The c.1617del variant of TMEM260 is identified as the most frequent single gene determinant for Japanese patients with a specific type of congenital heart disease.

Although the molecular mechanisms underlying congenital heart disease (CHD) remain poorly understood, recent advances in genetic analysis have facilitated the exploration of causative genes for CHD. We reported that the pathogenic variant c.1617del of TMEM260, which encodes a transmembrane protein, is highly associated with CHD, specifically persistent truncus arteriosus (PTA), the most severe cardiac outflow tract (OFT) defect. Using whole-exome sequencing, the c.1617del variant was identified in two siblings with PTA in a Japanese family and in three of the 26 DNAs obtained from Japanese individuals with PTA. The c.1617del of TMEM260 has been found only in East Asians, especially Japanese and Korean populations, and the frequency of this variant in PTA is estimated to be next to that of the 22q11.2 deletion, the most well-known genetic cause of PTA. Phenotype of patients with c.1617del appears to be predominantly in the heart, although TMEM260 is responsible for structural heart defects and renal anomalies syndrome (SHDRA). The mouse TMEM260 variant (p.W535Cfs*56), synonymous with the human variant (p.W539Cfs*9), exhibited truncation and downregulation by western blotting, and aggregation by immunocytochemistry. In situ hybridization demonstrated that Tmem260 is expressed ubiquitously during embryogenesis, including in the development of cardiac OFT implicated in PTA. This expression may be regulated by a ~ 0.8 kb genomic region in intron 3 of Tmem260 that includes multiple highly conserved binding sites for essential cardiac transcription factors, thus revealing that the c.1617del variant of TMEM260 is the major single-gene variant responsible for PTA in the Japanese population.

Functional characterization of variants found in Japanese patients with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant form of vascular dysplasia. Genetic diagnosis is made by identifying loss-of-function variants in genes, such as ENG and ACVRL1. However, the causal mechanisms of various variants of unknown significance remains unclear. In this study, we analyzed 12 Japanese patients from 11 families who were clinically diagnosed with HHT. Sequencing analysis identified 11 distinct variants in ACVRL1 and ENG. Three of the 11 were truncating variants, leading to a definitive diagnosis, whereas the remaining eight were splice-site and missense variants that required functional analyses. In silico splicing analyses demonstrated that three variants, c.526-3C > G and c.598C > G in ACVRL1, and c.690-1G > A in ENG, caused aberrant splicing, as confirmed by a minigene assay. The five remaining missense variants were p.Arg67Gln, p.Ile256Asn, p.Leu285Pro, and p.Pro424Leu in ACVRL and p.Pro165His in ENG. Nanoluciferase-based bioluminescence analyses demonstrated that these ACVRL1 variants impaired cell membrane trafficking, resulting in the loss of bone morphogenetic protein 9 (BMP9) signal transduction. In contrast, the ENG mutation impaired BMP9 signaling despite normal cell membrane expression. The updated functional analysis methods performed in this study will facilitate effective genetic testing and appropriate medical care for patients with HHT.

Novel pathogenic GATA6 variant associated with congenital heart disease, diabetes mellitus and necrotizing enterocolitis.

BACKGROUND: Pathogenic GATA6 variants have been associated with congenital heart disease (CHD) and a spectrum of extracardiac abnormalities, including pancreatic agenesis, congenital diaphragmatic hernia, and developmental delay. However, the comprehensive genotype-phenotype correlation of pathogenic GATA6 variation in humans remains to be fully understood. METHODS: Exome sequencing was performed in a family where four members had CHD. In vitro functional analysis of the GATA6 variant was performed using immunofluorescence, western blot, and dual-luciferase reporter assay. RESULTS: A novel, heterozygous missense variant in GATA6 (c.1403 G > A; p.Cys468Tyr) segregated with affected members in a family with CHD, including three with persistent truncus arteriosus. In addition, one member had childhood onset diabetes mellitus (DM), and another had necrotizing enterocolitis (NEC) with intestinal perforation. The p.Cys468Tyr variant was located in the c-terminal zinc finger domain encoded by exon 4. The mutant protein demonstrated an abnormal nuclear localization pattern with protein aggregation and decreased transcriptional activity. CONCLUSIONS: We report a novel, familial GATA6 likely pathogenic variant associated with CHD, DM, and NEC with intestinal perforation. These findings expand the phenotypic spectrum of pathologic GATA6 variation to include intestinal abnormalities. IMPACT: Exome sequencing identified a novel heterozygous GATA6 variant (p.Cys468Tyr) that segregated in a family with CHD including persistent truncus arteriosus, atrial septal defects and bicuspid aortic valve. Additionally, affected members displayed extracardiac findings including childhood-onset diabetes mellitus, and uniquely, necrotizing enterocolitis with intestinal perforation in the first four days of life. In vitro functional assays demonstrated that GATA6 p.Cys468Tyr variant leads to cellular localization defects and decreased transactivation activity. This work supports the importance of GATA6 as a causative gene for CHD and expands the phenotypic spectrum of pathogenic GATA6 variation, highlighting neonatal intestinal perforation as a novel extracardiac phenotype.

Flow pattern analysis of right ventricular outflow tract in repaired tetralogy of Fallot through 4D flow MRI.

Cardiac magnetic resonance imaging (CMR) often shows discrepancies between right ventricular outflow tract (RVOT) flow and left ventricular outflow tract flow in patients with late-stage repaired tetralogy of Fallot (rTOF), leading to potential errors in pulmonary regurgitation fraction (PRF) assessment. This study aimed to identify the conditions under which RVOT flow can be acutely evaluated using four-dimensional (4D) flow CMR. Twenty-seven consecutive patients with rTOF underwent both two-dimensional phase-contrast (2D PC) and 4D flow CMR between 2016 and 2018, excluding those with peripheral pulmonary artery stenosis, RVOT conduit replacement, unknown surgical method, and an aortic valve regurgitation greater than 20%. Seven healthy controls also underwent only 4D Flow CMR. All healthy controls and fifteen patients with rTOF showed laminar RVOT flow, while seven patients exhibited helical, and four patients exhibited vortical RVOT flow in 4D flow CMR visualization. Flow-volume concordance between the pulmonary artery and aortic flow was significantly lower in patients with rTOF and PRF > 40% in 2D PC CMR. This concordance rate in the suprapulmonary valve was high in both the TOF and control groups, comparing at five RVOT locations in 4D flow CMR. Regarding RVOT flow regurgitation in 4D flow, the whole bulk evaluation exhibited greater variation depending on the flow type compared to the whole pixel-wise evaluation. The study confirmed the flow volume at the upper section of the pulmonary valve as the most accurate correlate of aortic flow volume. Furthermore, the 4D flow CMR using the pixel-wise method demonstrated superior accuracy compared to the traditional bulk flow method.

Human Genetics of Truncus Arteriosus.

Integrated human genetics and molecular/developmental biology studies have revealed that truncus arteriosus is highly associated with 22q11.2 deletion syndrome. Other congenital malformation syndromes and variants in genes encoding TBX, GATA, and NKX transcription factors and some signaling proteins have also been reported as its etiology.

Genetic and functional analyses of TBX4 reveal novel mechanisms underlying pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease, with approximately 10% of cases associated with genetic variants. Recent genetic studies have reported pathogenic variants in the TBX4 gene in patients with PAH, especially in patients with childhood-onset of the disease, but the pathogenesis of PAH caused by TBX4 variant has not been fully uncovered. METHODS: We analysed the TBX4 gene in 75 Japanese patients with sporadic or familial PAH using a PCR-based bidirectional sequencing method. Detected variants were evaluated using in silico analyses as well as in vitro analyses including luciferase assay, immunocytochemistry and chromatin immunoprecipitation (ChIP) whether they have altered function. We also analysed the function of TBX4 using mouse embryonic lung explants with inhibition of Tbx4 expression. RESULTS: Putative pathogenic variants were detected in three cases (4.0%). Our in vitro functional analyses revealed that TBX4 directly regulates the transcriptional activity of fibroblast growth factor 10 (FGF10), whereas the identified TBX4 variant proteins failed to activate the FGF10 gene because of disruption of nuclear localisation signal or poor DNA-binding affinity. Furthermore, ex vivo inhibition of Tbx4 resulted in insufficiency of lung morphogenesis along with specific downregulation of Tie2 and Kruppel-like factor 4 expression. CONCLUSION: Our results implicate variants in TBX4 as a genetic cause of PAH in a subset of the Japanese population. Variants in TBX4 may lead to PAH through insufficient lung morphogenesis by disrupting the TBX4-mediated direct regulation of FGF10 signalling and pulmonary vascular endothelial dysfunction involving PAH-related molecules.

Risk Factors of Coronary Artery Aneurysms in Kawasaki Disease with a Low Risk of Intravenous Immunoglobulin Resistance: An Analysis of Post RAISE.

OBJECTIVE: To detect risk factors of coronary artery aneurysm (CAA) development in patients with Kawasaki disease determined to have a low risk for resistance to primary intravenous immunoglobulin (IVIG) treatment based on the Kobayashi score. STUDY DESIGN: This study included 1757 predicted IVIG responders from Prospective Observational study on STRAtified treatment with Immunoglobulin plus Steroid Efficacy for Kawasaki disease (Post RAISE), a large-scale, multicenter, prospective cohort study of Kawasaki disease in Japan. Predicted IVIG responders were defined as patients with Kawasaki disease with a Kobayashi score of <5, a predictive scoring system for IVIG resistance created in Japan. The primary outcome was CAA development at 1 month after disease onset. CAA was defined as a Z score of ≥2.5. Multivariable logistic regression was used to identify the independent risk factors of CAA. The variables for inclusion were identified based on univariate analysis results and previously reported risk factors of CAA. RESULTS: Among 1632 patients who had complete coronary outcome data, CAA developed in 90 patients (5.5%) at 1 month after disease onset. Multivariable analysis found that a baseline maximum Z score of >2.5, age of <12 months at fever onset, and nonresponsiveness to IVIG were significant, independent risk factors of CAA development at 1 month after disease onset. Among the risk factors, a baseline maximum Z score of >2.5 was most strongly associated with CAA development (OR, 7.1; 95% CI, 4.1-12.2; P ≤ .001). CONCLUSIONS: Predicted IVIG responders with CAA risk factors identified in this study may be candidates for future clinical trials of intensified primary IVIG treatment with prednisolone, cyclosporine or infliximab.

Exercise Stress Electrocardiography Using the Two-Minute Jump Test in Children.

Although the treadmill and cycle ergometer are commonly used for exercise stress electrocardiography (ECG) testing, they are often difficult to perform with children. We herein evaluated the utility and safety of the 2-minute jump test (2MJT) as a simple, alternative exercise test. One hundred patients, including 60 male patients, with an average age at study commencement of 10.7±3.5 years (mean±standard deviation) and with no exercise restriction who underwent a cardiac check-up between November 2020 and March 2022 at the study center were included. After recording their resting ECG, they jumped for 2 minutes during ECG recording, and the change in heart rate (HR), ECG findings, and occurrence of adverse events were investigated. As a result, patients jumped 185±60 times in two minutes, and their HR increased from 76±13 beats/min at rest to 172±18 beats/min at peak during the test. Ninety (90%) patients attained the ideal target HR of > 150 beats/minute. During the recovery period after loading, five patients had abnormal ECG findings (ventricular extrasystoles, second-degree atrioventricular block, and atrial extrasystoles in two, two, and one patient, respectively) but completely resolved spontaneously within three minutes. Our findings suggested that the 2MJT is a useful and safe exercise test capable of inducing sufficient increase in HR in a short time in children.

Successful Preoperative Partial Splenic Artery and Aneurysm Embolization for Thrombocytopenia Associated with Failed Fontan Circulation.

Long-term complications after the Fontan procedure are important concerns for patients with pediatric and adult congenital heart disease. Although thrombocytopenia due to portal hypertension and hypersplenism is a well-known complication of the Fontan circulation, few studies have reported on its management. Herein we describe a young adult Fontan patient with thrombocytopenia and a splenic artery aneurysm caused by conduit stenosis. The patient required conduit replacement due to high venous pressure. We performed partial splenic artery embolization (PSE) and embolization of the aneurysm preoperatively to reduce the risk of bleeding, resulting in successful subsequent cardiac surgery. Preoperative evaluation of the splenic artery aneurysm was informative, and PSE was a safe and effective treatment option for thrombocytopenia to avoid bleeding during open-heart surgery in this patient.

Inositol 1,4,5-trisphosphate receptor 2 as a novel marker of vasculature to delineate processes of cardiopulmonary development.

Congenital heart diseases (CHDs) involving the outflow tract (OFT), such as persistent truncus arteriosus (PTA), lead to mortality and morbidity with implications not only in the heart, but also in the pulmonary vasculature. The mechanisms of pulmonary artery (PA) development and the etiologies underlying PA disorders associated with CHD remain poorly understood partly because of a specific marker for PA development is nonexistent. The three subtypes of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) are intracellular Ca2+ channels that are essential for many tissues and organs. We discovered that IP3R2 was expressed in the vasculature and heart during development using transgenic mice, in which a LacZ marker gene was knocked into the IP3R2 locus. Whole-mount and section LacZ staining showed that IP3R2-LacZ-positive cells were detectable exclusively in the smooth muscle cells, or tunica media, of PA, merging into αSMA-positive cells during development. Furthermore, our analyses suggested that IP3R2-LacZ positive PA smooth muscle layers gradually elongate from the central PA to the peripheral PAs from E13.5 to E18.5, supporting the distal angiogenesis theory for the development of PA, whereas IP3R2-LacZ was rarely expressed in smooth muscle cells in the pulmonary trunk. Crossing IP3R-LacZ mice with mice hypomorphic for Tbx1 alleles revealed that PTA of Tbx1 mutants may result from agenesis or hypoplasia of the pulmonary trunk; thus, the left and right central to peripheral PAs connect directly to the dorsal side of the truncus arteriosus in these mutants. Additionally, we found hypercellular interstitial mesenchyme and delayed maturation of the lung endoderm in the Tbx1 mutant lungs. Our study identifies IP3R2 as a novel marker for clear visualization of PA during development and can be utilized for studying cardiopulmonary development and disease.

Evaluation of a Kawasaki Disease Risk Model for Predicting Coronary Artery Aneurysms in a Japanese Population: An Analysis of Post RAISE.

OBJECTIVES: To test the performance of the Son risk score, which was created to predict coronary artery abnormalities from baseline variables in North American patients with Kawasaki disease. STUDY DESIGN: The dataset from Post RAISE, the largest prospective cohort study of Japanese patients with Kawasaki disease to date, was used for the present study. With high risk defined as ≥3 points, sensitivity, specificity, positive predictive value, and negative predictive value for coronary artery abnormality development were calculated. To evaluate the effect of each risk factor in the Son score, the OR and 95% CIs were calculated using logistic regression analysis with the presence of coronary artery abnormality at 1 month after disease onset. RESULTS: Post RAISE enrolled 2628 consecutive patients with Kawasaki disease, and 304 patients had a high-risk score, of whom 15.1% showed coronary artery abnormality. At the cutoff ≥3 points, the sensitivity was 37.7%, and the specificity was 87.2%. The maximum z score at baseline ≥2.0 (OR 3.5, 95% CI 2.3-5.2) and age <6 months at disease onset (OR 3.2, 95% CI 1.9-5.4), were significantly associated with coronary artery abnormality development. However, a high concentration of C-reactive protein was not associated with coronary artery abnormality. The area under the receiver operating characteristic curve for the Son score was 0.65 (95% CI 0.59-0.71). CONCLUSIONS: The Son score had insufficient sensitivity and good specificity in a Japanese cohort of patients with Kawasaki disease. Among the variables comprising the Son score, a large baseline z score and young age at disease onset were significant, independent predictors of coronary artery abnormality development.

Echocardiographic Left Ventricular Z-Score Utility in Predicting Pulmonary-Systemic Flow Ratio in Children With Ventricular Septal Defect or Patent Ductus Arteriosus.

BACKGROUND: The correlation between the Z-score of the left ventricular (LV) diameter and the LV volume-overload due to pulmonary over-circulation in children with ventricular septal defect (VSD) or patent ductus arteriosus (PDA) remains unclear.Methods and Results:The present, retrospective study enrolled 70 children (aged 0.3-16.8 years; 33 males, 37 females) with a diagnosis of isolated VSD and/or PDA who underwent cardiac catheterization (CC) between 2015 and 2019. Patients with chromosomal/genetic anomalies, growth disorder, right-ventricular enlargement or other conditions causing LV enlargement were excluded. Echocardiographic parameters were retrospectively evaluated from the medical records, converted to a Z-score, then compared with CC data. The pulmonary-systemic flow ratio on CC (cQp/Qs) correlated significantly with the Z-score of both the LV end-diastolic diameter (Zd) (r=0.698, P<0.0001) and LV end-systolic diameter (r=0.593, P<0.0001). Regression analysis and curve-fitting were used to predict the cQp/Qs based on the Zd, and a significant regression equation was found on cubic regression (R2of 0.524, P<0.0001) showing a strong correlation with the cQp/Qs (r=0.724, P<0.0001). CONCLUSIONS: The Z-score of the LV diameter can be a useful, non-invasive marker for evaluating LV volume overload and determining the surgical indications in children with VSD or PDA because of its strong correlation with the cQp/Qs.

Outcomes of idiopathic pulmonary arterial hypertension in Japanese children: a retrospective cohort study.

Recently, targeted therapy has been developed for idiopathic pulmonary arterial hypertension (IPAH). Studies evaluating the prognosis of IPAH have been conducted in adults. However, there is no nationwide survey of pediatric patients with IPAH regarding the long-term prognosis in Japan. Therefore, we investigated the clinical outcomes of Japanese pediatric patients with IPAH and risk factors for a poor prognosis. This multi-center, retrospective cohort study included pediatric patients with IPAH under the age of 15 years, who were gleaned from the nationwide network of Japanese Society of Pediatric Cardiology and Cardiac Surgery (JSPCCS). The questionnaire was sent to members of JSPCCS in 2015. Patients who were diagnosed with IPAH from 1994 to 2014 were included. The primary endpoint was death or lung transplantation. Ninety-five patients were finally enrolled. Both the mean age at diagnosis and the mean follow-up duration were 7 years. Ninety-five percent of patients had received targeted therapy for IPAH during follow-up. The overall 1, 3, 5, and 10-year event free rate, estimated using Kaplan-Meier survival estimate, was 96, 91, 83, and 74%, respectively. The prognosis was significantly poorer in patients with increased right ventricular systolic pressure (RVp), mean pulmonary artery pressure (mPAP) (≥ 52 mmHg), cardiothoracic ratio (≥ 55%), and levels of B-type natriuretic peptide (BNP) during follow-up (≥ 300 pg/mL) than in those without these parameters. In conclusion, in Japanese children with IPAH, the event-free rate for death or lung transplantation was found to be good. Greater RVp, mPAP, BNP levels during follow-up, and cardiothoracic ratio may be predictive indicators for a poor prognosis.

Pregnancy and Delivery in Patients With Repaired Congenital Heart Disease　- A Retrospective Japanese Multicenter Study.

BACKGROUND: Although advances in cardiac surgery have led to an increased number of survivors with congenital heart disease (CHD), epidemiological data regarding the pregnancies and deliveries of patients with repaired CHD are scarce.Methods and Results:In this study, we retrospectively reviewed the clinical outcomes of pregnancies and deliveries of women with repaired CHD. Overall, 131 women with repaired CHD were enrolled and there were 269 gestations. All patients were classified as New York Heart Association (NYHA) Class I or II. The prevalence of cesarean sections was higher in patients with (CyCHD) than without (AcyCHD) a past history of cyanosis (51% vs. 19%, respectively; P<0.01). There were 228 offspring from 269 gestations and the most prevalent neonatal complication was premature birth (10%), which was more frequent in the CyCHD than AcyCHD group (15.7% vs. 5.6%, respectively; P<0.01). Five maternal cardiac complications during delivery were observed only in the CyCHD group (8%); these were classified as NYHA Class II and none was fatal. CONCLUSIONS: Delivery was successful in most women with repaired CHD who were classified as NYHA Class I or II, although some with CyCHD and NYHA Class II required more attention. Cesarean sections were more common in the CyCHD than AcyCHD group, and CyCHD may be a potential risk for preterm deliveries.

Histone H1 quantity determines the efficiency of chromatin condensation in both apoptotic and live cells.

Although chromatin condensation is a well-known hallmark of apoptosis, the generation mechanism has not been clarified. Histone H1, a positively-charged abundant nuclear protein, is located in the linker region of chromatin. There are several Histone H1 subtypes that are encoded by variant genes. Using serial histone H1-deletion mutant cells established from the chicken B-cell leukemia line DT40, we found that apoptotic chromatin condensation was decreased in relation to histone H1 protein level and that the chromatin in nuclei prepared from the live null mutant cells had a high accessibility of DNases and transposase. This indicated that linker histone H1 was the general chromatin condensation factor and that the loss of histone H1 generated open chromatin in both apoptotic and live cells.

Polysplenia Syndrome as a Risk Factor for Early Progression of Pulmonary Hypertension.

BACKGROUND: Recent progress in surgical and intensive care has improved the prognosis of congenital heart disease (CHD) associated with heterotaxy syndrome. Less is known, however, about pulmonary vascular complications in these patients. Methods and Results: We reviewed medical records of 236 patients who were diagnosed with polysplenia syndrome at 2 institutions for pediatric cardiology in Japan from 1978 to 2015. We selected and compared the clinical records of 16 patients with polysplenia who had incomplete atrioventricular septal defect (AVSD) as the polysplenia group, and 22 age-matched patients with incomplete AVSD without any syndromes including polysplenia as the control group. Although the severity of systemic to pulmonary shunt was not significantly different between the groups, mean pulmonary artery pressure (mPAP) and pulmonary vascular resistance index (PVRI) were significantly higher in the polysplenia group than the control (mPAP, 37.3 vs. 19.1 mmHg, P=0.001; PVRI, 5.7 vs. 1.4 WU∙m2, P=0.014) before surgical intervention. On regression analysis, polysplenia influenced the development of pulmonary hypertension (PH) regardless of age at evaluation or degree of systemic to pulmonary shunt in the patients with incomplete AVSD. CONCLUSIONS: Polysplenia syndrome is an independent risk factor for CHD-associated PH. Earlier intervention may be required to adjust the pulmonary blood flow in polysplenia syndrome with CHD to avoid the progression of PH.

Chronotropic incompetence to exercise in anorexia nervosa patients during the body-weight recovery phase as an index of insufficient treatment.

Resting bradycardia is an important symptom for early diagnosis of anorexia nervosa (AN) during weight loss, and it improves with body-weight recovery. However, chronotropic incompetence (CI) in exercise is observed in some patients with AN despite amelioration of resting bradycardia in the recovery phase. We examined the relationship between CI in exercise and other parameters in patients with AN during the recovery phase. Ninety-two girls with AN (aged 13-20 years, median 15 years) performed cardiopulmonary exercise tolerance tests with a bicycle ergometer in the post-treatment recovery phase. Subjects with a peak-heart rate (HR) of < 160 beats/min (bpm) on subjective maximum loading were assigned to the CI+ group (n = 7), and those with a peak-HR of ≥ 160 bpm were assigned to the CI- group (n = 85). The peak-oxygen uptake (VO2) of both groups was below the normal range. Although there was no difference in peak-VO2 between these groups, both the resting-HR and ΔHR (peak-HR - resting-HR) were significantly lower in the CI+ group than in the CI- group (82 ± 8 vs. 93 ± 16 bpm, respectively; 72 ± 14 vs. 89 ± 13 bpm, respectively), suggesting lower exercise tolerance in patients with CI during the recovery phase of AN. Interestingly, the ΔVO2/ΔHR value was higher in the CI+ group than in the CI- group (0.31 ± 0.13 vs. 0.26 ± 0.06, respectively), suggesting excessive stroke volume for maintaining the cardiac output in patients with CI during their recovery phase. These data suggest that CI could be an index of insufficient recovery of AN and utilized for ideal exercise treatments of patients with AN during the recovery phase.

Type 2 inositol 1,4,5-trisphosphate receptor inhibits the progression of pulmonary arterial hypertension via calcium signaling and apoptosis.

Pulmonary arterial hypertension (PAH) is a progressive disease associated with vasoconstriction and remodeling. Intracellular Ca2+ signaling regulates the contraction of pulmonary arteries and the proliferation of pulmonary arterial smooth muscle cells (PASMCs); however, it is not clear which molecules related to Ca2+ signaling contribute to the progression of PAH. In this study, we found the specific expression of type 2 inositol 1,4,5-trisphosphate receptor (IP3R2), which is an intracellular Ca2+ release channel, on the sarco/endoplasmic reticulum in mouse PASMCs, and demonstrated its inhibitory role in the progression of PAH using a chronic hypoxia-induced PAH mouse model. After chronic hypoxia exposure, IP3R2-/- mice exhibited the significant aggravation of PAH, as determined by echocardiography and right ventricular hypertrophy, with significantly greater medial wall thickness by immunohistochemistry than that of wild-type mice. In IP3R2-/- murine PASMCs with chronic hypoxia, a TUNEL assay revealed the significant suppression of apoptosis, whereas there was no significant change in proliferation. Thapsigargin-induced store-operated Ca2+ entry (SOCE) was significantly enhanced in IP3R2-/- PASMCs in both normoxia and hypoxia based on in vitro fluorescent Ca2+ imaging. Furthermore, the enhancement of SOCE in IP3R2-/- PASMCs was remarkably suppressed by the addition of DPB162-AE, an inhibitor of the stromal-interacting molecule (STIM)-Orai complex which is about 100 times more potent than 2-APB. Our results indicate that IP3R2 may inhibit the progression of PAH by promoting apoptosis and inhibiting SOCE via the STIM-Orai pathway in PASMCs. These findings suggest a previously undetermined role of IP3R in the development of PAH and may contribute to the development of targeted therapies.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.