Rare mutations of ADAM17 from TOFs induce hypertrophy in human embryonic stem cell-derived cardiomyocytes via HB-EGF signaling.

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.

Impairment of mitochondrial respiratory chain enzyme activities in tetralogy of Fallot.

BACKGROUND: During the last decade, disorders of the respiratory chain, so-called mitochondrial disorders, have emerged as a major clinical entity. Tetralogy of fallot (TOF) children>2 month of age are at risk for postoperative myocardial contractile failure. Myocardial ischemia is associated with a reduction in mitochondrial enzyme activity and have impaired metabolism resulting in decreased postoperative myocardial adenosine triphosphate (ATP) concentrations and increased lactate levels. With this in view, we measured the mitochondrial energy system (respiration and OXPHOS) and to study morphological changes from the right ventricular outflow tract (RVOT) muscle of patients with TOF. METHODS: 30 infants with TOF were studied with age-matched control group consisted of 12 normal patients who died due to extracardiac causes. Mitochondrial respiratory chain complexes, OXPHOS, cytochrome content and ATPase activity were measured by documented standard procedure. Morphological changes examined with a transmission electron microscope. RESULTS: In the presence of glutamate and succinate as substrates, the rate of mitochondrial oxygen consumption was significantly lower in RVOT muscles (p<0.001) by using with and without addition of ADP. The ADP/O ratio indices for glutamate and succinate were not significantly affected. The activities of rotenone-sensitive NADH cytochrome c reductase (complexes I+III), cytochrome c oxidase (complex IV) and the ratio of I and III to II and III complexes (complex I) were significantly lower in TOF (p<0.001). A significant reduction of total cytochrome content and ATPase activity (p<0.001) was noted in study group. Morphological changes were also seen in study group as compared with control. CONCLUSIONS: OXPHOS, mitochondrial respiratory chain complex I, I+III and IV, cytochrome content and ATPase activity are more impaired in RVOT muscles in patients with TOF.

Expression of adenylyl cyclase V/VI mRNA and protein is upregulated in cyanotic infant human myocardium.

We have previously demonstrated that both basal and isoproterenol-stimulated activities of myocardial adenylyl cyclase were greater in cyanotic patients with tetralogy of Fallet (TOF) than those in acyanotic patients. However, it was not determined whether increased enzyme activity was related to a similar increase in adenylyl cyclase protein and mRNA expression. In the current study, we examined the mRNA and protein expression of cardiac adenylyl cyclase, types V and VI, in cyanotic and acyanotic patients with TOF. Ribonuclease protection assays and immunoblotting were performed on myocardial specimens obtained from cyanotic patients with TOF and acyanotic patients with TOF or ventricular septal defect. We demonstrated that in both cyanotic and acyanotic patients, there was more type V adenylyl cyclase mRNA than type VI. Types V and VI cardiac adenylyl cyclase mRNA were significantly increased in myocardium of the cyanotic group compared to the acyanotic group. Protein expression of both V and VI adenylyl cyclases was correspondingly upregulated in cyanotic patients compared to acyanotic patients. Our results indicate that gene and protein expression of cardiac adenylyl cyclases, types V and VI, is increased in the cyanotic myocardium. These results suggest that chronic hypoxemia may regulate the expression of adenylyl cyclase enzymes.

Nitric oxide synthase expression in lungs of pulmonary hypertensive patients with heart disease.

Since little is known about the contribution of endothelial nitric oxide synthase (e-NOS) to the mechanism of pulmonary vasospasm and the development of pulmonary vascular occlusive disease, we elucidate how e-NOS is expressed in lung biopsy specimens obtained from operative patients with pulmonary hypertension. Lung biopsy specimens were obtained from 17 patients who underwent open-heart operations for various heart diseases. A piece of normal lung specimen was also obtained from the resected lungs of three lung cancer patients as a control. e-NOS expression was visualized with a monoclonal antibody against e-NOS, and the level of expression was partially quantified. Significantly high levels of e-NOS expression were seen in adult patients, whose preoperative mean pulmonary arterial pressures were greater than 20 mm Hg. In contrast, e-NOS expression in pediatric patients with the same levels of mean pulmonary arterial pressure was the same as that in the controls and in low pulmonary arterial pressure. There was a statistically significant positive correlation between the level of e-NOS expression and Heath--Edwards grading. These data suggest that the e-NOS expression in lung tissue is induced when pulmonary vascular obstructive diseases progress.

The myocardial profile of the cytosolic isozymes of creatine kinase is apparently not related to cyanosis in congenital heart disease.

BACKGROUND: CKMB, the cardiac-specific heterodimer of cytosolic creatine-kinase (CK), is developmentally and physiologically regulated, tissue hypoxia being a proposed regulator. In patients with cyanotic heart disease the myocardium is perfused with partially saturated blood. We questioned whether the myocardium of cyanotic subjects contains higher proportions of CKMB. MATERIALS AND METHODS: CK activity, the distribution of cytosolic CK isozymes, activity of lactic dehydrogenase (LDH), and tissue protein content were determined in obstructive tissues removed at corrective surgery of patients with congenital heart defects. Cyanotic (n = 13) and acyanotic (n = 12) subjects were compared. RESULTS: In cyanotic and acyanotic patients, CK activity was 8.4 +/- 0.6 and 7.6 +/- 0.6 IU/mg protein and the proportion of CKMB was 21 +/- 1.4 and 22 +/- 2. 0% (mean +/- S.E.M), respectively. In the two groups of patients, the activity related to the B subunit corresponded to the steady-state level of the CKBmRNA. The tissue content of protein and the activities of CK and LDH were similar in cyanotic and acyanotic subjects and increased with the age. CONCLUSIONS: The lack of difference in CKMB distribution between the cyanotic and acyanotic patients may either indicate that hypooxygenation is not a regulator of CK isozyme expression, or may be attributed to the already high proportion of this isozyme in hypertrophied, obstructive tissues. Recruitment of additional CKMB, in the cyanotic hearts, may thus not be required.

Mitochondrial respiratory chain enzyme activities in tetralogy of Fallot.

It has been suggested that myocardial ischemia is associated with a reduction in mitochondrial complex I activity. Respiratory chain enzyme activities were measured in right ventricular biopsies of eight infants with tetralogy of Fallot (TF), left ventricular biopsies of one of the infants with TF, right and left ventricular tissue of seven transplant recipients with atherosclerotic coronary artery disease (CAD), and in right ventricular biopsies of one infant without cardiac pathology (normal control). In right ventricular tissue the specific activity of complexes I+III was significantly lower in TF than in CAD (3.8 +/- 2.7 vs 23 +/- 12 nmol min-1 mg-1 non-collagen protein). In the right ventricular control specimen the activity of complexes I+III was 13.7-fold standard deviation higher than in TF and 1.5-fold higher than in CAD. Left ventricular respiratory chain enzyme activities measured in one patient with TF were lower than those in patients with CAD. Enzyme activities of left ventricular tissue were not significantly different from those of the right ventricle in CAD. The activity of the mitochondrial matrix enzyme citrate synthase did not differ between groups. The data indicate that the prominent reduction of complex I activity found in myocardial ischemia due to CAD is even more pronounced in myocardial hypoxemia due to TF.

Effect of oxygen tension and cardiovascular operations on the myocardial antioxidant enzyme activities in patients with tetralogy of Fallot and aorta-coronary bypass.

Since the chronically cyanotic myocardium appears to be more susceptible to reperfusion injury after cardiac operations than the noncyanotic myocardium, we studied the association between the preoperative arterial oxygen tension and the myocardial superoxide dismutase, catalase, and glutathione peroxidase activities. Fourteen patients with tetralogy of Fallot scheduled for elective operations had baseline arterial blood gas measurements done before operation. During the operation right ventricular biopsy specimens were taken for enzyme analysis immediately before cold blood cardioplegic arrest and 20 minutes after crossclamp removal. The tissue antioxidant enzyme activities of the patients with tetralogy of Fallot were compared with the myocardial results in 15 adults with stable angina pectoris having elective aorta-coronary artery bypass graft operations. Myocardial tissues removed from two patients with hypertrophic obstructive cardiomyopathy who had corrective operations were analyzed for antioxidant activities. There were no changes in myocardial antioxidant enzyme activities during the operation in the patients with tetralogy of Fallot and coronary artery bypass graft. The myocardial superoxide dismutase, catalase, and glutathione peroxidase activities correlated (0.82, 0.68, and 0.89, respectively) significantly (p values were less than 0.01, 0.05, and 0.01, respectively) with the preoperative arterial oxygen tensions in the patients with tetralogy of Fallot. The myocardial glutathione peroxidase activities were at least four times higher in the myocardium of patients with coronary artery bypass graft and hypertrophic obstructive cardiomyopathy than in that of those with tetralogy of Fallot. This study provides putative evidence that the myocardium of patients with tetralogy of Fallot is a risk of oxygen-derived free radical injury during and immediately after corrective cardiovascular operations.

Different activities of energy metabolism enzymes in children's cardiac atria and ventricles.

Tissue samples from the right atrium and ventricle of the same heart were obtained during surgery on 45 children operated on for congenital heart disease (tetralogy of Fallot, ventricular septal defect). Significant enzyme activity differences were found between atrial and ventricular muscle. Aerobic metabolism enzymes (citrate synthase, malate dehydrogenase), with lactate metabolism (lactate dehydrogenase) and fatty acid oxidation (hydroxyacyl-SoA dehydrogenase) showed significantly higher activities in ventricular muscle tissue. In contrast, hexokinase, the enzyme responsible for glucose phosphorylation was significantly higher in the atria. Hence, the right ventricle can utilize and oxidize to the full all the main nutrients (fatty acids, glucose and lactate) while the right atrium utilizes primarily glucose. These atrio-ventricular differences are independent of the type of congenital heart disease and their existence can be presumed in healthy persons, too.

[Differences in the activity of enzymes associated with energy metabolism in the heart atria and ventricles in children]. / Rozdílné aktivity enzymu energetického metabolismu v srdecních síních a komorách u detí.

Tissue specimens from the right atrium and ventricle of the same heart were obtained during surgery in 45 children operated on account of congenital heart disease (tetralogy of Fallot and ventricular septal defect). Significant differences were found in the enzyme activities between the atrial and ventricular musculature. The activities of enzymes associated with aerobic metabolism (citrate synthase, malate dehydrogenase, with lactate metabolism) lactate dehydrogenase (and the fatty acid oxidation) hydroxyacyl-SoA-dehydrogenase) were significantly higher in the ventricular musculature. Hexokinase, the enzyme responsible for glucose phosphorylation was on the other hand, significantly higher in the atria. From this ensues that the right ventricle can utilize and oxidize to a full extent all main nutrients (fatty acids, glucose and lactate), while the right atrium utilizes above all glucose. These atrio-ventricular differences do not depend on the type of the congenital heart disease and it may be assumed that they exist also in healthy subjects.

Effect of oxygen tension on the anti-oxidant enzyme activities of tetralogy of Fallot ventricular myocytes.

Since the chronically cyanotic tetralogy of Fallot (TOF) myocardium is more sensitive to reperfusion injury after cardiac surgery than the adult myocardium, we decided to study the regulation of myocardial superoxide dismutase (SOD), catalase and glutathione peroxidase by oxygen tension. TOF myocytes were cultured at a Po2 of 150 mmHg for 30 days to establish the culture. The cells were then cultured at Po2 of 150 and 40 mmHg and the myocyte antioxidant enzymes measured at days 3, 7, 14 and 21. On day 21 the myocytes cultured at Po2 of 40 mmHg were then cultured at 150 mmHg and SOD and catalase activities measured on days 28 and 35. Although there were no differences in the rates of incorporation of 35S-methionine into the myocytes at either Po2 on these days, the myocytes scavenger enzyme levels were significantly higher by day 14 when cultured at a Po2 of 150 mmHg than at a Po2 of 40 mmHg. With the increase in oxygen tension from 40 to 150 mmHg, SOD and catalase activities increased significantly by day 35. The myocytes cultured at Po2 40 mmHg were more sensitive by day 7 to an hypoxanthine-xanthine oxidase generated free radical injury than the Po2 150 mmHg cultured cells. The regulation of these enzyme activities by oxygen tension and the increased sensitivity to free radical injury of the myocytes cultured at an oxygen tension of 40 mmHg provide putative evidence that the chronically cyanotic myocardium may be less well protected than the normally perfused myocardium against oxygen-mediated free radical injury and be at higher risk for cardiovascular surgery.

Long-term administration of prostaglandin E1: report of two cases with tetralogy of Fallot and esophageal atresia.

The authors continuously administered prostaglandin E1 (PGE1) iv to 2 infants with tetralogy of Fallot (TOF) and esophageal atresia with tracheoesophageal fistula (TEF) for more than 30 days, and observed side effects which could be attributed to the long-term administration of PGE1. After division of the TEF and anastomosis of the esophagus, leakage from the anastomosis developed in both cases. Because of the infectious foci in the thorax, Blalock's procedure was postponed and PGE1 was continued for 49 and 37 days. The authors believe radiographs of long bones and ribs demonstrated cortical hyperosteosis in both cases. Bone abnormalities became apparent approximately 30 days after the start of PGE1 and were associated with increases in serum alkaline phosphatase (peak value of about 2000 IU/L). Roentgenographic changes reverted toward normal and alkaline phosphatase values decreased after the cessation of PGE1 in both cases.