Fig 2: CM-specific overexpression of dn-c-kit resulted in CM hypertrophy, leading to a thicker LV wall, a smaller LV cavity and higher ejection fraction. (a) Expression of Kit (c-Kit) mRNA in male P196 dn-c-kit-Tg (Tg; n = 11) and NTL (n = 6) hearts; differences analysed by Student’s t-test. (b,c) Examples (white arrowheads) of BrdU+/cTnT+ (b) or H3P+/cTnT+ (c) CMs isolated from P121 Tg (b, right panel, and c) or NTL (b, left panel) hearts after 9 days of BrdU (10 mg/kg/day) infusion by osmotic mini-pump. Scale bar: 50 µm. (d) Left panels, representative photographs, and right panel, quantitation of surface area showing hypertrophy of isolated CMs from P121 Tg (682 CMs analysed from n = 3 mice) relative to NTL hearts (1015 CMs analysed from n = 3 mice); differences analysed by Student’s t-test. Scale bar: 100 µm. (e) Abundance of Myh6 (encoding a-MHC) and Acta1 (a-skeletal actin) mRNAs was similar, while abundance of other mRNAs (Nppa (ANP), Nppb (BNP), Myh7 (ß-MHC)) and the Myh7:Myh6 (ß-:a-MHC) ratio were significantly increased in male P196 dn-c-kit-Tg relative to NTL hearts (n = 7); differences analysed by Student’s t-test. (f) Increased LV wall thickness to chamber radius (h/r) ratio at end-diastole in P = 196 dn-c-kit-Tg (n = 10–11) relative to NTL (n = 9) hearts; differences analysed by two-way ANOVA with Tukey’s multiple comparison test. (g) Increased ejection fraction in P = 196 dn-c-kit-Tg (n = 10–11) relative to NTL (n = 9) hearts; differences analysed by two-way ANOVA with Tukey’s multiple comparison test.

Fig 3: MSCs exposure to 5'-aza-2'-deoxycytidine (5-aza-dC) induces in vitro differentiation to a “myogenic phenotype” in P1 (first passage) but this effect is lost at P4. The morphology of MSCs changed after 5-aza-dC exposure from fibroblast-like to form a stick-like morphology and several primitive myofilaments were detected in P1 (A). The mRNA levels (qPCR) of the specific marker cardiac myosin (ß-MHC) were significantly augmented after 5-aza-dC treatment in P1 without changes in cardiac troponin (cTnT) levels. No changes were detected in P4 (B). The expression of ß-MHC and cTnT was enhanced at the protein level (Western blot; C,D) and by immunofluorescence analysis, using DAPI (nuclear) and phalloidin (actin) staining (E,F), after 5-aza-dC in P1 but was missing in P4. *p < 0.05; **p < 0.01 vs. the untreated cells (0 h).

Fig 4: PARIS inhibited myogenic differentiation.a The expression of PARIS, Myogenin, MHC and PGC-1a was analyzed by immunoblotting. ß-Tubulin serves as a loading control. b Immunofluorescence staining of MHC (red) in pCMV- or pCMV-PARIS-expressing C2C12 cells. Nuclei were visualized by DAPI (blue). Scale bar = 100 µm. c, d The percentage of nuclei and myotubes containing indicated myonuclei number was determined (n = 3 per group, 356 ~ 411 (pCMV) and 377 ~ 401 (PARIS) cells per sample were counted, respectively). e The expression of MHC, TnT, MyoD, and PARIS proteins in pCMV- or pCMV-PARIS-overexpressing C2C12 cells was analyzed by immunoblotting. f Quantification of the relative protein levels (three sets per group). g Immunofluorescence staining of MHC (red) in Scrambled (Scr)- or PARIS siRNA (siPARIS)-expressing C2C12 cells. Scale bar = 100 µm. h, i Quantification of the percentage of nuclei and myotubes containing indicated myonuclei (n = 3 per group, 635 ~ 1017 (pCMV) and 789 ~ 924 (PARIS) cells per sample were counted, respectively). j The expression of MHC, TnT, MyoD, and PARIS was analyzed by immunoblotting with Scr- or siPARIS-expressing C2C12 cells. k Quantification of the relative protein levels of MHC and TnT (three sets per group). Significant difference was determined by Student t-test (*p < 0.05, **p < 0.01).

Fig 5: Disruption of TOP2B in hPSC-derived cardiomyocytes decreases sensitivity to doxorubicin-induced cell death.(a) Schematic of the TOP2B gene showing location of single guide RNAs (sgRNAs) designed to recognize the 3' region of exon 3. Exonic sequence is shown in uppercase and intronic sequence in lowercase. (b) Sequences of two clones transfected with CRISPR-Cas9 targeting TOP2B gene showing either wild type sequence (WT), or bi-allelic deletions resulting in gene knock-out (KO). (c,d) Flow cytometry analysis of cTnT expression in wild type (c) and KO (d) clones. (e) Relative gene expression levels of TOP2B mRNA in the genome edited-cardiomyocytes. Data represent the mean Ct values of duplicate measurements normalized against the values obtained for ß actin for the same sample. (f) After cardiomyocyte differentiation, cell viability of TOP2B edited-clones treated with doxorubicin for 24 hours was measured using CellTiter-Glo® luminescent assay. (g) Wild type and knock out TOP2B cardiomyocytes were treated with doxorubicin and stained with ?-H2AX to identify double strand DNA breaks. (h) Quantification of data in panel (g). Graphs represent the means of three independent experiments ± SD **p < 0.01 Scale bar: 10 µ m.