Editorial
Cardiovascular Research (2009) 83, 613–614 doi:10.1093/cvr/cvp227
Cx43 phosphorylation and cardioprotection Antonio Rodrı´guez-Sinovas* Laboratorio de Cardiologı´a Experimental, Institut de Recerca Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain Online publish-ahead-of-print 3 July 2009
This editorial refers to ‘Phosphorylation of connexin-43 at serine 262 promotes a cardiac injury-resistant state’ by W. Srisakuldee et al., pp. 672–681, this issue Connexin-43 (Cx43), a transmembrane protein able to oligomerize into hexamers around a central pore, is the main protein-forming gap junctional channels in ventricular cardiomyocytes. Altered Cx43 expression or distribution has been shown to impair propagation of electrical impulses and promote arrhythmias during ischaemia and reperfusion.1 However, in the past few years, it has become evident that the role of Cx43 in myocardial ischaemia pathophysiology goes beyond its role in arrhythmogenesis. Gap junction uncouplers have been shown to limit infarct size after ischaemia in different tissues, including heart2 and brain,3 thus supporting a role for gap junctions in intercellular spreading of injury during ischaemia–reperfusion. These findings were further strengthened by studies in heterozygous Cx43-deficient mice (Cx43þ/2 ) submitted to chronic ischaemia without reperfusion,4 which showed reduced infarctions in mutant animals. However, the involvement of Cx43 in the pathophysiology of ischaemia–reperfusion injury is not restricted to the spreading of injury, and several studies suggest an important role in cardioprotection.5,6 In this context, the study by Srisakuldee et al. 7 adds important information on the cardioprotective role of Cx43. The authors demonstrate that both ischaemic preconditioning and pretreatment with diazoxide are able to induce, in isolated rat hearts, specific phosphorylation of Cx43 at serines 262 and 368 without modifying total Cx43 expression. Furthermore, hearts treated with another cardioprotective agent, fibroblast growth factor 2 (FGF-2), displayed similar increases in P-S262-Cx43 and P-S368-Cx43 levels when given during normoxia, before ischaemia or at the onset of reperfusion, and showed reduced Cx43 lateralization during ischaemia. Importantly, a link between these three protective manoeuvres may exist, as they have been proposed to protect hearts by activating PKC1, which also phosphorylates Cx43. However, the study by Srisakuldee et al. 7 goes beyond this initial descriptive The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology. * Corresponding author. Tel: þ34 93 489 4134; fax: þ34 934894032. E-mail address:
[email protected]
data and adds important mechanistic information that can help to understand the cardioprotective role of Cx43. Thus, in elegant experiments carried out in isolated neonatal cardiomyocytes, authors demonstrated that overexpression of wild-type Cx43 was able to reduce injury after simulated ischaemia, whereas overexpression of mutated S262A-Cx43, incapable of phosphorylation at that site, increased injury and abolished protection by FGF-2 and by overexpression of PKC1. In view of these results, Srisakuldee et al. 7 propose that phosphorylation of Cx43 at serine 262 induces an injury-resistant state that can be reached after several protective manoeuvres like ischaemic or pharmacological preconditioning, or treatment with FGF-2. Although the exact mechanism by which Cx43 phosphorylation at S262 contributes to cardioprotection is not explained (i.e. whether it affects channel gating, kinetics, or Cx43 distribution), this study is the first to address the question of the potential molecular modifications of Cx43 involved in protection. The study leaves open several important questions. It is not known whether the lack of protection seen in neonatal cardiomyocytes overexpressing mutant S262A-Cx43 can be generalized to the entire heart. Transgenic mice models expressing this defect should be developed to provide a clear answer. In addition, it is not known whether Cx43 mutations other than at S262 would also affect protection by ischaemic or pharmacological preconditioning. Furthermore, the authors suggest a dominant-negative effect of S262A-Cx43 overexpression over endogenous Cx43 in isolated neonatal cardiomyocytes, but no clear proof of the presence of this effect is given. Moreover, it is not known whether all forms of endogenous protection are linked to Cx43 phosphorylation at S262. Although authors conclude that Cx43 phosphorylation at S262 likely mediates PKC1dependent protection, more studies have to be carried out to clearly confirm this statement. A critical question is how S262 phosphorylation can affect cardioprotection; at present, the mechanisms by which Cx43 mediates cardioprotection are not known. Two main groups of possibilities have been proposed. One involves a reduction in chemical coupling induced by treatments such as ischaemic preconditioning.8 According to the other proposal, Cx43 may mediate cardioprotection through gap junction-independent mechanisms. This hypothesis is supported by experiments performed in isolated
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009. For permissions please email:
[email protected].
614
cardiomyocytes obtained from heterozygous, Cx43-deficient mice (Cx43þ/2 ). As occurred with isolated mice hearts,5 isolated cardiomyocytes obtained from these mutant mice cannot be preconditioned.6 In fact, both hypotheses are not mutually exclusive, and it is possible that both may contribute to some degree to cardioprotection. Little is known about the gap junction-independent effects of Cx43 on protection.9 Unapposed hemichannels that open under ischaemic conditions may mediate the release of several cardioprotective mediators to the extracellular space. Unapposed hemichannels have been considered to be important mediators of protection in other tissues or cells such as astrocytes.10 Deficiency of mitochondrial Cx43 has been shown to abolish protection by diazoxide, a protective drug that supposedly specifically interacts with mitochondrial KATP channels, and to reduce production of reactive oxygen species, known mediators of protection, in response to this drug.11 Importantly, replacement of Cx43 by a different connexin isoform, Cx32, in a knock-in mice model, modifies mitochondrial potassium uptake,12 highlighting the importance of Cx43 in mitochondrial physiology . At present, it is not known whether the link between Cx43 phosphorylation at S262 and cardioprotection described in Srisakuldee et al. 7 affects the complete population of Cx43 or whether it is restricted to Cx43 located at gap junctions, hemichannels, or mitochondria. Previous work has shown that most of the Cx43 located both in gap junctions and mitochondria is in the phosphorylated state, resulting in electrophoretic migration at 43–45 kDa, which corresponds with the mobility described for P-S262-Cx43. Thus, both Cx43 pools are candidates for being involved in protective mechanisms. Regulation of Cx43 goes beyond a single amino acid phosphorylation. More than 20 serine or tyrosine residues are able to be phosphorylated by different protein kinases. Furthermore, it involves regulation by factors like pH, intracellular Ca2þ levels, and post-translational modifications, including S-nitrosylation. The work by Srisakuldee et al. 7 is the first to address this important question and opens an intriguing new field of research.
Editorial
Funding Partially supported by the Spanish Ministries of Science (SAF2008-03067 and RETICS-RECAVA). Antonio Rodrı´guezSinovas is a recipient of a contract from the Generalitat de Catalunya (Programa d’estabilitzacio ´ d’investigadors, Departament de Salut, Direccio ´ d’Estrate `gia i Coordinacio ´).
References 1. Sato T, Ohkusa T, Honjo H, Suzuki S, Yoshida MA, Ishiguro YS et al. Altered expression of connexin43 contributes to the arrhythmogenic substrate during the development of heart failure in cardiomyopathic hamster. Am J Physiol Heart Circ Physiol 2008;294:H1164–H1173. 2. Garcia-Dorado D, Inserte J, Ruiz-Meana M, Gonzalez MA, Solares J, Julia M et al. Gap junction uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion. Circulation 1997;96:3579–3586. 3. Rami A, Volkmann T, Winckler J. Effective reduction of neuronal death by inhibiting gap junctional intercellular communication in a rodent model of global transient cerebral ischemia. Exp Neurol 2001;170:297–304. 4. Kanno S, Kovacs A, Yamada KA, Saffitz JE. Connexin43 as a determinant of myocardial infarct size following coronary occlusion in mice. J Am Coll Cardiol 2003;41:681–686. 5. Schwanke U, Konietzka I, Duschin A, Li X, Schulz R, Heusch G. No ischemic preconditioning in heterozygous connexin43-deficient mice. Am J Physiol Heart Circ Physiol 2002;283:H1740–H1742. 6. Li X, Heinzel FR, Boengler K, Schulz R, Heusch G. Role of connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions. J Mol Cell Cardiol 2004;36:161–163. 7. Srisakuldee W, Jeyaraman MM, Nickel BE, Tanguy S, Jiang ZS, Kardami E. Phosphorylation of connexin-43 at serine 262 promotes a cardiac injury-resistant state. Cardiovasc Res 2009;83:672–681. 8. Miura T, Ohnuma Y, Kuno A, Tanno M, Ichikawa Y, Nakamura Y et al. Protective role of gap junctions in preconditioning against myocardial infarction. Am J Physiol Heart Circ Physiol 2003;286:H214–H221. 9. Rodriguez-Sinovas A, Cabestrero A, Lopez D, Torre I, Morente M, Abellan A et al. The modulatory effects of connexin 43 on cell death/survival beyond cell coupling. Prog Biophys Mol Biol 2007;94:219–232. 10. Lin JH, Lou N, Kang N, Takano T, Hu F, Han X et al. A central role of connexin 43 in hypoxic preconditioning. J Neurosci 2008;28:681–695. 11. Ruiz-Meana M, Rodriguez-Sinovas A, Cabestrero A, Boengler K, Heusch G, Garcia-Dorado D. Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia-reperfusion injury. Cardiovasc Res 2008;77:325–333. 12. Miro-Casas E, Ruiz-Meana M, Agullo E, Stahlhofen S, Rodriguez-Sinovas A, Cabestrero A et al. Connexin43 in cardiomyocyte mitochondria contributes to mitochondrial potassium uptake. Cardiovasc Res 2009; 83:747–756.
