Transforming growth factor beta3 (TGFB3) gene mutations in patients of arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD1) and Loeys-Dietz syndrome-5 (LDS5)/Rienhoff syndrome are associated with cardiomyopathy, cardiac arrhythmia, cardiac fibrosis, cleft palate, aortic aneurysms, and valvular heart disease. Although the developing heart of embryos express Tgfb3, its overarching role remains unclear in cardiovascular development and disease. We used histological, immunohistochemical, and molecular analyses of Tgfb3−/− fetuses and compared them to wildtype littermate controls. The cardiovascular phenotypes were diverse with approximately two thirds of the Tgfb3−/− fetuses having one or more cardiovascular malformations, including abnormal ventricular myocardium (particularly of the right ventricle), outflow tract septal and alignment defects, abnormal aortic and pulmonary trunk walls, and thickening of semilunar and/or atrioventricular valves. Ventricular septal defects (VSD) including the perimembranous VSDs were observed in Tgfb3−/− fetuses with myocardial defects often accompanied by the muscular type VSD. In vitro studies using TGFβ3-deficient fibroblasts in 3-D collagen lattice formation assays indicated that TGFβ3 was required for collagen matrix reorganization. Biochemical studies indicated the ‘paradoxically’ increased activation of canonical (SMAD-dependent) and noncanonical (MAP kinase-dependent) pathways. TGFβ3 is required for cardiovascular development to maintain a balance of canonical and noncanonical TGFβ signaling pathways.

AimsCalcific aortic valve disease (CAVD) is a progressive heart disease that is particularly prevalent in elderly patients. The current treatment of CAVD is surgical valve replacement, but this is not a permanent solution, and it is very challenging for elderly patients. Thus, a pharmacological intervention for CAVD may be beneficial. In this study, we intended to rescue aortic valve (AV) calcification through inhibition of TGFβ1 and SMAD3 signaling pathways.Methods and ResultsThe klotho gene, which was discovered as an aging-suppressor gene, has been observed to play a crucial role in AV calcification. The klotho knockout (Kl–/–) mice have shorter life span (8–12 weeks) and develop severe AV calcification. Here, we showed that increased TGFβ1 and TGFβ-dependent SMAD3 signaling were associated with AV calcification in Kl–/– mice. Next, we generated Tgfb1- and Smad3-haploinsufficient Kl–/– mice to determine the contribution of TGFβ1 and SMAD3 to the AV calcification in Kl–/– mice. The histological and morphometric evaluation suggested a significant reduction of AV calcification in Kl–/–; Tgfb1± mice compared to Kl–/– mice. Smad3 heterozygous deletion was observed to be more potent in reducing AV calcification in Kl–/– mice compared to the Kl–/–; Tgfb1± mice. We observed significant inhibition of Tgfb1, Pai1, Bmp2, Alk2, Spp1, and Runx2 mRNA expression in Kl–/–; Tgfb1± and Kl–/–; Smad3± mice compared to Kl–/– mice. Western blot analysis confirmed that the inhibition of TGFβ canonical and non-canonical signaling pathways were associated with the rescue of AV calcification of both Kl–/–; Tgfb1± and Kl–/–; Smad3± mice.ConclusionOverall, inhibition of the TGFβ1-dependent SMAD3 signaling pathway significantly blocks the development of AV calcification in Kl–/– mice. This information is useful in understanding the signaling mechanisms involved in CAVD.

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.

The gold standard of arterial stiffness measurements in isolated aorta is the stress-strain (SS) relationship. Wall thickness (WT) and artery inner diameter (ID) are variables that plot the SS curve and should be normalized to the dimensions of the individual subject. Previously, we used measurements from the current literature, but the data collected were not accurate. Therefore, we hypothesized that the measurements of the vascular structure using echocardiogram (ECHO) should be applied to evaluate tissue puller to obtain SS data. With that, we also hypothesized that aorta from spontaneously hypertensive rats (SHR) has greater stress and lower strain levels than normotensive Wistar rats. To test this hypothesis, thoracic aorta parameters from 3-month-old male Wistar (WM) (body weight: 449±6.87g, n=4), SHR male (SM) (body weight: 295±3.34g, n=4), Wistar Female (WF) (body weight: 250±8.50g, n=4), and SHR Female (SF 169.4±2.69g, n=4) rats were measured from an ECHO. After, animals were killed and aortas were cut in 2 mm segments and mounted on Tissue Puller to measure SS. The inner diameter (ID) and wall thickness (WT) were measured by the ECHO for all animals: [WM (WT: 0.519±0.014mm, ID: 2.042±0.11mm), SM (WT: 0.485±0.026mm, ID: 2.023±0.055mm), WF (WT: 0.504±0.01mm, ID: 1.628±0.092mm), SF (WT: 0.521±0.011mm, ID: 1.64±0.099mm)] and there were no significant difference between these values. We then normalized the SS curves to each of the arteries’ parameters for ECHO and we found there were significant decreases in the strain values in aorta from hypertensive rats, regardless of sex (WM: 1.775±0.108 vs SM: 1.370±0.058; p=0.0146), and (WF: 2.056±0.218 vs. SF: 1.713±0.224; p=0.0517). We found a trend in the stress from female rats (WF: 1367±61 mN/mm2 vs SF 2361±391 mN/mm2; p=0.0597). There were no differences between male rats (WM:1.269±153 mN/mm2 vs SM: 1319±80 mN/mm2; p=0.671).Overall, these data show that ECHO should be used to accurately measure rat aorta structural parameters under the tissue puller. They also show SHR and Wistar were different in both sexes in strain, but only female SHR vs Wistar showed differences in stress. National Institutes of Health - R00GM118885, R01HL149762, and R00HL151889. and McNair Junior Fellows (MJF) Program This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.