POU2F1 promotes hypertensive cardiac fibrosis by regulating mitochondrial homeostasis through PINK1/Parkin‑dependent mitophagy

Figures

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  Figure 1.. POU2F1 upregulation and mitochondrial damage in myocardial fibrosis of SHRs. (A) Overlap between transcription factors and differentially expressed genes in cardiac tissues of SHRs compared with WKY rats. (B) Heatmap representing the relative levels of differentially expressed transcription factors between WKY rats and SHRs. (C) mRNA levels of POU2F1 in cardiac tissues were detected by reverse transcription-quantitative PCR. (D) Protein levels of POU2F1 in cardiac tissues were detected by western blotting (n=6). (E) Representative immunohistochemical staining images of POU2F1 in left ventricular myocardial tissues from WKY rats and SHR. (scale bar, 100 µm). (F) Transmission electron microscopy was used to observe mitochondrial structure in cardiac tissue from WKY rats and SHRs (scale bar, 500 nm). White arrows indicate cristae disorganization; red arrows indicate damaged mitochondria. (G) Seahorse assay profile of OCRs in CFs following sequentially treatment with oligomycin, FCCP and AA + ROT. Quantitative analysis of mitochondrial function parameters (basal respiration, maximal respiration and ATP production) is shown in the bar charts (n=3). (H) Protein levels of PINK1 and…

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  Figure 2.. POU2F1 upregulation and mitochondrial damage in Ang II-induced CFs. (A) CF proliferation was assessed using an EdU assay (scale bar, 200 µm). (B) Representative immunofluorescence staining of α-SMA in CFs (scale bar, 100 µm). (C) mRNA levels of POU2F1 in CFs were detected by reverse transcription-quantitative PCR (n=6). (D) Protein levels of POU2F1 in CFs were detected by western blotting (n=6). (E) α-SMA fluorescence intensity was positively correlated with the protein expression levels of POU2F1 in CFs (Spearman correlation, n=12). (F) MitoSOX staining showing levels of mitochondrial superoxide in CFs (scale bar, 200 µm). (G) Protein levels of PINK1 and Parkin in CFs were detected by western blotting (n=6). (H) Protein expression levels of PINK1 were positively correlated with POU2F1 expression in CFs (Spearman correlation, n=12). Data are presented as the mean ± SD. (C and D) Unpaired two-tailed Student's t-test were used for statistical analysis. ****P<0.0001. α-SMA, α-smooth muscle actin; Ang II, angiotensin II; CF, cardiac fibroblast; PINK1, PTEN-induced kinase 1; POU2F1, POU domain class 2 transcription factor 1.

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  Figure 3.. POU2F1 knockdown reduces PINK1-mediated mitophagy and mitochondrial dysfunction in Ang II-induced CFs. (A) MitoSOX staining showing levels of mitochondrial superoxide in Ang II-induced CFs infected with shPOU2F1 or shNC (scale bar, 200 µm). (B) Transmission electron microscopy was used to observe mitochondrial structure (scale bar, 500 nm). White arrows indicate cristae disorganization; red arrows indicate damaged mitochondria. (C) Quantification of MitoSOX fluorescence intensity was performed (n=6). Quantification of abnormal mitochondria was performed using low-magnification TEM images (×5,300 magnification; n=4). (D) Seahorse assay profile of OCRs in CFs following treatment with oligomycin, FCCP and AA + ROT. Quantitative analysis of mitochondrial function parameters (basal respiration, maximal respiration and ATP production) is shown in the bar charts (n=3). (E) Western blotting was utilized to determine the protein expression levels of PINK1, Parkin, LC3B and P62 (n=4). (F) CF proliferation was assessed using an EdU assay (scale bar, 200 µm). (G) Representative immunofluorescence images of α-SMA (scale bar, 100 µm). (H) Quantification of EdU-positive cells and α-SMA…

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  Figure 4.. PINK1 knockdown reverses oePOU2F1-induced myocardial fibrosis. (A) Proliferation of Ang II-induced cardiac fibroblasts was assessed following different interventions using an EdU assay (scale bar, 200 µm). (B) Representative immunofluorescence images of α-SMA (scale bar, 100 µm). (C) Rate of EdU-positive cells was quantified (n=6). (D) Quantification of the average intensity of α-SMA fluorescence (n=6). (E) Western blotting was utilized to determine the protein expression levels of collagen I and α-SMA. (F) Semi-quantification of collagen I and α-SMA protein expression (n=4). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. α-SMA, α-smooth muscle actin; Ang II, angiotensin II; oe, overexpression; PINK1, PTEN-induced kinase 1; POU2F1, POU domain class 2 transcription factor 1; sh, short hairpin.

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  Figure 5.. PINK1 knockdown reduces mitophagy and myocardial fibrosis independent of POU2F1 expression in Ang II-induced cardiac fibroblasts. Western blotting was utilized to determine the protein expression levels of (A) PINK1 and (B) POU2F1 (n=3). Expression levels of (C) PINK1, Parkin, LC3B and P62, as well as (D) collagen I and α-SMA were determined using western blotting (n=3). **P<0.01; ***P<0.001; ****P<0.0001. α-SMA, α-smooth muscle actin; Ang II, angiotensin II; ns, not significant; NC, negative control; PINK1, PTEN-induced kinase 1; POU2F1, POU domain class 2 transcription factor 1; sh, short hairpin.

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  Figure 6.. POU2F1 directly binds the PINK1 gene promoter to enhance PINK1 transcription. (A) Schematic of the binding site between POU2F1 and the PINK1 promoter region. (B) Chromatin immunoprecipitation-qPCR was performed to determine the binding of POU2F1 to the promoter sequence of PINK1 (n=3). IgG was applied as a negative control. (C) 293T cells were transfected with WT or MUT PINK1 plasmids and then transfected with vector or oePOU2F1 plasmid. A dual-luciferase reporter assay was performed (n=3). (D) Electrophoretic mobility shift assay was employed to analyze the interaction between POU2F1 and the PINK1 promoter fragment. (E) Following transfection with oePOU2F1 or empty vector, the mRNA levels of PINK1 in cardiac fibroblasts were quantified by reverse transcription-qPCR (n=3). (F) Protein levels of POU2F1 and PINK1 were detected by western blotting (n=3). Data are presented as the mean ± SD. (B, C, E and F) Unpaired two-tailed Student's t-test was used for statistical analysis. *P<0.05; ***P<0.001; ****P<0.0001. MUT, mutant; oe, overexpression; PINK1, PTEN-induced kinase 1; POU2F1, POU domain class 2 transcription factor 1; qPCR, quantitative PCR; WT, wild-type.

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  Figure 7.. POU2F1 knockdown reduces PINK1-mediated mitochondrial dysfunction and cardiac fibrosis in SHRs. Western blot analysis of (A) POU2F1, and (B) PINK1 and Parkin in SHRs infected with AAV9-shPOU2F1 or AAV9-shNC (n=4). (C) Transmission electron microscopy was used to observe the mitochondrial structure in cardiac tissue from SHRs infected with AAV9-shPOU2F1 or AAV9-shNC (scale bar, 500 nm). White arrows indicate cristae disorganization; red arrows indicate damaged mitochondria. (D) Seahorse assay profile of OCRs in CFs following treatment with oligomycin, FCCP and AA + ROT. Quantitative analysis of mitochondrial function parameters (basal respiration, maximal respiration and ATP production) is shown in the bar charts (n=3). (E) Interstitial myocardial fibrosis (blue) assessed via Masson's trichrome staining (scale bar, 100 µm; n=4). (F) Western blot analysis of collagen I and α-SMA protein expression in two groups (n=4). (G) Representative echocardiographic images of SHRs following different interventions. (H) Echocardiographic parameters, including IVSD, IVSS, LVPWD, LVPWS, LVIDD and LVIDS, were measured in the two groups (n=4). Data are presented as the mean ± SD. (A-F and H)…

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  Figure 8.. POU2F1 promotes hypertensive cardiac fibrosis by regulating mitochondrial homeostasis through PINK1/Parkin-dependent mitophagy. α-SMA, α-smooth muscle actin; Ang II, angiotensin II; CF, cardiac fibroblast; PINK1, PTEN-induced kinase 1; POU2F1, POU domain class 2 transcription factor 1.