Ndufs1 Deficiency Aggravates the Mitochondrial Membrane Potential Dysfunction in Pressure Overload‐Induced Myocardial Hypertrophy

Figure 1. scRNA-seq clustering by the Seurat package and functional enrichment analysis. (a) UMAP visualization after batch correction. Cells are colored according to the clusters. (b) GO enrichment analysis of differentially expressed genes, including biological process, cellular component, and molecular function categories. (c) KEGG: oxidative phosphorylation pathway enrichment map of time series analysis during the development of a TAC model. (d) The patterns of the GSVA-calculated scores of the KEGG: oxidative phosphorylation pathway at various stages of cardiac hypertrophy and after sham operation. (e) The patterns of the GSVA-calculated scores of the KEGG: oxidative phosphorylation pathway across various cell clusters.

Figure 2. Identification of candidate biomarkers involved in TAC-induced CH. (a) Volcano plot of the differentially expressed genes in cluster 2 based on the p value and expression fold changes. (b) Spearman correlation analysis of genes from cluster 2 and GSVA scores of the KEGG: oxidative phosphorylation pathway in cluster 2. OxP score: GSVA scores of KEGG: oxidative phosphorylation. (c) The expression of Ndufs1 in mouse cardiac tissue based on the data of GSE24454. Mice were subjected to 4 weeks of aortic banding (AB) or sham procedure (sAB), banding and subsequent debanding (DB3), or sham procedure and subsequent debanding (sDB3) and sacrificed 3 days after debanding. (d) Protein-protein interaction network analysis of NDUFS1 based on the ComPPI database. (e) Protein-protein interaction network analysis of NDUFS1 in mitochondria. (f) Functional enrichment analysis of mitochondrial proteins interacting with NDUFS1.

Figure 3. Ndufs1 was downregulated in the hypertrophic heart of mice. (a) H&E staining of mouse heart tissue in the sham and TAC groups. (b) The cardiomyocyte area was evaluated in the sham and TAC groups. The heart was dissected, and the heart weight (c), HMI (d), LVMI (e), and LV/TL (f) in the sham and TAC groups were determined. (g) Representative images of echocardiography in the sham and TAC groups, and the thickness of IVS (h) and LVPW (i) in the sham and TAC groups according to echocardiography. (j–l) The mRNA expression levels of ANP, BNP, and β-MHC in the heart tissue of the sham and TAC groups determined by qRT-PCR. (m, n) The mRNA and protein expression levels of Ndufs1 in the heart tissue of the sham and TAC groups were determined by qRT-PCR and western blot assays, respectively. (o) Representative TEM images of the heart tissue of the sham and TAC groups. (p, q) The mitochondrial volume and number were analyzed based on TEM images. N = 6. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 4. Ang II induced downregulation of Ndufs1 in rat cardiomyocytes. The mRNA and protein expression levels of ANP (a, b), BNP (c, d), β-MHC (e, f), and Ndufs1 (g, h) in control and Ang II-treated cardiomyocytes were determined by qRT-PCR and western blot assay. N = 3. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 5. Effects of Ndufs1 knockdown on the expression of hypertrophic markers and mitochondrial function. (a, b) The mRNA and protein expression levels of Ndufs1 in cardiomyocytes transfected with scrambled siRNA or Ndufs1 siRNA were determined by qRT-PCR and western blot assays. (c) The relative cardiomyocyte area was determined in cardiomyocytes transfected with scrambled siRNA or Ndufs1 siRNA. The mRNA and protein expression levels of ANP (d, e), BNP (f, g), and β-MHC (h, i) in cardiomyocytes transfected with scrambled siRNA or Ndufs1 siRNA were determined by qRT-PCR and western blot assays. The mitochondrial DNA content (j), mitochondrial ROS production (k), ROS-positive cardiomyocytes (l), MMP (m), and mitochondrial mass (n) were evaluated in cardiomyocytes transfected with scrambled siRNA or Ndufs1 siRNA. N = 3. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 6. Ndufs1 overexpression attenuated Ang II-induced cardiac hypertrophy and mitochondrial dysfunction in cardiomyocytes. (a, b) The mRNA and protein expression levels of Ndufs1 in cardiomyocytes transfected with pcDNA3.1 or pcDNA3.1-Ndufs1 were determined by qRT-PCR and western blot assays. (c) The relative cardiomyocyte area was determined in cardiomyocytes subjected to various treatments. The mRNA and protein expression levels of ANP (d, e), BNP (f, g), and β-MHC (h, i) in cardiomyocytes subjected to various treatments were determined by qRT-PCR and western blot assays. The mitochondrial DNA content (j), mitochondrial ROS production (k), ROS-positive cardiomyocytes (l), MMP (m), and mitochondrial mass (n) were evaluated in cardiomyocytes subjected to various treatments. N = 3. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.