Fig 2: Analysis of DNA methylation in cardiomyocytes isolated from newborn and adult murine hearts.(a) Hematoxylin–eosin staining (upper panels; scale bar, 1 mm) of mouse hearts 1 day after birth (newborn), at 8 weeks of age (adult) and 3 weeks after chronic pressure overload in adult mice (failing). WGA-stained ventricular sections (middle panels; scale bar, 20 µm) to determine cardiomyocyte cross-sectional areas (n=10 hearts per group). Nuclei were stained with propidium iodide (PI). (b) Identification of cardiomyocyte nuclei by PCM1 immunostaining in adult mouse cardiac tissue (insert, arrows; scale bar, 20 µm) and purification of cardiomyocyte nuclei (red) by flow cytometry (histogram). (c) Percentage of cardiomyocyte nuclei in ventricular biopsies identified by PCM1 flow cytometry in newborn, adult healthy and failing hearts and at postnatal days 5–28 (n=3–6 hearts per group). (d) IGV (integrative genomics viewer) traces of CpG methylation of the myosin-6 (Myh6) and myosin-7 (Myh7) gene region in ES cells, newborn, adult and failing cardiomyocytes and in murine heart tissue. (e) Differentially methylated CpGs during fetal and postnatal development in percent of assessed CpGs. (f) Density of CpG methylation, histone H3K4me1 and H3K27ac at transcription factor (TF)-binding sites (±5 kbp). Data are shown as mean±s.e.m., ***P<0.001, analysis of variance, Bonferroni post hoc test.

Fig 3: Myonuclear-specific Cre-mediated recombination. a Schematic diagram of the RosaCAG-lsl-ntdTomato construct before and after Cre-mediated recombination. Representation of primers used for quantitative RT-PCR analysis showing amplicon of recombinant DNA and amplicon of internal control for normalization. b Representative flow cytometry plots depicting the gating strategy for sorting of myonuclei. Isolated nuclei were analyzed by side scatter (SSC) and Hoechst. Hoechst+ PCM1+ single nuclei were sorted for further analysis. Purity of sorted myonuclei is shown in the right panel. c RT-PCR performed on sorted Hoechst+ PCM1+ nuclei. DNA isolated from satellite cells from Pax7CreERT2/+; RosaCAG-lsl-ntdTomato/+ mice with the presence of one recombined allele in each satellite cell served as a 100% reference (positive control). d Schematic diagram of the RosamTmG construct before and after Cre-mediated recombination. Representation of primers used for quantitative RT-PCR analysis showing amplicon of recombinant DNA and amplicon of internal control for normalization. e RT-PCR performed on genomic DNA isolated from bulk muscle. DNA isolated from tamoxifen injected HSAiCre/+; RosamTmG/+ mice with the presence of one recombined allele in each myonucleus, served as a 100% reference (positive control). Recombination rates were calculated from the relative expression of recombined levels normalized for internal control. Statistics, one-way ANOVA test with Tukey correction for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001). Each dot represents a single mouse. Bar graphs represent mean ± SEM (error bars)

Fig 4: Acute SB2 Treatment Increases ß-Catenin Protein in Human LV Slices(A) Western blot of ß-catenin and GAPDH levels with quantification based on band density relative to GAPDH shows an increase in ß-catenin (31% average increase) in SB2-treated slices. (B) Both chromatin-bound ß-catenin and nonphosphorylated ß-catenin levels increased with respect to histone H3 in slices cultured with SB2. In contrast to other subcellular fractions, chromatin-bound ß-catenin shows multiple bands of lower molecular weights in addition to the primary band at 92 kDa. (C) ABC (green) is present in human myocyte nuclei, marked by PCM1 (red) and DAPI (blue), in baseline, 3-hour control, and 3-hour SB2 conditions. White arrowheads indicate ß-catenin–positive cardiomyocyte nuclei, whereas white asterisks mark ß-catenin–negative cardiomyocyte nuclei. Scale = 50 µm. Statistics were performed using repeated-measures analysis of variance followed by Tukey’s post hoc test for multiple pairwise comparisons. *P < 0.05. ABC = active ß-catenin; DAPI = 4',6-diamidino-2-phenylindole; non-P = nonphosphorylated; other abbreviations as in Figure 2.

Fig 5: Sarcomeric cardiomyopathy is associated with increased cardiomyocyte (CM) aneuploidy.(A) Representative immunofluorescence images of fluorescent in situ hybridization (FISH) assay for chromosome 8 (Chrom. 8) (red, arrowhead) from control (Ctl) and Mybpc3-/- left ventricular (LV) tissue at postnatal day (P) 25. CMs were identified with pericentriolar material 1 (PCM1) (green), with nuclei labeled with 4',6-diamidino-2-phenylindole (DAPI) (blue). Bar=5 µm. (B) Percentage of aneuploid nuclei (3 or 5 foci) per total CM nuclei from Ctl (n=6) and Mybpc3-/- (n=6). (C) Percentage of aneuploid nuclei per noncardiomyocyte (Non-CM) nuclei from Ctl (n=6) and Mybpc3-/- (n=6). (D) Representative immunofluorescence images of FISH assay for Chrom. 8 (red, arrowhead) from TNNT2WT and TNNT2I79N LV tissue at P25. CMs were identified with PCM1 (green), and nuclei were labeled with DAPI (blue). Bar=5 µm. (E) Percentage of aneuploid nuclei per total CM nuclei from TNNT2WT (n=4) and TNNT2I79N (n=4). (F) Percentage of aneuploid nuclei per total Non-CM nuclei from TNNT2WT (n=4) and TNNT2I79N (n=4). (G) Representative immunofluorescence images of FISH assay for Chrom. 8 (green, arrowhead) from Ctl or hypertrophic cardiomyopathy (HCM) human LV tissue. CMs were identified with PCM1 (red), and nuclei were labeled with DAPI (blue). Bar=5 µm. (H) Percentage of aneuploid nuclei per total CM nuclei from Ctl (n=4) and HCM (n=6) LV tissue. (I) Percentage of aneuploid nuclei per total Non-CM nuclei from Ctl (n=4) and HCM (n=6) LV tissue. Minimum 50 PCM+ or PCM– nuclei/sample were analyzed. All results are shown as mean±SEM. (J) Mechanistic overview for how sarcomere dysfunction leads to replication stress-induced DNA damage and selective DNA damage response (DDR) pathway activation, which modifies pathological LV remodeling and CM genome stability. ATR indicates ataxia telangiectasia and rad3 related; and N.D., not detectable.