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

Summary

Myocardial fibrosis after myocardial infarction is promoted by endothelial-to-mesenchymal transition (EndoMT) driven by TGF-β1. We investigated whether cardiomyocyte heat shock protein B1 (HSPB1) shapes this pathway. In mouse infarction models, cardiomyocyte-targeted HSPB1 overexpression reduced collagen deposition and preserved ventricular function, whereas HSPB1 knockdown exacerbated fibrosis and EndoMT activation. In endothelial assays, HSPB1 attenuated TGF-β1–induced Smad2/3 phosphorylation and mesenchymal marker expression. Mechanistically, HSPB1 modulated redox conditions to restrain disulfide-bond formation during pro-TGF-β1 maturation, reducing the secretion of mature TGF-β1. These results link cardiomyocyte redox homeostasis with paracrine control of endothelial plasticity and support HSPB1 as a therapeutic entry point to limit post-infarction fibrotic remodeling.

Graphical abstract

Highlights

• •Cardiomyocyte HSPB1 acts as a redox-sensitive paracrine regulator after MI
• •HSPB1 limits pro-TGF-β1 disulfide bonds to reduce mature TGF-β1 secretion
• •Reduced TGF-β1/Smad2/3 signaling restrains endothelial EndoMT activation
• •HSPB1 attenuates post-MI fibrosis and preserves cardiac function in vivo

Cardiovascular medicine; Molecular biology; Molecular biology experimental approach

Introduction

Post-myocardial infarction fibrosis is a major pathological feature of cardiovascular diseases and is closely associated with the incidence and mortality of heart failure.¹ Fibrosis arises from an imbalance between collagen and extracellular matrix deposition and degradation during myocardial repair. Its excessive progression increases ventricular stiffness, disrupts electrical conduction, and impairs pump function, ultimately elevating the risk and mortality of heart failure.^2,3 At present, no specific antifibrotic drugs are available in clinical practice, highlighting the urgent need to identify new therapeutic targets for precise fibrosis regulation to improve long-term outcomes in patients with myocardial infarction.

Endothelial-to-mesenchymal transition (EndoMT), defined as the phenotypic conversion of endothelial cells into mesenchymal cells, represents an important source of fibroblast activation.^4,5 Endothelial cells are the most abundant stromal cell type in the heart,⁶ and their excessive EndoMT activation is a critical contributor to myocardial fibrosis.^7,8,9 In a mouse model of cardiac pressure overload, nearly 30% of fibroblasts originate from endothelial cells, and pathological EndoMT significantly aggravates fibrosis.¹⁰ Thus, inhibiting excessive EndoMT activation may represent a promising therapeutic approach for myocardial fibrosis.

The TGF-β1 signaling pathway is a central driver of EndoMT and is broadly involved in immune regulation, tissue repair, and cardiovascular homeostasis.¹¹ However, the direct inhibition of this pathway can cause adverse effects, including immune dysregulation, impaired tissue repair (e.g., delayed wound healing), and ventricular dilation, limiting its application in antifibrotic therapy.¹² The maturation and secretion of TGF-β1 are tightly controlled by the formation of disulfide bonds, which are essential for the protein’s maturation, stability, and functional activation.¹³ Specifically, the interchain disulfide bond (Cys77–Cys77′) and multiple intrachain disulfide bonds (Cys7–Cys16, Cys15–Cys78, Cys44–Cys109) play crucial roles in stabilizing the structure of TGF-β1.^14,15 Additionally, the disulfide bond between Cys33 and cysteine residues on LTBP or GARP anchors TGF-β1 to the extracellular matrix or cell surface, further regulating its bioactivity.^16,17 This suggests that targeting disulfide bond formation provides a more refined strategy for modulating TGF-β1 signaling, enabling the selective inhibition of pathological activation while preserving its physiological functions. Given that the redox environment governs disulfide bond formation, proteins that regulate oxidative balance may indirectly control TGF-β1 maturation and secretion.

Small heat shock protein beta-1 (HSPB1), a 205-amino-acid protein of approximately 27 kDa, is a member of the heat shock protein family.^18,19 Owing to its cytoprotective effects and regulatory role in fibrosis, HSPB1 has become a focus of recent research.^20,21 In cardiovascular pathology, HSPB1 functions as an essential regulator with broad biological activity, particularly during myocardial repair after infarction.^22,23 Notably, HSPB1 harbors a conserved cysteine residue (Cys137 in human; Cys141 in mouse) that enables it to regulate intracellular redox balance and disulfide-bond dynamics, thereby influencing protein folding and cytokine secretion.^22,24,25 Given this biochemical property, we postulate that HSPB1 may modulate the redox-dependent maturation of pro-TGF-β1 and, in turn, affect EndoMT and fibrotic remodeling.

Emerging evidence suggests that cardiomyocytes not only serve as contractile units but also act as key regulators of the cardiac microenvironment through paracrine signaling, influencing endothelial activation and fibrotic remodeling.^26,27 Given that HSPB1 participates in maintaining redox balance and cytokine secretion,^28,29,30 we hypothesized that cardiomyocyte-derived HSPB1 may indirectly regulate endothelial phenotype and myocardial remodeling by modulating TGF-β1 activation within the cardiac microenvironment.

Although previous studies have highlighted the cardioprotective and anti-apoptotic roles of HSPB1 after ischemic injury, its involvement in EndoMT and post-infarction fibrosis remains unclear. Addressing this gap, our study explores the redox-dependent regulation of TGF-β1 maturation by HSPB1 and its impact on endothelial-mesenchymal transition and myocardial fibrosis, thereby providing new mechanistic insight and a potential therapeutic target for antifibrotic intervention.

Results

Expression pattern of heat shock protein B1 in cardiac tissue

To clarify the distribution of HSPB1 in different tissues and cardiac cell populations, we performed a multi-level transcriptomic analysis using publicly available datasets (BioGPS, Human Protein Atlas, and Human Heart Atlas). Data were accessed through the corresponding public portals and visualized as provided by each resource. As shown in Figure S1, HSPB1 was highly expressed in the heart and skeletal muscle, indicating a muscle-enriched pattern (Figure S1A). Within cardiac tissue, HSPB1 was broadly distributed among cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts, with limited expression in immune cells (Figure S1B). Single-cell RNA-seq analysis further confirmed that HSPB1 expression was predominantly localized to ventricular cardiomyocytes, while immune and stromal cells exhibited minimal expression (Figure S1C). These findings indicate that HSPB1 is not endothelial-specific and is predominantly expressed in cardiomyocytes, supporting our focus on cardiomyocytes in subsequent experiments. Notably, this basal expression pattern under physiological conditions suggests that HSPB1 participates in maintaining myocardial homeostasis even in the absence of pathological stress.

Given that HSPB1 is abundantly expressed in cardiomyocytes and other cardiac structural cells, we next sought to determine whether its expression is altered following myocardial infarction and to elucidate its potential role in post-infarction remodeling.

Upregulation of heat shock protein B1 expression in the peri-infarct area of AMI mice

Eight-week-old male C57BL/6 mice underwent left anterior descending (LAD) coronary artery ligation to establish the AMI model. Cardiac function was assessed by echocardiography on postoperative day 28. After euthanasia via cervical dislocation, heart tissue was collected for TTC staining and immunohistochemical analysis. TTC staining results (Figures 1A and 1B) showed that the infarct area in the AMI group (44.63 ± 8.45%) was significantly larger than in the Sham group (5.19 ± 2.05%), with a statistically significant difference. Western blot analysis (Figures 1C and 1D) revealed a significant upregulation of HSPB1 expression in the AMI group compared to the Sham group. Furthermore, immunohistochemical staining demonstrated that HSPB1 expression was markedly increased in the peri-infarct zone of AMI mice, compared to the control group (Figures 1E and 1F). These findings suggest that HSPB1 may play a crucial role in the myocardial remodeling process following myocardial infarction (MI).

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.

The effect of heat shock protein B1 on myocardial fibrosis following myocardial infarction

To explore the role of HSPB1 in myocardial fibrosis after MI, we constructed an MI model in C57BL/6J mice by injecting adeno-associated virus (AAV9-HSPB1-RNAi) to silence HSPB1 (experimental group) or a control virus (CON534) (control group) via tail vein injection for 4 weeks. Cardiac function and myocardial fibrosis were assessed 28 days post-surgery. As shown in Figures 2A and 2B, AAV9-HSPB1-RNAi effectively reduced HSPB1 expression in myocardial tissue. Echocardiographic results (Figures 2C–2F) demonstrated a significant decrease in left ventricular fractional shortening (LVFS%) (26.01 ± 1.549 vs. 47.66 ± 1.845), left ventricular ejection fraction (LVEF%) (44.15 ± 2.247 vs. 60.55 ± 1.728), and left ventricular mass (LV mass) (131.1 ± 2.510 vs. 113.2 ± 4.075) in the experimental group compared to the control group. Histological examination revealed that, in the experimental group, myocardial cell alignment was more disorganized, with enlarged cells, significantly increased intercellular spaces, and prominent myocardial fibrosis lesions. Masson’s trichrome staining indicated a substantial increase in collagen deposition around myocardial microvessels and within the myocardial interstitium, with the fibrotic area accounting for 10.01 ± 0.76% (Figures 2G and 2H). These results suggest that HSPB1 plays an anti-fibrotic role in myocardial fibrosis following MI.

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.

The regulatory role of heat shock protein B1 in endothelial-to-mesenchymal transition

Excessive activation of EndoMT is a key feature of myocardial fibrosis.²⁴ Given the observed anti-fibrotic effect of HSPB1, we next explored whether HSPB1 influences EndoMT activation in vivo. Immunohistochemical analysis showed that the AMI+AAV9-HSPB1-RNAi group exhibited a significantly larger α-SMA-positive area in the cardiac interstitial and perivascular fibrotic regions, particularly around blood vessels, indicating a marked increase in myofibroblast expression (Figures 3A and 3B). Western blot analysis revealed a significant increase in α-SMA expression and a decrease in the endothelial marker CD31 in the AMI+AAV9-HSPB1-RNAi group (Figures 3C–3E). These data suggest that HSPB1 silencing aggravates EndoMT and fibrosis in the infarcted myocardium.

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.

To further verify the mechanistic role of HSPB1 in regulating endothelial responses to TGF-β1, stable HUVEC models with HSPB1 knockdown and overexpression were established (Figures 4A and 4B). As shown in Figures 4C and 4D, HSPB1 silencing enhanced the inhibitory effect of TGF-β1 on endothelial migration, whereas HSPB1 overexpression partially reversed this effect. Tube formation assays further revealed that HSPB1 knockdown markedly impaired TGF-β1–induced angiogenic capacity, while HSPB1 overexpression restored it (Figures 4E–4H). These findings indicate that HSPB1 modulates endothelial sensitivity to TGF-β1 signaling through its redox-regulatory activity, thereby supporting the concept that cardiomyocyte-derived HSPB1 may indirectly stabilize endothelial phenotype and restrain EndoMT within the infarct microenvironment.

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.

Heat shock protein B1 modulates endothelial-to-mesenchymal transition by regulating TGF-β1 maturation and secretion

Given that EndoMT is largely driven by TGF-β signaling, we next examined whether HSPB1 modulates this pathway at the level of ligand maturation and secretion. To this end, high-throughput RNA sequencing was performed in cardiomyocytes following HSPB1 overexpression or knockdown to analyze differential gene expression profiles. Gene Set Enrichment Analysis (GSEA) revealed that HSPB1 overexpression significantly suppressed TGF-β signaling activity, whereas HSPB1 knockdown enhanced this pathway (Figures 5A and 5B).

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 Bonferroni post hoc test.

As shown in Figures 5C–5H, under hypoxic conditions, HSPB1 overexpression decreased the phosphorylation of Smad2/3 (p-Smad2/3) and downregulated mesenchymal markers (N-Cadherin and Vimentin) while maintaining endothelial markers (CD31 and VE-Cadherin). In contrast, HSPB1 knockdown led to elevated -Smad2/3 phosphorylation and a shift toward a mesenchymal phenotype, consistent with enhanced EndoMT activation. Furthermore, ELISA assays demonstrated that HSPB1 silencing markedly increased TGF-β1 secretion, whereas HSPB1 overexpression reduced its release (Figure 5I). These findings indicate that HSPB1 suppresses EndoMT by limiting TGF-β1 signaling activation.

The activation of latent TGF-β1 depends on the formation of pro-TGF-β1 from pre-pro-TGF-β1 through the establishment of disulfide bonds, a critical step for its conversion into the active form.²⁷ Because HSPB1 is known to regulate disulfide bond stability via its unique cysteine residue (Cys137), we next examined whether HSPB1 affects pro-TGF-β1 dimer formation and redox state. High-resolution mass spectrometry under non-reducing conditions identified characteristic disulfide-linked peptides of pro-TGF-β1, with distinct peaks at approximately m/z 200 and m/z 600. Compared to controls, HSPB1 knockdown increased the intensity of the 600 m/z peak, indicating enhanced disulfide bond formation, whereas HSPB1 overexpression reduced these peaks (Figure 6A).

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.

Consistently, non-reducing SDS-PAGE analysis showed that the HSPB1-C137S mutant promoted stronger disulfide bond formation in pro-TGF-β1, while overexpression of wild-type HSPB1 inhibited this disulfide bond formation, leading to the accumulation of unlinked SH2 forms (Figures 6B and 6C). Together, these results demonstrate that HSPB1 restrains TGF-β1 activation by destabilizing pro-TGF-β1 disulfide bonds, thereby attenuating downstream EndoMT signaling and myocardial fibrosis.

Discussion

This study demonstrates that HSPB1 plays a pivotal regulatory role in EndoMT and post-myocardial infarction fibrosis. Under physiological conditions, HSPB1 is constitutively expressed in cardiomyocytes, where it maintains protein homeostasis and redox balance,^21,22,28 but its regulatory function becomes markedly enhanced following cardiac injury. Using both gain- and loss-of-function approaches, we confirmed that HSPB1 suppresses excessive EndoMT activation and attenuates myocardial fibrosis progression. Importantly, our findings reveal that cardiomyocyte-derived HSPB1 exerts indirect paracrine control over endothelial phenotype by regulating TGF-β1 maturation and secretion. This discovery identifies a novel cardiomyocyte-endothelial crosstalk mechanism, positioning HSPB1 as a key molecular link between redox regulation, cytokine homeostasis, and fibrotic remodeling.

EndoMT is widely recognized as an important source of fibroblasts during cardiac remodeling and fibrosis.^5,31 Our results are consistent with previous studies showing that excessive EndoMT aggravates pathological fibrosis, particularly during the early stages after MI.^32,33,34 While endothelial dysfunction has long been considered a major driver of fibrosis,^35,36,37 our findings extend this concept by demonstrating that cardiomyocytes actively influence endothelial behavior through redox-dependent paracrine signaling. Specifically, loss of HSPB1 in cardiomyocytes disrupts oxidative balance and enhances the secretion of active TGF-β1, thereby promoting EndoMT and fibroblast expansion in the peri-infarct myocardium. These results identify HSPB1 as a central homeostatic regulator that maintains intercellular communication fidelity between cardiomyocytes and endothelial cells under stress conditions. The observed upregulation of HSPB1 in peri-infarct regions likely reflects a stress-adaptive response to oxidative and mechanical injury, potentially mediated through the p38 MAPK-HSF1 signaling pathway, as suggested by previous studies.^38,39 Such stress-induced induction of HSPB1 enables cardiomyocytes to restore redox homeostasis and stabilize protein folding, thereby attenuating excessive TGF-β1 activation and subsequent EndoMT.

Mechanistically, the TGF-β1/Smad signaling pathway is a core driving factor in EndoMT.^11,40 TGF-β1 is initially produced in the form of pre-pro-TGF-β1, which undergoes a series of disulfide bond formations to convert into pro-TGF-β1, and is then processed through conformational regulation and enzymatic cleavage to mature into biologically active TGF-β1.^41,42 Recent structural analyses indicate that dynamic conformational regulation is essential for the precise activation of TGF-β1 signaling, providing theoretical support for exploring the relationship between dynamic allostery and specific residues, such as cysteine residues, in signal regulation.⁴³ Our study provides the first evidence that HSPB1 regulates the redox stability of disulfide bonds in pro-TGF-β1, potentially through its reactive cysteine residue (Cys137), although the precise mechanism of this regulation requires further investigation (see proposed model in Figure 7).

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.

These findings carry important clinical implications. Current antifibrotic strategies targeting TGF-β1 signaling are limited by systemic toxicity and impaired physiological repair.^12,44 In contrast, HSPB1 serves as an endogenous checkpoint molecule that selectively inhibits pathological TGF-β1 activation, while preserving its physiological functions in tissue repair and immune regulation. Thus, the therapeutic modulation of HSPB1 expression or activity may enable precise control of fibrotic remodeling without compromising essential healing processes. With its dual cytoprotective and antifibrotic properties, HSPB1 represents a promising molecular target for next-generation antifibrotic therapy in cardiovascular disease.

In conclusion, this study identifies HSPB1 as a redox-sensitive checkpoint that bridges cardiomyocyte homeostasis and endothelial transition. Through redox-dependent control of TGF-β1 maturation, HSPB1 restrains pathological EndoMT and fibrotic remodeling. These findings reveal a previously unrecognized redox-paracrine regulatory axis between cardiomyocytes and endothelial cells and highlight HSPB1 as a potential therapeutic entry point for antifibrotic intervention in cardiovascular disease.

Limitations of the study

However, this study has certain limitations. Although our data support a link between HSPB1 expression and TGF-β1 regulation, it remains to be determined whether HSPB1 directly affects the formation of the Cys77–Cys77′ interchain disulfide bond or instead alters the stability of other disulfide bonds during pro-TGF-β1 maturation, thereby limiting the secretion of mature TGF-β1. In addition, our conclusions are based on a mouse myocardial infarction model and cultured endothelial cells; validation in human cardiac tissue and more human-relevant systems will be important to establish clinical relevance. Finally, future studies using endothelial-specific genetic manipulation and complementary structural or biochemical approaches will be important to distinguish cardiomyocyte-derived paracrine effects from endothelial cell-autonomous regulation and to clarify the molecular basis of HSPB1-mediated control of TGF-β1 maturation.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xiehong Liu (dolly9918@hunnu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •The proteomics data generated in this study have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD072325 and are publicly available at: http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD072325.
• •The high-throughput sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1402128 and are publicly available at: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1402128.
• •Any additional data supporting the findings of this study are available from the corresponding author upon reasonable request. This study did not generate custom code.

Acknowledgments

Generative AI tools were used to assist with language editing and to improve the readability of this article. The authors assume full responsibility for the integrity, accuracy, and originality of the work. This study was funded by the Key Project of the Hunan Provincial Department of Education (25A0103), the Natural Science Foundation of Hunan Province (2022JJ30340 and 2023JJ30352), the Hunan Provincial Department of Education Scientific Research Project (23C0451), the Research Project of the Health Commission of Hunan Province (202203013494), the Changsha Natural Science Foundation (kq2208116), and the ESI Discipline Construction Special Project of Changsha Medical University (2022CYY040).

Author contributions

J. W. and A. F.: writing – original draft, software, methodology, data curation, and conceptualization. P. D.: methodology and investigation. G. T.: methodology, formal analysis, and data curation. H. Y.: visualization, validation, and investigation. J. Z.: validation, resources, and data curation. Q. Z.: writing – review and editing and supervision. J. P.: writing – review and editing, supervision, resources, project administration, and data curation. X. L.: writing – review and editing, supervision, project administration, funding acquisition, data curation, and conceptualization.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
HSPB1	Santa Cruz Biotechnology	sc-13132
CD31	Zen-BioScience	347526
α-SMA	Zen-BioScience	M50132
phospho-Smad2/3	Zen-BioScience	251795
Smad2/3	Zen-BioScience	382472
E-cadherin	Zen-BioScience	340341
N-cadherin	Zen-BioScience	680441
TGF-β (detects precursor proteins)	Cell Signaling Technology	Cat# 3711; RRID: AB_2147209
β-actin	Sigma-Aldrich	Cat# A5316; RRID: AB_476744
Experimental models: cell lines
Human umbilical vein endothelial cells (HUVECs)	ATCC	Primary cells, pooled donors
Experimental models: organisms/strains
C57BL/6 mice (male)	Slyke Jingda Experimental Animal Co., Ltd.	6–8 weeks old
Recombinant DNA
pAAV9-cTnT-HSPB1-RNAi	GeneChem (Shanghai, China)	N/A
Lentiviral vectors for HSPB1 modulation	GeneChem (Shanghai, China)	N/A
Deposited data
Proteomics data	ProteomeXchange Consortium via iProX partner repository	PXD072325
High-throughput sequencing data	NCBI Sequence Read Archive (SRA)	BioProject: PRJNA1402128
Software and algorithms
GraphPad Prism	GraphPad Software	N/A
ImageJ	NIH	RRID: SCR_003070
Vevo Lab	FUJIFILM VisualSonics	N/A

Experimental model and study participant details

Animals

Male C57BL/6 mice (6–8 weeks old, 18–22 g) were purchased from Slyke Jingda Experimental Animal Co., Ltd. (Changsha, Hunan, China). Animals were housed in standard cages under controlled environmental conditions (temperature: 20°C–26 °C; relative humidity: 30–70%; 12 h light/dark cycle), with free access to water and certified pellet diet. All animal experiments were performed in compliance with national regulations on the use of laboratory animals. The study protocol was reviewed and approved by the Ethics Committee of Hunan Normal University and the Animal Ethics Committee of Hunan Provincial People’s Hospital (The First Affiliated Hospital of Hunan Normal University) (Approval No. 158, 2025).

Cell lines

Human umbilical vein endothelial cells (HUVECs; primary, pooled donors; ATCC) were purchased through an authorized distributor in China and originally sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell line authentication was performed by the supplier, and cells were routinely tested and confirmed to be free of mycoplasma contamination prior to use in experiments. Information regarding donor sex was not provided by ATCC and therefore could not be assessed in this study.

Method details

Myocardial infarction model

Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending (LAD) coronary artery.⁴⁵ Mice were anesthetized with isoflurane (3–5% for induction, 1.5–2.5% for maintenance) and placed on a temperature-controlled surgical table. After a left thoracotomy, the LAD was ligated 1–2 mm below its origin with a 6-0 polypropylene suture to produce a moderate mid-apical infarction. Successful ligation was confirmed by blanching of the left ventricular apex and loss of regional wall motion under direct visualization. Sham-operated mice underwent the same procedure without LAD ligation. All animals received cefotaxime (25 mg/kg, intraperitoneally) postoperatively and were monitored until the end of the study. Cardiac tissue was collected 28 days after surgery for histological, functional, and molecular analyses.

Viral vector injection

For cardiac-specific silencing of HSPB1, pAAV9-cTnT-HSPB1-RNAi vectors were administered via tail vein injection at a dose of 5 × 10¹⁰–1 × 10¹¹ viral genomes per mouse (1 × 10¹² vg/mL). The AAV9-cTnT system predominantly targets cardiomyocytes, enabling selective assessment of cardiomyocyte-derived effects. Knockdown efficiency was verified in myocardial tissue by RT-qPCR and Western blot analyses.

Cell culture and viral transduction

HUVECs were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37°C in a humidified incubator with 5% CO₂. For stable HSPB1 overexpression or knockdown, lentiviral vectors carrying HSPB1 cDNA (LV-HSPB1) or HSPB1-targeting RNAi (LV-HSPB1-RNAi) were used, and all lentiviral vectors were obtained from GeneChem (Shanghai, China). Transduced cells were selected with G418 or puromycin to establish stable cell lines for subsequent experiments.

TTC staining

Infarct size was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Excised hearts were rinsed in cold phosphate-buffered saline (PBS), sliced into 1–2 mm transverse sections, and incubated in 1% TTC solution at 37 °C for 30 min. Viable myocardium stained red, whereas infarcted regions remained pale. Sections were fixed in 4% paraformaldehyde, imaged, and infarct size was quantified using ImageJ software as the percentage of infarcted area relative to total left ventricular area. To minimize baseline pallor caused by transient ischemia during thoracotomy, all procedures were performed under identical ischemic exposure and temperature conditions.

HSPB1 immunohistochemistry

Heart tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 4 μm. Sections were incubated with anti-HSPB1 antibody (1:500, Santa Cruz) at room temperature, followed by HRP-conjugated secondary antibody. Detection was performed using DAB chromogen, and images were captured by light microscopy. HSPB1-positive areas were quantified using ImageJ software.

Histology staining

For hematoxylin and eosin (H&E) staining, hearts were fixed in 10% formalin for 48 h, embedded in paraffin, and sectioned at 4 μm. Sections were stained using standard protocols and evaluated by light microscopy.

For fibrosis assessment, Masson’s trichrome staining was performed on paraffin-embedded sections (4 μm). Collagen fibers appeared blue and muscle fibers appeared red. Fibrotic area was quantified using ImageJ software and expressed as the percentage of total myocardial area.

Western blot

Tissue and cell lysates were prepared in RIPA buffer, and protein concentrations were determined using a BCA assay. Equal amounts of protein (20–40 μg) were separated by SDS–PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies against HSPB1 (1:500, Santa Cruz) and β-actin (1:1000, Sigma). After incubation with HRP-conjugated secondary antibodies (1:2000), signals were detected using enhanced chemiluminescence reagents and quantified with ImageJ software.

Echocardiography

Cardiac function was assessed using a Vevo 2100 ultrasound system. Mice were anesthetized with isoflurane and placed on a heated platform. M-mode images were acquired in the short-axis view of the left ventricle. Left ventricular ejection fraction (LVEF), fractional shortening (LVFS), and left ventricular mass were calculated using Vevo Lab software. Measurements were independently verified by two blinded operators.

Tube formation assay

Matrigel (10 μL per well) was added to 96-well plates and polymerized at 37 °C for 30 min. HUVECs (2 × 10⁵ cells/mL, 50 μL per well) were seeded onto Matrigel in serum-free medium. After 6 h, cells were stained with Calcein AM (6.25 μg/mL) for 30 min in the dark. Images were captured by fluorescence microscopy, and tube formation was quantified using ImageJ software.

Transwell migration assay

HUVECs (5 × 10⁴ cells per well) in serum-free medium were seeded into the upper chambers of Transwell inserts. The lower chambers contained medium with chemoattractants. After 24 h, non-migrated cells were removed, and migrated cells on the lower surface were fixed with methanol, stained with crystal violet, and counted in three random microscopic fields per insert.

Mass spectrometry

HUVECs were transfected with adenoviral vectors for HSPB1 overexpression, knockdown, or control. After 48 h, cells were exposed to hypoxia (3% O₂) for an additional 48 h. Proteins were extracted under non-reducing conditions, and free cysteines were alkylated with iodoacetamide (IAA). Proteins were digested with trypsin overnight at 37 °C. Peptides were desalted and analyzed using a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nLC 1200 system. Spectra were acquired in positive ion mode (m/z 100–1000). Intensities of disulfide bond–containing peptides of pro–TGF-β1 were normalized across experimental groups.

Quantification and statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism (version 9.0). For comparisons between two groups, unpaired two-tailed Student’s t-tests were used. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests was applied. A p value <0.05 was considered statistically significant.

For animal experiments, n represents the number of individual mice analyzed per group. For cell-based assays, n represents the number of independent biological replicates performed on different days using separately prepared cell cultures. The exact value of n and the corresponding statistical tests used for each experiment are specified in the figure legends.

Additional resources

No additional resources were generated in this study.

Published: February 13, 2026