ErbB2 is required for cardiomyocyte proliferation in murine neonatal hearts

Abstract

It has been long recognized that the mammalian heart loses its proliferative capacity soon after birth, yet, the molecular basis of this loss of cardiac proliferation postnatally is largely unknown. In this study, we found that cardiac ErbB2, a member of the epidermal growth factor receptor family, exhibits a rapid and dramatic decline in expression at the neonatal stage. We further demonstrate that conditional ablation of ErbB2 in the ventricular myocardium results in upregulation of negative cell cycle regulators and a significant reduction in cardiomyocyte proliferation during the narrow neonatal proliferative time window. Together, our data reveal a positive correlation between the expression levels of ErbB2 with neonatal cardiomyocyte proliferation and suggest that reduction in cardiac ErbB2 expression may contribute to the loss of postnatal cardiomyocyte proliferative capacity.

Keywords: Cardiomyocyte; ErbB2; Neonatal; Proliferation.

Fig. 1

The expression profile of ErbB2 in the heart correlates with cardiac proliferative capacity. (A) A schematic illustration showing the difference of cardiac proliferative capacity over time. (B) qRT-PCR analysis of ErbB2, ErbB4, Nrg1 mRNA expression in murine hearts of different ages. (C) The relative expression of ErbB4 to ErbB2 from late embryonic to adult stages. (D) Western blots and quantification of ErbB2 protein expression in postnatal murine hearts. GADPH is used as a loading control. Data are presented as mean ± SEM. Statistical significance was determined by ANOVA test. *p < 0.05, **p < 0.01.

Fig. 2

ErbB2 is required to neonatal CM proliferation. (A) qRT-PCR to determine the expression level of ErbB2 in CMs from ErbB2^Cko. (B) Immunofluorescence analysis of CM proliferation of the ErbB2^fl/fl and ErbB2^Cko neonatal hearts by double staining of Ki67 (green) and cTnT (Red) on P0. Representative images obtained under 10× and 40× magnification respectively. (C) Quantification of immunofluorescence analysis in (B). (D) Immunofluorescence analysis of CM karyokinesis of the ErbB2^fl/fl and ErbB2^Cko hearts by double staining of pH 3 (green) and cTnT (Red) at P0. Representative images obtained under 10× and 40× magnification respectively. (E) Quantification of immunofluorescence analysis in (D). Data are presented as mean ± SEM. Statistical significance was determined by student’s t-test (between two groups). *p < 0.05, **p < 0.01.

Fig. 3

ErbB2 ablation results in an upregulation in the expression of the negative cell cycle regulators. (A) qRT-PCR analysis of the expression of selected CDKIs in ErbB2^fl/fl and ErbB2^Cko CMs at P0. (B) qRT-PCR analysis of the expression of negative regulators of cell cycle in ErbB2^fl/fl and ErbB2^Cko CMs at P0. (C) qRT-PCR analysis of the expression of positive regulators of cell cycle in ErbB2^fl/fl and ErbB2^Cko CMs at P0. Data are presented as mean ± SEM. Statistical significance was determined by student’s t-test (between two groups). *p < 0.05, **p < 0.01.