Paradigm of genetic mosaicism and lone atrial fibrillation: physiological characterization of a connexin 43-deletion mutant identified from atrial tissue.

BACKGROUND: Atrial fibrillation (AF) is the most common sustained arrhythmia observed in otherwise healthy individuals. Most lone AF cases are nonfamilial, leading to the assumption that a primary genetic origin is unlikely. In this study, we provide data supporting a novel paradigm that atrial tissue-specific genetic defects may be associated with sporadic cases of lone AF. METHODS AND RESULTS: We sequenced the entire coding region of the connexin 43 (Cx43) gene (GJA1) from atrial tissue and lymphocytes of 10 unrelated subjects with nonfamilial, lone AF who had undergone surgical pulmonary vein isolation. In the atrial tissue of 1 patient, we identified a novel frameshift mutation caused by a single nucleotide deletion (c.932delC) that predicted 36 aberrant amino acids followed by a premature stop codon, leading to truncation of the C-terminal domain of Cx43. The mutation was absent from the lymphocyte DNA of the patient, indicating genetic mosaicism. Protein trafficking studies demonstrated intracellular retention of the mutant protein and a dominant-negative effect on gap junction formation of both wild-type Cx43 and Cx40. Electrophysiological studies revealed no electrical coupling of cells expressing the mutant protein alone and significant reductions in coupling when coexpressed with wild-type connexins. CONCLUSIONS: This study reports atrial tissue genetic mosaicism of a novel loss-of-function Cx43 mutation associated with lone AF. These findings implicate somatic genetic defects of Cx43 as a potential cause of AF and support the paradigm that sporadic, nonfamilial cases of lone AF may arise from genetic mosaicism that creates heterogeneous coupling patterns, predisposing the tissue to reentrant arrhythmias.

Advances in Pluripotent Stem Cells: History, Mechanisms, Technologies, and Applications.

Over the past 20 years, and particularly in the last decade, significant developmental milestones have driven basic, translational, and clinical advances in the field of stem cell and regenerative medicine. In this article, we provide a systemic overview of the major recent discoveries in this exciting and rapidly developing field. We begin by discussing experimental advances in the generation and differentiation of pluripotent stem cells (PSCs), next moving to the maintenance of stem cells in different culture types, and finishing with a discussion of three-dimensional (3D) cell technology and future stem cell applications. Specifically, we highlight the following crucial domains: 1) sources of pluripotent cells; 2) next-generation in vivo direct reprogramming technology; 3) cell types derived from PSCs and the influence of genetic memory; 4) induction of pluripotency with genomic modifications; 5) construction of vectors with reprogramming factor combinations; 6) enhancing pluripotency with small molecules and genetic signaling pathways; 7) induction of cell reprogramming by RNA signaling; 8) induction and enhancement of pluripotency with chemicals; 9) maintenance of pluripotency and genomic stability in induced pluripotent stem cells (iPSCs); 10) feeder-free and xenon-free culture environments; 11) biomaterial applications in stem cell biology; 12) three-dimensional (3D) cell technology; 13) 3D bioprinting; 14) downstream stem cell applications; and 15) current ethical issues in stem cell and regenerative medicine. This review, encompassing the fundamental concepts of regenerative medicine, is intended to provide a comprehensive portrait of important progress in stem cell research and development. Innovative technologies and real-world applications are emphasized for readers interested in the exciting, promising, and challenging field of stem cells and those seeking guidance in planning future research direction.

Gain-of-function mutation of Nav1.5 in atrial fibrillation enhances cellular excitability and lowers the threshold for action potential firing.

Genetic mutations of the cardiac sodium channel (SCN5A) specific only to the phenotype of atrial fibrillation have recently been described. However, data on the biophysical properties of SCN5A variants associated with atrial fibrillation are scarce. In a mother and son with lone atrial fibrillation, we identified a novel SCN5A coding variant, K1493R, which altered a highly conserved residue in the DIII-IV linker and was located six amino acids downstream from the fast inactivation motif of sodium channels. Biophysical studies of K1493R in tsA201 cells demonstrated a significant positive shift in voltage-dependence of inactivation and a large ramp current near resting membrane potential, indicating a gain-of-function. Enhanced cellular excitability was observed in transfected HL-1 atrial cardiomyocytes, including spontaneous action potential depolarizations and a lower threshold for action potential firing. These novel biophysical observations provide molecular evidence linking cellular "hyperexcitability" as a mechanism inducing vulnerability to this common arrhythmia.

Restoring Motor Neurons in Spinal Cord Injury With Induced Pluripotent Stem Cells.

Spinal cord injury (SCI) is a devastating neurological disorder that damages motor, sensory, and autonomic pathways. Recent advances in stem cell therapy have allowed for the in vitro generation of motor neurons (MNs) showing electrophysiological and synaptic activity, expression of canonical MN biomarkers, and the ability to graft into spinal lesions. Clinical translation, especially the transplantation of MN precursors in spinal lesions, has thus far been elusive because of stem cell heterogeneity and protocol variability, as well as a hostile microenvironment such as inflammation and scarring, which yield inconsistent pre-clinical results without a consensus best-practice therapeutic strategy. Induced pluripotent stem cells (iPSCs) in particular have lower ethical and immunogenic concerns than other stem cells, which could make them more clinically applicable. In this review, we focus on the differentiation of iPSCs into neural precursors, MN progenitors, mature MNs, and MN subtype fates. Previous reviews have summarized MN development and differentiation, but an up-to-date summary of technological and experimental advances holding promise for bench-to-bedside translation, especially those targeting individual MN subtypes in SCI, is currently lacking. We discuss biological mechanisms of MN lineage, recent experimental protocols and techniques for MN differentiation from iPSCs, and transplantation of neural precursors and MN lineage cells in spinal cord lesions to restore motor function. We emphasize efficient, clinically safe, and personalized strategies for the application of MN and their subtypes as therapy in spinal lesions.

A teleost in vitro reporter gene assay to screen for agonists of the peroxisome proliferator-activated receptors.

Several contaminants detected in aquatic ecosystems are agonists of peroxisome proliferator-activated receptors (PPARs). Peroxisome proliferator-activated receptors interact with the retinoid X receptor (RXR) to activate the transcription of genes that control a variety of physiological functions. We cloned and sequenced partial cDNA fragments of rainbow trout (Oncorhynchus mykiss) PPARalpha and PPARbeta from rainbow trout (rt) gill-W1 cells, a cell line derived from rainbow trout gills; predicted amino acid identities are 77% and 82% compared with their respective human homologs and 83 to 88% and 91 to 98% identical to fish homologs. A reporter gene assay was developed by transfecting rt-gill-W1 cells with a reporter gene construct containing the peroxisome proliferator response element (PPRE) of the rat liver 3-ketoacyl-CoA thiolase B (TB) gene, which drives luciferase expression. Agonists of both PPARalpha (WY14,643 and gemfibrozil) and PPARbeta (bezafibrate) induced luciferase activity, while rosiglitazone, a PPARgamma agonist, was not effective. The fibrate drug, bezafibrate increased luciferase activity in a dose-dependent manner, but addition of 50 nM 9-cis-retinoic acid to the transfected rt-gill-W1 cell culture maximized the sensitivity of the assay so that bezafibrate could be detected at concentrations as low as 6 nM. Extracts from treated domestic wastewater containing fibrate drugs induced luciferase activity in the transfected gill cells. This in vitro reporter gene assay shows promise as a rapid and sensitive technique for screening environmental samples for PPAR-active substances.

Use of induced pluripotent stem cell derived neurons engineered to express BDNF for modulation of stressor related pathology.

Combined cell and gene-based therapeutic strategies offer potential in the treatment of neurodegenerative and psychiatric conditions that have been associated with structural brain disturbances. In the present investigation, we used a novel virus-free re-programming method to generate induced pluripotent stem cells (iPSCs), and then subsequently transformed these cells into neural cells which over-expressed brain derived neurotrophic factor (BDNF). Importantly, the infusion of iPSC derived neural cells (as a cell replacement and gene delivery tool) and BDNF (as a protective factor) influenced neuronal outcomes. Specifically, intracerebroventricular transplantation of iPSC-derived neural progenitors that over-expressed BDNF reversed the impact of immune (lipopolysaccharide) and chronic stressor challenges upon subventricular zone adult neurogenesis, and the iPSC-derived neural progenitor cells alone blunted the stressor-induced corticosterone response. Moreover, our findings indicate that mature dopamine producing neurons can be generated using iPSC procedures and appear to be viable when infused in vivo. Taken together, these data could have important implications for using gene-plus-cell replacement methods to modulate stressor related pathology.

The Role of the Val66Met Polymorphism of the Brain Derived Neurotrophic Factor Gene in Coping Strategies Relevant to Depressive Symptoms.

Disturbances of brain derived neurotrophic factor (BDNF) signalling have been implicated in the evolution of depression, which likely arises, in part, as a result of diminished synaptic plasticity. Predictably, given stressor involvement in depression, BDNF is affected by recent stressors as well as stressors such as neglect experienced in early life. The effects of early life maltreatment in altering BDNF signalling may be particularly apparent among those individuals with specific BDNF polymorphisms. We examined whether polymorphisms of the Val66Met genotype might be influential in moderating how early-life events play out with respect to later coping styles, cognitive flexibility and depressive features. Among male and female undergraduate students (N = 124), childhood neglect was highly related to subsequent depressive symptoms. This outcome was moderated by the BDNF polymorphism in the sense that depressive symptoms appeared higher in Met carriers who reported low levels of neglect than in those with the Val/Val allele. However, under conditions of high neglect depressive symptoms only increased in the Val/Val individuals. In effect, the Met polymorphism was associated with depressive features, but did not interact with early life neglect in predicting later depressive features. It was further observed that among the Val/Val individuals, the relationship between neglect and depression was mediated by emotion-focused styles and diminished perceived control, whereas this mediation was not apparent in Met carriers. In contrast to the more typical view regarding this polymorphism, the data are consistent with the perspective that in the presence of synaptic plasticity presumably associated with the Val/Val genotype, neglect allows for the emergence of specific appraisal and coping styles, which are tied to depression. In the case of the reduced degree of neuroplasticity expected in the Met carriers, early life adverse experiences are not tied to coping styles, and hence less likely to be translated into depressive states.

Growth hormone-induced diacylglycerol and ceramide formation via Galpha i3 and Gbeta gamma in GH4 pituitary cells. Potentiation by dopamine-D2 receptor activation.

Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH(4)ZR(7) somatomammotroph cells was examined. GH (EC(50) = 5 nm) stimulated diacylglycerol (DAG) and ceramide formation in parallel by over 10-fold within 15 min and persisting for >3 h. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating G(i)/G(o) proteins and was potentiated 1.5-fold by activation of G(i)/G(o)-coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant Galpha(i)3, not Galpha(o) or Galpha(i)2, rescued GH-induced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing Gbetagamma blocker GRK-ct. In GH(4)ZR(7) cells, GH induced phosphorylation of JAK2 and STAT5, which was blocked by PTX and mimicked by ceramide analogue C2-ceramide or sphingomyelinase treatment to increase endogenous ceramide. We conclude that in GH(4) pituitary cells, GH induces formation of DAG/ceramide via a novel Galpha(i)3/Gbetagamma-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function.

Bradykinin stimulates ceramide production by activating specific BK-B(1) receptor in rat small artery.

Bradykinin (BK), a proinflammatory factor and vasodilator, causes functional change of the small artery. However, it is not clear whether any of these changes induced by BK are mediated by N-acetyl-D-sphingosine (ceramide). Therefore, we investigated whether BK affects the hydrolysis of sphingomyelin and generation of ceramide in the intact rat small artery. Our results suggest that BK induces sphingomyelin hydrolysis and increases ceramide production in a time- and dose-dependent manner. Relative to controls, BK causes a 50% decrease in sphingomyelin levels. Ceramide levels increase in response to BK with the highest level being obtained with 10(-8) M BK as well as similar amounts of ceramide are generated when exogenous sphingomyelinase (SMase) is added. We then determined which of the two BK receptors (BK-B(1) antagonist Lys-Des-Arg(9)-Leu(8)-BK or the BK-B(2) antagonist HOE-140) are implicated in the BK-induced generation of ceramide. The BK-B(2) antagonist did not alter the effect of BK on ceramide generation, whereas the BK-B(1) antagonist blocked the BK-induced production of ceramide. Although ceramide had no effect on KCl-induced constrictions, ceramide dilated preconstricted (phenylephrine) small pressurized rat mesenteric arteries by approximately 40%. These results suggest that the activation of the BK-B(1) receptor mediates the BK-induced activation of SMase and of the production of ceramide. In conclusion, BK-mediated effects on vascular tone may be due, at least in part, to the increased production of ceramide.

Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gbeta gamma -subunits in Balb/c-3T3 cells.

Diacylglycerol (DAG) and ceramide are important second messengers affecting cell growth, differentiation, and apoptosis. Balb/c-3T3 fibroblast cells expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a model of G protein-mediated cell growth and transformation. In Balb-D2S cells, apomorphine (EC(50) = 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at 1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating G(i)/G(o) proteins. To address which G proteins are involved, Balb-D2S clones expressing individual PTX-insensitive Galpha(i) proteins were treated with PTX and tested for apomorphine-induced responses. Neither PTX-insensitive Galpha(i2) nor Galpha(i3) rescued D2S-induced DAG or ceramide formation. Both D2S-induced DAG and ceramide signals required Gbetagamma-subunits and were blocked by inhibitors of phospholipase C [1-(6-[([17beta]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) and partially by D609]. The similar G protein specificity of D2S-induced calcium mobilization, DAG, and ceramide formation indicates a common Gbetagamma-dependent phospholipase C-mediated pathway. Both D2 agonists and ceramide specifically induced mitogen-activated protein kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of D2S-induced ERK1/2 activation, leading to cell growth.