ABSTRACT
Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart defects (CHDs). The right ventricular hypertrophy is associated with the survival rate of patients with repaired TOF. However, very little is known concerning its genetic etiology. Based on mouse model studies, a disintergrin and metalloprotease 10/17 (ADAM10 and ADAM17) are the key enzymes for the NOTCH and ErbB pathways, which are critical pathways for heart development. Mutations in these two genes have not been previously reported in human TOF patients. In this study, we sequenced ADAM10 and ADAM17 in a Han Chinese CHD cohort comprised of 80 TOF patients, 286 other CHD patients, and 480 matched healthy controls. Three missense variants of ADAM17 were only identified in 80 TOF patients, two of which (Y42D and L659P) are novel and not found in the Exome Aggregation Consortium (ExAC) database. Point mutation knock-in (KI) and ADAM17 knock-out (KO) human embryonic stem cells (hESCs) were generated by CRISPR/Cas9 and programmed to differentiate into cardiomyocytes (CMs). Y42D or L659P KI cells or complete KO cells all developed hypertrophy with disorganized sarcomeres. RNA-seq results showed that phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt), which is downstream of epidermal growth factor receptor (EGFR) signaling, was affected in both ADAM17 KO and KI hESC-CMs. In vitro experiments showed that these two mutations are loss-of-function mutations in shedding heparin-binding EGF-like growth factor (HB-EGF) but not NOTCH signaling. Our results revealed that CM hypertrophy in TOF could be the result of mutations in ADAM17 which affects HB-EGF/ErbB signaling.
Subject(s)
ADAM17 Protein/genetics , Cardiomegaly/genetics , Heparin-binding EGF-like Growth Factor/metabolism , Human Embryonic Stem Cells/enzymology , Loss of Function Mutation , Mutation, Missense , Myocytes, Cardiac/enzymology , Tetralogy of Fallot/genetics , ADAM17 Protein/metabolism , Animals , COS Cells , Cardiomegaly/enzymology , Cardiomegaly/pathology , Case-Control Studies , Cell Differentiation , Child , Child, Preschool , Chlorocebus aethiops , Female , Genetic Predisposition to Disease , HEK293 Cells , Human Embryonic Stem Cells/pathology , Humans , Infant , Male , Myocytes, Cardiac/pathology , Phenotype , Signal Transduction , Tetralogy of Fallot/diagnosis , Tetralogy of Fallot/enzymologyABSTRACT
OBJECTIVES: This study was performed to investigate the influence of traditional Chinese medicines in the Zhenju antihypertensive compound (ZJAHC) on the oral absorption of hydrochlorothiazide (HCT) both in vitro and in vivo. METHODS: Caco-2 cells and the in situ closed loop system were used to investigate the possible mechanism of the Chinese-Western medicine interaction on the transepithelial transport and uptake of HCT. The influence of TCMs on the pharmacokinetics and bioavailability of HCT was also studied to reveal the possible interaction in vivo. KEY FINDINGS: In an in situ intestinal perfusion study, the cumulative amount of HCT of ZJAHC group (506.05 µg ± 96.03) was 2.2-fold, 2.18-fold and 1.38-fold higher compared to that of the HCT group (228.29 µg ± 23.39), HCT-clonidine (CLO) group (232.13 ± 54.79 µg) and HCT-rutin (RT) group (366.08 ± 21.97 µg), respectively, after 120 min of perfusion. A pharmacokinetic analysis showed a significant increase in area under the plasma concentration-time curve (AUC) of HCT in the ZJAHC group by 2.14-fold, 2.01-fold and 1.32-fold compared to the HCT, HCT-CLO and HCT-RT groups, respectively. As a P-gp inhibitor, RT could contribute to the enhanced oral absorption of HCT in ZJAHC. CONCLUSION: The combination of traditional Chinese medicines and chemical drugs may provide a promising strategy and unique advantages to reduce the dosage and side effects of chemical drugs while maintaining an effect on hypertension.
