Skip to main content

Advertisement

SpringerLink
Log in

Activation of RISK and SAFE pathways is not involved in the effects of Cx43 deficiency on tolerance to ischemia–reperfusion injury and preconditioning protection

  • Original Contribution
  • Published:
Basic Research in Cardiology Aims and scope Submit manuscript

Abstract

Connexin 43 (Cx43) deficiency increases myocardial tolerance to ischemia–reperfusion injury and abolishes preconditioning protection. It is not known whether modifications in baseline signaling through protective RISK or SAFE pathways or in response to preconditioning may contribute to these effects. To answer this question we used Cx43Cre−ER(T)/fl mice, in which Cx43 expression is abolished after 4-hydroxytamoxifen (4-OHT) administration. Isolated hearts from Cx43Cre−ER(T)/fl mice, or from Cx43fl/fl controls, treated with vehicle or 4-OHT, were submitted to global ischemia (40 min) and reperfusion. Cx43 deficiency was associated with reduced infarct size after ischemia–reperfusion (11.17 ± 3.25 % vs. 65.04 ± 3.79, 59.31 ± 5.36 and 65.40 ± 4.91, in Cx43fl/fl animals treated with vehicle, Cx43fl/fl mice treated with 4-OHT, and Cx43Cre−ER(T)/fl mice treated with vehicle, respectively, n = 8–9, p < 0.001). However, the ratio phosphorylated/total protein expression for Akt, ERK-1/2, GSK3β and STAT3 was not increased in normoxic samples from animals lacking Cx43. Instead, a reduction in the phosphorylation state of GSK3β was observed in Cx43-deficient mice (ratio: 0.15 ± 0.02 vs. 0.56 ± 0.11, 0.77 ± 0.15, and 0.46 ± 0.14, respectively, n = 5–6, p < 0.01). Furthermore, ischemic preconditioning (IPC, 4 cycles of 3.5 min of ischemia and 5 min of reperfusion) increased phosphorylation of ERK-1/2, GSK3β, and STAT3 in all hearts without differences between groups (n = 5–6, p < 0.05), although Cx43 deficient mice were not protected by either IPC or pharmacological preconditioning with diazoxide. Our data demonstrate that modification of RISK and SAFE signaling does not contribute to the role of Cx43 in the increased tolerance to myocardial ischemia–reperfusion injury and in preconditioning protection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Azarashvili T, Baburina Y, Grachev D, Krestinina O, Evtodienko Y, Stricker R, Reiser G (2011) Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43. Am J Physiol Cell Physiol 300:C707–C720. doi:10.1152/ajpcell.00061.2010

    Article  PubMed  CAS  Google Scholar 

  2. Boengler K, Dodoni G, Rodriguez-Sinovas A, Cabestrero A, Ruiz-Meana M, Gres P, Konietzka I, Lopez-Iglesias C, Garcia-Dorado D, Di Lisa F, Heusch G, Schulz R (2005) Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning. Cardiovasc Res 67:234–244. doi:10.1016/j.cardiores.2005.04.014

    Article  PubMed  CAS  Google Scholar 

  3. Boengler K, Hilfiker-Kleiner D, Heusch G, Schulz R (2010) Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion. Basic Res Cardiol 105:771–785. doi:10.1007/s00395-010-0124-1

    Article  PubMed  CAS  Google Scholar 

  4. Boengler K, Ruiz-Meana M, Gent S, Ungefug E, Soetkamp D, Miro-Casas E, Cabestrero A, Fernandez-Sanz C, Semenzato M, Di Lisa F, Rohrbach S, Garcia-Dorado D, Heusch G, Schulz R (2012) Mitochondrial connexin 43 impacts on respiratory complex I activity and mitochondrial oxygen consumption. J Cell Mol Med 16:1649–1655. doi:10.1111/j.1582-4934.2011.01516.x

    Article  PubMed  CAS  Google Scholar 

  5. Cohen MV, Downey JM (2011) Ischemic postconditioning: from receptor to end-effector. Antioxid Redox Signal 14:821–831. doi:10.1089/ars.2010.3318

    Article  PubMed  CAS  Google Scholar 

  6. Danik SB, Rosner G, Lader J, Gutstein DE, Fishman GI, Morley GE (2008) Electrical remodeling contributes to complex tachyarrhythmias in connexin43-deficient mouse hearts. FASEB J 22:1204–1212. doi:10.1096/fj.07-8974com

    Article  PubMed  CAS  Google Scholar 

  7. Desplantez T, Dupont E, Severs NJ, Weingart R (2007) Gap junction channels and cardiac impulse propagation. J Membr Biol 218:13–28. doi:10.1007/s00232-007-9046-8

    Article  PubMed  CAS  Google Scholar 

  8. Eckardt D, Theis M, Degen J, Ott T, van Rijen HV, Kirchhoff S, Kim JS, de Bakker JM, Willecke K (2004) Functional role of connexin 43 gap junction channels in adult mouse heart assessed by inducible gene deletion. J Mol Cell Cardiol 36:101–110. doi:10.1016/j.yjmcc.2003.10.006

    Article  PubMed  CAS  Google Scholar 

  9. Fan WJ, van Vuuren D, Genade S, Lochner A (2010) Kinases and phosphatases in ischaemic preconditioning: a re-evaluation. Basic Res Cardiol 105:495–511. doi:10.1007/s00395-010-0086-3

    Article  PubMed  CAS  Google Scholar 

  10. Garcia-Dorado D, Inserte J, Ruiz-Meana M, Gonzalez MA, Solares J, Julia M, Barrabes JA, Soler–Soler J (1997) Gap junction uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion. Circulation 96:3579–3586. doi:10.1161/01.CIR.96.10.3579

    Article  PubMed  CAS  Google Scholar 

  11. Garcia-Dorado D, Rodriguez-Sinovas A, Ruiz-Meana M (2004) Gap junction-mediated spread of cell injury and death during myocardial ischemia-reperfusion. Cardiovasc Res 61:386–401. doi:10.1016/j.cardiores.2003.11.039

    Article  PubMed  CAS  Google Scholar 

  12. Haugan K, Marcussen N, Kjolbye AL, Nielsen MS, Hennan JK, Petersen JS (2006) Treatment with the gap junction modifier rotigaptide (ZP123) reduces infarct size in rats with chronic myocardial infarction. J Cardiovasc Pharmacol 47:236–242. doi:10.1097/01.fjc.0000200990.31611.6e

    Article  PubMed  CAS  Google Scholar 

  13. Hausenloy DJ, Tsang A, Mocanu MM, Yellon DM (2005) Ischemic preconditioning protects by activating prosurvival kinases at reperfusion. Am J Physiol Heart Circ Physiol 288:H971–H976. doi:10.1152/ajpheart.00374.2004

    Article  PubMed  CAS  Google Scholar 

  14. Hawat G, Helie P, Baroudi G (2012) Single intravenous low-dose injections of connexin 43 mimetic peptides protect ischemic heart in vivo against myocardial infarction. J Mol Cell Cardiol 53:559–566. doi:10.1016/j.yjmcc.2012.07.008

    Article  PubMed  CAS  Google Scholar 

  15. Heinzel FR, Luo Y, Li X, Boengler K, Buechert A, Garcia-Dorado D, Di Lisa F, Schulz R, Heusch G (2005) Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in connexin 43 deficient mice. Circ Res 97:583–586. doi:10.1161/01.RES.0000181171.65293.65

    Article  PubMed  CAS  Google Scholar 

  16. Hennan JK, Swillo RE, Morgan GA, Rossman EI, Kantrowitz J, Butera J, Petersen JS, Gardell SJ, Vlasuk GP (2009) GAP-134 ([2S,4R]-1-[2-aminoacetyl]4-benzamidopyrrolidine-2-carboxylic acid) prevents spontaneous ventricular arrhythmias and reduces infarct size during myocardial ischemia/reperfusion injury in open-chest dogs. J Cardiovasc Pharmacol Ther 14:207–214. doi:10.1177/1074248409340779

    Article  PubMed  CAS  Google Scholar 

  17. Heusch G, Boengler K, Schulz R (2008) Cardioprotection: nitric oxide, protein kinases, and mitochondria. Circulation 118:1915–1919. doi:10.1161/CIRCULATIONAHA.108.805242

    Article  PubMed  Google Scholar 

  18. Heusch G, Boengler K, Schulz R (2010) Inhibition of mitochondrial permeability transition pore opening: the Holy Grail of cardioprotection. Basic Res Cardiol 105:151–154. doi:10.1007/s00395-009-0080-9

    Article  PubMed  Google Scholar 

  19. Heusch G, Buchert A, Feldhaus S, Schulz R (2006) No loss of cardioprotection by postconditioning in connexin 43-deficient mice. Basic Res Cardiol 101:354–356. doi:10.1007/s00395-006-0589-0

    Article  PubMed  CAS  Google Scholar 

  20. Heusch G, Musiolik J, Gedik N, Skyschally A (2011) Mitochondrial STAT3 activation and cardioprotection by ischemic postconditioning in pigs with regional myocardial ischemia/reperfusion. Circ Res 109:1302–1308. doi:10.1161/CIRCRESAHA.111.255604

    Article  PubMed  CAS  Google Scholar 

  21. Ishikawa S, Kuno A, Tanno M, Miki T, Kouzu H, Itoh T, Sato T, Sunaga D, Murase H, Miura T (2012) Role of connexin-43 in protective PI3K-Akt-GSK-3beta signaling in cardiomyocytes. Am J Physiol Heart Circ Physiol 302:H2536–H2544. doi:10.1152/ajpheart.00940.2011

    Article  PubMed  CAS  Google Scholar 

  22. Kanno S, Kovacs A, Yamada KA, Saffitz JE (2003) Connexin43 as a determinant of myocardial infarct size following coronary occlusion in mice. J Am Coll Cardiol 41:681–686. doi:10.1016/S0735-1097(02)02893-0

    Article  PubMed  CAS  Google Scholar 

  23. Li X, Heinzel FR, Boengler K, Schulz R, Heusch G (2004) Role of connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions. J Mol Cell Cardiol 36:161–163. doi:10.1016/j.yjmcc.2003.10.019

    Article  PubMed  CAS  Google Scholar 

  24. Lofgren B, Povlsen JA, Rasmussen LE, Stottrup NB, Solskov L, Krarup PM, Kristiansen SB, Botker HE, Nielsen TT (2010) Amino acid transamination is crucial for ischaemic cardioprotection in normal and preconditioned isolated rat hearts–focus on l-glutamate. Exp Physiol 95:140–152. doi:10.1113/expphysiol.2009.049452

    Article  PubMed  CAS  Google Scholar 

  25. Miro-Casas E, Ruiz-Meana M, Agullo E, Stahlhofen S, Rodriguez-Sinovas A, Cabestrero A, Jorge I, Torre I, Vazquez J, Boengler K, Schulz R, Heusch G, Garcia-Dorado D (2009) Connexin 43 in cardiomyocyte mitochondria contributes to mitochondrial potassium uptake. Cardiovasc Res 83:747–756. doi:10.1093/cvr/cvp157

    Article  PubMed  CAS  Google Scholar 

  26. Mocanu MM, Bell RM, Yellon DM (2002) PI3 kinase and not p42/p44 appears to be implicated in the protection conferred by ischemic preconditioning. J Mol Cell Cardiol 34:661–668. doi:10.1006/jmcc.2002.2006

    Article  PubMed  CAS  Google Scholar 

  27. Murphy E, Steenbergen C (2008) Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol Rev 88:581–609. doi:10.1152/physrev.00024.2007

    Article  PubMed  CAS  Google Scholar 

  28. Rodriguez-Sinovas A, Boengler K, Cabestrero A, Gres P, Morente M, Ruiz-Meana M, Konietzka I, Miro E, Totzeck A, Heusch G, Schulz R, Garcia-Dorado D (2006) Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res 99:93–101. doi:10.1161/01.RES.0000230315.56904.de

    Article  PubMed  CAS  Google Scholar 

  29. Rodriguez-Sinovas A, Garcia-Dorado D, Ruiz-Meana M, Soler–Soler J (2004) Enhanced effect of gap junction uncouplers on macroscopic electrical properties of reperfused myocardium. J Physiol 559:245–257. doi:10.1113/jphysiol.2004.065144

    Article  PubMed  CAS  Google Scholar 

  30. Rodriguez-Sinovas A, Sanchez JA, Gonzalez-Loyola A, Barba I, Morente M, Aguilar R, Agullo E, Miro-Casas E, Esquerda N, Ruiz-Meana M, Garcia-Dorado D (2010) Effects of substitution of Cx43 by Cx32 on myocardial energy metabolism, tolerance to ischemia and preconditioning protection. J Physiol 588:1139–1151. doi:10.1113/jphysiol.2009.186577

    Article  PubMed  CAS  Google Scholar 

  31. Ruiz-Meana M, Garcia-Dorado D, Hofstaetter B, Piper HM, Soler–Soler J (1999) Propagation of cardiomyocyte hypercontracture by passage of Na(+) through gap junctions. Circ Res 85:280–287. doi:10.1161/01.RES.85.3.280

    Article  PubMed  CAS  Google Scholar 

  32. Ruiz-Meana M, Inserte J, Fernandez-Sanz C, Hernando V, Miro-Casas E, Barba I, Garcia-Dorado D (2011) The role of mitochondrial permeability transition in reperfusion-induced cardiomyocyte death depends on the duration of ischemia. Basic Res Cardiol 106:1259–1268. doi:10.1007/s00395-011-0225-5

    Article  PubMed  CAS  Google Scholar 

  33. Sanchez JA, Rodriguez-Sinovas A, Fernandez-Sanz C, Ruiz-Meana M, Garcia-Dorado D (2011) Effects of a reduction in the number of gap junction channels or in their conductance on ischemia-reperfusion arrhythmias in isolated mouse hearts. Am J Physiol Heart Circ Physiol 301:H2442–H2453. doi:10.1152/ajpheart.00540.2011

    Article  PubMed  CAS  Google Scholar 

  34. Schwanke U, Konietzka I, Duschin A, Li X, Schulz R, Heusch G (2002) No ischemic preconditioning in heterozygous connexin 43-deficient mice. Am J Physiol Heart Circ Physiol 283:H1740–H1742. doi:10.1152/ajpheart.00442.2002

    PubMed  CAS  Google Scholar 

  35. Stewart S, Lesnefsky EJ, Chen Q (2009) Reversible blockade of electron transport with amobarbital at the onset of reperfusion attenuates cardiac injury. Transl Res 153:224–231. doi:10.1016/j.trsl.2009.02.003

    Article  PubMed  CAS  Google Scholar 

  36. Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM (2004) Postconditioning: a form of “modified reperfusion” protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ Res 95:230–232. doi:10.1161/01.RES.0000138303.76488.fe

    Article  PubMed  CAS  Google Scholar 

  37. Wang N, De Vuyst E, Ponsaerts R, Boengler K, Palacios-Prado N, Wauman J, Lai CP, De Bock M, Decrock E, Bol M, Vinken M, Rogiers V, Tavernier J, Evans WH, Naus CC, Bukauskas FF, Sipido KR, Heusch G, Schulz R, Bultynck G, Leybaert L (2013) Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury. Basic Res Cardiol 108:309. doi:10.1007/s00395-012-0309-x

    Google Scholar 

  38. Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, Cohen MV (2004) Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardiol 44:1103–1110. doi:10.1016/j.jacc.2004.05.060

    Article  PubMed  Google Scholar 

  39. Yasui K, Kada K, Hojo M, Lee JK, Kamiya K, Toyama J, Opthof T, Kodama I (2000) Cell-to-cell interaction prevents cell death in cultured neonatal rat ventricular myocytes. Cardiovasc Res 48:68–76. doi:10.1016/S0008-6363(00)00145-0

    Article  PubMed  CAS  Google Scholar 

  40. Yoshioka J, Chutkow WA, Lee S, Kim JB, Yan J, Tian R, Lindsey ML, Feener EP, Seidman CE, Seidman JG, Lee RT (2012) Deletion of thioredoxin-interacting protein in mice impairs mitochondrial function but protects the myocardium from ischemia-reperfusion injury. J Clin Invest 122:267–279. doi:10.1172/JCI44927

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by the Spanish Ministries of Science (SAF2008-03067 and RETICS-RECAVA, RD06/0014/0025) and Sociedad Española de Cardiología (Proyecto de Investigación de la SEC para Investigación Básica 2011). Antonio Rodríguez-Sinovas is a recipient of a contract from the Generalitat de Catalunya (Programa d’estabilització d’investigadors, Departament de Salut, Direcció d’Estratègia i Coordinació). Jose A. Sánchez is supported by the International Research Training Group 1566 on Protecting the Heart from Ischemia (PROMISE).

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Author information

Authors and Affiliations

  1. Laboratorio de Cardiología Experimental, Vall d’Hebron University Hospital and Research Institute, Universitat Autònoma de Barcelona, Pg. Vall d’Hebron 119-129, 08035, Barcelona, Spain

    Jose A. Sánchez, Antonio Rodríguez-Sinovas, Ignasi Barba, Elisabet Miró-Casas, Celia Fernández-Sanz, Marisol Ruiz-Meana, Juan J. Alburquerque-Béjar & David García-Dorado

Authors
  1. Jose A. Sánchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Antonio Rodríguez-Sinovas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ignasi Barba
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elisabet Miró-Casas
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Celia Fernández-Sanz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marisol Ruiz-Meana
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Juan J. Alburquerque-Béjar
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David García-Dorado
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Antonio Rodríguez-Sinovas.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 1593 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sánchez, J.A., Rodríguez-Sinovas, A., Barba, I. et al. Activation of RISK and SAFE pathways is not involved in the effects of Cx43 deficiency on tolerance to ischemia–reperfusion injury and preconditioning protection. Basic Res Cardiol 108, 351 (2013). https://doi.org/10.1007/s00395-013-0351-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00395-013-0351-3

Keywords

Search

Navigation