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.

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.

Reliability and validity of a short Japanese version of the UPPS-P Impulsive Behavior Scale.

OBJECTIVE: This study aimed to verify the reliability and validity of a Japanese version of the S-UPPS-P Impulsive Behavior Scale. This is expected to facilitate comparisons of findings between international and Japanese samples in studies of impulsivity. METHODS: Two surveys were conducted. In the first survey, 632 participants, aged 20-44 years old, completed a translated version of the Japanese S-UPPS-P Impulsive Behavior Scale, the Motor Impulsiveness Scale, a short form of the Big-Five scale, the short Grit scale, and the brief version of the self-control scale. Two weeks later, the second survey containing the S-UPPS-P and the motor impulsiveness scale were completed by 450 participants who had completed the first survey to examine test-retest reliability. RESULTS: In the first survey, an exploratory factor analysis was performed on the S-UPPS-P responses. A four-factor solution was the most suitable solution, with the factors of "Lack of Perseverance," "Lack of Premeditation," "Sensation Seeking," and "Negative-Positive Urgency." Then, a confirmatory factor analysis was performed. The conformity index of the original five-factor model was slightly better than that of the four-factor model. We also compared the five-factor model's conformity index with three other models that had been examined in the original and other foreign language versions of the S-UPPS-P. The five-interrelated factor model had the best model fit. The reliability of the five scales was confirmed. The scales exhibited internal consistency with α coefficients ranging from 0.65 to 0.79, in addition to the test-retest reliability ranging from 0.74 to 0.80. The convergent validity of each S-UPPS-P scale was supported by high relationships with the four personality scales, with the highest correlation coefficients ranging from 0.37 to -0.67. CONCLUSION: The reliability and validity of the Japanese version of the S-UPPS-P were confirmed, despite the minor limitations of the exploratory factor analysis providing a four-factor solution instead of a five-factor solution, and the α reliability coefficients of two scales being acceptable but rather low. Thus, comparisons of findings between international and Japanese studies on impulsivity could be facilitated.

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.

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 I(K1) and I(Kr) were expressed before differentiation. After differentiation, ion channels underlying ICa,L and If were expressed first, followed by ion channels mediating I(to) and I(K,ATP). Differentiated CMG cells expressed beta-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.

A new synbiotic consisting of Lactobacillus casei subsp. casei and dextran improves milk production in Holstein dairy cows.

To evaluate the effects of a new synbiotic consisting of Lactobacillus casei subsp. casei (Lcc) and dextran (Dex) on milk production, a total of 58 Holstein dairy cows, which became pregnant and gave birth to calves at regular intervals and lactated steadily and continuously, were selected. The study had a completely randomized design, and the animals were divided into two groups. Group A was fed with a basic diet only, and Group B was fed with a basic diet supplemented with the synbiotic consisting of freeze-dried Lcc and mixed feed containing Dex for one year from August 2004. After supplementation with the synbiotic, milk yields and components of Group B were compared with those of Group A in the August, December of 2004, April and August of 2005. Milk yields of Group B were greater than those of Group A. There were significant differences (p<0.01 or 0.05) between these groups for all values. Furthermore, total amounts of fat, protein and solid non-fat in Group B significantly increased in comparison with those of Group A. In addition, the somatic cell counts of Group A significantly increased in August of 2004 and 2005 in comparison with those of Group B. Thus, the new synbiotic consisting of Lcc and Dex can increase the milk production of Holstein dairy cows throughout the year.

Radical trifluoromethylation of ketone silyl enol ethers by activation with dialkylzinc.

[reaction: see text] The radical trifluoromethylation of ketone silyl enol ethers gave alpha-CF(3) ketones in good yields with wide scope of the ketonic substrates including acyclic ketones and cyclopentanone. The use of dialkylzinc to activate the silyl enol ethers is the key to the efficient radical trifluoromethylation.

Bone marrow-derived regenerated cardiomyocytes (CMG Cells) express functional adrenergic and muscarinic receptors.

BACKGROUND: We recently reported that cardiomyocytes could be differentiated from bone marrow mesenchymal stem cells in vitro by 5-azacytidine treatment. In native cardiomyocytes, adrenergic and muscarinic receptors play crucial roles in mediating heart rate, conduction velocity, contractility, and cardiac hypertrophy. We investigated whether these receptors are expressed in differentiated CMG cells, and if so, whether they have downstream signaling systems. METHODS AND RESULTS: Reverse transcription-polymerase chain reaction revealed that CMG cells had already expressed alpha(1A)-, alpha(1B)-, and alpha(1D)-adrenergic receptor mRNA before 5-azacytidine treatment, whereas expression of beta(1)-, beta(2)-adrenergic and M(1)-, M(2)-muscarinic receptors was first detected at 1 day. Phenylephrine dose-dependently induced phosphorylation of ERK1/2, which was completely inhibited by prazosin, and significantly increased cell size. Isoproterenol augmented cAMP by 38-fold, which was fully inhibited by propranolol. Isoproterenol (10(-7) mol/L) increased the spontaneous beating rate by 47.6% (basal, 127+/-16 bpm), and propranolol and CGP20712A (beta(1)-selective blocker) reduced it by 79.0% and 71.0%, respectively, whereas ICI118551 (beta(2)-selective blocker) induced slight reduction. Cell motion, percent shortening, and contractile velocity were increased by 37.5%, 26.9%, and 50.6%, respectively, in response to isoproterenol. Phenylephrine and isoproterenol augmented ANP and BNP gene expressions. Carbachol increased IP(3) by 32-fold, which was markedly inhibited by atropine as well as AFDX116 (M(2)-selective blocker) measured by radioimmunoassay. CONCLUSIONS: These findings indicate that CMG cells expressed alpha(1A), alpha(1B), and alpha(1D) receptors before differentiation and expressed beta(1), beta(2), M(1), and M(2) receptors after they obtained the cardiomyocyte phenotype. These receptors had functional signal transduction pathways and could modulate cell function.

Selective involvement of p130Cas/Crk/Pyk2/c-Src in endothelin-1-induced JNK activation.

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 (CasDeltaSD), 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 CasDeltaSD, 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.

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.