's to the jSR anchors and a possible positioning of Jct.In the model the triad is seen in a view parallel to the T-tubule axis (as in Figs. 2 B–C and 3 B, D and F) and the RyR array is modeled as seen in this orientation. The proportion between RyR heights and their spacing is appropriate, as suggested to us by Dr M. Samso [64]. Clusters (polymers) of six Tdn molecules, modeled roughly according to the topology proposed by Knudson et al., (1993) and Marty et al., (1995) are located between the RyRs as initially suggested by Fan et al. (1995). Their aggregated mass is responsible for the visible anchors, but the exact ratio of Tdns to anchors is not known. Individual triadin molecules are connected to RyRs and to a long linear CASQ polymer on the luminal side of the SR. The latter corresponds to the fine line visible in the EM images at the tips of anchors (see Fig. 2 C and inset). The jSR lumen is filled by long CASQ polymers that randomly intersect each other [15]. Junctin is depicted as monomers associated with RyRs [19], [21]. Although we cannot visualize them directly, it is likely that individual Jct molecules are positioned as indicated along the jSR face and also possibly at the sites where CASQ is linked to the lateral sides of the SR.

Fig 4: Effect of Tdn and Jct ablation on SR Ca2+ content of cultured myotubes.A) Average peak fluorescent amplitude of caffeine-induced Ca2+ transients of Fura-4F loaded myotubes from WT (black, n = 59 cells), Tdn-null (blue, n = 52 cells), Jct-null (green, n = 60 cells) and Tdn-/Jct null (red, n = 59 cells) mice. B) Representative traces of CPA-induced Ca2+ transients of WT (black), Jct-null (green), Tdn-null (blue) and Tdn/Jct-null (red) myotubes loaded with Fura-2 used to estimate SR Ca2+ content. C) Comparison of average peak Ca2+ transient amplitude induced by 10 µM CPA. Numbers in the bars indicate the number of cells analyzed per condition. Data presented as mean ± SEM. **p<0.01, ***p<0.001 (ANOVA, One-way analysis of variance, Tukey's multiple comparison test).

Fig 5: Gene expression profiling in skeletal muscle from delACAG mutants. a Hierarchical clustering and heatmap visualization of RNA-seq data (selected DEGs) resulting from analysis of skeletal muscle RNA samples isolated from 3 wt and 3 delACAG P15 mice. b Number of genes upregulated (red) and downregulated (green) among selected 233 DEGs. c Gene Ontology (GO) term enrichment analysis showing pathways and biological processes over-represented in selected DEGs. Gene count is shown on the X-axis (Supplementary Table 3a). d Relative expression analysis of the indicated genes by qRT-PCR using TaqMan expression assays in gastrocnemius RNA of 1.5-months old mice. Values are normalized to Tbp expression and are represented as fold change of the mean value of wt expression. Graphs are mean ± s.d. followed by one-way ANOVA with Tukey post-hoc test. n = 3. e Representative immunoblots showing protein levels of CDKN1A, ANKRD1, CASP3 and cleaved CASP3 in gastrocnemius from 2–3-months old delACAG and dupA mice and corresponding wt littermates. f Representative immunoblots for SLN and TRDN in protein extracts from the soleus (SLN) or gastrocnemius (TRDN) from delACAG, dupA and wt mice demonstrating increased expression of both proteins in delACAG mutants.Vinculin served as loading control in e and f. n = 6(wt), 3(delACAG), 3(dupA) mice for all proteins excepting SLN (n = 7(wt), 4(delACAG), 3(dupA) mice)