Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

Figures

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  Figure 1. Characterization of HSPB1 expression in the AMI infarction model (A) Myocardial infarction was induced in mice by ligating the left anterior descending artery (LAD), and TTC staining was performed 28 days post-surgery to visualize the infarcted area. Red tissue represents non-infarcted myocardium, while pale tissue indicates infarcted myocardium (n = 9). (B) Quantification of the infarcted area was performed using ImageJ software. (C and D) Western blot analysis of HSPB1 and β-actin expression in cardiac tissue, with statistical data showing the grayscale ratio of HSPB1 to β-actin. (E) Immunohistochemical staining for HSPB1 expression in myocardial tissue (scale bars, 100 μm). (F) Quantification of HSPB1 expression area using ImageJ software. Data are presented as mean ± SD (n = 6). p values shown in the graphs were calculated using unpaired two-tailed Student’s t test.

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  Figure 2. Targeted silencing of HSPB1 in the heart alleviates myocardial fibrosis in MI mice (A) Tail vein injection of AAV9-HSPB1-RNAi or CON534 virus (with cTnT promoter) was performed at a concentration of 1.0×1012 virus particles/ml and a volume of 150 μL for 4 weeks, followed by Western blot analysis to validate HSPB1 silencing. (B) Grayscale ratio of HSPB1 to β-actin. (C–H) Following AAV9-HSPB1-RNAi or CON534 virus injection and MI model construction, cardiac function and fibrosis were assessed 4 weeks post-surgery: (C) cardiac function parameters by small animal echocardiography, (D) left ventricular fractional shortening (LVFS%), (E) left ventricular ejection fraction (LVEF%), (F) left ventricular mass (LV mass), (G) H\&E and Masson staining for myocardial injury and fibrosis, and (H) collagen fiber area percentage in myocardial tissue (scale bars, 200 μm). Data are presented as mean ± SD (n = 6). Exact p values are indicated in the graphs. Statistical analyses were performed using unpaired two-tailed Student’s t test.

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  Figure 3. HSPB1 gene silencing exacerbates EndoMT in the heart of MI Mice (A) Immunohistochemical staining shows α-SMA expression in myocardial tissue (heart cross-sections). (B) Quantification of the α-SMA-positive area using ImageJ software. (C) Western blot analysis of CD31, α-SMA, and β-actin protein expression in different groups. (D) Statistical analysis shows the ratio of CD31 to β-actin. (E) Statistical analysis shows the ratio of α-SMA to β-actin. Data are presented as mean ± SD (n = 6). p values shown in the graphs were calculated using unpaired two-tailed Student’s t test.

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  Figure 4. Regulatory role of HSPB1 in endothelial cell EndoMT (A) Western blot shows HSPB1 expression in HUVECs following lentiviral-mediated overexpression (LV-HSPB1) or knockdown (LV-HSPB1-RNAi); β-actin served as a loading control. (B) Quantification of HSPB1/β-actin ratio shows significant differences between groups. (C) Representative images of Transwell migration assays evaluating the effect of HSPB1 on TGF-β1–induced endothelial migration (scale bars, 100 μm). (D) Quantification of migrated cells per field. (E) Representative tube formation images showing the effect of HSPB1 modulation on TGF-β1–induced angiogenic activity (scale bars, 200 μm). (F–H) Quantitative analysis of tube formation parameters, including the number of branches (F), loops (G), and total tube length (H), measured using ImageJ software. Data are presented as mean ± SD (n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

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  Figure 5. Effects of HSPB1 on signaling pathways and TGF-β secretion in HUVECs under hypoxic conditions (A and B) HUVECs were transfected with adenoviral vectors for HSPB1 overexpression (OE) or knockdown (KD) and cultured for 48 h before RNA extraction. Gene expression analysis was performed using RNA sequencing. Gene set enrichment analysis (GSEA) assessed the regulatory roles of HSPB1 in processes such as heart development, angiogenesis, and cell proliferation (A). Further analysis using Hallmark gene sets explored HSPB1 signaling pathway activation (B). (C–G) Following transfection, HUVECs were cultured for 24 h and subjected to hypoxic conditions (3% O2) for 48 h. Western blot analysis of the indicated proteins was performed. (D) pSmad2/3/Smad2/3 ratio, (E) quantification of CD31 protein expression, (F) quantification of E-cadherin expression, (G) quantification of α-SMA expression, and (H) quantification of N-cadherin expression were measured relative to β-actin. (I) TGF-β levels were measured by ELISA in cell supernatants. Data are presented as mean ± SD (n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a…

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  Figure 6. HSPB1 regulation of Pro-TGF-β1 disulfide bond formation (A) HUVEC cells were transfected with adenoviral vectors for HSPB1 overexpression or silencing and cultured for 48 h, followed by an additional 48-h incubation under hypoxic conditions (3% O2). After enzymatic digestion, protein samples were analyzed for peptide-level disulfide bond formation using high-resolution mass spectrometry. (B and C) HUVEC cells were transfected with adenoviral vectors for HSPB1 overexpression or silencing, cultured for 24 h, and the HSPB1-silenced group was subsequently transfected with the HSPB1C137S mutant. After 48 h, cells were subjected to 48-h hypoxic induction (3% O2). The redox status of pro-TGF-β1 was analyzed by non-reducing SDS-PAGE (B), and the stability of pro-TGF-β1 disulfide bonds was assessed by electrochemical potential (Eh), calculated using the Nernst equation27(C). Data are presented as mean ± SD (n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

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  Figure 7. Proposed model of HSPB1-mediated redox regulation of TGF-β1 maturation during post-MI fibrosis. During myocardial fibrosis following myocardial infarction, the expression of HSPB1 is markedly upregulated in the peri-infarct region. Upon activation, HSPB1 exposes its reactive cysteine residue (Cys137), which may interact with critical cysteine sites within pre-pro-TGF-β1, thereby influencing its redox-dependent folding and disulfide bond formation. This interaction potentially interferes with the maturation and secretion of active TGF-β1 into the extracellular space. Reduced secretion of mature TGF-β1 limits Smad2/3 phosphorylation and endothelial-to-mesenchymal transition, ultimately alleviating myocardial fibrosis. The red dashed box highlights the hypothesized redox regulatory interaction between HSPB1 and pre-pro-TGF-β1, which requires further biochemical validation.