Cx43 as a transducer of signals linked to cardioprotection and DNA synthesis

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Jeyaraman, Maya
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Background and Rationale: Connexin-43 (Cx43) is an integral membrane phosphoprotein and the major constituent of cardiac gap junctions, intercellular channels mediating metabolic and electrical coupling. In addition, Cx43 is involved in the regulation of cell proliferation, acting as an inhibitor of DNA synthesis, and is suggested to be essential for the ability of the heart to become resistant to injury in response to ischemic or pharmacological preconditioning. Cx43 is the target of several different signal transduction cascades culminating in its phosphorylation at several sites located at its C-terminal tail. Previous work in my mentor’s laboratory provided evidence that: cardioprotection of isolated perfused rat hearts by ischemic preconditioning or fibroblast growth factor 2 (FGF-2) administration was closely correlated with above-physiological Cx43 phosphorylation at protein kinase C (PKC) target sites such as serine S262; (ii), the FGF-2-induced cardiac Cx43 phosphorylation at S262 was mediated by protein kinase Cε (PKCε), and this cancelled the ability of Cx43 to inhibit DNA synthesis in neonatal cardiomyocytes. The overall purpose of this study was to further investigate the relationship between Cx43, its phosphorylation at PKCε-target sites such as S262, and PKCε-mediated end-points such as cytoprotection/cardioprotection, and stimulation of DNA synthesis. The main hypotheses were that (a) Cx43 phosphorylation at S262 mediates cardiomyocyte protection from ischemic injury by FGF-2, PKC and ischemic preconditioning, and (b) Cx43-associated inhibition of DNA synthesis is caused by activation of the transforming growth factor beta (TGFβ) pathway. Materials and Methods: Primary cultures of neonatal rat ventricular cardiomyocytes as well as the Cx43-deficient cell line HEK293 were used as in vitro models. Simulated ischemia was achieved in the presence of a low pH medium and incubation in a hypoxia chamber. Cell injury or cell death was assessed using the lactate dehydrogenase or TUNEL assay kits, respectively. Protein expression and localization were examined by western blotting and immunofluorescence using well characterized antibodies. The fraction of cells synthesizing DNA was obtained by determining the BrdU labeling index. Gene transfer in cardiomyocytes was achieved by infection with various adenoviruses carrying: wild type Cx43 or the Cx43 phosphorylation mutants such as S262A-Cx43, or a Cx43 mutant containing a serine to aspartate (S262D-) substitution; dominant negative Smad2 or TGFRII; wild type PKCε. Gene transfer in HEK293 cells was achieved using the TransIT reagent. Results: In the cytoprotection experiments, a modest overexpression of wild type Cx43, which localized predominantly to cell-cell contact sites, increased the resistance of cardiomyocytes to ischemic injury. In contrast, expression of a C-terminal-HA tagged Cx43, which showed localization to perinuclear sites, or of the channel domain-deficient C-terminal fragment of Cx43 (Cx43CT), which localized to cytosol and nucleus, rendered cardiomyocytes more vulnerable to ischemic injury. Preventing Cx43 phosphorylation at S262 by overexpressing S262A-Cx43 also rendered cardiomyocytes more vulnerable to ischemic injury, and fully abolished the ability of either FGF2, or PKC to confer cytoprotection. Conversely, the protective effect of ischemic preconditioning was only partially prevented by S262A-Cx43 expression. In addressing the mechanism by which Cx43 inhibits DNA synthesis in cardiomyocytes we found that the inhibition of several components of TGF-associated signal transduction including TGFβRI (with SB431542), or TGFβRII (by overexpressing dominant negative TGFβRII), or Smad2 (by overexpressing dominant negative Smad2) had no effect on Cx43-mediated inhibition of DNA synthesis. Treatment of cardiomyocytes with TGFβ prevented the FGF-2-induced Cx43 phosphorylation at S262, detected by phospho-specific antibodies. In HEK293 cells, we found that expression of Cx43CT, lacking the channel-forming domain, retained the ability to inhibit DNA synthesis, and that this event was dependent on S262: expression of S262A-Cx43CT elicited maximal inhibition of DNA synthesis, while S262D-Cx43CT (simulating constitutive phosphorylation) had no inhibitory effect. Conclusions: Phosphorylation of Cx43 at S262 is likely a mediator of FGF-2-induced and PKC-mediated cardiomyocyte resistance to ischemic injury, while protection by ischemic preconditioning may be only partially dependent on Cx43 and its phosphorylation at S262. The mechanism of cytoprotection by Cx43 phosphorylation at S262 is linked to preservation of membrane targeting and intercellular channel formation, as it was abolished in the absence of channel-forming ability (Cx43CT) or aberrant Cx43 localization (Cx43-HA). The mechanism by which Cx43 inhibits DNA synthesis is not dependent on downstream activation of TGF signal transduction. It is possible, however, that a component of TGF-triggered inhibition of DNA synthesis includes prevention of mitogen-induced Cx43 phosphorylation at S262. Furthermore, inhibition of DNA synthesis by Cx43, and its regulation by S262 phosphorylation are independent of channel-forming ability or subcellular localization. Targeting Cx43 and its phosphorylation at S262 may provide a novel strategy to improve cardiac response to injury, by decreasing tissue loss through increased resistance as well as improving a regenerative response by disinhibiting cardiomyocyte proliferation.
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