Fas Apoptosis Inhibitory Molecule 2 Inhibits Pathological Cardiac Hypertrophy by Regulating the MAPK Signaling Pathway

Figures

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  Figure 1. Fas apoptotic inhibitory molecule 2 (FAIM2) protein level was decreased in cardiac hypertrophic models in vivo and in vitro. (A) The mRNA level of FAIM2 in mice hearts after 4 weeks of transverse aortic constriction (TAC) or sham surgery (n=5 mice per group). (B) The mRNA level of FAIM2 in control and phenylephrine‐treated neonatal rat ventricular myocytes (NRVMs; phenylephrine, 50 μmol 24 hours). (C) Western blot (upper) and quantification result (lower) of FAIM2 protein in mice hearts after 4 weeks of TAC or sham surgery (n=3 mice per group). (D) Western blot (upper) and quantification result (lower) of FAIM2 protein in control and phenylephrine‐treated NRVMs. (E) Immunohistochemical staining of FAIM2 in mice hearts after 4 weeks of TAC or sham surgery (n=4 mice per group). Scale bar, 25 μm. **P<0.01. * indicates between‐group differences between the control and experimental groups. Normality test and independent samples test were used for all of the statistical methods. All of the cell experiments were completed in 3 independent experiments. n.s. indicates not significant.

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  Figure 2. Fas apoptotic inhibitory molecule 2 (FAIM2) overexpression ameliorated phenylephrine‐induced myocardial dysfunction and apoptosis in vitro. (A) Western blot analysis showing the overexpression effects of Faim2 gene overexpression adenovirus (AdFaim2) in neonatal rat ventricular myocytes (NRVMs). (B) Representative images (left) and cell surface area's statistical results (right) of NRVMs harboring control or Faim2 overexpression adenovirus subjected to phenylephrine treatment or PBS. α‐actinin (the red signal in the figure) was used to stain the cardiomyocyte skeleton, anti‐Faim2 antibody (the green signal in the figure) was used to detect the expression of Faim2 in cells, and DAPI (the blue signal in the figure) was used to stain the cardiomyocyte nucleus. Scale bar=20 μm. (C) Real‐time quantitative polymerase chain reaction (RT‐qPCR) results of atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), β‐myosin heavy chain (Myh7), and actin α1 (Acta1) in NRVMs harboring control or Faim2 overexpression adenovirus subjected to phenylephrine treatment or PBS. (D) RT‐qPCR results of BCL2‐associated X protein (Bax) and B‐cell lymphoma 2 (Bcl2) in NRVMs harboring control or…

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  Figure 3. Fas apoptotic inhibitory molecule 2 (FAIM2) knockdown aggravated phenylephrine‐induced myocardial dysfunction and apoptosis in vitro. (A) Western blot analysis showing the knockdown efficiency of short hairpin RNA against Faim2 adenoviral constructs (AdshFaim2) adenovirus in neonatal rat ventricular myocyte (NRVMs). (B) Representative images (left) and cell surface area's statistical results (right) of NRVMs harboring control or Faim2 knockdown adenovirus subjected to phenylephrine treatment or PBS. α‐actinin (the red signal in the figure) was used to stain the cardiomyocyte skeleton, anti‐Faim2 antibody (the green signal in the figure) was used to detect the expression of Faim2 in cells, and DAPI (the blue signal in the figure) was used to stain the cardiomyocyte nucleus. Scale bar=20 μm. (C) Real‐time quantitative polymerase chain reaction (RT‐qPCR) results of atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), β‐myosin heavy chain (Myh7), and actin α1 (Acta1) in NRVMs harboring control or Faim2 knockdown adenovirus subjected to phenylephrine treatment or PBS. (D) RT‐qPCR results of BCL2‐associated X protein (Bax) and B‐cell lymphoma 2 (Bcl2) in NRVMs harboring…

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  Figure 4. Fas apoptotic inhibitory molecule 2 (FAIM2) depletion aggravated transverse aortic constriction (TAC)–induced cardiac dysfunction in vivo. (A) Western blot analysis showing the knockout (KO) efficiency of Faim2 in mice hearts (n=3 mice per group). (B–F) Statistical results of body weight (BW; B), heart weight (HW; C), HW/BW (D), lung weight (LW)/BW (E), and HW/tibia length (TL; F) in indicated groups (n=10 mice per group). (G–N) Echocardiography measurements of heart rate (HR; G), left ventricular (LV) internal diameter at end‐diastole (LVEDd; H), LV internal diameter at end‐systole (LVESd; I), ejection fraction (EF; J), fractional shortening (FS; K), interventricular septum diameter in diastole (IVSd; L), LV posterior wall diameter in diastole (LVPWd; M), and corrected LV mass (LV mass cor; N) in indicated groups (n=10 mice per group). The asterisk signs indicate the significant difference between the WT sham group and the WT TAC group, while the pound signs indicate the significant difference between the WT TAC group and Faim2‐KO TAC group. * or # P<0.05, ** or ## P<0.01. *denotes comparisons between the WT sham and WT TAC groups, and # denotes comparisons between the KO sham…

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  Figure 5. Fas apoptotic inhibitory molecule 2 (FAIM2) deficiency aggravated transverse aortic constriction (TAC)–induced cardiac hypertrophy, fibrosis, and apoptosis in vivo. (A) Representative images of gross hearts, hematoxylin–eosin staining (left) and statistical results of cardiomyocytes' areas (right) in mice hearts of different groups (n=6 mice per group). Scale bars: 0.3 cm, 25 μm. (B) Real‐time quantitative polymerase chain reaction (RT‐qPCR) results of Anp (atrial natriuretic peptide), Bnp (brain natriuretic peptide), Myh7 (β‐myosin heavy chain), and Acta1 (actin α1) in the mice heart of designated groups (n=3 mice per group). (C) Representative images of picrosirius red staining (left) and statistical results of collagen volume (right) in sections of mice hearts from different groups (n=6 mice per group). Scale bar=50 μm. (D) RT‐qPCR results of collagen type I alpha 1 chain (Col1α1), collagen type III alpha 1 chain (Col3α1), connective tissue growth factor (Ctgf), and tissue inhibitor matrix metalloproteinase 1 (Timp1) in the mice hearts of designated groups (n=3 mice per group). (E) RT‐qPCR results of BCL2‐associated X protein (Bax) and B‐cell lymphoma 2 (Bcl2) in the mice…

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  Figure 6. RNA‐sequencing (RNA‐Seq) analysis showing that Fas apoptotic inhibitory molecule 2 (FAIM2) deficiency aggravates transverse aortic constriction (TAC)–induced heart dysfunction and fibrosis. (A) Hierarchical clustering dendrogram showing the distribution of samples from wild‐type (WT) and Faim2‐knockout (KO) mice hearts subjected to TAC surgery. (B) Enrichment analysis of biological processes of Gene Ontology data sets, including heart function, protein processing and fibrosis, in the RNA‐Seq results from WT and Faim2‐KO mice hearts subjected to TAC surgery. (C) Heatmaps showed the differentially expressed genes involved in the heart function, protein processing, and fibrosis in the RNA‐Seq results from WT and Faim2‐KO mice hearts subjected to TAC surgery.

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  Figure 7. Fas apoptotic inhibitory molecule 2 (FAIM2) regulates the mitogen‐activated protein kinase (MAPK) signaling pathway through transforming growth factor β–activated kinase 1 (TAK1) phosphorylation. (A) Kyoto Encyclopedia of Genes and Genomes analysis of RNA sequencing results from wild‐type (WT) and Faim2‐knockout (KO) mice hearts subjected to transverse aortic constriction (TAC) surgery. (B) Western blot (upper) and quantification results (lower) of representative MAPKs in WT and Faim2‐KO mice hearts subjected to TAC surgery (n=3 mice per group). (C) Western blot (upper) and quantification results (lower) of representative MAPKs in neonatal rat ventricular myocytes (NRVMs) harboring control or Faim2 knockdown adenovirus subjected to phenylephrine treatment. (D) Western blot (upper) and quantification results (lower) of representative MAPKs in NRVMs harboring control or Faim2 overexpression adenovirus subjected to phenylephrine treatment. (E) Western blot (upper) and quantification results (lower) of representative MAPK kinase kinases in WT and Faim2‐KO mice hearts subjected to TAC surgery (n=3 mice per group). (F) Western blot (upper) and quantification results (lower) of…

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  Figure 8. The exacerbating role of Fas apoptotic inhibitory molecule 2 (Faim2) knockdown in cardiac hypertrophy in vitro is mediated by the activation of transforming growth factor β–activated kinase 1 (TAK1). (A) Endogenous immunoprecipitation (IP) assays proved the interaction between FAIM2 and TAK1 in cardiomyocytes. (B) Western blot (left) and quantification results (right) verified the inhibitory effects of 5Z‐7‐oxozeaenol (5Z‐7‐ox) on TAK1 activity. The neonatal rat ventricular myocytes (NRVMs) were treated with 1 μmol of 5Z‐7‐ox for 24 hours in the presence of phenylephrine (50 μmol) pressure. (C) Representative images (left) and cell surface area's statistical results (right) of NRVMs harboring nontargeting short hairpin RNA (AdshRNA) and short hairpin RNA against Faim2 adenoviral constructs (AdshFaim2) adenovirus with 5Z‐7‐ox or control (CT) subjected to phenylephrine treatment. α‐actinin was used to stain the cardiomyocyte skeleton, and DAPI was used to stain the cardiomyocyte nucleus. Scale bar=20 μm. (D) Real‐time quantitative polymerase chain reaction (RT‐qPCR) results of Anp (atrial natriuretic peptide), Bnp (brain natriuretic peptide), Myh7 (β‐myosin heavy chain), and Acta1…