Dystrophin-deficient cardiomyocytes exhibit progressive telomere shortening in two phases: proliferation dependent and proliferation independent. (A) Telomere length (TelC) was evaluated by immunofluorescence staining relative to DAPI staining in cardiac troponin T–positive cardiomyocytes. White arrowheads indicate nuclei within a cardiomyocyte from a mdx^4cv/mTR^G2 animal. (B) Intensity of telomere staining relative to DAPI of cardiomyocytes is shown for 1-, 4-, 8-, and 32-wk-old hearts (n = 3 per genotype). The number of nuclei (N) scored was mdx^4cv/mTR^Het (n = 560, 318, 475, and 353), mdx^4cv/mTR^G2 (n = 558, 292, 370, and 284), and mTR^G2 (n = 513, 287, 419, and 349) for 1-, 4-, 8-, and 32-wk-old hearts, respectively. (C) Percentages of telomere shortening are shown for preadult (1 vs 8 wk old), adult phases (8 vs 32 wk old), and proliferation-independent phase (1 vs 32 wk old). (Scale bars, 10 μm.) Data are represented as mean ± SEM. Statistical analyses entailed nonparametric Kruskal-Wallis test corrected with Dunn’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

Analysis of proliferation in cardiomyocytes by K_i-67. Representative micrographs for cardiomyocytes from 1, 8, and 32 wk of age heart sections from mdx^4cv/mTR^G2, mdx^4cv/mTR^Het, and mTR^G2 mice immunostained for (A) K_i-67 at the single-cell level and (B) by flow cytometry at the population level determined relative to IgG control (gray). (Scale bars, 10 μm.) Data are represented as mean ± SEM.

Analysis of cell cycle phase in cardiomyocytes by Phospho-Histone 3 (pH3). Representative micrographs for cardiomyocytes from 1, 8, and 32 wk of age heart sections from mdx^4cv/mTR^G2, mdx^4cv/mTR^Het, and mTR^G2 mice immunostained (A) for pH3 at the single-cell level and (B) by flow cytometry at the population level determined relative to IgG control (gray). (Scale bars, 10 μm.) Data are represented as mean ± SEM.

Analysis of mitosis in cardiomyocytes by BrdU labeling. Analysis of BrdU incorporation (24 h pulse) by flow cytometry performed in mdx^4cv/mTR^G2, mdx^4cv/mTR^Het, and mTR^G2 cardiomyocytes. (A) FACS gating strategy of cardiac troponin T–positive cardiomyocytes. (B) BrdU incorporation in cardiomyocytes from in 8- and 32-wk-old animals and (C) quantification of FACS data of BrdU labeling relative to IgG control. Proliferative spleen from mdx^4cv/mTR^G2 served as a positive control for BrdU labeling. Data are represented as mean ± SEM.

Cardiomyocytes of DMD mouse model exhibit telomere dysfunction. (A) Schematic of telomeric ends protected by shelterin proteins. (B) Endogenous expression levels of telomere capping genes were determined by RT-qPCR in primary cardiomyocytes: Trf1, Trf2, Tpp1, Tin2, Pot1a, and Pot1b (n = 4–5 mice per genotype; technical replicates n = 2). (C) Percentage of DNA damage response marker 53bp1 positivity was quantified by flow cytometry relative to IgG control (gray). (D) 53bp1-positive cardiomyocytes (percentage total) are shown (n = 3 mice per genotype; n = 5,000 cardiac troponin T–positive cells per sample). All assays were performed using 8-wk-old animals. Data are represented as mean ± SEM. Statistical analyses entailed one-way ANOVA with post hoc Bonferroni correction. *P < 0.05; **P < 0.01; ****P < 0.0001.

Critically short telomeres lead to activation of p53, leading to decreased mitochondrial biogenesis. (A) Cell lysates from cardiomyocytes were immunoblotted for p53. Histone 3 (H3) protein expression was used as loading control. (B) Percentage of p53 positivity was quantified by flow cytometry at the population level. (C) Percentage of p53-positive cardiomyocytes determined by area under the curve relative to IgG control (gray; n = 3 mice per genotype; n = 5,000 cardiac troponin T–positive cardiomyocytes per condition). (D) Expression levels of p53 target gene, p21, were determined by RT-qPCR in cardiomyocytes (n = 4–5 mice per genotype; technical replicates n = 2). (E) p53 activation evaluated as a function of p21 protein levels by flow cytometry in cardiomyocytes. (F) Percentage of p21-positive cardiomyocytes determined by area under the curve relative to IgG control (gray; n = 3 mice per genotype; n = 5,000 cardiac troponin T–positive cells per sample). (G and H) Reduced expression levels of Pgc1α and Pgc1β, known p53 targets, determined by RT-qPCR (n = 4–5 mice per genotype; technical replicates n = 2). All assays were performed using 8-wk-old mice. Data are represented as mean ± SEM. Statistical analyses entailed one-way ANOVA with post hoc Bonferroni correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Critically short telomeres result in mitochondrial dysfunction in cardiomyocytes of DMD mouse model. (A) Scheme for mitochondrial measurements. (B) Mitochondria copy number assessed as mitochondrial gene (Nd2) to nuclear DNA (Nrf1) in mdx^4cv/mTR^G2, mdx^4cv/mTR^Het, and mTR^G2 cardiomyocytes (n = 5–8 mice per genotype; technical n = 2). (C) Mitochondrial superoxide species (MitoSox Red) and (D) intracellular reactive oxygen species (CellROX Deep Red) were measured in cardiomyocytes (n = 4–5 mice per genotype; technical replicates n = 4–5). (E) Mitochondrial membrane potentials (∆ψm) were detected by fluorogenic probe TMRM in cardiomyocytes (n = 4–5 mice per genotype; technical replicates n = 4–5). (F) Real-time oxygen consumption rate was measured in freshly isolated mdx^4cv/mTR^G2, mdx^4cv/mTR^Het, and mTR^KO cardiomyocytes. Cells were exposed to 5 mM glucose, 167 μM Palmitate, or assay medium alone (n = 5 mice per genotype; technical n = 8). (G) Mean ATP levels normalized to total protein in cardiomyocyte lysates (n = 4–5 mice per genotype). (H) Mitochondrial superoxide species (MitoSox Red). (I) intracellular reactive oxygen species (CellROX Deep Red), (J) mitochondrial membrane potentials (∆ψm), and (K) real-time oxygen consumption rate were measured in freshly isolated mdx^4cv/mTR^G2 cardiomyocytes treated with either MnTBAP or saline vehicle (n = 8 mice per treatment; technical n = 4–8). All assays performed using 8-wk-old mice. Data are represented as mean ± SEM. Statistical analyses entailed one-way ANOVA with post hoc Bonferroni correction, except for the MnTBAP rescue experiments, for which a two-tailed Student’s t test was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Mitochondrial protein changes assayed by proteomic mass spectrometry. (A) Schematic of sample preparation and multiplex labeling before proteomic analysis (n = 2 per genotype). (B) Differentially up-regulated and (C) down-regulated proteins in mdx^4cv/mTR^G2 relative to mTR^G2 or wildtype were categorized by GO terms designated by molecular function and biological and cellular components.