Myocardial TGFβ2 Is Required for Atrioventricular Cushion Remodeling and Myocardial Development

Abstract

Among the three transforming growth factor beta (TGFβ) ligands, TGFβ2 is essential for heart development and is produced by multiple cell types, including myocardium. Heterozygous mutations in TGFB2 in patients of connective tissue disorders result in congenital heart defects and adult valve malformations, including mitral valve prolapse (MVP) with or without regurgitation. Tgfb2 germline knockout fetuses exhibit multiple cardiac defects but the role of myocardial-TGFβ2 in heart development is yet to be elucidated. Here, myocardial Tgfb2 conditional knockout (CKO) embryos were generated by crossing Tgfb2flox mice with Tgfb2+/−; cTntCre mice. Tgfb2flox/− embryos were normal, viable. Cell fate mapping was done using dual-fluorescent mT/mG+/− mice. Cre-mediated Tgfb2 deletion was assessed by genomic PCR. RNAscope in situ hybridization was used to detect the loss of myocardial Tgfb2 expression. Histological, morphometric, immunohistochemical, and in situ hybridization analyses of CKOs and littermate controls at different stages of heart development (E12.5–E18.5) were used to determine the role of myocardium-derived TGFβ2 in atrioventricular (AV) cushion remodeling and myocardial development. CKOs exhibit a thin ventricular myocardium, AV cushion remodeling defects and developed incomplete AV septation defects. The loss of myocardial Tgfb2 resulted in impaired cushion maturation and dysregulated cell death. Phosphorylated SMAD2, a surrogate for TGFβ signaling, was “paradoxically” increased in both AV cushion mesenchyme and ventricular myocardium in the CKOs. Our results indicate that TGFβ2 produced by cardiomyocytes acting as cells autonomously on myocardium and via paracrine signaling on AV cushions are required for heart development.

Hippo Signaling Mediates TGFβ-Dependent Transcriptional Inputs in Cardiac Cushion Mesenchymal Cells to Regulate Extracellular Matrix Remodeling

“…The cushion mesenchymal cell line used in this study is a very well-characterized and unique cell line to perform in vitro investigation to study cushion mesenchymal cell proliferation, apoptosis, ECM remodeling and cardiac cushion remodeling, differentiation, and maturation [28]. We chose TGFβ2 for the in vitro investigation in this study because a loss of TGFβ2 leads to major cardiovascular defects, including cardiac cushion remodeling defects [32][33][34]. Zhang et al [22] studied the role of YAP1 in 'endothelial cells' in TGFβ1induced endothelial to mesenchymal transition (EMT).…”

“…Increased Acta2, Ctgf (Ccn2), and Col1a1 expression is involved in tissue fibrosis [9,18] and tissue regeneration [41]. The loss of Ccn1 or Tgfb2 causes cardiac septal defects in humans and mice [33,34,42,43], suggesting a potential genetic and molecular interaction between the TGFβ2 and Hippo pathways. Mechanosensitive and profibrotic signaling pathways, such as YAP/TGFβ, are also implicated in multiple aspects of valvular heart disease and therefore considered potential targets for therapeutic interventions and prognostic biomarkers with the implications to improve the management of valvular heart disease [44].…”

Increased TGFβ1 and SMAD3 Contribute to Age-Related Aortic Valve Calcification

“…Endogenous peroxidases were blocked with freshly prepared 0.5% H 2 O 2 /methanol for 30 min, followed by non-specific epitope blocking with 5% goat serum/0.1% Tween/0.02% sodium azide in PBS for 20 min. Avidin and Biotin blocking was done as per the manufacturer’s recommendation (Cat# SP-2001, Vector Labs, Burlingame, CA, United States), followed by overnight incubation at 4°C in anti-pSMAD2 (1:3000, Millipore, Burlington, MA, United States) ( 61 ). Slides were then washed and incubated with appropriate biotinylated secondary antibody (1:200) for 30 min, followed by Avidin-Biotin complex (Cat# PK-6100, Vectastain Elite ABC HRP kit) for 30 min, washed in PBS, and finally developed with DAB/H 2 O 2 .…”

“…Before starting each study, we collected the tail at the age of 3 weeks and genomic DNAs were extracted for each animal and confirmed the genotype using gene-specific primers for Kl, Tgfb1, and Smad3 (Table 1). PCR genotyping was done as described earlier (60)(61)(62).…”