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Simple Summary In this study, the induced pluripotent stem cell-derived cardiomyocyte model from a patient with long QT syndrome harboring a heterozygous Nav1.5 R1623Q mutation exhibited prolonged field potential duration corrected by Fridericia’s formula (FPDcF, analogous to QTcF). FPDcF was shortened with mexiletine treatment and increased the frequency of arrhythmia-like EAD events following E4031, an Ikr blocker, administration. These characteristics partly reflect the patient phenotypes. As the R1623Q mutation is related to severe congenital LQT syndrome in fetuses and neonates, the effect of the neonatal variants on the electrophysiological properties of the R1623Q mutant was examined using an automated patch-clamp system. Our results demonstrated that both R1623Q and neonatal R1623Q delayed inactivation of INa and increased late Na current. We speculated that neonatal Nav1.5 ameliorates QTc prolongation. Developmental switching of neonatal/adult Nav1.5 isoforms might play a role in the mechanisms underlying severe long QT syndrome in fetuses and neonates. Abstract The SCN5A R1623Q mutation is one of the most common genetic variants associated with severe congenital long QT syndrome 3 (LQT3) in fetal and neonatal patients. To investigate the properties of the R1623Q mutation, we established an induced pluripotent stem cell (iPSC) cardiomyocyte (CM) model from a patient with LQTS harboring a heterozygous R1623Q mutation. The properties and pharmacological responses of iPSC-CMs were characterized using a multi-electrode array system. The biophysical characteristic analysis revealed that R1623Q increased open probability and persistent currents of sodium channel, indicating a gain-of-function mutation. In the pharmacological study, mexiletine shortened FPDcF in R1623Q-iPSC-CMs, which exhibited prolonged field potential duration corrected by Fridericia’s formula (FPDcF, analogous to QTcF). Meanwhile, E4031, a specific inhibitor of human ether-a-go-go-related gene (hERG) channel, significantly increased the frequency of arrhythmia-like early after depolarization (EAD) events. These characteristics partly reflect the patient phenotypes. To further analyze the effect of neonatal isoform, which is predominantly expressed in the fetal period, on the R1623Q mutant properties, we transfected adult form and neonatal isoform SCN5A of control and R1623Q mutant SCN5A genes to 293T cells. Whole-cell automated patch-clamp recordings revealed that R1623Q increased persistent Na+ currents, indicating a gain-of-function mutation. Our findings demonstrate the utility of LQT3-associated R1623Q mutation-harboring iPSC-CMs for assessing pharmacological responses to therapeutic drugs and improving treatment efficacy. Furthermore, developmental switching of neonatal/adult Nav1.5 isoforms may be involved in the pathological mechanisms underlying severe long QT syndrome in fetuses and neonates.

Background Pancreatic cancer is one of the most aggressive human malignancies. The development of a novel drug to treat pancreatic cancer is imperative, and it is thought that complementary and alternative medicine (CAM) could yield such a candidate. Agaricus blazei Murrill is a CAM that has been tested as an anticancer drug, but its efficacy against pancreatic cancer is poorly understood. To study the potential of A. blazei in the treatment of pancreatic cancer, we examined the effects of its hot water extract on the proliferation and global gene expression profile of human pancreatic cancer cells. Methods Three distinct human pancreatic cancer cell lines, MIAPaCa-2, PCI-35, and PK-8, and the immortalized human pancreatic duct-epithelial cell line, HPDE, were employed. The cells were incubated with the appropriate growth medium supplemented with the hot water extract of A. blazei at final concentrations of 0.005, 0.015%, or 0.045%, and cellular proliferation was assessed for five consecutive days using an MTT assay. Apoptosis was examined by using flow cytometry and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Caspase-dependent apoptosis was assayed using immunoblotting. Global gene expression profiles were examined using a whole human genome 44 K microarray, and the microarray results were validated by using real-time reverse transcription PCR. Results The hot water extract of A. blazei significantly inhibited the proliferation of cultured pancreatic cancer cells through the induction of G0/G1 cell cycle arrest and caspase-dependent apoptosis; the effect was the smallest in HPDE cells. Furthermore, significant alterations in the global gene expression profiles of pancreatic cancer cells occurred following treatment with the hot water extract of A. blazei. Genes associated with kinetochore function, spindle formation, and centromere maintenance were particularly affected, as well as cyclins and cyclin-dependent kinases that are essential for cell cycle progression. In addition, proapoptotic genes were upregulated. Conclusions The hot water extract of A. blazei may be useful for the treatment of pancreatic cancer and is a potential candidate for the isolation of novel, active compounds specific for mitotic spindle dysfunction. Electronic supplementary material The online version of this article (10.1186/s12906-018-2385-4) contains supplementary material, which is available to authorized users.

The two cardiac progenitor cell lineages, cardiac neural crest cells (cNCCs) and the second heart field (SHF), play key roles in the development of the cardiac outflow tract (OFT). Both cardiac progenitor cells interact with each other and contribute to OFT formation cooperatively. The neurovascular guiding molecule, semaphorin 3c (Sema3c), is thought to serve as a key attractant for the migration of cNCCs. A previous study reported that Tbx1 null mice showed a significant reduction in Sema3c expression in the OFT region [1]. However, the regulatory effect of Tbx1 on Sema3c was unclear. Here, we show that Sema3c plays key roles in cNCCs-SHF interactions through the regulation by Tbx1 and other molecules during OFT development [2].

Although some studies have attempted to find useful prognostic factors in hypertrophic cardiomyopathy (HCM), those results are not fully helpful for use in actual clinical practice. Furthermore, several genetic abnormalities associated with HCM have been identified. However, the genotype-phenotype correlation in HCM remains to be elucidated. Here, we attempted to assess patients with different types of gene mutations causing HCM and investigate the prognosis. A total of 140 patients with HCM underwent a screening test for myofilament gene mutations by direct sequencing of eight sarcomeric genes. Patients with a single mutation in cardiac troponin T, cardiac troponin I, α-tropomyosin, and regulatory and essential light chains were excluded from the study because the number of cases was too small. The clinical presentations and outcomes of the remaining 127 patients with HCM, 31 β-myosin heavy chain (MYH7) mutation carriers, 19 cardiac myosin-binding protein C (MYBPC3) mutation carriers, and 77 mutation non-carriers were analyzed retrospectively. MYBPC3 mutation carriers had a high frequency of ventricular arrhythmia and syncope. Kaplan-Meier curves revealed no significant difference in prognosis among the three groups, but a lack of family history of sudden death (SD) and a past history of syncope were significantly related to poor prognosis. An absence of family history of SD and past history of syncope are useful prognostic factors in patients with HCM. MYH7 and MYBPC3 mutations did not significantly influence prognosis compared to non-carriers. However, patients with the MYBPC3 mutation should be closely followed for the possibility of SD.

Long QT syndrome (LQTS) can cause syncope, ventricular fibrillation, and death. Recently, several disease-causing mutations in ion channel genes have been identified, and compound mutations have also been detected. It is unclear whether children who are carriers of compound mutations exhibit a more severe phenotype than those with single mutations. Although predicting phenotypic severity is clinically important, the availability of prediction tools for LQTS is unknown. To determine whether the severity of the LQTS phenotype can be predicted by the presence of compound mutations in children is needed. We detected 97 single mutations (Group S) and 13 compound mutations (Group C) between 1998 and 2012, age at diagnosis ranging 0-19 years old (median age is 9.0) and 18.0 years of follow-up period. The phenotypes and Kaplan-Meier event-free rates of the two groups were compared for cardiac events. This study investigated phenotypic severity in relation to the location of mutations in the protein sequence, which was analyzed using two sequence homology-based tools. In results, compound mutations in children were associated with a high incidence of syncope within the first decade (Group S: 32 % vs. Group C: 61 %), requiring an ICD in the second decade (Group S: 3 % vs. Group C: 56 %). Mortality in these patients was high within 5 years of birth (23 %). Phenotypic prediction tools correctly predicted the phenotypic severity in both Groups S and C, especially by using their coupling method. The coupling prediction method is useful in the initial evaluation of phenotypes both with single and compound mutations of LQTS patients. However, it should be noted that the compound mutation makes more severe phenotype.

Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ~9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.

Common periodical health check-ups include several clinical test items with affordable cost. However, these standard tests do not directly indicate signs of most lifestyle diseases. In order to detect such diseases, a number of additional specific clinical tests are required, which increase the cost of the health check-up. This study aims to enrich our understanding of the common health check-ups and proposes a way to estimate the signs of several lifestyle diseases based on the standard tests in common examinations without performing any additional specific tests. In this manner, we enable a diagnostic process, where the physician may prefer to perform or avoid a costly test according to the estimation carried out through a set of common affordable tests. To that end, the relation between standard and specific test results is modeled with a multivariate kernel density estimate. The condition of the patient regarding a specific test is assessed following a Bayesian framework. Our results indicate that the proposed method achieves an overall estimation accuracy of 84%. In addition, an outstanding estimation accuracy is achieved for a subset of high-cost tests. Moreover, comparison with standard artificial intelligence methods suggests that our algorithm outperforms the conventional methods. Our contributions are as follows: (i) promotion of affordable health check-ups, (ii) high estimation accuracy in certain tests, (iii) generalization capability due to ease of implementation on different platforms and institutions, (iv) flexibility to apply to various tests and potential to improve early detection rates.

Agaricus blazei (A. blazei) Murrill mycelia-dikaryon has attracted the attention of scientists and clinicians worldwide owing to its potential for the treatment of cancer. However, little is known about its effect on other pathologies. This study sought to extend the potential medical usefulness of A. blazei for preventing vascular damage and to unravel its mechanism of action. The A. blazei extract showed estrogen-like activity in both gene expression profiling and a luciferase assay. Indeed, the extract inhibited oxidized low-density lipoprotein-stimulated activation of Erk1/2, Akt and p38 in HUVECs and macrophage-derived TIB-67 cells. Moreover, the extract enhanced transcription of the glutathione peroxidase 3 (GPX3), α-synuclein (SNCA) and endothelial nitrogen-oxide synthase (eNOS) genes. Furthermore, atherosclerotic lesions in rabbits were reduced by intake of A. blazei powder. Therefore, A. blazei may be useful for preventing atherosclerosis via dual roles in cell signaling, suppression of macrophage development and the recovery of endothelial cells from vascular damage.

Background: The genetic basis of most congenital heart defects (CHDs), especially non-syndromic and non-familial conditions, remains largely unknown. Methods and Results: DNA samples were collected from immortalized cell lines and original genomes of 256 non-syndromic, non-familial patients with cardiac outflow tract (OFT) defects. Genes encoding NKX2.5, GATA4, GATA6, MEF2C, and ISL1, essential for heart development, were analyzed using PCR-based bidirectional sequencing. The transcriptional activity of proteins with identified sequence variations was analyzed using a luciferase assay. A novel sequence variant (A103V in MEF2C) was identified, in addition to 4 unreported non-synonymous sequence variants in 3 known causative genes (A6V in NKX2.5, T330R and S339R in GATA4, and E142K in GATA6) in 5 individuals. None of these was found in 500 controls without CHDs. In vitro functional assay showed that all proteins with identified sequence variations exhibited significant changes in transcriptional activity and/or synergistic activity with other transcription factors. Furthermore, overexpression of the A103V MEF2C variant in a fish system disturbed early cardiac development. Conclusions: New mutations in the transcription factors NKX2.5, GATA4, GATA6, and MEF2C that affect their protein function were identified in 2.3% (6/256) of patients with OFT defects. Our results provide the first demonstration of MEF2C mutation and suggest that disturbances in the regulatory circuits involving these cardiac transcription factors may cause a subset of non-syndromic and non-familial CHDs. (Circ J 2012; 76: 1703–1711)

Background Previously published papers showed that cardiac stem cells (CSCs) form (cardio)sphere. However, recent studies questioned the significance of the sphere-formation as one of the characteristics of CSCs. We isolated c-kit-positive cardiac stem cells, cultured as bulk (CSC-BC) and characterized them previously. Among them, CSC-BC21 formed an extraordinary number of spheres. Using a clone derived from this bulk culture, we investigated the effect of sphere-formation on differentiation and performed proteomics analysis comparing two statuses, cardiosphere and dish substrate attachment. Methods We performed sphere-forming assay to compare the sphere-forming ability among CSC-BCs. The cloned cells from CSC-BC21, which had distinct sphere-forming ability, were cultured in a differentiation medium (DM) to induce cardiac myocyte differentiation. We performed RT-PCR analysis to investigate if cardiosphere-formation affects cardiac myocyte gene expression level. Furthermore, proteome analysis was performed to compare floating cardiosphere (flCS) and dish-attached cardiosphere-derived cells (daCS). Results One of the cloned cells, CSC-21E expressed higher troponin I message than CSC-BC21. Moreover, the message level of troponin I was enhanced when they had experienced cardiosphere prior to the treatment of myocyte differentiation medium. The change from flCS to daCS accompanied up-regulation of chaperones and down regulation of glycolytic and other metabolic enzymes. Calreticulin and Hsp 90 were among the up-regulated chaperons. Calreticulin is known to be an essential component of cardiogenesis. Conclusion These results suggest that the switch from aggregated sphere to the cell attachment, is important for advancing the cardiac cell differentiation.

A child with vein of Galen aneurysmal malformation (VGAM) presented with cardiac failure in the neonatal period. The family history revealed his mother to have hereditary hemorrhagic telangiectasia. The child underwent an endoglin genetic analysis after the newborn period, which eventually demonstrated an endoglin mutation. The pathogenesis of VGAM is currently unknown. The findings of this case suggest that an endoglin mutation might be linked with VGAM.

We have shown previously that PI3K/Akt pathway is active after cell differentiation in HL60 cells. In the present study, we have investigated whether additional molecules, such as protein kinase C (PKC), are involved in the regulation, not only of telomerase, but also of leukemia cell differentiation. We show that PKC activates telomerase and is, itself, activated following VD3- or ATRA-induced differentiation of HL60 cells, as was observed for PI3K/Akt. To clarify the significance of PI3K/Akt and PKC pathway activation in leukemia cell differentiation, we examined the active proteins in either the downstream or upstream regulation of these pathways. In conjunction with the activation of Akt or PKC, mTOR and S6K were phosphorylated and the protein expression levels of Rictor were increased, compared with Raptor, following cell differentiation. Silencing by Rictor siRNA resulted in the attenuation of Akt phosphorylation on Ser473 and PKCα/βII phosphorylation, as well as the inhibition of Rictor itself, suggesting that Rictor is an upstream regulator of both Akt and PKC. In addition, in cells induced to differentiate by ATRA or VD3, Nitroblue-tetrazolium (NBT) reduction and esterase activity, were blocked either by LY294002, a PI3K inhibitor, or by BIM, a PKC inhibitor, without affecting cell surface markers such as CD11b or CD14. Intriguingly, the silencing of Rictor by its siRNA also suppressed the reducing ability of NBT following VD3-induced cell differentiation. Taken together, our results show that Rictor associated with mTOR (mTORC2) regulates the activity of both Akt and PKC that are involved in cell functions such as NBT reduction and esterase activity induced by leukemia cell differentiation.

Many stem cell studies have focused on the subject of cell fate and the signal molecules that modulate the regulatory switches for a given differentiation pathway. Genome-wide screens for cell fate determination signals require a cell source that differentiates purely into a single cell type. From adult rat left atrium, we established LA-PCs that differentiates into cardiac/skeletal myocytes or adipocytes with almost 100% purity. In this study, we compared gene expression profiles of undifferentiated LA-PCs with those of differentiated cells [adipocytes (Adi) or cardiac/skeletal myocytes (Myo)] to identify the signals that set the regulatory switch for adipocyte or myocyte differentiation. Microarray analysis verified the feasibility of genome-wide screening by this method. Using a pathway analysis screen, we found that members of the TGF-beta superfamily signal transduction pathways modulate the adipocyte/myocyte differentiation switch. Further analysis determined that recombinant TGF-beta inhibits adipogenesis and induces myogenesis simultaneously in a dose-dependent manner. Moreover, noggin induces differentiation into fully developed beating cardiac myocytes in vitro. These results provided new insight into the molecules that modulate the differentiation switch and validated a screening method for their identification.

Previous studies have revealed c-kit-positive (c-kit(+)) cardiac stem cells (CSCs) in the adult mammalian heart and these cells could be a suitable cell source for heart regeneration therapy. However, these cells have not been fully evaluated in terms of characterization and effect of long-term culture, which is necessary for their safe and optimal usage. Therefore, we isolated c-kit(+) CSCs from adult rat hearts to characterize these cells and investigate stability over long-term culture. We performed isolations of c-kit(+) CSCs 11 times and passaged them 40 times in a bulk culture system; we termed these cultures, bulk culture CSCs (CSC-BC). c-kit(+) CSCs expressed stemness genes and exhibited stem cell properties of single cell-derived clone formation, cardiosphere generation, and potential to differentiate into the three main cardiac lineages: cardiomyocyte, smooth muscle, and endothelial cells in vitro. Over long-term culture, some CSC-BC up-regulated GATA-4 expression, which resulted in enhanced cardiomyocyte differentiation, suggesting that the GATA-4 high c-kit(+) CSCs have potent cardiac regenerative potential. We also observed the spontaneous differentiation into cells other than cardiac lineages, such as adipocyte and skeletal myocyte. This effect of long-term culture on the c-kit(+) CSCs has not been previously reported. Interestingly, when c-kit(+) CSCs were co-cultured with adult rat cardiomyocytes, we found increased cardiomyocyte survival, and the growth factors, insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF), appeared to be responsible factors. The present study suggests that c-kit(+) CSCs have great therapeutic potential yet should be further investigated and optimized as a cell source for regenerative therapies prior to transplantation.

Here we report on a patient with multiple lentigines, hypertelorism, short stature, arachnodactyly, scoliosis, dissecting aneurysm, hypertrophic cardiomyopathy and developmental delay, and a family history of Marfan syndrome. The patient is affected with both Marfan and LEOPARD syndromes. Mutational screening of the FBN1 gene showed a c.1464TA (p.C488X) mutation and screening of the PTPN11 gene showed a c.836AG (p.Y279C) mutation. We conclude that each mutation contributed independently to individual features in the ocular and cardiovascular systems, although short stature was more significantly influenced by the p.Y279C change in PTPN11 rather than the mutation in FBN1. To our knowledge, this is the first report of mutations in both FBN1 and PTPN11 with combined phenotypes of Marfan and LEOPARD syndromes.

Congenital heart diseases (CHD) occur in nearly 1% of all live births and are the major cause of infant mortality and morbidity. Although an improved understanding of the genetic causes of CHD would provide insight into the underlying pathobiology, the genetic etiology of most CHD remains unknown. Here we show that mutations in the gene encoding the transcription factor GATA6 cause CHD characteristic of a severe form of cardiac outflow tract (OFT) defect, namely persistent truncus arteriosus (PTA). Two different GATA6 mutations were identified by systematic genetic analysis using DNA from patients with PTA. Genes encoding the neurovascular guiding molecule semaphorin 3C (SEMA3C) and its receptor plexin A2 (PLXNA2) appear to be regulated directly by GATA6, and both GATA6 mutant proteins failed to transactivate these genes. Transgenic analysis further suggests that, in the developing heart, the expression of SEMA3C in the OFT/subpulmonary myocardium and PLXNA2 in the cardiac neural crest contributing to the OFT is dependent on GATA transcription factors. Together, our data implicate mutations in GATA6 as genetic causes of CHD involving OFT development, as a result of the disruption of the direct regulation of semaphorin-plexin signaling.

A number of nuclear complexes modify chromatin structure and operate as functional units. However, the in vivo role of each component within the complexes is not known. ATP-dependent chromatin remodeling complexes form several types of protein complexes, which reorganize chromatin structure cooperatively with histone modifiers. Williams syndrome transcription factor (WSTF) was biochemically identified as a major subunit, along with 2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH, an ISWI-type complex. Here, WSTF −/− mice were generated to investigate its function in chromatin remodeling in vivo. Loss of WSTF expression resulted in neonatal lethality, and all WSTF −/− neonates and ≈10% of WSTF +/− neonates suffered cardiovascular abnormalities resembling those found in autosomal-dominant Williams syndrome patients. Developmental analysis of WSTF −/− embryos revealed that Gja5 gene regulation is aberrant from E9.5, conceivably because of inappropriate chromatin reorganization around the promoter regions where essential cardiac transcription factors are recruited. In vitro analysis in WSTF −/− mouse embryonic fibroblast (MEF) cells also showed impaired transactivation functions of cardiac transcription activators on the Gja5 promoter, but the effects were reversed by overexpression of WINAC components. Likewise in WSTF −/− MEF cells, recruitment of Snf2h, an ISWI ATPase, to PCNA and cell survival after DNA damage were both defective, but were ameliorated by overexpression of WICH components. Thus, the present study provides evidence that WSTF is shared and is a functionally indispensable subunit of the WICH complex for DNA repair and the WINAC complex for transcriptional control.

Using three different differentiation agents (1α, 25 dihydroxyvitamin D3, all-trans-retinoic acid, and Am80), down-regulation of telomerase activity was found to be a common response during the monocytic or granulocytic differentiation of human acute myeloblastic leukemia cell line 60 (HL60) cells. Rapid down-regulation of telomerase transcription occurred during early differentiation of HL60 cells prior to G1 arrest. Akt kinase activity was suppressed after 6 h of differentiation along with inhibition of telomerase activity, and the extent of the suppression that occurred while maintaining telomerase protein expression suggested the post-translational regulation of telomerase activity. Recombinant Akt dose-dependently increased telomerase activity, and telomerase was inhibited at the transcriptional and post-translational levels by LY294002, suggesting that PI-3K/Akt is one of the key signaling proteins involved in telomerase regulation. Each of the three differentiation agents caused a significant increase of signaling proteins (including Akt) at 3 days after the initiation of differentiation. Changes of acetyl-histone H4, which regulates transcription of the telomerase gene, were observed before the activation of Akt. This finding suggests that epigenetic control of telomerase transcription occurs before activation of Akt during the late stage of differentiation. These results indicate that telomerase activity is regulated by at least two mechanisms during granulocytic and monocytic differentiation, with one mechanism being transcriptional and the other being post-translational.

Chromosomal 8p23 deletion syndrome is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. The responsible gene for the heart defects in this syndrome has been identified as GATA4 on 8p23.1. Two patients with interstitial deletions of 8p23.1 were investigated; one patient showed moderate developmental delay and Ebstein anomaly, and the other showed mild delay and typical atrioventricular septum defect. The precise deletion sizes, 17 and 2.9 Mb, were determined by FISH analyses using BAC clones as probes. The latter deletion was the smallest deletion including GATA4 in the previously reported patients, and the critical regions and genes for clinical manifestation of 8p23 deletion syndrome, including facial anomalies, microcephaly, behavioral abnormality, and developmental delay, were discussed.

Background Mutations of the bone morphogenetic protein receptor II gene (BMPR2), and 1 mutation of the activin receptor-like kinase 1 gene (ALK1) have been reported in patients with pulmonary arterial hypertension (PAH). Methods and Results A genomic study of ALK1 and BMPR2 was conducted in 21 PAH probands under 16 years of age to study the relationship between the clinical features of the patients and these genes. In all 4 familial aggregates of PAH, 3 ALK1 or 1 BMPR2 mutations were identified. Among 17 probands aged between 4 and 14 years with idiopathic PAH, 2 ALK1 mutations (2/17: 11.8%) and 3 BMPR2 mutations (3/17: 17.6%; 5 mutations in total: 5/17: 29.4%) were found. Conclusion Each proband with the ALK1 mutation developed PAH, as did the probands with the BMPR2 mutation. Hence, it is proposed that ALK1 plays as notable a role as BMPR2 in the etiology of PAH. Furthermore, asymptomatic carriers with the ALK1 mutation within the serine - threonine kinase domain are at risk of developing PAH and hereditary hemorrhagic telangiectasia, so close follow-up is recommended for those individuals. (Circ J 2008; 72: 127 - 133)

Junctophilin subtypes, designated as JPH1 approximately 4, are protein components of junctional complexes and play essential roles in cellular Ca2+ signaling in excitable cells. Knockout mice lacking the cardiac-type Jph2 die of embryonic cardiac arrest, and the mutant cardiac myocytes exhibit impaired formation of peripheral couplings and arrhythmic Ca2+ signaling caused by functional uncoupling between dihydropyridine and ryanodine receptor channels. Based on these observations, we hypothesized that mutations of JPH2 could cause human genetic cardiac diseases. Among 195 Japanese patients (148 index cases and 47 affected family members) with hypertrophic cardiomyopathy (HCM), two heterozygous nonsynonymous nucleotide transitions, G505S and R436C, were newly found in JPH2. When Fisher's exact test was used to compare index cases with HCM to unrelated Japanese healthy controls in the frequencies of mutant alleles, only the G505S mutation showed statistical significance (4/296 HCM patients and 0/472 control individuals, P=0.022). This result was still significant after Bonferroni's correction for multiple comparisons (P=0.044). To the best of our knowledge, this is the first report on JPH2 mutation associated with HCM.

Oxygen-sensitive, voltage-gated potassium channels (Kv) may contribute to the determination of the membrane potential in smooth muscle cells of the ductus arteriosus (DA), and thus to regulation of contractile tone in response to oxygen. Developmental changes in Kv during gestation may be related to closure of the DA after birth. This study investigated developmental changes in the expression of Kv in the DA and compared it with that of the pulmonary artery (PA) and the aorta (Ao). The DA, PA, and Ao were isolated from fetal rats at days 19 and 21 of gestation (term: 21.5 days). The expression of Kv1.2, Kv1.5, Kv2.1, and Kv3.1, putative oxygen-sensitive Kv channels that open in response to oxygen, was evaluated at both the mRNA and protein levels, using quantitative real-time polymerase chain reaction and immunohistochemistry. In the Kv family studied, Kv1.5 mRNA was expressed most abundantly in the DA, PA, and Ao in both day-19 and day-21 fetuses. Although the expression levels of Kv1.2, Kv1.5, Kv2.1, and Kv3.1 did not change much with development in the PA and Ao, in the DA they decreased with development. The decrease in the expression of Kv channels may enhance DA closure after birth by eliminating the opening of Kv channels when oxygen increases.

The voltage-gated potassium channels (Kv) are partially responsible for the contraction/relaxation of blood vessels in response to changes in the Po(2) level. The present study determined the expression of Kvbeta1 and four oxygen-sensitive Kvalpha subunits (Kv1.2, Kv1.5, Kv2.1, and Kv9.3) in the ductus arteriosus (DA), the aorta (Ao), and the pulmonary artery (PA) in porcine neonates immediately after birth. We cloned three Kvbeta1 transcript variants (Kvbeta1.2, Kvbeta1.3, and Kvbeta1.4), Kv1.2, Kv1.5, and Kv9.3 from piglets. Three Kvbeta1 transcripts, Kv1.2, Kv1.5, and Kv9.3, encode predicted proteins of 401, 408, 202, 499, 600, and 491 residues. These Kv showed a high degree of sequence conservation with the corresponding Kv in human. Northern and quantitative real-time PCR (qr-PCR) analyses showed that Kvbeta1.2 expression was high in the DA and Ao but low in the PA. Kv1.5 expression was high in the Ao and PA but low in the DA. Expression of Kvbeta1.3, Kvbeta1.4, Kv1.2, Kv2.1, and Kv9.3 was low in these blood vessels. The inactivation property of Kvbeta1.2 against Kv1.5 was confirmed using Xenopus laevis oocytes. Our findings suggest that the molecular basis for the differential electrophysiological characteristics including opposing response to oxygen in the DA and the PA are partially due to diversity in expression of Kv1.5 and Kvbeta1.2 subunits. The high expression of Kvbeta1.2 and relatively low expression of Kv1.5 in the DA might be partially responsible for the ductal closure after birth.

Functional coupling between the sarcolemmal membrane and the sarcoplasmic reticulum is based on distinct structures called junctional membrane complexes (JMCs). Recently, junctophilins are found to be responsible for normal formation of JMCs. In the present study, we found that junctophilin type 2 (JP-2), a unique isoform in the heart, was localized in caveolin-rich membranes, and that the expression of JP-2 was up-regulated during normal development and down-regulated in a hypertrophic or a dilated cardiomyopathic mouse model. The expression levels of JP-2 may be associated with the development of T-tubules and impaired Ca2+-induced Ca2+ release in the heart.

To determine the pathogenicities of mutated mtDNAs in patients with respiration defects, the possible involvement of nuclear DNA mutations has to be excluded, since respiratory function is controlled by both nuclear DNA and mtDNA. This was achieved by showing that the mutated mtDNAs and respiration defects were co-transferred from patients to mtDNA-less human cells, and the resultant cybrid clones carrying mutated mtDNAs expressed respiration defects. To decide whether the cybrid clones expressed respiration defects, in this study the lowest limits of normal respiratory function were evaluated by transfer of mtDNAs from 54 normal subjects to mtDNA-less HeLa cells. The resultant cybrid clones showed that 71% respiratory function was the lowest limit of mtDNAs from normal subjects. On the other hand, cybrid clones carrying pathogenic mtDNAs from patients with mitochondrial diseases showed 0-64% respiratory function, suggesting that less than 71% respiratory function in cybrid clones should be a reliable indicator of whether the mutated mtDNAs of the patients were pathogenic.

Congenital heart defects (CHDs) are the most common developmental anomaly and are the leading non-infectious cause of mortality in newborns. Only one causative gene, NKX2-5, has been identified through genetic linkage analysis of pedigrees with non-syndromic CHDs. Here, we show that isolated cardiac septal defects in a large pedigree were linked to chromosome 8p22-23. A heterozygous G296S missense mutation of GATA4, a transcription factor essential for heart formation, was found in all available affected family members but not in any control individuals. This mutation resulted in diminished DNA-binding affinity and transcriptional activity of Gata4. Furthermore, the Gata4 mutation abrogated a physical interaction between Gata4 and TBX5, a T-box protein responsible for a subset of syndromic cardiac septal defects. Conversely, interaction of Gata4 and TBX5 was disrupted by specific human TBX5 missense mutations that cause similar cardiac septal defects. In a second family, we identified a frame-shift mutation of GATA4 (E359del) that was transcriptionally inactive and segregated with cardiac septal defects. These results implicate GATA4 as a genetic cause of human cardiac septal defects, perhaps through its interaction with TBX5.

Purpose: To identify the relationship between specific genes and phenotypic features of Williams syndrome. Methods: Subjects were selected based on their deletion status determined by fluorescence in situ hybridization using a panel of 24 BACs and cosmids spanning the region commonly deleted and single gene analysis using Southern blotting. From the cohort of subjects, three had atypical deletions. Physical examinations and cognitive tests were administered to the three subjects and the results were compared to those from a cohort of typical WS subjects. Results: The molecular results indicate smaller deletions for each subject. In all three cases, typical Williams facies were absent and visual spatial abilities were above that of full deletion WS subjects, particularly in the qualitative aspects of visual spatial processing. Conclusions: Combining the molecular analysis with the cognitive results suggest that the genes GTF2IRD1 and GTF2I contribute to deficits on visual spatial functioning.

Phospholamban is an endogenous inhibitor of sarcoplasmic reticulum calcium ATPase and plays a prime role in cardiac contractility and relaxation. Phospholamban may be a candidate gene responsible for cardiomyopathy. We investigated genome sequence of phospholamban in patients with cardiomyopathy. PCR-based direct sequence was performed for the promoter region and the whole coding region of phospholamban in 87 hypertrophic, 10 dilated, and 2 restricted cardiomyopathic patients. We found a heterozygous single nucleotide transition from A to G at −77-bp upstream of the transcription start site in the phospholamban promoter region of one patient with familial hypertrophic cardiomyopathy. This nucleotide change was not found in 296 control subjects. Using neonatal rat cardiomyocytes, the mutation, −77A→G, increased the phospholamban promoter activity. No nucleotide change in the phospholamban coding region was found in 99 patients with cardiomyopathy. We suspect that the mutation plays an important role in the development of hypertrophic cardiomyopathy.

Intracellular Ca2+ regulation is critical in the normal cardiac function and development of pathologic hearts. Phospholamban, an endogenous inhibitor of sarcoplasmic reticulum Ca2+ ATPase in the sarcoplasmic reticulum, plays an important role in Ca2+ cycling in heart. Recently, sarcolipin has been identified as having a similar function as phospholamban in skeletal muscle. Because phospholamban is differentially expressed in atrial and ventricular myocardia and its expression is often altered in diseased hearts, we investigated the cardiac chamber specificity of sarcolipin expression and its regulation during development and hypertrophic remodeling. Northern blot analysis revealed that the expression of mouse sarcolipin mRNA was most abundant in the atria and was undetectable in the ventricles, indicating an atrial chamber-specific expression pattern. Atrial chamber-specific expression of sarcolipin mRNA was increased during development. These findings were confirmed by in situ hybridization studies. In addition, sarcolipin expression was down-regulated in the atria of hypertrophic heart when induced by ventricular specific overexpression of the activated H-ras gene. In humans, sarcolipin mRNA was also expressed in the atria but not detected in the ventricles, although sarcolipin expression was most abundant in skeletal muscle. Taken together, sarcolipin is likely to be an atrial chamber-specific regulator of Ca2+ cycling in heart.

The mammalian heart is known to contain only two isoformic myosin heavy chain (MHC) genes, α and β. A previously uncharacterized MHC gene was isolated in Syrian hamster hearts (McCully et al., J Mol Biol 1991). We identified the novel MHC gene as a hamster embryonic skeletal MHC gene based on the developmental stage- and tissue-specific expression pattern: the restricted expression of mRNA to striated muscles was highest in embryonic skeletal muscle and was developmentally down-regulated. We confirmed that the embryonic skeletal MHC gene exhibited higher expression in cardiomyopathic than in normal hamster hearts, and was up-regulated during the development of cardiomyopathy. The sporadic expression was highly localized in the endocardium. The present study identified that a very small number of undifferentiated myogenic cells existed in adult hamster endocardium. Moreover, using RT-PCR, a homologue of embryonic skeletal MHC mRNA was also expressed in human embryonic, but not adult ventricles. Our data provide a new insight into the regulatory mechanisms of MHCs in the cardiomyopathic hamster heart.

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodelling of the heart. In rat cardiac ventricles, pressure and volume overload are well known to be associated with changes in cardiac myosin heavy chain (MHC) isoforms. To study the effects of HHE on the MHC profile in the ventricles, 83 male Wistar rats were housed in a chamber at the equivalent of 5500 m altitude for 1-8 weeks. Pulmonary arterial pressure, right ventricular free wall (RVFW) weight, the ratio of RVFW weight over body weight (BW), the ratio of left ventricular free wall (LVFW) weight over BW, and myocyte diameter in both ventricles showed significant increases after 1 week, 2 weeks, 1 week, 6 weeks, and 4 weeks of HHE, respectively. Semi-quantitative reverse transcriptase-polymerase chain reaction revealed that beta-MHC mRNA expression was increased significantly in both ventricles at 6 and 8 weeks of HHE, whereas alpha-MHC mRNA expression was decreased significantly at 6 and 8 weeks of HHE in the right ventricle (RV) and at 6 weeks of HHE in the left ventricle (LV). The percentage of myosin containing the beta-MHC isoform was increased significantly at 4-8 weeks of HHE in RV and at 6 weeks of HHE in LV. In situ hybridization showed that the area of strong staining for beta-MHC mRNA was increased in both ventricles at 8 weeks of HHE, and showed a decrease from RVFW to cardiac septum, and from cardiac septum to LVFW. These results suggest that HHE has a significant effect on the expression of both MHC mRNA and protein in the heart, particularly in RV. These changes may reflect a role for cardiac MHC in the response to pulmonary hypertension in HHE.

Background A high incidence of cardiovascular (CV) risk factors has been reported in adults with Williams-Beuren syndrome (WS). However, the prevalence of these factors in children and adolescents with WS is unknown. Therefore, the purpose of this study was to evaluate the prevalence of CV risk factors in these patients. Methods Thirty-two WS patients aged 0.65 mm) and low FMD (

The progression of cardiomyopathy to congestive heart failure is often associated with the expression of fetal cardiac-specific genes. In mice, the basic helix-loop-helix transcription factors, dHAND and eHAND , are expressed in a cardiac chamber-specific fashion and are essential for fetal cardiac development, but are down-regulated in the adult. Their expression in specific chambers of healthy and diseased human hearts has not been studied previously. Human dHAND and eHAND were mapped to human chromosomes 4q33 and 5q33, respectively, by fluorescent in situ hybridization. RNA from the four chambers of healthy human adult hearts, and from hearts of patients with several forms of cardiomyopathy, was obtained and assayed for dHAND and eHAND expression. Unlike in mice, dHAND expression was observed in all four chambers of the healthy human adult heart, but was diminished in the right atrium. In contrast, eHAND was expressed in the right and left ventricles, but was downregulated in both atrial chambers. We examined tissue from 15 human cardiomyopathic hearts obtained during cardiac transplantation or by endomyocardial biopsy for alterations in HAND gene expression. dHAND expression was unchanged in all forms of cardiomyopathy tested. However, cardiac expression of eHAND was severely down-regulated in six of six patients with ischemic cardiomyopathy and six of six patients with dilated cardiomyopathy. This study demonstrates that human dHAND and eHAND have unique spatial patterns of expression within human cardiac chambers. Downregulation of eHAND in ischemic and dilated cardiomyopathy suggests a correlation between eHAND dysregulation and the evolution of a subset of cardiomyopathies.

Objectives The current study evaluated the hypoxic induction of adrenomedullin gene expression and secretion, and its mechanism in cultured human umbilical vein endothelial cells (HUVEC). Methods HUVEC were exposed to hypoxia or normoxia as controls for 1 to 24 h. Using Northern blot analysis and a radioimmunoassay, we evaluated adrenomedullin expression in HUVEC. The transcriptional component of adrenomedullin gene regulation was assessed by nuclear run-off experiments, and adrenomedullin mRNA half-life was measured by actinomycin D experiments. Results We found that hypoxic conditions (1 -3% oxygen) significantly increased adrenomedullin mRNA and protein in HUVEC. This increase was inversely proportional to oxygen tension and was reversible upon re-exposure to a 21% oxygen environment Nuclear run-off experiments revealed the enhanced transcriptional rate of adrenomedullin gene. Next, actinomycin D experiments revealed the enhanced adrenomedullin mRNA stability. Conclusions These results indicate that hypoxia increases adrenomedullin gene expression and secretion in HUVEC by transcriptional and post-transcriptional mechanisms. Hypoxic induction of adrenomedullin may play a pathophysiological role in the vascular systems.