Gap Junction Protein Phenotypes of the Human Heart and Conduction System

Davis, Lloyd M.; Rodefeld, Mark E.; Green, Karen; Beyer, Eric C.; Saffitz, Jeffrey E.

doi:10.1111/j.1540-8167.1995.tb00357.x

Cited by 187 publications

(118 citation statements)

References 36 publications

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“…Therefore, it is expected that implementing standardized protocols for antibody labeling and analysis should better permit reliable comparison of the efficiency of cardiomyogenesis and subtype identity among protocols. As the field continues to establish robust protocols for efficient cardiomyogenesis, future efforts to more fully define the cells generated from such protocols will include the analysis of maturation stage and chamber-restricted markers such as hairy-related transcription factors 1 and 2 (HRT1, HRT2 38,39 ), gap junction alpha-5 protein (GJA5, CX40 40 ), sarcolipin (SLN 41 ) natriuretic peptides A (NPPA 42,43 ), potassium voltage-gated channel subfamily E member 1 (KCNE1 44 ), T-box transcription factor TBX3 (TBX3 45 ),heart-and neural crest derivatives-expressed protein(HAND1, HAND2 [46][47][48] ), and potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4 [49][50][51] ), among others. For marker analysis, it is recommended that both mRNA and protein levels be measured when possible, as protein modifications, stability and turnover can affect the relationship between gene expression and protein abundance (e.g., TNNT2 in Figures 1B, 2E) and, during development, patterns of expression differ between mRNA and protein for some markers (reviewed in Franco et al 20 ).…”

Section: Discussionmentioning

confidence: 99%

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

Bhattacharya

Burridge

Kropp

et al. 2014

JoVE

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There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle "in a dish" for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

Bhattacharya

Burridge

Kropp

et al. 2014

JoVE

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“…Cx40, Cx43 and Cx45 are the most abundant connexin isoforms of the working mammalian myocardial cells and the conduction system. Cx40 is mainly expressed in the atrium and in the conduction system [44][45][46][47][48][49], while Cx43 can be found both in atrial and ventricular cells but not in sinoatrial and atrioventricular nodes [46,[48][49][50][51][52][53][54][55]. Cx45 is preferentially expressed in sinoatrial and atrioventricular nodes, His-bundle and bundle branches [56][57][58].…”

Section: The Distribution Of Gap Junctionsmentioning

confidence: 99%

Role of Gap Junction Channel in the Development of Beat-to-Beat Action Potential Repolarization Variability and Arrhythmias

Magyar

Bányász

Szentandrássy

et al. 2014

CPD

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The short-term beat-to-beat variability of cardiac action potential duration (SBVR) occurs as a random alteration of the ventricular repolarization duration. SBVR has been suggested to be more predictive of the development of lethal arrhythmias than the action potential prolongation or QT prolongation of ECG alone. The mechanism underlying SBVR is not completely understood but it is known that SBVR depends on stochastic ion channel gating, intracellular calcium handling and intercellular coupling.Coupling of single cardiomyocytes significantly decreases the beat-to-beat changes in action potential duration (APD) due to the electrotonic current flow between neighboring cells. The magnitude of this electrotonic current depends on the intercellular gap junction resistance. Reduced gap junction resistance causes greater electrotonic current flow between cells, and reduces SBVR.Myocardial ischaemia (MI) is known to affect gap junction channel protein expression and function. MI increases gap junction resistance that leads to slow conduction, APD and refractory period dispersion, and an increase in SBVR. Ultimately, development of reentry arrhythmias and fibrillation are associated post-MI. Antiarrhythmic drugs have proarrhythmic side effects requiring alternative approaches. A novel idea is to target gap junction channels. Specifically, the use of gap junction channel enhancers and inhibitors may help to reveal the precise role of gap junctions in the development of arrhythmias. Since cell-to-cell coupling is represented in SBVR, this parameter can be used to monitor the degree of coupling of myocardium.

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“…1 Cx43 is abundant in atrial and ventricular myocardium. 2,3 Cx40 is expressed in atrial tissue and in the atrioventricular conducting system. Cx45 is observed in the sinoatrial and atrioventricular nodes, and small amounts colocalize with Cx43 in adult ventricular myocardium.…”

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confidence: 99%

Effects of Mechanical Forces and Mediators of Hypertrophy on Remodeling of Gap Junctions in the Heart

Saffitz

Kléber

2004

Circulation Research

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Abstract-This review article focuses on remodeling of gap junctions in response to chemical mediators of ventricular hypertrophy, mechanical forces, and alterations in cell-to-cell adhesion. Signaling mediated by mechanical forces is likely to be involved in the upregulation of cardiac gap junctions during the early phase of cardiac hypertrophy and the subsequent downregulation in cardiac failure. Several signaling pathways involving cAMP, angiotensin II, transforming growth factor-␤, vascular endothelial growth factor, and integrin-mediated regulators have been shown to affect expression of gap junction proteins. However, a comprehensive view of regulation of gap junction trafficking, synthesis, and degradation is still lacking. In addition to gap junction regulation by extracellular mechanical forces, there is a close relation between gap junctions and adhesion junctions and their linkage to the cytoskeleton. This can be inferred from experiments on neoformation of cell-to-cell coupling, concomitant upregulation of adherens and gap junctions after mechanical stretch, and human cardiomyopathies caused by genetic defects in cell-cell adhesion junction proteins. The molecular mechanisms responsible for the interaction between mechanical and functional cell-to-cell coupling remain to be elucidated. Key Words: gap junctions Ⅲ adhesion junctions Ⅲ mechanical signaling Ⅲ remodeling Ⅲ cardiac hypertrophy and failure C onnexin proteins form intercellular pores in many tissues, such as myocardium, vascular endothelium, and brain. 1 Twenty distinct connexin genes have been identified in the human genome. Three types of connexins, connexin43 (Cx43), Cx40, and Cx45, are expressed in heart. 1 Cx43 is abundant in atrial and ventricular myocardium. 2,3 Cx40 is expressed in atrial tissue and in the atrioventricular conducting system. Cx45 is observed in the sinoatrial and atrioventricular nodes, and small amounts colocalize with Cx43 in adult ventricular myocardium. 4 Six connexin proteins oligomerize to form a hemichannel (connexon) that migrates to the cell surface membrane and becomes incorporated at the periphery of existing junctional plaques, 5,6 where it combines with a corresponding hemichannel in an adjacent cell to form a complete gap junction channel. Colocalization of different connexin proteins in gap junction plaques, observed immunohistochemically, 7-9 probably reflects formation of heterotypic or heteromeric gap junction channels, as shown in expression systems (oocytes or immortalized cell lines). 10 -12 A major role of gap junctions in the myocardium is to enable rapid and coordinated electrical excitation, a prerequisite for normal rhythmic cardiac function, and probably also to facilitate intercellular exchange of small molecules, such as regulatory proteins. Because diffusion of molecules across gap junctions is possible up to a molecular weight of Ϸ1000 Over the past years, many of the molecular and biophysical properties of gap junction channels have been described. For example, it has been shown that ...

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Gap Junction Protein Phenotypes of the Human Heart and Conduction System

Cited by 187 publications

References 36 publications

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

Role of Gap Junction Channel in the Development of Beat-to-Beat Action Potential Repolarization Variability and Arrhythmias

Effects of Mechanical Forces and Mediators of Hypertrophy on Remodeling of Gap Junctions in the Heart

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