[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.