Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

FIGURE 1. Deletion of Ercc1, a DNA repair gene, in striated muscle leads to premature death. (a) qRT‐PCR measurement of Ercc1 mRNA levels in the heart, skeletal muscle, liver, and kidney from the tissue specific‐mutant Ckmm‐Cre +/− ;Ercc1 −/fl mice and wild‐type (WT) animals. See Table S4 for primer sets. See Table S5 for detailed relative expression levels of target genes. Data represent the mean ± SD (n = 5 mice, 2–3 months old), two‐tailed, paired Student' s t‐test, **p < 0.01, ns = non‐significant. The mRNA expression unit is fold change normalized to the control group. (b) Kaplan–Meier survival curve demonstrating that Ckmm‐Cre +/− ;Ercc1 −/fl mice have a significantly reduced lifespan (n = 15 mutant mice, median lifespan 4.9 months) compared to WT animals (n = 19, median lifespan 23.4 months), p < 0.0001, Log‐rank (Mantel‐Cox) test. See Figure S3 for sex‐specific differences in lifespan.

FIGURE 2. Deletion of Ercc1 in striated muscle leads to dilated cardiomyopathy. (a) Representative images of hearts from Ckmm‐Cre +/− ;Ercc1 −/fl and WT mice at 6‐months of age (Scale bar: 2 mm). (b) Representative images of H&E stained sagittal section of the heart from a Ckmm‐Cre +/− ;Ercc1 −/fl and WT at 6 months of age. (Scale bar: 2.5 mm). (c) Measures of heart weight to body weight ratio and cardiac function (ejection fraction (EF), fractional shortening (FS), left ventricular end‐systolic volume (LV ESV), and left ventricular end‐diastolic volume (LV EDV)) calculated from echocardiography data from male mutant mice and littermate controls at 6 months of age. Graphs indicate the mean with error bars representing ± SD (n = 6–9 mice per group); two‐tailed, unpaired Student's t‐test *p < 0.05, **p < 0.01, ***p < 0.001. (d). Same as c for female mice at 6 months of age (controls n = 14 and Ckmm‐Cre +/− ;Ercc1 −/fl n = 8 mice). A summary of all echo data can be found in Table S2. (e) Representative images of heart (left) and lung (right) sections stained with H&E or picrosirius red with Fast Green counterstain (SR/FG) to detect fibrosis. Ckmm‐Cre +/− ;Ercc1 −/fl mice and littermate…

FIGURE 3. Deletion of Ercc1 in striated muscle induces elevated levels of p53, markers of DNA damage, and apoptosis in isolated cardiac myocytes. (a) Immunocytochemical detection of p53 in cardiac myocytes isolated from 3‐month‐old WT, Ckmm‐Cre +/− ;Ercc1 −/fl and Ercc1 /D mice, which have tissue‐specific or systemic depletion of Ercc1 expression, respectively. (b) Protein lysates from 6‐month‐old mouse hearts of indicated genotypes were analyzed by immunoblot to detect p21, p53, or (phospho) p‐Ser15‐p53. Tubulin was used as a loading control. See Figure S16 for uncropped immunoblots. (c) Quantification of protein levels in b. One‐way ANOVA with Tukey's multiple comparison test, *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. (d) Representative images of immunofluorescence detection of γH2AX in doxorubicin‐treated (2.5 μM) cardiac myocytes isolated from 3‐month‐old WT, Ckmm‐Cre +/‐ ;Ercc1 −/fl and Ercc1 −/D mice at two time points post‐drug treatment. (Scale bar: 40 μM; NT = not treated). (e) Graphic representation of the fraction of γH2AX foci‐positive cells/field. Five independent fields per biological replicate were used for counting. Data represent the mean ± SD. (n = 3 biological…

FIGURE 4. Apoptosis of Ercc1‐deficient cardiac myocytes is p53‐dependent. (a) Representative images of cardiac myocytes isolated from 3‐month‐old mice of the indicated genotypes, which were not treated (NT), or with treated with Pifithrin‐α (an inhibitor of p53) treated, doxorubicin (a genotoxin), doxorubicin + Pifithrin‐α, UV, or UV+ Pifithrin‐α for 24 h and assessed for cell viability (rods = viable vs. rounds = dying or dead; numbers reported for each image in bottom left) (Scale bar: 100 μM). (b) Quantification of rod‐shaped cardiac myocytes (%) for the indicated genotypes and treatments groups. Data represent the mean ± SD, n = 3 animals per genotype, two‐way ANOVA in mutant mice and WT mice. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 and ns = non‐significant. Black font for ns (non‐significant) and * denotes the comparison of a treated group to the non‐treated group (NT). Green font and bars denote the comparison between cells treated with a genotoxin +/− the p53 inhibitor. (c) Caspase activity in cardiac myocytes for the indicated genotypes and treatment groups. Apoptosis was measured 24 h postdoxorubicin treatment using a Caspase 3/7 Glow assay. Graphed is the mean ± SD, n…

FIGURE 5. p53‐mediated oxidative stress contributes to heart failure in Ercc1‐deficient mice. (a) EPR detection of ascorbate free radical abundance (Asc·−) in 4‐ to 6‐month‐old Ckmm‐Cre +/‐ ;Ercc1 −/fl mice compared to littermate controls. Data represent the mean ± SD, n = 10–14 per group, two‐tailed, unpaired Student's t‐test ****p < 0.0001. ARI, Ascorbyl Radical Intensity. (b, c) The ratio of reduced to oxidized glutathione (GSH/GSSG) in cardiac tissue from 2–3‐month‐old and 4‐ to 6‐month‐old Ckmm‐Cre +/− ;Ercc1 −/fl mice compared to littermate controls and in Ercc1 −/D and WT mice of the same age. Values represent the mean ± SD, n = 3–4 per group, *p < 0.05 and ns = non‐significant determined by two‐tailed, unpaired Student's t‐test. (d–f) Relative expression of Cat, Hmox1, and Nqo1, antioxidant p53‐target genes, in cardiac tissue of mice of the indicated genotypes and age groups per above, measured by qRT‐PCR. The data represent the mean ± SD, n = 5 per group, two‐way ANOVA, *p < 0.05; **p < 0.001; ***p < 0.001; ****p < 0.0001 and ns = non‐significant. Blue asterisks indicate differences compared to young WT. The mRNA expression unit is fold changes normalized to the control group. (g,…

FIGURE 6. Model for the mechanism by which DNA damage drives heart failure. Unrepaired endogenous DNA damage leads to transcriptional activation of p53. If genotoxic stress and p53 activation occur chronically, antioxidant defenses are overcome. The direct cause of cardiac myocyte loss and ultimately cardiomyopathy in response to genotoxic stress is oxidative stress and p53‐dependent apoptosis as inhibition of either rescues cardiac myocyte viability. Figure created using bioRENDER.com (https://biorender.com/).