Yuichi Tomita

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell–, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0–Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells—and under the right conditions—differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.

Sema3a maintains normal heart rhythm through sympathetic innervation patterning

Sympathetic innervation is critical for effective cardiac function. However, the developmental and regulatory mechanisms determining the density and patterning of cardiac sympathetic innervation remain unclear, as does the role of this innervation in arrhythmogenesis. Here we show that a neural chemorepellent, Sema3a, establishes cardiac sympathetic innervation patterning. Sema3a is abundantly expressed in the trabecular layer in early-stage embryos but is restricted to Purkinje fibers after birth, forming an epicardial-to-endocardial transmural sympathetic innervation patterning. Sema3a−/− mice lacked a cardiac sympathetic innervation gradient and exhibited stellate ganglia malformation, which led to marked sinus bradycardia due to sympathetic dysfunction. Cardiac-specific overexpression of Sema3a in transgenic mice (SemaTG) was associated with reduced sympathetic innervation and attenuation of the epicardial-to-endocardial innervation gradient. SemaTG mice demonstrated sudden death and susceptibility to ventricular tachycardia, due to catecholamine supersensitivity and prolongation of the action potential duration. We conclude that appropriate cardiac Sema3a expression is needed for sympathetic innervation patterning and is critical for heart rate control.

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.

Selective Involvement of p130Cas/Crk/Pyk2/c-Src in Endothelin-1-Induced JNK Activation

Abstract— Both integrin‐based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds on which the G protein‐coupled receptor (GPCR)‐induced signaling complex might assemble. We have recently reported that Ca2+‐sensitive tyrosine kinase, Pyk2, and epidermal growth factor receptor (EGFR) act as independently regulated scaffolds in cardiomyocytes. In this report, we investigated the activation and regulation of p130Cas, Crk, Pyk2, and c‐Src by a well‐known hypertrophic agonist, endothelin‐1 (ET), and determined their contributions to the activation of c‐Jun NH2‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK) in cardiomyocytes. Like Pyk2, ET‐induced tyrosine phosphorylation of p130Cas was significantly inhibited by either chelating intracellular Ca2+ ([Ca2+]i) or a protein kinase C inhibitor, calphostin C. This activation of p130Cas was also abrogated by the tetrapeptide RGDS, which disrupts integrin heterodimerization; cytochalasin D, which depolymerizes the actin cytoskeleton; or a selective Src family kinase inhibitor, PP2, but not by an EGFR inhibitor, AG1478. We also observed ET‐induced temporal associations of Pyk2 with active c‐Src, followed by p130Cas with Pyk2, c‐Src, and Crk. Overexpression of a dominant‐negative mutant of p130Cas (Cas[DELTA]SD), Crk (CrkSH2m), Pyk2 (PKM), or C‐terminal Src kinase (Csk), but not of a deletion mutant of EGFR (533delEGFR), attenuated ET‐induced JNK activation. Similarly, an ET‐induced increase in c‐jun promoter luciferase activity was inhibited by overexpression of Cas[DELTA]SD, CrkSH2m, PKM, or Csk. In contrast, ET‐induced ERK activation and c‐fos gene expression were predominantly regulated by EGFR. Collectively, the focal adhesion‐dependent p130Cas/Crk/Pyk2/c‐Src‐mediated pathway is selectively involved in ET‐induced JNK activation in cardiomyocytes.

Application of mesenchymal stem cell-derived cardiomyocytes as bio-pacemakers: current status and problems to be solved

Bone marrow mesenchymal stem cells (CMG cells) are multipotent and can be induced by 5-azacytidine to differentiate into cardiomyocytes. We characterized the electrophysiological properties of these cardiomyocytes and investigated their potential for use as transplantable bio-pacemakers. After differentiation, action potentials in spontaneously beating cardiomyocytes were initially sinus node-like, but subsequently became ventricular cardiomyocyte-like. RT-PCR established that ion channels mediating IK1 and IKr were expressed before differentiation. After differentiation, ion channels underlying ICa,L and If were expressed first, followed by ion channels mediating Ito and IK,ATP. Differentiated CMG cells expressed β-adrenergic receptors and increased their beat rate in response to isoproterenol. CMG cardiomyocytes were purified using GFP fluorescence and transplanted into the free walls of the left ventricles of mice. The transplanted cardiomyocytes survived and connected to surrounding recipient cardiomyocytes via intercalated discs. Although further innovation is required, the present findings provide evidence of the potential for bone marrow-derived cardiomyocytes to be used as bio-pacemakers.

Cardiomyocytes undergo cells division following myocardial infarction is a spatially and temporally restricted event in rats

Dividing cardiomyocytes are observed in autopsied human hearts following recent myocardial infarction, however there is a lack of information in the literature on the division of these cells. In this study we used a rat model to investigate how and when adult mammalian cardiomyocytes proliferate by cell division after myocardial infarction. Myocardial infarction was induced in Wistar rats by ligation of the left coronary artery. The rats were sacrificed periodically up to 28 days following induced myocardial infarction, and the hearts subjected to microscopic investigation. Cardiomyocytes entering the cell cycle were assayed by observation of nuclear morphology and measuring expression of Ki-67, a proliferating cell marker. Ki-67 positive cardiomyocytes and dividing nuclei were observed initially after 1 day. After 2 days dividing cells gradually increased in number at the ischemic border zone, reaching a peak increase of 1.12% after 3 days, then gradually decreasing in number. Dividing nuclei increased at the ischemic border zone after 3 days, peaked by 0.14% at day 5, and then decreased. In contrast, Ki-67 positive cells and dividing nuclei were limited in number in the non-ischemic area throughout all experiments. In conclusion, mitogenic cardiomyocytes are present in the adult rat heart following myocardial infarction, but were spatially and temporally restricted.

Statistical test for peri-stimulus time histograms in assessing motor neuron activity

The peri-stimulus time histogram is a valuable tool for evaluating neural connections in humans. To detect the degree to which a conditioning stimulus to a sensory nerve modulates motor neuron activity, a histogram of motor unit spike intervals after a conditioning stimulus is measured. This histogram allows the effect of the conditioning stimulus to be visualised. By comparison with a reference histogram of motor unit spike intervals after a sham stimulus, the noise caused by spontaneous firing sway can be removed. However, no valid statistical test has yet been developed to separate the physiological effect from the spontaneous sway and statistical noise. A computational method has been proposed to detect modulation caused by a conditioning stimulus. To clarify the effect of a conditioning stimulus, this new method used reference histograms to calculate a confidence interval. A simulated experiment demonstrated that about 2000 re-samplings were sufficient to estimate a confidence interval for a histogram with 1 ms bin width constructed from 300 triggers. Testing of the experimental data, measured from the tibialis anterior muscles during the elicitation of the excitatory spinal reflex, confirmed that significant peaks were produced at 30, 34, 35 and 38 ms after the conditioning stimulus. These correspond appropriately to the delay of the spinal reflex.

Development of regenerated cardiomyocyte for the cardiovascular tissue engineering

We have isolated a cardiomyogenic cell line (CMG cell) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneous beating cells were repeatedly screened for. The cells showed a f ibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in approximately 30% of the cells : they connected with adjoining cells after 1 week, formed myotube-like structures and began spontaneous beating after 2 weeks, and beat synchronously after 3 weeks. They expressed ANP and BNP. Electron microscopy revealed a cardiomyocyte-like ultrastructure including typical sarcomeres and atrial granules. These cells had several types of action potentials : sinus node like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain and α-actin, indicated that their muscle phenotype was similar to fetal ventricular cardiomyocytes. These cells expressed Nkx 2.5/Csx, GATA 4, TEF-1 and MEF-2 C mRNA before 5-azacytidine treatment, and expressed MEF-2 A and MEF-2 D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.