Fig 2: Flexiblity is inversely correlated with myonuclear number.a A comparison of the cross-sectional area of tibialis anterior muscle in ?2w and ?3w mice at postnatal (P) day 42 revealed similar effects on cell size, despite differences in myonuclear number (n = 3–7 biologically independent animals). b Assessment of the myonuclear domain in isolated EDL myofibers from ?3w mice at P42 (n = 3 biologically independent animals). c Total RNA levels normalized to muscle weight in the various groups of mice (n = 5–10 biologically independent animals). d Normalized RNA levels from (c) were normalized again to the average number of nuclei/myofiber (n = 5–10 biologically independent animals). A significant increase in the amount of RNA per myonucleus is observed in ?2w muscle, but remains unchanged in ?3w muscle. e Concentration of transcripts coding for key skeletal muscle structural genes (Acta1, Myh1, Myh4, Tnnt3, and Tnnc2) on a per nuclear basis are increased in ?2w and ?3w muscle compared to controls (n = 3–8 biologically independent animals). ?2w muscle has a larger increase in transcript concentration per nucleus than ?3w, despite having fewer myonuclei per fiber. To obtain these values, relative transcript levels were determined by semi-quantitative qPCR from P42 samples, which were then normalized to total RNA and average myonuclear numbers for each genotype. f Abundance of transcripts on a per nuclear basis in wild-type tibialis anterior across developmental time points (n = 3–7 biologically independent animals). Statistical analyses and data presentation: a, two-sided unpaired t-test. c, d One-way ANOVA with a Tukey correction for multiple comparisons; significance compared to control group. e, Acta1 (both groups), Myh1 (?2w only): Mann–Whitney test. Myh1 (?3w only), Myh4, Tnnt3, and Tnnc2 (both groups): two-sided unpaired t-test with Welch’s correction. f One-way ANOVA with Tukey correction for multiple comparisons. Acta1 was analyzed with one-way ANOVA with Kruskal–Wallis correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SD. Source data are provided as a Source Data file.

Fig 3: Myotube formation was accompanied by the observation of lateral striations indicative of sarcomeric structures. a Myotubes at 4d, 5d, 9d (DM1) and 42d (DM1) of differentiation were immunostained for the presence of sarcomeric a-actin (Sigma A2172, 1:500 dilution). b Sarcomeric lateral striations were visible in H&E stained myotubes differentiated for 5d, derived from P3 and P5 longissimus dorsi myoblasts. Bar = 50 µm for all panels

Fig 4: ADRCs induced with six factors express multiple myocardial genes and have the characteristics of cardiac differentiation accessed by bioinformatics analysis(A) qPCR analysis of cardiac marker genes in 6F-ADRCs and mock induced ADRCs 3 weeks after transfection. Myh6 (n = 5), Actc1 (n = 5), Tnnt2 (n = 6), Ttn (n = 8), Fhod3 (n = 6), Actn2 (n = 8) (B) Representative images of GFP+ADRCs immunostaining for a-sarcomeric actin after induction of six factors. The secondary antibody was labeled with Alexa 594 (red). Scale bars, 500 µm (left panel) and 50 µm (right panels) (C) Heatmap image of RNA sequencing analysis illustrating selected cardiac genes among uninduced ADRCs and GFP+ 6F-ADRCs (FACS sorted 3 weeks after transfection). Log10(RPKM+1) values obtained from RNA-seq are shown (n = 2). Lists of genes are shown in Table S5.(D) MA plot of differentially regulated genes between GFP+ 6F-ADRCs (G2) and uninduced ADRCs (G1). M=log2(G2)-log2(G1), A=(log2(G2)+log2(G1))/2. Strawberry colored plots show differentially expressed genes (DEGs) with tagwise dispersions FDR p value cutoff <0.05. Lists of DEGs are shown in Table S6.(E) Gene ontology analysis for significantly upregulated genes (RNA-seq analysis) in GFP+ 6F-ADRCs. Lists of genes are shown in Figure S4 and Table S7.(F) Heatmap image of RNA-seq analysis illustrating 23 cardiac genes among uninduced ADRCs, GFP+ 6F-ADRCs, and 12.9 weeks C57BL/6J adult ventricular cardiomyocytes (GSM2746289, GSM2746290).(G) RNA-seq-based 21 cardiac genes expression levels among uninduced ADRCs, GFP+ 6F-ADRCs, and adult ventricular cardiomyocytes (GSM2746289, GSM2746290). Log10(RPKM+1) values obtained from RNA-seq are shown (n = 2).(H) Uniform manifold approximation and projection (UMAP) clustering of six factors ADRCs 3 weeks after transfection, mixed with uninduced ADRCs. FeaturePlots of each gene expression showing (I) EGFP, (J) Myh6, (K) Actc1, (L) Tnnc1, (M) Tnnt2, (N) Myl3, (O) Myl7 expression.Data are represented as mean ± SEM (A, G). Statistical significance was determined with the Student’s t-test between two groups (A). ***p < 0.01.