[Molecular mechanisms of the TRPV4-RhoA/ROCK1 signaling axis in mediating mechanical stress-induced myocardial fibrosis in fetuses with tetralogy of Fallot].

Objective

To explore the role and underlying mechanisms of transient receptor potential vanilloid 4 (TRPV4) in pressure overload-induced myocardial fibrosis in fetal tetralogy of Fallot (TOF).

Methods

Fetal cardiac samples diagnosed by prenatal echocardiography or postmortem examination were retrospectively collected from Qingdao University Affiliated Women and Children's Hospital between May 2022 and July 2024, including 4 cases in the Tetralogy of Fallot (TOF) group and 4 cases in the structurally normal heart control group. Histological analysis of myocardial hypertrophy, fibrosis, and collagen content was performed on collected right ventricular wall tissues using hematoxylin-eosin (HE), Masson＇s trichrome, and Sirius red staining. Immunohistochemistry was used to detect the expression levels of TRPV4, Ras homolog gene family member A (RhoA), and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) in myocardial tissues, while Western blot was applied to measure the expression of Collagen Ⅰ, Collagen Ⅲ, and α-smooth muscle actin (α-SMA). In vitro, human umbilical vein endothelial cells (HUVEC) at passages 3 to 6 were seeded on hydrogel-coated plates with different stiffnesses (8 kPa, 50 kPa) and divided into 8 kPa, 50 kPa, and 50 kPa+TRPV4 inhibitor (pretreated with a TRPV4 antagonist) groups. Small interfering RNA targeting TRPV4 (siTRPV4) and negative control siRNA (siNC) were transfected into HUVECs to establish TRPV4-knockdown models. These cells were then seeded on hydrogel plates of varying stiffness and divided into 8 kPa, 50 kPa, siNC, and siTRPV4 groups. Western blot was used to assess the expression levels of TRPV4, RhoA, ROCK1, Collagen Ⅰ, Collagen Ⅲ, α-SMA, CD31, vascular endothelial cadherin (VE-cadherin), and endothelial-mesenchymal transition (EndMT)-related regulatory proteins (p-Smad2 and the transcription factor Slug).

Results

HE staining revealed cardiomyocyte hypertrophy in the right ventricular wall of the TOF group compared with the control group, accompanied by varying degrees of myocardial disarray. Masson＇s trichrome staining indicated a higher percentage of fibrotic area in the right ventricular myocardium of the TOF group, and Sirius red staining showed a larger collagen deposition area (all P<0.05). Compared with the control group, the TOF group exhibited significantly higher protein expression of Collagen Ⅰ, Collagen Ⅲ, and α-SMA, along with enhanced positive signal intensity for TRPV4, RhoA, and ROCK1 (all P<0.05). In vitro experiments, compared with the 50 kPa group, both the 8 kPa group and the 50 kPa+TRPV4 inhibitor group had lower expression levels of Collagen Ⅰ, Collagen Ⅲ, α-SMA, p-Smad2, Slug, TRPV4, RhoA, and ROCK1, while expression levels of CD31 and VE-cadherin were higher (all P<0.05). Similarly, the siTRPV4 group showed lower expression of Collagen Ⅰ, Collagen Ⅲ, α-SMA, TRPV4, RhoA, ROCK1, Slug and p-Smad2, and higher expression of CD31 and VE-cadherin compared with the 50 kPa group (all P<0.05). In contrast, no significant differences in the expression of these proteins were observed between the siNC group and the 50 kPa group (all P>0.05).

Conclusions

The right ventricle myocardium of TOF fetuses exhibits evident myocardial fibrosis. Mechanical stimulation induced by high-pressure load activates TRPV4, which may mediate EndMT in cardiac vascular endothelial cells through regulation of the RhoA/ROCK1 signaling pathway. This mechanism may represent one of the primary pathological contributors to myocardial fibrosis development.