The cause of schizophrenia is unknown, but it has a significant genetic component. Pharmacologic studies, studies of gene expression in man, and studies of mouse mutants suggest involvement of glutamate and dopamine neurotransmitter systems. However, so far, strong association has not been found between schizophrenia and variants of the genes encoding components of these systems. Here, we report the results of a genomewide scan of schizophrenia families in Iceland; these results support previous work, done in five populations, showing that schizophrenia maps to chromosome 8p. Extensive fine-mapping of the 8p locus and haplotype-association analysis, supplemented by a transmission/disequilibrium test, identifies neuregulin 1 (NRG1) as a candidate gene for schizophrenia. NRG1 is expressed at central nervous system synapses and has a clear role in the expression and activation of neurotransmitter receptors, including glutamate receptors. Mutant mice heterozygous for either NRG1 or its receptor, ErbB4, show a behavioral phenotype that overlaps with mouse models for schizophrenia. Furthermore, NRG1 hypomorphs have fewer functional NMDA receptors than wild-type mice. We also demonstrate that the behavioral phenotypes of the NRG1 hypomorphs are partially reversible with clozapine, an atypical antipsychotic drug used to treat schizophrenia.

Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. We report here analysis of Tbx20 expression during murine cardiac development and assessment of DNA-binding and transcriptional properties of Tbx20 isoforms. Tbx20 was expressed in myocardium and endocardium, including high levels in endocardial cushions. cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carried strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bound specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacted with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, led to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behaviour in an in vivo context. We propose that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development.

These beneficial effects make rhNRG-1 promising as a broad-spectrum therapeutic for the treatment of heart failure due to a variety of common cardiac diseases.

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program,defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical,repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20,underlie both congenital heart disease and adult cardiomyopathies.

Abstract-Store-operated Ca 2ϩ entry was investigated in isolated mouse sinoatrial nodes (SAN) dissected from right atria and loaded with Ca 2ϩ indicators. Incubation of the SAN in Ca 2ϩ -free solution caused a substantial decrease in resting intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and stopped pacemaker activity. Reintroduction of Ca 2ϩ in the presence of cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca 2ϩ pump inhibitor, led to sustained elevation of [Ca 2ϩ ] i , a characteristic of store-operated Ca 2ϩ channel (SOCC) activity. Two SOCC antagonists, Gd 3ϩ and SKF-96365, inhibited 72Ϯ8% and 65Ϯ8% of this Ca 2ϩ influx, respectively. SKF-96365 also reduced the spontaneous pacemaker rate to 27Ϯ4% of control in the presence of CPA. Because members of the transient receptor potential canonical (TRPC) gene family may encode SOCCs, we used RT-PCR to examine mRNA expression of the 7 known mammalian TRPC isoforms. Transcripts for TRPC1, 2, 3, 4, 6, and 7, but not TRPC5, were detected. Immunohistochemistry using anti-TRPC1, 3, 4, and 6 antibodies revealed positive labeling in the SAN region and single pacemaker cells. 4,5 In ventricular myocytes, PLC responses are generally modest with only small amounts of IP 3 being produced. 6 Thus release of Ca 2ϩ through IP 3 R is probably too small to modify excitation-contraction coupling. However, atria myocytes express functional IP 3 R at 6 to 10 times higher levels than those in ventricules, and Ca 2ϩ release from IP 3 R may be involved in the generation of arrhythmias. 7 Furthermore, recent studies have shown that activation of PLC in the heart leads to Ca 2ϩ release from perinuclear IP 3 R and thereby regulates nuclear-cytoplasmic cycling of transcription factors and alters gene expression. 8 A similar role may be played by IP 3 and the IP 3 R in skeletal muscle and SOCCs have been implicated in IGF-1 induced muscle hypertrophy. 5,9 The identity of the genes that encode SOCCs remains uncertain. Studies of the transient receptor potential (TRP) gene from Drosophila showed that it encodes a PLC-activated Ca 2ϩ permeable channel. 10 Subsequently, 7 TRP channel homologues in mammals, termed TRPC1-TRPC7, have been identified and there is considerable evidence to indicate that TRPC1 can encode a SOCC. 11 In addition, a recent study showed that overexpression of TRPC3 substantially enhanced SOCC activity in the heart. 5 The importance of Ca 2ϩ release from stores in cardiac pacemaking is now widely accepted. [12][13][14] In a previous study, we found that activation of the P2Y 1 purinergic receptor by ATP results in modulation of pacemaker firing attirbutable to receptor-coupled PLC activation and depletion of SR Ca 2ϩ stores. 15 Because activation of SOCCs also involves PLC, we speculated that SOCCs might be present in pacemaker tissue.In mammals, the sinoatrial node (SAN) is a heterogeneous tissue. The shape of the action potential and the rate of rise of the pacemaker potential change progressively from the periphery to the center, the latter being the leadin...

Rationale:The cardiac gene regulatory network (GRN) is controlled by transcription factors and signaling inputs, but network logic in development and it unraveling in disease is poorly understood. In development, the membrane-tethered signaling ligand Neuregulin (Nrg)1, expressed in endocardium, is essential for ventricular morphogenesis. In adults, Nrg1 protects against heart failure and can induce cardiomyocytes to divide.Objective: To understand the role of Nrg1 in heart development through analysis of null and hypomorphic Nrg1 mutant mice. Methods and Results:Chamber domains were correctly specified in Nrg1 mutants, although chamber-restricted genes Hand1 and Cited1 failed to be activated. The chamber GRN subsequently decayed with individual genes exhibiting decay patterns unrelated to known patterning boundaries. Both trabecular and nontrabecular myocardium were affected. Network demise was spatiotemporally dynamic, the most sensitive region being the central part of the left ventricle, in which the GRN underwent complete collapse. Other regions were partially affected with graded sensitivity. In vitro, Nrg1 promoted phospho-Erk1/2-dependent transcription factor expression, cardiomyocyte maturation and cell cycle inhibition. We monitored cardiac pErk1/2 in embryos and found that expression was Nrg1-dependent and levels correlated with cardiac GRN sensitivity in mutants. Conclusions:The chamber GRN is fundamentally labile and dependent on signaling from extracardiac sources.Nrg1-ErbB1/4 -Erk1/2 signaling critically sustains elements of the GRN in trabecular and nontrabecular myocardium, challenging our understanding of Nrg1 function. Transcriptional decay patterns induced by reduced Nrg1 suggest a novel mechanism for cardiac transcriptional regulation and dysfunction in disease, potentially linking biomechanical feedback to molecular pathways for growth and differentiation. (Circ Res. 2010;107:715-727.) Key Words: neuregulin 1 Ⅲ cardiac gene regulation Ⅲ heart development Ⅲ cardiac gene regulatory network A n early patterning event in vertebrate heart development is the specification of myocardium of the atrial and ventricular chambers, a specialized muscle adapted to pumping blood through a closed circulatory system at high pressure. 1 Luminal myocytes of the cardiac chambers develop sponge-like convolutions termed trabeculae, which in development serve as a morphological marker for chamber specification. The trabecular zone is also marked by a unique set of genes. Chamber muscle is an electric syncytium through which action potentials spread via gap junctions, guided by caudal pacemaker myocytes and their conduction and Purkinje fiber tracts.Nontrabecular myocardium of the atrium, inner curvature, atrioventricular (AV) canal and outflow tract (OFT) is less specialized for contraction and gives rise to the myogenic layers of the outflow and inflow vessels, and cells of the proximal conduction system including the sinoatrial (SA) and AV nodes. 1 Nontrabecular myocardium also plays a critical role in induct...

OBJECTIVETo elucidate the mechanism of the unique beneficial effect of intravitreal steroid therapy on diabetic macular edema, we investigated the effect of locally administered triamcinolone acetonide (TA) on the expression of vascular endothelial growth factor (VEGF)-A and its receptors in retinas of rats with streptozotocin (STZ)-induced diabetes. We then correlated the expression of these proteins with breakdown of the blood-retinal barrier (BRB).RESEARCH DESIGN AND METHODSThirty-two eyes of 16 diabetic and nondiabetic rats were divided into four groups. TA was injected into the vitreous of the right eye, and saline was injected into the left eye (control) 3.5 weeks after induction of diabetes. Retinas were harvested 48 h following treatment. mRNA and protein expression of VEGF-A, VEGF-A receptor 1 (fms-like tyrosine kinase [FLT]-1), and VEGF-A receptor 2 (fetal liver kinase [FLK]-1) were determined by real-time RT-PCR and immunohistochemistry. BRB permeability was quantitated by measuring extravasated endogenous albumin and retinal thickness.RESULTSDiabetes-induced retinal thickness and albumin extravasation were significantly reduced in TA-treated diabetic retinas to a level similar to that in sham-treated nondiabetic eyes. A close correlation between albumin leakage and increased expression of both Vegf-a and Flk-1 was noted in the diabetic retinas. TA downregulated the expression of Vegf-a and Flk-1 but upregulated the expression of Flt-1. TA did not alter the expression of these genes in nondiabetic retinas.CONCLUSIONSIntravitreal injection of TA stabilizes the BRB in association with regulation of Vegf-a, Flk-1, and Flt-1 expression in retinas in the early stages of diabetes.

Rationale: Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) have been implicated in the generation of arrhythmias and cardiac muscle nuclear signaling. However, in the mammalian sinoatrial node (SAN), where the heart beat originates, the expression and functional activity of IP 3 Rs have not been investigated. Objectives:To determine whether SAN express IP 3 Rs and which isoforms are present. To examine the response of the SAN to IP 3 R agonists and antagonist, and the potential role played by IP 3 Rs in cardiac pacemaking. Methods and Results:The expression and distribution of IP 3 Rs were studied by reverse-transcription polymerase chain reaction, Western blotting, and immunolabeling. Ca 2؉ signaling and electric activity in intact mouse SAN were measured with Ca 2؉ -sensitive fluorescent dyes. We found that although the entire SAN expressed three IP 3 R mRNA isoforms, the type II IP 3 R (IP 3 R2) was the predominant protein isoform detected by Western blot using protein extracts from the SAN, atrioventricular node, and atrial tissue. Immunohistochemistry studies also showed that IP 3 R2 was expressed in the central SAN region. Studies using isolated single pacemaker cells revealed that IP 3 R2 (but not IP 3 R1) was located with a similar distribution to the sarcoplasmic reticulum marker protein SERCA2a with some labeling adjacent to the surface membrane. The application of membranepermeable IP 3 (IP 3 -butyryloxymethyl ester) increased Ca 2؉ spark frequency and the pacemaker firing rate in single isolated pacemaker cells. In intact SAN preparations, IP 3 R agonists, endothelin-1 and IP 3 -butyryloxymethyl ester both increased intracellular Ca 2؉ and the pacemaker firing rate, whereas the IP 3 R antagonist, 2-aminoethoxydiphenyl borate decreased Ca 2؉ and the firing rate. Both of these effects were absent in the SAN from transgenic IP 3 R2 knockout mice. Key Words: pacemaker activity Ⅲ sinoatrial node F or many years cardiac pacemaker activity was believed to be determined solely by the electric activity of voltage-dependent membrane currents, particularly the hyperpolarization-activated cation current (I f ). 1 However, the discovery that ryanodine, an antagonist of sarcoplasmic reticulum (SR) Ca 2ϩ release channels (ryanodine receptors), slows the heart rate 2-6 suggested that Ca 2ϩ release from the SR also plays an important role in determining the heart rate. [7][8][9][10][11] Although ryanodine receptors (RyRs) are the main source of SR Ca 2ϩ release, another class of Ca 2ϩ release channels, inositol 1,4,5-trisphosphate receptors (IP 3 Rs), have been found in cardiac SR membranes. 12-14 Although the expression level of IP 3 Rs is much lower than RyRs, Ca 2ϩ release from IP 3 Rs modulates the excitation-contraction coupling in both atrial and ventricular myocytes, 15,16 and several lines of evidence suggest that arrhythmogenic activity in the atria may be associated with increased IP 3 and IP 3 R expression. [17][18][19][20] In addition, new important roles for IP 3 Rs in controlling nuclear transcription ...