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. 2020 Jun 2;9(11):e015574.
doi: 10.1161/JAHA.119.015574. Epub 2020 May 30.

Pressure Overload Greatly Promotes Neonatal Right Ventricular Cardiomyocyte Proliferation: A New Model for the Study of Heart Regeneration

Lincai Ye  1   2   3 , Shoubao Wang  1   4 , Yingying Xiao  1 , Chuan Jiang  1   2   3 , Yanhui Huang  5 , Huiwen Chen  1 , Haibo Zhang  1 , Hao Zhang  1   3 , Jinfen Liu  1 , Zhuoming Xu  1 , Haifa Hong  2
Affiliations

Pressure Overload Greatly Promotes Neonatal Right Ventricular Cardiomyocyte Proliferation: A New Model for the Study of Heart Regeneration

Lincai Ye et al. J Am Heart Assoc. .

Abstract

Background Current mammalian models for heart regeneration research are limited to neonatal apex amputation and myocardial infarction, both of which are controversial. RNAseq has demonstrated a very limited set of differentially expressed genes between sham and operated hearts in myocardial infarction models. Here, we investigated in rats whether pressure overload in the right ventricle, a common phenomenon in children with congenital heart disease, could be used as a better animal model for heart regeneration studies when considering cardiomyocyte proliferation as the most important index. Methods and Results In the rat model, pressure overload was induced by pulmonary artery banding on postnatal day 1 and confirmed by echocardiography and hemodynamic measurements at postnatal day 7. RNA sequencing analyses of purified right ventricular cardiomyocytes at postnatal day 7 from pulmonary artery banding and sham-operated rats revealed that there were 5469 differentially expressed genes between these 2 groups. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that these genes mainly mediated mitosis and cell division. Cell proliferation assays indicated a continuous overproliferation of cardiomyocytes in the right ventricle after pulmonary artery banding, in particular for the first 3 postnatal days. We also validated the model using samples from overloaded right ventricles of human patients. There was an approximately 2-fold increase of Ki67/pHH3/aurora B-positive cardiomyocytes in human-overloaded right ventricles compared with nonoverloaded right ventricles. Other features of this animal model included cardiomyocyte hypotrophy with no fibrosis. Conclusions Pressure overload profoundly promotes cardiomyocyte proliferation in the neonatal stage in both rats and human beings. This activates a regeneration-specific gene program and may offer an alternative animal model for heart regeneration research.

Keywords: RNA‐seq; cardiomyocyte; pressure overload; proliferation; right ventricle.

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Figures

Figure 1
Figure 1. Establishment of right ventricular pressure overload model.
A, Representative echocardiographic images at P7 after sham and PAB surgery in neonatal rats. B, Graphical comparison of the PPG across PA constriction measured by transthoracic echocardiography between the sham and PAB groups. Data are presented as means±SD. Statistical analysis was performed using 2‐tailed Student t tests. C, H&E‐stained 2‐chamber cross sections of a P7 heart (Scale bar: 2 mm). D, High magnification of H&E‐stained sections of P7 RV. E, High magnification of H&E‐stained sections of P7 right ventricle (Scale bar: 50 μm). H&E indicates hematoxylin and eosin; LPA, left pulmonary artery; LV, left ventricle; PA, pulmonary artery; PAB, pulmonary artery banding; PPG, peak pressure gradient; RPA, right pulmonary artery; and RV, right ventricle.
Figure 2
Figure 2. Pressure overload greatly changes gene expression of cardiomyocytes.
A, Volcano map of differentially expressed genes. There were 5469 differentially expressed genes between PAB (Operation‐1 week, Opt1w) and sham (control‐1 week, Con1w) groups, among which, 2902 genes were upregulated, and 2567 genes were downregulated. B, Venn diagram of differentially expressed genes. There were 13 257 genes expressed in both groups, 624 genes expressed only in the PAB group, and 695 genes expressed only in the sham group. C, Cluster analysis of differentially expressed genes. Every group had 6 rats. The clusters of genes in each animal in the same group were similar to each other but quite different from the other group. OWOP is from the PAB group; OWCON is from the sham group. OWCON indicates Operationer Wang‐Control; OWOP, Operationer Wang‐operationl; and PAB, pulmonary artery banding.
Figure 3
Figure 3. GO analysis indicates that the differentially expressed genes mainly mediate mitosis and cell division.
A, From the results of the GO enrichment analysis, the most significant 30 terms are displayed. The abscissa is the GO Term, and the ordinate is the significance level of GO Term enrichment. The higher the value, the more significant, and the different colors represent 3 different GO subclasses: biological process (BP), (CC), and MF. B, From the results of the GO enrichment analysis, we selected the most significant 30 terms to draw scatterplots for display. The abscissa is the ratio of the number of differentially expressed genes on the GO Term to the total number of differentially expressed genes, the ordinate is the GO Term, the size of the dots represents the number of genes annotated to the GO Term, and the color from red to purple represents the significance level of GO Term enrichment. C, A network diagram of the top 5 most significant GO Terms in BP. D, A network diagram of the top 5 most significant GO Terms in BP in MF in CC. E, A network diagram of the top 5 most significant GO Terms in biological processes in molecular functions. BP indicates biological processes; CC, cellular components; GO, gene ontology; and MF, molecular function.
Figure 4
Figure 4. KEGG pathway analysis indicates that the differentially expressed genes mainly mediate mitosis and cell division.
A, From the KEGG enrichment results, the most significant 20 KEGG pathways are displayed. The abscissa is the KEGG pathway, and the ordinate is the significance level of pathway enrichment. The higher the value, the more significant. B, From the KEGG enrichment results, the most significant 20 KEGG pathways were selected for the scatterplots. The abscissa is the ratio of the number of differentially expressed genes on the KEGG pathway to the total number of differentially expressed genes, the ordinate is the KEGG pathway, the size of the dots represents the number of genes annotated to the KEGG pathway, and the color from red to purple the represents significance level of KEGG pathway enrichment. C, The Network of the top 5 most significantly enriched KEGG pathways. KEGG indicates Kyoto Encyclopedia of Genes and Genomes.
Figure 5
Figure 5. Immunofluorescence staining confirms that pressure overload greatly promotes RVCM proliferation.
A, Immunofluorescence staining for Ki67 (green), cardiac troponin T (red), and DAPI (blue) in rats at P7. B, Quantification of Ki67‐positive cardiomyocytes at P7, n=10 samples. C, Immunofluorescence staining for pHH3 (green), cTnT (red), and DAPI (blue) in rats at P7. Arrows indicate proliferating cardiomyocytes. D, Quantification of pHH3‐positive cardiomyocytes, n=10 samples. E, Immunofluorescence staining for aurora B (green), cTnT (red), and DAPI (blue) in rats at P7. F, Quantification of aurora B‐positive cardiomyocytes at P7. G, Representative Ki67‐positive cardiomyocytes from patients with RV pressure overload. Ki67 (green), cardiac troponin T (red), and DAPI (blue). Arrows indicate proliferating cardiomyocytes. H, Representative pHH3‐positive cardiomyocytes from patients with RV pressure overload. pH3 (red), cTnT (white), and DAPI (blue). Arrows indicate proliferating CMs. I, Quantification of Ki67/pHH3‐positive cardiomyocytes in human samples. J, Representative Ki67‐positive cardiomyocytes from patients with RV pressure overload. Aurora B (green), cTnT (red), and DAPI (blue). K, Quantification of aurora B‐positive cardiomyocytes in human samples. CMs indicates cardiomyocytes; cTnT, cardiac troponin T DAPI, 4′,6‐diamidino‐2‐phenylindole; PAB, pulmonary artery banding; pHH3, phospho‐histone H3; RV, right ventricle; and RVCM, right ventricular cardiomyocyte.
Figure 6
Figure 6. Pressure overload promotes RVCM proliferation associated with rat age.
Dash LINE indicates scale bar. A, 25 μm; B, 100 μm, above panel. Arrow indicates proliferating CMs. A, Immunofluorescence staining for Ki67 (green), cardiac troponin T (red), and DAPI (blue) in rats at P3. B, Immunofluorescence staining for pH3 (green), cardiac troponin T (red), and DAPI (blue) in rats at P14. C, Quantification of Ki67/pHH3‐positive cardiomyocytes at different ages. DAPI indicates 4′,6‐diamidino‐2‐phenylindole; PAB, pulmonary artery banding; pHH3, phospho‐histone H3; and RVCM, right ventricular cardiomyocyte.
Figure 7
Figure 7. Cardiomyocytes hypertrophy without fibrosis in pressure overload model.
A, Heart sections were stained with Alexa 488‐conjugated WGA (green), cardiac troponin T (red), and DAPI (blue). B, Quantification of cell size. C, qPCR analysis of mRNA expression levels for cardiac hypertrophy markers ANP and BNP. D, Trichrome green–stained sections of RV. Scale bar, 50 μm. E, qPCR analysis of mRNA expression levels for cardiac fibrosis markers Col1a2 and Col3a1. F, Detection of collagen II and IV from P7 rat hearts by Western blot. G, Quantification of Western blot bands from (F). ANP indicates atrial natriuretic peptide; BNP, brain natriuretic peptide; DAPI, 4′,6‐diamidino‐2‐phenylindole; PAB, pulmonary artery banding; qPCR, quantitative real‐time polymerase chain reaction; and RV, right ventricle.
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