A total of 23 patients with HF aged 50–80 years who were hospitalized at the Cardiovascular Center of The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School (Suzhou, China) between March 2022 and May 2022 were enrolled in the present study. The inclusion criteria were based on the Chinese Guidelines for the Diagnosis and Treatment of Heart Failure 2018 (19). Briefly, the inclusion criteria were as follows: i) Dyspnea, fatigue and fluid retention; ii) left ventricular ejection fraction <40%; and iii) elevated natriuretic peptide with a left ventricular ejection fraction of ≥40% and at least one of the following: i) Left ventricular hypertrophy and/or left atrial enlargement; and ii) abnormal cardiac diastolic function. Patients with acute infection, serious primary disease, trauma, pregnancy, cancer, drug use or surgery in the month before blood collection were excluded. During the same period, 21 volunteers who underwent routine physical examination but did not have heart disease, and these subjects were similar to patients in terms of age, sex ratio and body mass index at the Suzhou Physical Examination Center (The Affiliated Suzhou Hospital of Nanjing Medical University) were recruited as the control group. The present study was approved by the Ethics Committees of Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School (Suzhou, China; approval no. K-2021-GSKY20210202). Blood samples (2 ml) were collected from patients and controls using procoagulant blood collection vessels, and the sera were collected after centrifugation at 3,000 × g for 5 min at 4°C. The serum levels of Cdc42 and E2 were measured using human Cdc42 ELISA kit (cat. no. YX-76511. Sinobestbio, Co., Ltd.) and the human 17β-E2 ELISA kit (cat. no. YK90121. Sinobestbio, Co., Ltd.) according to the manufacturer's instructions, and the results were obtained using a Varioskan Lux multifunctional microplate reader (Thermo Fisher Scientific, Inc.) by measuring the absorbance at a wavelength of 425 nm.

Immortalized MCFs (subsequently referred to as MCFs in the present study) purchased from Shanghai Yilei Information Technology Co., Ltd. were cultured at 37°C with 5% CO₂ in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 100 U/ml streptomycin, 100 U/ml penicillin and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). After the MCFs were cultured for 24 h and had grown to 50–70% confluence, they were treated with 20 ng/ml TGF-β1 (PeproTech, Inc.) for 0, 12, 24, 48 or 72 h at 37°C. Finally, the cultured cells were collected, and the protein expression levels of α-SMA, collagen I, collagen III and Cdc42 were examined via western blotting.

MCFs were divided into the negative control (NC)-OE, Cdc42-OE, negative control for siRNA (siNC), TGF-β1 + siNC, siRNA of Cdc42 (siCdc42)-1 and siCdc42-2 groups. For the NC-OE and Cdc42-OE groups, the pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin vector (Shanghai Genechem Co., Ltd.) was used for the OE experiment, and for the NC-OE group, an empty vector with no sequence inserted at the multiple cloning site (Shanghai Genechem Co., Ltd.) was used for lentiviral transfection. Cell lines stably transfected with lentivirus were generated according to the manufacturer's instructions. Briefly, the lentivirus (1×10⁸ TU/ml) was added to cells that had grown to 20–40% confluence in 6-well plates. After 16 h of infection at 37°C, the medium was replaced with fresh complete medium, and stable cell lines were selected with 10 µg/ml puromycin at 72 h after infection and maintained with 2 µg/ml puromycin. The generated cell lines stably transfected with lentivirus were frozen for later use or cultured with fresh complete medium for 24 h for subsequent experiments. For the siNC, TGF-β1 + siNC, siCdc42-1 and siCdc42-2 groups, MCFs were cultured in 6-well plates for 24 h grown to 50–70% confluence and transfected with 7.5 pmol siRNA according to the manufacturer's instructions (Shanghai GenePharma, Co., Ltd..). Briefly, serum-free medium was divided into two parts, and siRNA was added to one half, while Lipofectamine^® 3000 reagent (Thermo Fisher Scientific, Inc.) was added to the other half. After 5 min, the two halves were mixed. The mixture was allowed to rest for 20 min before being added to cells at 50 to 70%. After 24 h of transfection at 37°C, the medium was replaced with fresh medium, and 20 ng/ml TGF-β1 was added to the TGF-β1 + siNC group, which was subsequently incubated for another 24 h at 37°C. The following siRNA sequences were used: siRNA-1 sense, 5′-GCUAAGUUAUCCACAGACATT-3′ and antisense, 5′-UGUCUGUGGAUAACUUAGCTT-3′; siRNA-2 sense, 5′-GCUUGUUGGGACCCAAAUUTT-3′ and antisense, 5′-AAUUUGGGUCCCAACAAGCTT-3′; and non-targeted siRNA control sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. Tgfb1 mRNA expression levels were detected by quantitative PCR (qPCR), and the protein expression levels of α-SMA, collagen I and III, Pak1, JNK, c-Jun and TGF-β1 were detected by western blotting. Immunofluorescence staining was used to observe α-SMA expression. The activity of the Tgfb1 promoter (−1,799/+55) (20) was evaluated using a dual-luciferase reporter assay.

After MCFs were cultured for 24 h, they were divided into untreated control, DMSO and MLS-573151-treated groups. The MCFs were treated with vehicle (0.04% DMSO) or 10 µM MLS-573151 for 24 h at 37°C, and the protein expression of total and phosphorylated (p-)Cdc42, Pak1, JNK, c-Jun and TGF-β1 was detected by western blotting.

After MCFs were cultured for 24 h, they were divided into the TGF-β1 + control, TGF-β1 + NC and TGF-β1 + E2-treated (5, 10 and 50 nM) groups. The MCFs were pretreated with 20 ng/ml TGF-β1 for 6 h at 37°C, after which vehicle (0.04% DMSO) or E2 (5 or 10 or 50 nM, MedChemExpress Co., Ltd.) was added for another 48 h at 37°C, and the protein expression levels of α-SMA, collagen I, collagen III and Cdc42 were determined via western blotting. Subsequently, the cells were divided into the control, TGF-β1, TGF-β1 + E2 and TGF-β1 + E2 + siCdc42-1 groups. The MCFs were pretreated with 20 ng/ml TGF-β1 for 6 h at 37°C, and then 10 nM E2 was added for another 48 h at 37°C. MCFs in the TGF-β1 + E2 + siCdc42 group were transfected with siCdc42-1 (Shanghai GenePharma Co., Ltd.) according to the manufacturer's instructions as aforementioned with addition of E2, and MCFs in the remaining groups were transfected with siNC, as aforementioned. The protein expression levels of α-SMA, collagen I, collagen III, Pak1, JNK and c-Jun were detected by western blotting. Immunofluorescence staining was used to observe α-SMA expression.

After the two stable MCF lines, NC-OE and Cdc42-OE, were cultured for 24 h, the cells were divided into the control, TGF-β1, TGF-β1 + Cdc42-OE and TGF-β1 + E2 groups. The cells in the TGF-β1 + Cdc42-OE group were the Cdc42-OE cells, and the cells in the other groups were the NC-OE cells. The MCFs were pretreated with 20 ng/ml TGF-β1 for 6 h at 37°C, and 10 nM E2 was added for another 48 h at 37°C. The protein expression levels of α-SMA, collagen I, collagen III, Cdc42, Pak1, JNK and c-Jun were detected by western blotting. Immunofluorescence staining was used to observe α-SMA expression.

Cell slides were added to 24-well plates before cell culture. After the MCFs had grown to 20–30% confluence, the medium containing the treated cells in the plate was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton X-100 at room temperature for 10 min and blocked with 1% BSA (Beyotime Institute of Biotechnology) at room temperature for 45 min. An α-SMA antibody (1:200; cat. no. CY5295. Shanghai Abways Biotechnology Co., Ltd.) was added to the glass slide and the cells were incubated at 4°C overnight in the dark. The CoraLite488-conjugated goat anti-rabbit IgG(H+L) (1:500; cat. no. SA00013-2. Proteintech Group, Inc.) was added to each well for incubation at room temperature for 60 min. The secondary antibodies were discarded, DAPI was added at room temperature for 20 min and the cells were washed. The glass slide in each well was removed and sealed with an anti-fluorescence quenching sealing solution. A confocal microscope (Carl Zeiss AG) was used to capture images of the cells. ImageJ software v1.8.0 (National Institutes of Health) was used to measure and analyze the mean fluorescence intensity.

MCFs were seeded into a 12-well plate and cultured for 24 h to 50–70% confluence. Subsequently, the cells were divided into the siNC + pGL3-basic + thymidine kinase (TK)-Renilla, siCdc42 + pGL3-basic + TK-Renilla, siNC + pGL3-TGF-β1 promoter + TK-Renilla and siCdc42-1 + pGL3-TGF-β1 promoter + TK-Renilla (Guangzhou RiboBio Co., Ltd.) groups. Briefly, 7.5 pmol siNC (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. Shanghai GenePharma Co., Ltd.) or siCdc42-1 (sense, 5′-GCUAAGUUAUCCACAGACATT-3′ and antisense, 5′-UGUCUGUGGAUAACUUAGCTT-3′. Shanghai GenePharma Co., Ltd.), 0.5 µg reporter plasmid pGL3-Basic (Guangzhou RiboBio Co., Ltd.) or the plasmid pGL3-TGF-β1 (−1,799/+55) (Guangzhou RiboBio Co., Ltd.) (20) and 0.05 µg control Renilla luciferase plasmid TK-Renilla (Guangzhou RiboBio Co., Ltd.) were simultaneously transfected using Lipofectamine^® 3000 reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cells were harvested 24 h after transfection. Luciferase activity was determined using a dual-luciferase reporter assay system (cat. no. E1910; Promega Corporation). Luciferase activity was normalized to the Renilla luciferase activity.

After total RNA was extracted from the cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.), the RNA concentration and purity were measured using a NanoDrop Life spectrophotometer (Thermo Fisher Scientific, Inc.). RNA (1,000 ng) was reverse transcribed into cDNA using PrimeScript™ RT Master Mix (Takara Bio, Inc.) at 37°C for 15 min and 85°C for 5 sec. qPCR was performed with TB Green^® Premix Ex Taq™ II (Takara Bio, Inc.) according to the manufacturer's instructions. The PCR program was as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The program was run using the LightCycler^® 480 II real-time PCR detection system (Roche Diagnostics) to detect Tgfb1 mRNA levels. The following primer sequences (Genewiz, Inc.) were used: TGF-β1 forward, 5′-CAACTTCTGTCTGGGACCCT-3′ and reverse, 5′-TAGTAGACGATGGGCAGTGG-3′; and GAPDH forward, 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse, 5′-TGTAGACCATGTAGTTGAGGTCA-3′. All measurements were performed in triplicate. Relative expression data were analyzed using the 2^−ΔΔCq method (21) and were normalized to the expression levels of GAPDH.

After total protein was extracted from MCFs using RIPA buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitors and quantified by BCA assay, protein samples (30 µg/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to polyvinylidene difluoride membranes, which were blocked with 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies at 4°C overnight. Primary antibodies against α-SMA (1:1,000; cat. no. CY5295. Shanghai Abways Biotechnology Co., Ltd.), collagen I (1:1,000; cat. no. A1352. ABclonal Biotech Co., Ltd.), Cdc42 (1:1,000; cat. no. 10155-1-AP. Proteintech Group, Inc.), p-Cdc42 (1:1,000; cat.no. YP1620. ImmunoWay Biotechnology Company), TGF-β1 (1:2,000; cat. no. 21898-1-AP. Proteintech Group, Inc.), Pak1 (1:3,000; cat. no. 21401-1-AP. Proteintech Group, Inc.), JNK (1:1,000; cat. no. CY5490. Shanghai Abways Biotechnology Co., Ltd.), c-Jun (1:1,000; cat. no. CY5290; Shanghai Abways Biotechnology Co., Ltd.) and mouse collagen III (1:1,000; cat. no. YM3123. ImmunoWay Biotechnology Company), GAPDH (1:5,000; cat. no. 60004-1-Ig. Proteintech Group, Inc.) were used. The next day, the membranes were washed three times with TBS-0.1% Tween-20 wash buffer for 10 min each. The sections were incubated with goat anti-mouse IgG (H+L) HRP (1:20,000; cat. no. 70-GAM007. MultiSciences (Lianke)Biotech Co., Ltd) or goat anti-rabbit IgG (H+L) HRP (1:20,000; cat. no. 70-GAR007. MultiSciences (Lianke)Biotech Co., Ltd) secondary antibodies for 1 h at room temperature. After the membranes were washed with TBS-0.1% Tween-20 wash buffer, protein expression levels were detected with an automatic Tanon 5200 Multi chemiluminescence image analyzer using Tanon™ High-sig ECL Western Blotting Substrate (Tanon Science and Technology Co., Ltd.). The gray areas were measured and analyzed using ImageJ software v1.8.0 (National Institutes of Health).

SPSS 21.0 statistical software (IBM Corp.) was used for statistical analysis, GraphPad Prism 8 (Dotmatics) was used for graph preparation. All data are presented as the mean ± SD of ≥2 repeats. Shapiro-Wilk test was used to evaluate the normality of the data. A standard unpaired Student's t-test was used to compare two groups of normally distributed quantitative data, Mann-Whitney U test was used to compare two groups of non-normally distributed quantitative data, and the Pearson χ² test, Fisher's exact test or Yates' correction was used to compare two groups of non-quantitative data. Comparisons of multiple groups of normally distributed quantitative data were performed using one-way ANOVA followed by Bonferroni post hoc correction. Spearman's analysis was used to assess linear correlations between groups. P<0.05 was considered to indicate a statistically significant difference.

Serum was isolated from 23 patients with HF and 21 controls for analysis. The baseline characteristics of the patients and controls are shown in Table I. There were no significant differences in terms of the baseline characteristics between the patients with HF and the controls.

The levels of Cdc42 and E2 in the serum of patients with HF and the controls were detected to examine the association between Cdc42 and estrogen in HF. The serum expression levels of Cdc42 and E2 were higher in patients with HF than in the control group (Table I; Fig. 1A and B), and the serum expression levels of Cdc42 and E2 were positively correlated in the patients with HF, with a correlation coefficient of r=0.4456 (Table I; Fig. 1C). These findings revealed links between HF and elevated Cdc42 protein levels, and between E2 and Cdc42 protein levels.

The results in Fig. 2A-C demonstrate that the protein expression levels of α-SMA, collagen I and collagen III were increased after MCFs were stimulated with 20 ng/ml TGF-β1 for 24 h; however, the protein expression levels of Cdc42 were decreased. These data suggested that TGF-β1 could stimulate MCF proliferation and differentiation into myofibroblasts, which highly express α-SMA, and these myofibroblasts could in turn cause collagen metabolism disorders and increase expression of collagen I and collagen III. Furthermore, the decrease in Cdc42 expression indicated that, with the activation of MCFs, Cdc42 expression was regulated by TGF-β1 and negatively associated with the activation level of MCFs.

To investigate the effect of Cdc42 on MCFs, Cdc42-OE lentivirus or siRNA was used to induce or reduce Cdc42 expression, respectively. The expression levels of α-SMA, collagen I and collagen III were reduced after Cdc42 OE (Fig. 2D and E). Furthermore, the knockdown of Cdc42 expression increased the expression levels of α-SMA, collagen I and collagen III, which was consistent with the stimulant effect of TGF-β1 (Fig. 2D and F). Immunofluorescence staining also confirmed that Cdc42-OE lentivirus transfection decreased α-SMA protein expression, while siCdc42 transfection increased α-SMA protein expression (Fig. 2G-I). These data suggested that Cdc42 could inhibit MCF differentiation and collagen synthesis, and that decreased Cdc42 expression could promote fibrosis.

To investigate whether Cdc42 inhibited the autocrine activity of TGF-β1, the present study examined TGF-β1 mRNA levels. The results showed that TGF-β1 transcript levels were decreased when Cdc42 was overexpressed, and TGF-β1 transcript levels were increased when Cdc42 was knocked down by siCdc42-1 (Fig. 3A and B). The dual-luciferase reporter assay demonstrated that activity of the Tgfb1 gene promoter was significantly enhanced after siCdc42-1 transfection of MCFs, indicating that Cdc42 knockdown promoted the activity of the Tgfb1 gene promoter (Fig. 3C). As shown in Fig. 3D-F, the expression levels of Pak1, JNK and c-Jun were higher and the expression level of TGF-β1 was lower in the Cdc42 OE group than those in the NC group. The inhibition of Cdc42 expression decreased the expression levels of Pak1, JNK and c-Jun, and increased the expression level of TGF-β1. (Fig. 3D-F). These data suggested that Cdc42 might exert its antifibrotic effects by regulating the Pak1/JNK/c-Jun signaling pathway and subsequently inhibiting the autocrine activity of TGF-β1.

To further demonstrate that Cdc42 might be involved in regulating the Pak1/JNK/c-Jun signaling pathway, MLS-573151 (22), an inhibitor of Cdc42, was used to reduce Cdc42 activity in MCFs. The phosphorylation of serine 71 in Cdc42 is known to negatively regulate Cdc42 activity, and thus, the increased expression of p-Cdc42 indicates a decrease in active (GTP-bound) Cdc42 (23). After Cdc42 activity was decreased, the expression levels of Pak1, JNK and c-Jun were decreased, and the expression level of TGF-β1 was increased (Fig. S1A and B), consistent with the effect of Cdc42 knockdown.

Fig. 4A-C shows that the protein expression levels of α-SMA, collagen I and collagen III were decreased and the protein expression levels of Cdc42 were increased compared with NC group after treatment of TGF-β1-stimulated MCFs with 5, 10 and 50 nM E2 for 24 h. Furthermore, the protein expression levels of Pak1, JNK and c-Jun were increased compared with those in the TGF-β1-stimulated group after treatment of TGF-β1-stimulated MCFs with 10 nM E2 for 24 h. However, the effect of E2 on the expression of those proteins in TGF-β1-stimulated MCFs was abolished after siCdc42 transfection (Fig. 4D-F). Immunofluorescence staining also confirmed that siCdc42 abolished the E2-mediated reversal of changes in α-SMA protein expression following TGF-β1 treatment (Fig. 4G and H). Given these results, we hypothesized that the ameliorative effects of E2 were mediated by an increase in Cdc42. Fig. 5A-C shows that similar effects were observed in the Cdc42-OE-treated group and the E2-treated group of TGF-β1-stimulated MCFs, namely, decreased expression of α-SMA, collagen I and collagen III and increased expression of Pak1, JNK and c-Jun compared with the TGF-β1-stimulated group. Immunofluorescence staining also confirmed that Cdc42 OE inhibited α-SMA protein expression, which was consistent with the effect of E2-treated group (Fig. 5D and E). The aforementioned results indicated that Cdc42 could exert ameliorative effects similar to those of E2.