Gap junctional remodeling by hypoxia in cultured neonatal rat ventricular myocytes

Article (PDF Available)inCardiovascular Research 66(1):64-73 · May 2005with24 Reads
DOI: 10.1016/j.cardiores.2005.01.014 · Source: PubMed
Abstract
Altered gap junctional coupling of ventricular myocytes plays an important role in arrhythmogenesis in ischemic heart disease. Since hypoxia is a major component of ischemia, we tested the hypothesis that hypoxia causes gap junctional remodeling accompanied by conduction disturbances. Cultured neonatal rat ventricular myocytes were exposed to hypoxia (1% O(2)) for 15 min to 5 h, connexin43 (Cx43) expression was analyzed, and conduction velocity was measured using the Micro-Electrode Array data acquisition system. After 15 min of hypoxia, conduction velocity was unaffected, while total Cx43, including the phosphorylated and nonphosphorylated isoforms, was increased. After 5 h of hypoxia, total Cx43 protein was decreased by 50%, while the nonphosphorylated Cx43 isoform was unchanged. Confocal analyses yielded a 55% decrease in the gap junctional Cx43 fluorescence signal, a 55% decrease in gap junction number, and a 26% decrease in size. The changes in Cx43 were not accompanied by changes in mRNA levels. The reduction in Cx43 protein levels was associated with a approximately 20% decrease in conduction velocity compared to normoxic cultures. Short-term hypoxia (5 h) decreases Cx43 protein and conduction velocity, thereby contributing to the generation of an arrhythmogenic substrate.

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Available from: Zaid Abassi, Jan 04, 2016
    • "In comparison, SCMs were selected for α-MHC expression using puromycin resistance and therefore, the resulting cell pool was free of any non-cardiac cells. Interestingly, pure SCM strands presented significantly lower CVs compared to pure PCMs, corroborating the results of other research groups (Satin et al., 2004; Zeevi-Levin et al., 2005; Mureli et al., 2013). In mixed cultures, we found that increasing the amount of PCMs resulted in an increase in CV. "
    [Show abstract] [Hide abstract] ABSTRACT: Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.
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    • "During short periods of hypoxia (up to 15 min) Cx43 content remains unchanged (Zeevi-Levin et al., 2005; Matsumura et al., 2006). With prolonged hypoxia (several hours), downregulation of Cx43 at gap junctions occurs (Danon et al., 2010), Cx43 is internalized (Sato et al., 2009 ) and subsequently the total cellular Cx43 content decreases (Zeevi-Levin et al., 2005). Metabolic inhibition activates c-Src kinase, an effect inhibited by 17ß-estradiol (Chung et al., 2009 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Connexins are widely distributed proteins in the body that are crucially important for heart and brain function. Six connexin subunits form a connexon or hemichannel in the plasma membrane. Interactions between two hemichannels in a head-to-head arrangement result in the formation of a gap junction channel. Gap junctions are necessary to coordinate cell function by passing electrical current flow between heart and nerve cells or by allowing exchange of chemical signals and energy substrates. Apart from its localisation at the sarcolemma of cardiomyocytes and brain cells, connexins are also found in mitochondria where they are involved in the regulation of mitochondrial matrix ion fluxes and respiration. Connexin expression is affected by age and gender as well as several pathophysiological alterations such as hypertension, hypertrophy, diabetes, hypercholesterolemia, ischemia, post-myocardial infarction remodelling or heart failure, and post-translationally connexins are modified by phosphorylation/de-phosphorylation and nitros(yl)ation which can modulate channel activity. Using knockout/knockin technology as well as pharmacological approaches, one of the connexins, namely connexin 43, has been identified to be important for cardiac and brain ischemia/reperfusion injury as well as protection from it. Therefore, the current review will focus on the importance of connexin 43 for irreversible injury of heart and brain tissue following ischemia/reperfusion and will highlight the importance of connexin 43 as an emerging therapeutic target in cardio- and neuroprotection. Copyright © 2015. Published by Elsevier Inc.
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    • "suggest the existence of underlying functional alterations that are not expressed morphologically, a circumstance that has been reported elsewhere (Friedman et al., 1973). Ischemia produces a reorganization of the gap junctions and, as a consequence, modifies the conduction velocity in the cardiomyocytes: the gap junctions close and the connexin-43 proteins are dephosphorylated and transferred from the intercalated discs to the cytoplasm and other membrane domains, that is, there is a redistribution (García Dorado et al., 2004; Oosthoek et al., 1993; Shimada et al., 2004; Suarez and Bravo, 2006; Zeevi-Levin et al., 2005). In the Purkinje fibers, and under normal conditions, these junctions are distributed throughout the entire fiber cell membrane, except in that adjoined to the connective tissue. "
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