Identify Tcea3 as a novel anti-cardiomyocyte hypertrophy gene involved in fatty acid oxidation and oxidative stress

Figures

• 

  Figure 1. Function annotations for deregulated genes in cardiac remodeling. (A) Venn diagram of the 17 downregulated and 78 upregulated DEGs based on the three GEO datasets. (B) The top 30 enriched gene ontology (GO) biological process (BP) terms BP.

• 

  Figure 2. Validation of the Tcea3 expression. (A) The box plot is based on 7 hypertrophy samples (marked in green), 7 HF samples (marked in blue) and 5 normal samples (marked in red) that revealed expression of Tcea3 in the left ventricle (wilcox.test) in GSE36074. Cardiac hypertrophy was defined as lung weight/tibia length ≤ 10.5 mg/mm and LAD ≤ 2.0 mm and HF was defined as lung weight/tibia length ≥ 15 mg/mm and LAD ≥ 2.4 mm. LAD, left atrial diameter. (B-D) Spearman correlation analysis of Tcea3 with the Nppa, Nppb and Myh7, respectively. (E) Relative mRNA expression level of Tcea3 in Sham or TAC group (n = 3). (F) Western blot and quantitation of Tcea3 protein level in the hearts of healthy individuals and DCM patients (n = 3). (G)Western blot and quantitation of Tcea3 protein level in the hearts of mouse subjected to sham or TAC surgery at 4 weeks (n = 3). (H) Western blot and quantitation of Tcea3 protein level in phenylephrine (PE) induced cardiomyocyte hypertrophy(n = 3). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01.

• 

  Figure 3. Silencing of Tcea3 in NRVMs aggravates cardiomyocyte hypertrophy. (A) Relative mRNA levels of Tcea3 in NRVMs. (B-E) Relative mRNA levels of cardiac hypertrophy marker genes in NRVMs transfected with control siRNA or Tcea3 siRNA followed by treatment with phenylephrine or PBS (20 μM) for 24 h (n = 3). (E,F) Representative α-actinin-stained immunofluorescence images (E) and Quantitative results of the cell surface area (B) of NRVMs (n = 3). The arrows show representative NRVMs with hypertrophy. Scale bar, 50 μm. Data are presented as mean ± SEM. *P < 0.05, ** P < 0.01, ***P < 0.001. PE, phenylephrine; DAPI, 4′,6-Diamidino-2′-phenylindole; ANP, atrial natriuretic peptide; BNP, b-type natriuretic peptide; MYH6, Myosin Heavy Chain 6; MYH7, Myosin Heavy Chain 7.

• 

  Figure 4. Tcea3 participates in lipid metabolism signaling pathways. (A) GSEA reveals fatty acid beta oxidation signaling pathway were enriched in the low-Tcea3 expression group. Top panels indicate the enrichment scores for each gene. Bottom panels show the ranking metrics of each gene. Y-axis: ranking metric values; X-axis: ranks for all genes. NES: normalized enrichment score. (B-E) Spearman correlation analysis of Tcea3 with the expression of Pla2g5, Ces1d, Pla2g4a, and Plcg2, respectively. (F-I) RT-PCR results of the four genes expression level in NRVMs transfected with control siRNA or Tcea3 siRNA followed by treatment with phenylephrine (20 μM) for 24 h. (n = 3). Data are presented as mean ± SEM. *P < 0.05, ** P < 0.01.

• 

  Figure 5. The impact of Tcea3 on fatty acid oxidation in NRVMs. (A) Oxygen consumption rate (OCR) in NRVMs transfected with control siRNA or Tcea3 siRNA followed by treatment with phenylephrine (20 μM) for 24 h. OCR was real-time detected in living NRVMs by Seahorse XFe24 Analyzer. (B-E) Quantification of basal respiration, maximal respiration, spare respiration capacity and ATP production. (F-H) Relative mRNA levels of genes involved in FAO in NRVMs (n = 3). Data are presented as mean ± SEM. *P < 0.05, ** P < 0.01, ***P < 0.001.

• 

  Figure 6. Effects of Tcea3 on oxidative stress in NRVMs. (A,B) Representative MitoSOX Red fluorescence images of NRVMs and the quantification of MitoSOX Red fluorescence intensity (n = 3). The arrows show representative NRVMs with oxidative stress. Scale bar, 20 μm. (C) Levels of GSH/GSSG in NRVMs of the four treatment groups (n = 5). (D)The level of NADPH/NADP+ in NRVMs transfected with control siRNA or Tcea3 siRNA and treated with phenylephrine (20 μM) or PBS for 24 h (n = 5). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01. PE, phenylephrine; GSH, glutathione; GSSG, glutathione disulfide; NADP+, Nicotinamide adenine dinucleotide phosphate.