Hot water extract of Agaricus blazei Murrill specifically inhibits growth and induces apoptosis in human pancreatic cancer cells.

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.

Prognostic predictive value of gene mutations in Japanese patients with hypertrophic cardiomyopathy.

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 ß-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.

Compound Mutations Cause Increased Cardiac Events in Children with Long QT Syndrome: Can the Sequence Homology-Based Tools be Applied for Prediction of Phenotypic Severity?

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.

Germline gain-of-function mutations in RAF1 cause Noonan syndrome.

Noonan syndrome is characterized by short stature, facial dysmorphia and a wide spectrum of congenital heart defects. Mutations of PTPN11, KRAS and SOS1 in the RAS-MAPK pathway cause approximately 60% of cases of Noonan syndrome. However, the gene(s) responsible for the remainder are unknown. We have identified five different mutations in RAF1 in ten individuals with Noonan syndrome; those with any of four mutations causing changes in the CR2 domain of RAF1 had hypertrophic cardiomyopathy (HCM), whereas affected individuals with mutations leading to changes in the CR3 domain did not. Cells transfected with constructs containing Noonan syndrome-associated RAF1 mutations showed increased in vitro kinase and ERK activation, and zebrafish embryos with morpholino knockdown of raf1 demonstrated the need for raf1 for the development of normal myocardial structure and function. Thus, our findings implicate RAF1 gain-of-function mutations as a causative agent of a human developmental disorder, representing a new genetic mechanism for the activation of the MAPK pathway.

High prevalence of cardiovascular risk factors in children and adolescents with Williams-Beuren syndrome.

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 <18 years were enrolled in the study. Oxidized low-density lipoprotein levels (n = 32), oral glucose tolerance test results (n = 20), plasma renin and aldosterone levels (n = 31), 24-h ambulatory blood pressure (ABP; n = 24), carotid artery intima-media thickness (IMT; n = 15), and brachial artery flow-mediated dilatation (FMD; n = 15) were measured and analyzed. RESULTS: The lipid profile revealed hypercholesterolemia in 22% and elevated oxidized low-density lipoprotein levels in 94% of the patients. Glucose metabolism abnormalities were found in 70% of the patients. Insulin resistance was observed in 40% of the patients. High plasma renin and aldosterone levels were detected in 45 and 39% of the patients, respectively. A mean systolic blood pressure above the 90th percentile was noted in 29% of patients. High IMT (>0.65 mm) and low FMD (<9%) were detected in 80 and 73% of patients, respectively. CONCLUSION: In patients with WS, CV risk factors are frequently present from childhood. In children with WS, screening tests for the early detection of CV risk factors and long-term follow-up are required to determine whether long-term exposure to these factors increases the risk for CV events in adulthood.

Ripply3, a Tbx1 repressor, is required for development of the pharyngeal apparatus and its derivatives in mice.

The pharyngeal apparatus is a transient structure that gives rise to the thymus and the parathyroid glands and also contributes to the development of arteries and the cardiac outflow tract. A typical developmental disorder of the pharyngeal apparatus is the 22q11 deletion syndrome (22q11DS), for which Tbx1 is responsible. Here, we show that Ripply3 can modulate Tbx1 activity and plays a role in the development of the pharyngeal apparatus. Ripply3 expression is observed in the pharyngeal ectoderm and endoderm and overlaps with strong expression of Tbx1 in the caudal pharyngeal endoderm. Ripply3 suppresses transcriptional activation by Tbx1 in luciferase assays in vitro. Ripply3-deficient mice exhibit abnormal development of pharyngeal derivatives, including ectopic formation of the thymus and the parathyroid gland, as well as cardiovascular malformation. Corresponding with these defects, Ripply3-deficient embryos show hypotrophy of the caudal pharyngeal apparatus. Ripply3 represses Tbx1-induced expression of Pax9 in luciferase assays in vitro, and Ripply3-deficient embryos exhibit upregulated Pax9 expression. Together, our results show that Ripply3 plays a role in pharyngeal development, probably by regulating Tbx1 activity.

Soluble ST2 and N-terminal pro-brain natriuretic peptide combination. Useful biomarker for predicting outcome of childhoodpulmonary arterial hypertension.

BACKGROUND: Some potential biomarkers have been reported recently in patients with pulmonary arterial hypertension (PAH), but the most clinically useful among these potential biomarkers, especially in childhood PAH, has not been identified. Therefore, this study investigated which biomarker is useful in assessing severity of and patient prognosis in childhood idiopathic PAH (IPAH)/heritable PAH (HPAH). METHODS AND RESULTS: Fifty-nine patients who were younger than 16 years at onset of IPAH/HPAH were selected. The following 10 biomarker candidates were quantified: high-sensitivity troponin T, human heart fatty acid-binding protein, N-terminal pro-brain natriuretic peptide (NT-proBNP), pentraxin-3, soluble ST2 (sST2), angiopoietin-2 (Ang-2), matrix metalloproteinase 2, tenascin C, endostatin (ES), and thymidine kinase. Functional characteristics and clinical outcomes were analyzed retrospectively. NT-proBNP, sST2, Ang-2, and ES correlated well with New York Heart Association class. On area under the receiver operating characteristic curve analysis, sST2 had a significantly good relationship with prognosis. On Kaplan-Meier curve and univariate Cox regression analyses, elevated sST2 and NT-proBNP level predicted poor outcome of the present patients with childhood IPAH/HPAH. Furthermore, patients with elevated sST2 had significantly worse prognosis among those with high NT-proBNP. CONCLUSIONS: The sST2 and NT-proBNP combination is a useful biomarker to predict clinical condition and outcome in patients with childhood IPAH/HPAH.

Mutations in the cardiac troponin T gene show various prognoses in Japanese patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder resulting from mutations in genes for at least 15 various sarcomere-related proteins including cardiac ß-myosin heavy chain, cardiac myosin-binding protein C, and cardiac troponin T. The troponin T gene (TNNT2) mutation has the third incidence of familial HCM, and the genotype-phenotype correlation of this gene still remains insufficient in Japanese familial HCM. Therefore, in the present study, we focused on screening the TNNT2 mutation in 173 unrelated Japanese patients with familial HCM, and found three reported mutations and a new mutation of TNNT2 in 11 individuals from four families. In these families, two individuals from one family had double mutations, Arg130Cys and Phe110Ile, six individuals from two other families had an Arg92Trp mutation, and one individual of another family had a new mutation, Ile79Thr, of TNNT2. The phenotype of each family was often different from reported cases, even if they had the same genetic mutation. In addition, families with the same genetic mutation showed a similar trend in the phenotype, but it was not exactly the same. However, sudden death in youth was observed in all of these families. Although the type of genetic mutation is not useful for predicting prognosis in HCM, the possibility of sudden cardiac death remains. Therefore, the prognosis of individuals bearing the TNNT2 mutation with familial HCM should be more carefully observed from birth.

Characterization of a novel KRAS mutation identified in Noonan syndrome.

Noonan syndrome (NS) is the most common non-chromosomal syndrome seen in children and is characterized by short stature, dysmorphic facial features, chest deformity, a wide range of congenital heart defects and developmental delay of variable degree. Mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathways cause about 70% of NS cases with a KRAS mutation present in about 2%. In a cohort of 65 clinically confirmed NS patients of Japanese origin, we screened for mutations in the RAS genes by direct sequencing. We found a novel mutation in KRAS with an amino acid substitution of asparagine to serine at codon 116 (N116S). We analyzed the biological activity of this mutant by ectopic expression of wild-type or mutant KRAS. NS-associated KRAS mutation resulted in Erk activation and active Ras-GTP levels, and exhibited mild cell proliferation. In addition, kras-targeted morpholino knocked-down zebrafish embryos caused heart and craniofacial malformations, while the expression of mutated kras resulted in maldevelopment of the heart. Our findings implicate that N116S change in KRAS is a hyperactive mutation which is a causative agent of NS through maldevelopment of the heart.

Genetic analysis of essential cardiac transcription factors in 256 patients with non-syndromic congenital heart defects.

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.

GATA6 mutations cause human cardiac outflow tract defects by disrupting semaphorin-plexin signaling.

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.

Distinct function of 2 chromatin remodeling complexes that share a common subunit, Williams syndrome transcription factor (WSTF).

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 approximately 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.

Myogenic differentiation in atrium-derived adult cardiac pluripotent cells and the transcriptional regulation of GATA4 and myogenin on ANP promoter.

We established cardiac pluripotent stem-like cells from the left atrium (LA-PCs) of adult rat hearts. These cells could differentiate not only into beating myocytes but also into cells of other lineages, including adipocytes and endothelial cells in the methylcellulose-based medium containing interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF). In particular, IL-3 and SCF contributed to the differentiation into cardiac troponin I-positive cells. Notably, small population of LA-PCs coexpressed GATA4 and myogenin, which are markers specific to cardiomyocytes and skeletal myocytes, respectively, and could differentiate into both cardiac and skeletal myocytes. Therefore, we investigated the involvement of these two tissue-specific transcription factors in the cardiac transcriptional activity. Coexpression of GATA4 and myogenin synergistically activated GATA4-specific promoter of the atrial natriuretic peptide gene. This combinatorial function was shown to be dependant on the GATA site, but independent of the E-box. The results of chromatin immunoprecipitation and electrophoretic mobility shift assays suggested that myogenin bound to GATA4 on the GATA elements and the C-terminal Zn-finger domain of GATA4 and the N-terminal region of myogenin were required for this synergistic activation of transcription. Taken together, these two transcription factors could be involved in the myogenesis of LA-PCs.

Identification of a novel TPM1 mutation in a family with left ventricular noncompaction and sudden death.

Left ventricular noncompaction (LVNC) is a cardiomyopathy morphologically characterized by 2-layered myocardium, numerous prominent trabeculations, and deep intertrabecular recesses communicating with the left ventricular cavity. The purpose of this study was to investigate patients with LVNC for possible disease causing mutations. We screened 4 genes (TAZ, LDB3, DTNA and TPM1) in 51 patients with LVNC for mutations by polymerase chain reaction and direct DNA sequencing. A novel missense substitution in exon 1 of TPM1 (c.109A>G: p.Lys37Glu) was identified in three affected members of a family with isolated LVNC. The substitution brings about a change in amino acid charge at a highly conserved residue and could result in aberrant mRNA splicing. This variant was not identified in 200 normal control samples. Pathologic analysis of a right ventricular myocardial specimen from the proband's maternal aunt revealed endocardial and subendocardial fibrosis with prominent elastin deposition, as well as the presence of adipose tissue between muscle layers, pathologic changes that are distinct from those seen in patients with HCM or DCM. Screening of the proband and her mother for variants in other sarcomeric protein-encoding candidate genes, MYH7, MYBPC3, TNNT2, TNNI3, ACTC, MYL2, and MYL3, did not identify any other non-synonymous variants or variants in splice donor-acceptor sequences that were potentially disease causing. We conclude TPM1 is a potential candidate disease-causing gene for isolated LVNC, especially in patients experiencing sudden death.

The impact of cardiac surgery in patients with trisomy 18 and trisomy 13 in Japan.

Congenital heart defects (CHD) are very common in patients with trisomy 18 (T18) and trisomy 13 (T13). The surgical indication of CHD remains controversial since the natural history of these trisomies is documented to be poor. To investigate the outcome of CHD in patients with T18 and T13, we collected and evaluated clinical data from 134 patients with T18 and 27 patients with T13 through nationwide network of Japanese Society of Pediatric Cardiology and Cardiac Surgery. In patients with T18, 23 (17%) of 134 were alive at this survey. One hundred twenty-six (94%) of 134 patients had CHDs. The most common CHD was ventricular septal defect (VSD, 59%). Sixty-five (52%) of 126 patients with CHD developed pulmonary hypertension (PH). Thirty-two (25%) of 126 patients with CHD underwent cardiac surgery and 18 patients (56%) have survived beyond postoperative period. While palliative surgery was performed in most patients, six cases (19%) underwent intracardiac repair for VSD. Operated patients survived longer than those who did not have surgery (P < 0.01). In patients with T13, 5 (19%) of 27 patients were alive during study period. Twenty-three (85%) of 27 patients had CHD and 13 (57%) of 27 patients had PH. Atrial septal defect was the most common form of CHD (22%). Cardiac surgery was done in 6 (26%) of 23 patients. In this study, approximately a quarter of patients underwent surgery for CHD in both trisomies. Cardiac surgery may improve survival in selected patients with T18.

Safety and efficacy of using heat-killed Lactobacillus plantarum L-137: High-dose and long-term use effects on immune-related safety and intestinal bacterial flora.

Heat-killed Lactobacillus plantarum L-137 (HK L-137) promotes immune function in animals. In healthy people, T-cell proliferation was shown to be enhanced by taking 10 mg HK L-137 daily for 12 weeks. However, the safety and efficacy of higher doses or longer treatments have not yet been investigated in humans. To investigate the high-dose and long-term use effects of HK L-137 on immune-related safety and on host intestinal bacterial flora, 15 healthy volunteers took a daily HK L-137 (50 mg) preparation for 4 weeks. An additional 29 participants who regularly visited a clinic for health care took HK L-137 (10 mg) daily for 12 months. Measures for anthropometrics, hematology, biochemistry, and urinalysis were taken at scheduled timepoints for all participants. Stool and blood samples were also collected and evaluated for microbes and short-chain fatty acids (SCFA); isolated T-cells were assessed for levels of proliferation induced by phytohemagglutinin in the long-term study. Adverse events or shifts in clinical measures from normal ranges due to the dietary intervention were not observed in the high-dose or long-term studies. Long-term intake also did not result in immune exhaustion due to any chronic immunostimulation; ex vivo T-cell proliferation was significantly greater at 12 months than at baseline (p < 0.01). In addition, the Firmicutes/Bacteroidetes ratio in stool samples was significantly lower at 12 months than at baseline (p < 0.05) due to the long-term intake of the HK L-137. Lastly, fecal SCFA concentrations were significantly greater (p < 0.05) at 6 months than at baseline. From these data, it can be concluded that the efficacy of HK L-137 is maintained with no overt adverse effects as a result of high-dose and/or long-term consumption.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes with SCN5A R1623Q Mutation Associated with Severe Long QT Syndrome in Fetuses and Neonates Recapitulates Pathophysiological Phenotypes.

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.

TGF-beta superfamily regulates a switch that mediates differentiation either into adipocytes or myocytes in left atrium derived pluripotent cells (LA-PCS).

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.

Akt and PKC are involved not only in upregulation of telomerase activity but also in cell differentiation-related function via mTORC2 in leukemia cells.

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/ßII 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.