Kouji Shimoda

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.

Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.

Expression of the rat calmodulin gene II in the central nervous system: a 294-base promoter and 68-base leader segment mediates neuron-specific gene expression in transgenic mice

Deletion analysis of the rat CaMII promoter demonstrated that the segment from -294 to +68 bases of CaMII was efficient as a promoter in NIH3T3 by transient assay. We developed transgenic mice carrying a fusion gene of this promoter segment and a beta-galactosidase reporter gene. This short CaMII promoter mediated the transgene expression in pyramidal cells of the cerebral neocortex, the pyriformcortex and the hippocampal regions CA1 to CA3, in granule cells of the dentate gyrus, in Purkinje cells of the cerebellum, and in neurons of the lateral vestibular nucleus of pons and the spinal cord of adult transgenic mice. The expression of endogenous CaMII was precisely analyzed by in situ hybridization in the nervous tissues. The localization of transgene expression was consistent with those of the endogenous CaMII in the adult transgenic mice. In the embryos at 13.5-15.5 days of gestation, the transgene was expressed in various neurons similarly to the endogenous CaMII but certain subtle differences were observed in the localization of expression. This short promoter of rat CaMII carried two sequence stretches highly conserved in the mouse, dog, chicken and Xenopus CaMII promoters. These conserved stretches may be involved in the observed neuron-specific expression of rat CaMII gene.

Dominant Negative Suppression of Rad Leads to QT Prolongation and Causes Ventricular Arrhythmias via Modulation of L-type Ca2+ Channels in the Heart

Disorders of L-type Ca2+ channels can cause severe cardiac arrhythmias. A subclass of small GTP-binding proteins, the RGK family, regulates L-type Ca2+ current (ICa,L) in heterologous expression systems. Among these proteins, Rad (Ras associated with diabetes) is highly expressed in the heart, although its role in the heart remains unknown. Here we show that overexpression of dominant negative mutant Rad (S105N) led to an increase in ICa,L and action potential prolongation via upregulation of L-type Ca2+ channel expression in the plasma membrane of guinea pig ventricular cardiomyocytes. To verify the in vivo physiological role of Rad in the heart, a mouse model of cardiac-specific Rad suppression was created by overexpressing S105N Rad, using the &agr;-myosin heavy chain promoter. Microelectrode studies revealed that action potential duration was significantly prolonged with visible identification of a small plateau phase in S105N Rad transgenic mice, when compared with wild-type littermate mice. Telemetric electrocardiograms on unrestrained mice revealed that S105N Rad transgenic mice had significant QT prolongation and diverse arrhythmias such as sinus node dysfunction, atrioventricular block, and ventricular extrasystoles, whereas no arrhythmias were observed in wild-type mice. Furthermore, administration of epinephrine induced frequent ventricular extrasystoles and ventricular tachycardia in S105N Rad transgenic mice. This study provides novel evidence that the suppression of Rad activity in the heart can induce ventricular tachycardia, suggesting that the Rad-associated signaling pathway may play a role in arrhythmogenesis in diverse cardiac diseases.

Expression of a MADS box gene, MEF2D, in neurons of the mouse central nervous system: implication of its binary function in myogenic and neurogenic cell lineages

MEF2D, a member of myocyte-specific enhancer binding factor 2 (MEF2) gene family, was shown by Northern blot hybridization to be strongly expressed in the head portion of mouse embryos at later stages of ontogenesis, in the cerebellum and the cerebrum of adult mice, in cultured cell lines of neuronal origin, and in skeletal and cardiac muscles. During ontogenesis, MEF2D transcripts were detected by in situ hybridization in the olfactory bulb, entorhinal cortex, pyriform cortex, and hippocampus, in Purkinje and granule cells, and in large neurons in both the ventral and dorsal horns of spinal cord. Adult mice continued to express MEF2D in these limited areas of the central nervous system. Thus, MEF2D seems to be involved in either the differentiation process or the function of these neurons.

Functional expression of the type 1 inositol 1,4,5-trisphosphate receptor promoter-lacZ fusion genes in transgenic mice

Abstract: Inositol 1,4,5‐trisphosphate receptor (IP3R) is an inositol 1,4,5‐trisphosphate (InsP3)‐gated Ca2+ release channel. Type 1 IP3R (IP3R1) is the neuronal member of the IP3R family in the CNS and is predominantly expressed in cerebellar Purkinje cells. To elucidate the molecular mechanisms responsible for coupling gene expression to neuronal InsP3/Ca2+ signaling, we have studied the structure and function of the 5′‐flanking region of the mouse IP3R1 gene. The cloned 5′‐flanking region has several sequences sharing identity with motifs for known transcriptional regulation. We have fused 5′‐flanking regions 1N from −528 to +169 and 4N from −4,187 to +169 to a β‐galactosidase gene (lacZ) as a reporter marker and have characterized their in vivo gene expression. Both 1N and 4N fusion genes functioned as a strong promoter in a neuroblastoma‐glioma hybrid cell line NG108‐15. Moreover, both 1N and 4N transgenic mouse lines carrying these 1N and 4N fusion genes showed characteristic patterns of β‐galactosidase activity in the CNS that are almost consistent with that of the endogenous IP3R1 protein, thereby suggesting that the 1N region from −528 to +169 contains sequence elements responsible for regulating gene expression in neurons and for specifying predominant expression in cerebellar Purkinje cells.

Fra‐1/AP‐1 Impairs Inflammatory Responses and Chondrogenesis in Fracture Healing

Inflammation inevitably follows injury of various tissues, including bone. Transgenic overexpression of Fra‐1, a component of the transcription factor activator protein‐1 (AP‐1), in various tissues progressively and globally enhances bone formation, but little is known about the possible effects of Fra‐1/AP‐1 on fracture healing. We created a transverse fracture of the mouse tibial diaphysis and examined fracture healing radiologically, histologically, and immunologically. Strikingly, fracture union was delayed even though the bone formation rate in callus was higher in Fra‐1 transgenic (Tg) mice. In these mice, chondrogenesis around the fracture site was impaired, resulting in accumulation of fibrous tissue, which interferes with the formation of a bony bridge across the callus. Curiously, immediately after fracture, induction of the inflammatory mediators TNF‐α, interleukin (IL)‐6, and Cox‐2 was significantly suppressed in Fra‐1 Tg mice followed, by the reduced expression of Sox‐9 and BMP‐2. Because serum prostaglandin E2 (PGE2) levels were dramatically low in these mice, we administered PGE2 to the fracture site using a slow‐release carrier. The accumulation of fibrous tissue in Fra‐1 Tg mice was significantly reduced by PGE2 administration, and chondrogenesis near the fracture site was partially restored. These data suggest that the Fra‐1‐containing transcription factor AP‐1 inhibits fracture‐induced endochondral ossification and bony bridge formation presumably through suppression of inflammation‐induced chondrogenesis.

Translocation of bacteria from the gastrointestinal tract in immunodeficient mice

Host defence mechanisms associated with the inhibition of translocation of bacteria from the gastrointestinal (GI) tract were investigated in SCID and beige mice after decontamination with oral antibiotics and colonization with Escherichia coli C25. SCID mice, which have impaired T and B cell function, tended to have a greater incidence of bacterial translocation from the GI tract up to 7 days after inoculation compared with controls. However, after 7 days both SCID and controls cleared the E. coli C25 from the liver, spleen, blood and peritoneal cavity. Beige mice, with impaired NK cell and polymorphonuclear leukocyte function, were not able to clear the inoculated bacteria from their liver by 14 days after inoculation although the controls were cleared by 7 days. Numbers of bacteria in the mesenteric lymph nodes (MLN) of beige mice did not decrease significantly by 14 days after inoculation, whereas numbers in SCID mice decreased markedly within 7 days. These results suggest that defence mechanisms other than T and B cell function are important in the inhibition of systemic infection from the GI tract.

Islet cell hyperplasia in transgenic mice overexpressing EAT/mcl-1, a bcl-2 related gene.

EAT/mcl-1 (EAT), a bcl-2 related anti-apoptotic gene, is up-regulated at the early stage of differentiation of human embryonal carcinoma cells; cells which serve as a model for early embryogenesis. We generated transgenic mice for the human EAT gene driven by the EF1 alpha promoter in order to elucidate its functional role in vivo. Histologically, these mice exhibited hyperplasia of Langerhans islet cells; pancreatic cell regions composed of both insulin- and glucagon-producing cells. Furthermore, Bax and Bag-1 -- possible heterodimeric partners for EAT in the anti-apoptotic process -- were up-regulated in islets isolated from the EAT transgenic mice. The insulin tolerance test exhibited no significant difference between the EAT transgenic mice and non-transgenic mice, indicating that islet cell hyperplasia was not due to insulin resistance. In conclusion, EAT transgenic mice exhibit hyperplasia of pancreatic beta cells. EAT may inhibit apoptosis of beta cells, allowing these cells to circumvent the process of apoptosis until the adult stage.

Stability of pathogenic bacteria from laboratory animals in various transport media

The stability of pathogenic bacteria from laboratory animals was investigated in various transport media at different temperatures. Bordetella bronchiseptica and Salmonella typhimurium survived for 8 days in phosphate-buffered saline (PBS, pH 7·0) at 37, 24, 4 and -20°C; Brucella canis at 24, 4 and -20°C; Corynebacterium kutscheri at 4 and -20°C; and Pseudomonas aeruginosa at all but -20°C. A marked decrease in numbers of Pasteurella multocida and Past. pneumotropica was observed in PBS at all temperatures. Skimmed milk in PBS improved the survival of Pasteurella spp. and Ps. aeruginosa at -20°C. Neither glycerin, ascorbic acid nor sodium thioglycollate improved survival. The numbers of viable B. canis, Ps. aeruginosa and S. typhimurium were maintained in blood or faecal specimens held for 8 days at 4°C. These results indicated that transport in PBS at 4°C was the only method satisfactory for all species of pathogenic organisms tested, but Pasteurella spp. were the most difficult to maintain.