Cyclin-dependent kinase-9 is a component of the p300/GATA4 complex required for phenylephrine-induced hypertrophy in cardiomyocytes.

A zinc finger protein GATA4 is one of the hypertrophy-responsive transcription factors and forms a complex with an intrinsic histone acetyltransferase, p300. Disruption of this complex results in the inhibition of cardiomyocyte hypertrophy and heart failure in vivo. By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9) as a novel GATA4-binding partner. Cdk9 also formed a complex with p300 as well as GATA4 and cyclin T1. We showed that p300 was required for the interaction of GATA4 with Cdk9 and for the kinase activity of Cdk9. Conversely, Cdk9 kinase activity was required for the p300-induced transcriptional activities, DNA binding, and acetylation of GATA4. Furthermore, the kinase activity of Cdk9 was required for the phosphorylation of p300 as well as for cardiomyocyte hypertrophy. These findings demonstrate that Cdk9 forms a functional complex with the p300/GATA4 and is required for p300/GATA4- transcriptional pathway during cardiomyocyte hypertrophy.

Cyclin-dependent kinase 9 forms a complex with GATA4 and is involved in the differentiation of mouse ES cells into cardiomyocytes.

The treatment of ES cells with trichostatin A (TSA), an HDAC inhibitor, induces the acetylation of GATA4 as well as histones, and facilitates their differentiation into cardiomyocytes. Recently, we demonstrated that cyclin-dependent kinase 9 (Cdk9), a core component of positive elongation factor-b, is a novel GATA4-binding partner. The present study examined whether Cdk9 forms a complex with GATA4 in mouse ES cells and is involved in their differentiation into cardiomyocytes. Mouse ES cells and Nkx2.5/GFP ES cells, in which green fluorescent protein (GFP) is expressed under the control of the cardiac-specific Nkx2.5 promoter, were induced to differentiate on feeder-free gelatin-coated plates. Immunoprecipitation/Western blotting in nuclear extracts from mouse ES cells demonstrated that Cdk9 as well as cyclin T1 interact with GATA4 during myocardial differentiation. TSA treatment increased Nkx2.5/GFP-positive cells and endogenous mRNA levels of Nkx2.5 and atrial natriuretic factor. To determine the role of Cdk9 in myocardial cell differentiation, we examined the effects of a dominant-negative form of Cdk9 (DN-Cdk9), which loses its kinase activity, and a Cdk9 kinase inhibitor, 5,6-dichloro-1-ß-ribofuranosyl-benzimidazole (DRB) on TSA-induced myocardial cell differentiation. The introduction of the DN-Cdk9 inhibited TSA-induced increase in GFP expression in Nkx2.5/GFP ES cells. The administration of DRB into ES cells significantly inhibited TSA-induced increase of endogenous Nkx2.5 mRNA levels in ES cells as well as GFP expression in Nkx2.5/GFP ES cells. These findings demonstrate that Cdk9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4.

Long-term outcome of sirolimus-eluting stent implantation for left main coronary artery stenosis in infancy.

Drug-eluting stents (DES) have markedly reduced the incidence of coronary in-stent restenosis. However, DES may be still vulnerable to coronary thrombus, and the long-term outcome remains unknown, especially in infancy. Here, we present a 9-month-old infant, who developed severe stenosis of the left main coronary artery after surgery for Brand-White-Garland syndrome, and was successfully treated with DES. He was healthy and his cardiac function had improved to the normal level at 6 years old. An angiographic examination and computed tomography showed the complete persistence of DES and no evidence of intimal thickening.

Neonatal mouse testis-derived multipotent germline stem cells improve the cardiac function of acute ischemic heart mouse model.

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. We previously reported that undifferentiated mGS cells are phenotypically similar to embryonic stem cells and that fetal liver kinase 1 (Flk1)(+) mGS cells have a similar potential to differentiate into cardiomyocytes and endothelial cells compared with Flk1(+) embryonic stem cells. Here, we transplanted these Flk1(+) mGS cells into an ischemic heart failure mouse model to evaluate the improvement in cardiac function. Significant increase in left ventricular wall thickness of the infarct area, left ventricular ejection fraction and left ventricular maximum systolic velocity was observed 4weeks after when sorted Flk1(+) mGS cells were transplanted directly into the hearts of the acute ischemic model mice. Although the number of cardiomyocytes derived from Flk1(+) mGS cells were too small to account for the improvement in cardiac function but angiogenesis around ischemic area was enhanced in the Flk1(+) mGS cells transplanted group than the control group and senescence was also remarkably diminished in the early phase of ischemia according to ß-galactosidase staining assay. In conclusion, Flk1(+) mGS cell transplantation can improve the cardiac function of ischemic hearts by promoting angiogenesis and by delaying host cell death via senescence.

The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells.

Developing effective drug therapies for arrhythmic diseases is hampered by the fact that the same drug can work well in some individuals but not in others. Human induced pluripotent stem (iPS) cells have been vetted as useful tools for drug screening. However, cardioactive drugs have not been shown to have the same effects on iPS cell-derived human cardiomyocytes as on embryonic stem (ES) cell-derived cardiomyocytes or human cardiomyocytes in a clinical setting. Here we show that current cardioactive drugs affect the beating frequency and contractility of iPS cell-derived cardiomyocytes in much the same way as they do ES cell-derived cardiomyocytes, and the results were compatible with empirical results in the clinic. Thus, human iPS cells could become an attractive tool to investigate the effects of cardioactive drugs at the individual level and to screen for individually tailored drugs against cardiac arrhythmic diseases.

MicroRNA-1 and MicroRNA-133 in spontaneous myocardial differentiation of mouse embryonic stem cells.

BACKGROUND: MicroRNAs (miRNAs) regulate various biological processes through inhibiting the translation of RNA transcripts. Although miRNA-1 (miR-1) and miRNA-133 (miR-133) are abundantly expressed in the adult heart and involved in cardiac hypertrophy, the roles of these miRNAs in spontaneous myocardial differentiation are unknown. METHODS AND RESULTS: The levels of miR-1 and miR-133 in mouse embryonic stem (ES) cells were increased during spontaneous differentiation by 2-dimensional culture, but reduced during forced myocardial differentiation by a histone deacetylase inhibitor, trichostatin A. The overexpression of miR-1 or miR-133 by lentiviral infection reduced the expression of a cardiac-specific gene, Nkx2.5, during differentiation of ES cells. In addition, miR-1 also inhibited alpha-myosin heavy chain expression. The results of luciferase assays revealed that miR-1 recognizes and targets the 3' untranslated region of cyclin-dependent kinase-9 (Cdk9) in ES cells. Overexpression of miR-1 decreased the protein amounts of Cdk9 without affecting the mRNA levels, indicating that miR-1 post-transcriptionally inhibits Cdk9 translation. CONCLUSIONS: miR-1 and miR-133 may play significant roles in the myocardial differentiation of mouse ES cells, and Cdk9 may be involved in this process as a target of miR-1.

Single-Needle Intensive Granulocyte and Monocyte Adsorptive Apheresis Is Suitable for Elderly Patients With Active Ulcerative Colitis Taking no Corticosteroids or Biologics.

Twice-weekly intensive granulocyte/monocyte adsorptive apheresis is effective and safe for ulcerative colitis, but maintaining two blood access routes is problematic. We previously reported that intensive granulocyte/monocyte adsorptive apheresis using a single needle in ulcerative colitis is effective and safe. We hypothesized that the efficacy and safety of single-needle intensive granulocyte/monocyte adsorptive apheresis for ulcerative colitis would especially benefit the elderly. We enrolled 17 elderly ulcerative colitis patients to receive single-needle intensive granulocyte/monocyte adsorptive apheresis, 27 elderly ulcerative colitis patients to receive double-needle intensive granulocyte/monocyte adsorptive apheresis, and 52 nonelderly ulcerative colitis patients to receive single-needle intensive granulocyte/monocyte adsorptive apheresis. Remission and mucosal healing rates after treatment did not differ significantly between elderly ulcerative colitis patients receiving single-needle apheresis and the other two groups. In addition, no serious adverse effects, including blood clots, were observed in single-needle intensive granulocyte/monocyte adsorptive apheresis patients. Single-needle intensive granulocyte/monocyte adsorptive apheresis might be a novel alternative therapeutic option for elderly ulcerative colitis patients before considering corticosteroids.

Effective cardiac resynchronization therapy for an adolescent patient with dilated cardiomyopathy seven years after mitral valve replacement and septal anterior ventricular exclusion.

Cardiac resynchronization therapy (CRT) is a new treatment for refractory heart failure. However, most heart failure patients treated with CRT are middle-aged or old patients with idiopathic or ischemic dilated cardiomyopathy. We treated a 17 year 11 month old girl with dilated cardiomyopathy after mitral valve replacement (MVR) and septal anterior ventricular exclusion (SAVE). Seven years after the SAVE procedure, she presented complaining of palpitations and general fatigue with normal activity. Her echocardiogram showed reduced left ventricular function. Despite of optimal medical therapy, her left ventricular function continued to decline and she experienced regular arrhythmias such as premature ventricular contractions. We thus elected to perform cardiac resynchronization therapy with defibrillator (CRT-D). After CRT-D, her clinical symptoms improved dramatically and left ventricular ejection fraction (LVEF) improved from 31.2% to 51.3% as assessed by echocardiogram. Serum BNP levels decreased from 448.2 to 213.6 pg/ml. On ECG, arrhythmias were remarkably reduced and QRS duration was shortened from 174 to 152 msec. In conclusion, CRT-D is an effective therapeutic option for adolescent patients with refractory heart failure after left ventricular volume reduction surgery.

Cell line-dependent differentiation of induced pluripotent stem cells into cardiomyocytes in mice.

AIMS: Mouse and human fibroblasts can be directly reprogrammed to pluripotency by the ectopic expression of four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) to yield induced pluripotent stem (iPS) cells. iPS cells can be generated even without the expression of c-Myc. The present study examined patterns of differentiation of mouse iPS cells into cardiomyocytes in three different cell lines reprogrammed by three or four factors. METHODS AND RESULTS: During the induction of differentiation on feeder-free gelatinized dishes, genes involved in cardiogenesis were expressed as in embryonic stem cells and myogenic contraction occurred in two iPS cell lines. However, in one iPS cell line (20D17) generated by four factors, the expression of cardiac-specific genes and the beating activity were extremely low. Treating iPS cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased Nkx2.5 expression in all iPS cell lines. While the basal Nkx2.5 expression was very low in 20D17, the TSA-induced increase was the greatest. TSA also induced the expression of contractile proteins in 20D17. Furthermore, we demonstrated the increased mRNA level of Oct3/4 and nuclear protein level of HDAC4 in 20D17 compared with the other two iPS cell lines. DNA microarray analysis identified genes whose expression is up- or down-regulated in 20D17. CONCLUSIONS: Mouse iPS cells differentiate into cardiomyocytes in a cell line-dependent manner. TSA induces myocardial differentiation in mouse iPS cells and might be useful to overcome cell line variation in the differentiation efficiency.

Generation of cardiac and endothelial cells from neonatal mouse testis-derived multipotent germline stem cells.

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. Here, we compared mGS, embryonic stem (ES), and embryonic germ (EG) cells with regard to their ability to differentiate into mesodermal cells, namely, cardiomyocytes and endothelial cells. The in situ morphological appearances of undifferentiated mGS, ES, and EG cells were similar, and 4 days after being induced to differentiate, approximately 30%-40% of each cell type differentiated into Flk1(+) cells. The sorted Flk1(+) cells differentiated efficiently into cardiomyocytes and endothelial cells. By day 10 after differentiation induction, the three cell types generated equal number of endothelial colonies. However, by day 13 after differentiation induction, the Flk1(+) mGS cells generated more contractile colonies than did the Flk1(+) ES cells, whereas the Flk1(+) EG cells generated equivalent numbers as the Flk1(+) mGS cells. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of differentiation markers such as Rex1, FGF-5, GATA-4, Brachyury, and Flk1 revealed that mGS cells expressed these markers more slowly during days 0-4 after differentiation induction than did ES cells, but that this mGS cell pattern was similar to that of the EG cells. RT-PCR analysis also revealed that the three differentiation cell types expressed various cardiac markers. Moreover, immunohistochemical analysis revealed that the contractile colonies derived from Flk1(+) mGS cells express mature cardiac cell-specific markers. In conclusion, mGS cells are phenotypically similar to ES and EG cells and have a similar potential to differentiate into cardiomyocytes and endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.