Takeshi Onizuka

G-CSF Promotes the Proliferation of Developing Cardiomyocytes In Vivo and in Derivation from ESCs and iPSCs

During a screen for humoral factors that promote cardiomyocyte differentiation from embryonic stem cells (ESCs), we found marked elevation of granulocyte colony-stimulating factor receptor (G-CSFR) mRNA in developing cardiomyocytes. We confirmed that both G-CSFR and G-CSF were specifically expressed in embryonic mouse heart at the midgestational stage, and expression levels were maintained throughout embryogenesis. Intrauterine G-CSF administration induced embryonic cardiomyocyte proliferation and caused hyperplasia. In contrast, approximately 50% of csf3r(-/-) mice died during late embryogenesis because of the thinning of atrioventricular walls. ESC-derived developing cardiomyocytes also strongly expressed G-CSFR. When extrinsic G-CSF was administered to the ESC- and human iPSC-derived cardiomyocytes, it markedly augmented their proliferation. Moreover, G-CSF-neutralizing antibody inhibited their proliferation. These findings indicated that G-CSF is critically involved in cardiomyocyte proliferation during development, and may be used to boost the yield of cardiomyocytes from ESCs for their potential application to regenerative medicine.

Nerve Growth Factor Is Critical for Cardiac Sensory Innervation and Rescues Neuropathy in Diabetic Hearts

Background— Molecular mechanisms regulating the cardiac sensory nervous system remain poorly understood. Cardiac sensory nerve impairment causes silent myocardial ischemia, a main cause of sudden death in diabetes mellitus (DM). The present study focused on the roles of nerve growth factor (NGF) in the regulation of the cardiac sensory nervous system and analyzed the mechanism of silent myocardial ischemia in DM. Methods and Results— We screened neurotrophic factors and found that cardiac sensory nerves developed in parallel with NGF synthesized in the heart. Cardiac nociceptive sensory nerves that were immunopositive for calcitonin gene-related peptide, dorsal root ganglia (DRG), and the dorsal horn were markedly retarded in NGF-deficient mice, whereas cardiac-specific overexpression of NGF rescued these deficits. DM was induced with streptozotocin in wild-type and transgenic mice overexpressing NGF in the heart. Downregulation of NGF, calcitonin gene-related peptide–immunopositive cardiac sensory denervation, and atrophic changes in DRG were observed in DM-induced wild-type mice, whereas these deteriorations were reversed in DM-induced NGF transgenic mice. Cardiac sensory function, measured by myocardial ischemia–induced c-Fos expression in DRG, was also downregulated by DM in the wild-type mice but not in NGF transgenic mice. Direct gene transfer of NGF in the diabetic rat hearts improved impaired cardiac sensory innervation and function, determined by electrophysiological activity of cardiac afferent nerves during myocardial ischemia. Conclusions— These findings demonstrate that the development and regulation of the cardiac sensory nervous system are dependent on NGF synthesized in the heart and that DM-induced NGF reduction causes cardiac sensory neuropathy.

G-CSF influences mouse skeletal muscle development and regeneration by stimulating myoblast proliferation

Granulocyte colony-stimulating factor and its receptor are needed for skeletal muscle development and injury-induced regeneration in mice.

Zac1 Is an Essential Transcription Factor for Cardiac Morphogenesis

Rationale: The transcriptional networks guiding heart development remain poorly understood, despite the identification of several essential cardiac transcription factors. Objective: To isolate novel cardiac transcription factors, we performed gene chip analysis and found that Zac1, a zinc finger–type transcription factor, was strongly expressed in the developing heart. This study was designed to investigate the molecular and functional role of Zac1 as a cardiac transcription factor. Methods and Results: Zac1 was strongly expressed in the heart from cardiac crescent stages and in the looping heart showed a chamber-restricted pattern. Zac1 stimulated luciferase reporter constructs driven by ANF, BNP, or &agr;MHC promoters. Strong functional synergy was seen between Zac1 and Nkx2-5 on the ANF promoter, which carries adjacent Zac1 and Nkx2-5 DNA-binding sites. Zac1 directly associated with the ANF promoter in vitro and in vivo, and Zac1 and Nkx2-5 physically associated through zinc fingers 5 and 6 in Zac1, and the homeodomain in Nkx2-5. Zac1 is a maternally imprinted gene and is the first such gene found to be involved in heart development. Homozygous and paternally derived heterozygous mice carrying an interruption in the Zac1 locus showed decreased levels of chamber and myofilament genes, increased apoptotic cells, partially penetrant lethality and morphological defects including atrial and ventricular septal defects, and thin ventricular walls. Conclusions: Zac1 plays an essential role in the cardiac gene regulatory network. Our data provide a potential mechanistic link between Zac1 in cardiogenesis and congenital heart disease manifestations associated with genetic or epigenetic defects in an imprinted gene network.

Common marmoset embryonic stem cell can differentiate into cardiomyocytes.

Common marmoset monkeys have recently attracted much attention as a primate research model, and are preferred to rhesus and cynomolgus monkeys due to their small bodies, easy handling and efficient breeding. We recently reported the establishment of common marmoset embryonic stem cell (CMESC) lines that could differentiate into three germ layers. Here, we report that our CMESC can also differentiate into cardiomyocytes and investigated their characteristics. After induction, FOG-2 was expressed, followed by GATA4 and Tbx20, then Nkx2.5 and Tbx5. Spontaneous beating could be detected at days 12-15. Immunofluorescent staining and ultrastructural analyses revealed that they possessed characteristics typical of functional cardiomyocytes. They showed sinus node-like action potentials, and the beating rate was augmented by isoproterenol stimulation. The BrdU incorporation assay revealed that CMESC-derived cardiomyocytes retained a high proliferative potential for up to 24 weeks. We believe that CMESC-derived cardiomyocytes will advance preclinical studies in cardiovascular regenerative medicine.

Distinct iPS Cells Show Different Cardiac Differentiation Efficiency

Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

Effect of preoperative evaluation by multidetector computed tomography in percutaneous coronary interventions of chronic total occlusions

BACKGROUND The prevalence of success of percutaneous coronary interventions (PCIs) of chronic total occlusions (CTOs) remains relatively low. We determined the effect of preoperative multidetector computed tomography coronary angiography (CTCA) in PCIs of CTOs. METHODS The study population was 100 consecutive patients who underwent PCIs of CTOs from January 2005 to December 2007 at Keio University School of Medicine. They were divided into two groups according to the absence (non-CT group, n=60) or presence (CT group, n=40) of preoperative CTCA. The effect of preoperative CTCA was assessed in the prevalence of success of the procedure, prevalence of complications, irradiation time, and the dose of contrast agents. RESULTS The prevalence of procedural success was similar in both groups (non-CT group vs CT group 80.0% vs 77.5%, p=0.76). Irradiation time and the dose of contrast agents were also similar between these groups. The prevalence of complications was significantly reduced in the CT group (23.3% vs 7.5%, p=0.039), especially coronary perforations, which required treatment only in the non-CT group (10.0% vs 0.0%, p=0.039). Multiple logistic regression analysis revealed that use of a rotablator (odds ratio [OR]: 4.40, 95% confidence interval [CI]: 1.19-16.27, p=0.027) and absence of preoperative CTCA (OR: 4.26, 95% CI: 1.04-17.49, p=0.044) were independent determinants of complications. CONCLUSION Preoperative CTCA does not affect the prevalence of procedural success, irradiation time and the dose of contrast agents, but may be useful to reduce the prevalence of complications during PCIs of CTOs.