Table 1.
Patients’ clinical information (Individual information provided in S1 Table).
Fig 1.
Oxidative DNA damage in different age hearts analyzed by mtDNA copy number.
qRT-PCR was used to analyze the levels of mtDNA in VSD patient hearts. mtDNA was not positively correlated with age, although group B (7–12 mo) had the highest amount of mtDNA. Bars indicate mean ±standard deviation. Both ANOVA and SNK were performed to evaluate statistical significance, n = 20, **p < 0.01.
Fig 2.
Oxidative DNA damage in hearts from different ages analyzed by 8-oxoG.
(A) Representative graph of groups A, B, and C. 8-oxoG was not positively correlated with age, and group B (7–12 mo) had the highest amount of 8-oxoG. Cardiac sarcomeric alpha actinin (red), 8-oxoG (green), and DAPI (blue) staining; Scale bar = 25 μm. (B) Quantification of 8-oxoG foci per cardiomyocyte in each group, n = 20, **p < 0.01.
Fig 3.
Oxidative DNA damage in hearts from different ages analyzed by p-ATM.
(A) Representative graph of groups A, B, and C. p-ATM was not positively correlated with ages, and group B (7–12 mo) had the highest amount of p-ATM. Cardiac troponin T (green), p-ATM (red), and DAPI (blue) staining; Scale bar = 25 μm. (B) Negtive control. Primary antibody was substituted with PBS. (C) Quantification of p-ATM foci per cardiomyocyte in each group, n = 20, **p < 0.01.
Fig 4.
Cardiomyocyte proliferation from different age groups analyzed via confocal microscopy.
Confocal microscopy of tissue sections using Ki67 and troponin T indicated that proliferating cardiomyocytes were decreased relative to age. (A) Representative Ki67-positive cardiomyocytes in Group B. (B) Representative Ki67-positive non-cardiomyocytes in Group B. (C) Quantification of Ki67-positive cardiomyocytes. (D) Quantification of Ki67-positive non-cardiomyocytes. Data presented as mean ±standard devation; *p < 0.05, **p < 0.01. Cardiac troponin T (red), Ki67 (green), and DAPI (blue) staining are shown. Arrows indicate proliferating cardiomyocytes and the triangle indicates non-cardiomyocytes.
Fig 5.
Cardiomyocyte proliferation from different age groups analyzed by cyclin D2.
Confocal microscopy of tissue sections using cyclin D2 and cardiac sarcomeric alpha actinin indicated that proliferating cardiomyocytes were decreased relative to age. (A) Representative cyclin D2-positive cardiomyocytes in Group B. (B) Representative cyclin D2-positive non-cardiomyocytes in Group B. (C) Quantification of cyclin D2-positive cardiomyocytes. (D) Quantification of cyclin D2-positive non-cardiomyocytes. Data presented as mean ± standard deviation; *p < 0.05, **p < 0.01. cardiac sarcomeric alpha actinin (red), cyclin D2 (green), and DAPI (blue) staining are shown. Arrows indicate proliferating cardiomyocytes and triangle indicates non-cardiomyocytes.
Fig 6.
Ki67 and cyclin D2 mRNA is significantly decreased relative to age.
qRT-PCR was used to analyze Ki67 and cyclin D2 mRNA levels. Both Ki67 (A) and cyclin D2 (B) were decreased with age. GAPDH served as a control. Bars indicate mean ±standard deviation. * p < 0.05, **p < 0.01, n = 20.
Fig 7.
(A) Representative graph of each group. Cardiac troponin T (red), WGA (green). (B) Quantification of the cell size for each group. Data presented as mean ±standard deviation; ***p < 0.001, n = 1000 cells for each group.