Cardiomyocytes in Young Infants With Congenital Heart Disease: a Three-Month Window of Proliferation

Abstract

Perinatal reduction in cardiomyocyte cell cycle activity is well established in animal models and humans. However, cardiomyocyte cell cycle activity in infants with congenital heart disease (CHD) is unknown, and may provide important information to improve treatment. Human right atrial specimens were obtained from infants during routine surgery to repair ventricular septal defects. The specimens were divided into three groups: group A (age 1-3 months); group B (age, 4-6 months); and group C (age 7-12 months). A dramatic fall in the number of Ki67-positive CHD cardiac myocytes occurred after three months. When cultured in vitro, young CHD myocytes (≤ 3 months) showed more abundant Ki67-positive cardiomyocytes and greater incorporation of EdU, indicating enhanced proliferation. YAP1 and NICD-important transcript factors in cardiomyocyte development and proliferation-decreased with age and β-catenin increased with age. Compared with those of older infants, cardiomyocytes of young CHD infants (≤ 3 months) have a higher proliferating capacity in vivo and in vitro. From the perspective of cardiac muscle regeneration, CHD treatment at a younger age (≤ 3 months) may be more optimal.

Figure 1. Percentage of Ki67-positive cardiomyocytes by flowcytometry.

(A) Representative flowcytometry data showing Ki67-positive cardiomyocytes at three different ages; (B) Quantification of Ki67-positive cardiomyocytes based on age (n = 6 per age group). Error bars represent SD. *P < 0.05.

Figure 2. Confocal microscopy of Ki67-positive cardiomyocytes in tissue sections.

(A) Representative Ki67-positive cardiomyocytes from different age groups. Cardiac Troponin T (red), Ki67 (green), and DAPI (blue). (B) Quantification of Ki67-positive cardiomyocytes in three age groups (n = 6 per age group). Error bars represent SD. **P < 0.01.

Figure 3. In vitro culture of cardiomyocytes.

(A) Representative graphs of different age groups. Cardiac Troponin T (red), Ki67 (green), and DAPI (blue). Magnification: x10. Arrow indicates proliferating cardiomyocytes, and the triangle indicates non-cardiomyocytes. (B) Quantification of Ki67-positive cardiomyocytes (n = 6). Error bars represent SD. **P < 0.01. (C) The volume of Ki67-positive cardiomyocytes. P < 0.01 (D) After four days of culture, human cardiomyocytes still showed cell cycle activity. Sarcomeric Alpha Actinin (red), EdU (green), and DAPI (blue).

Figure 4. Yap1 expression by Western blot analysis and YAP1 immunofluorescence in tissue sections.

(A) Representative Western blot (top) and mean data obtained by densitometry analysis (bottom) are shown. GAPDH served as a loading control. The bars indicate mean ± SD. ANOVA was performed to evaluate statistical significance of differences, n = 6, **P < 0.01. (B) Representative immunofluorescence in different age groups. Scale bar: 25 μm.

Figure 5. β-catenin expression in cardiomyocytes.

(A) Representative Western blot analysis (top) and mean data obtained by densitometry analysis (bottom) are shown. GAPDH served as loading control. The bars indicate mean ± SD. ANOVA was performed to evaluate statistical significance of differences, n = 6, **P < 0.01. (B) Representative immunohistochemistry in different age groups. Scale bar: Left panel, 100 μm; Right panel, 20 μm. (C) Representative immunofluorescence in different age group. Scale bar: 25 μm.

Figure 6. NICD expression in cardiomyocytes.

(A) Representative Western blot analysis (top) and mean data obtained by densitometry analysis (bottom) are shown. GAPDH served as loading control. The bars indicate mean ± SD. ANOVA was performed to evaluate significance of differences, n = 6, **P < 0.01. (B) Representative immunohistochemistry in different age groups. Scale bar: Left panel, 100 μm; Right panel, 20 μm. (C) Representative immunofluorescence in different age groups. Scale bar: 25 μm.