Skip to main content

Advertisement

SpringerLink
Log in

Cardiac fibroblasts inhibit β-adrenoceptor-dependent connexin43 expression in neonatal rat cardiomyocytes

  • Original Article
  • Published:
Naunyn-Schmiedeberg's Archives of Pharmacology Aims and scope Submit manuscript

Abstract

Cardiac fibroblasts play an important role in adverse cardiac remodelling. As in many cardiac diseases connexin43 (Cx43) is altered, we wanted to elucidate whether fibroblasts may influence cardiac Cx43 expression. We used four different cell culture systems of neonatal rat cardiomyocytes (CM) and fibroblasts (FB): type 1, pure CM culture; type 2, co-culture of CM/FB; type 3, pure FB culture; type 4, Transwell® system: CM/FB co-cultured but separated by a microporous membrane. Stimulation of types 1–3 cell culture models with isoprenaline significantly enhanced Cx43-protein and Cx43-mRNA expression as well as phosphorylation of ERK and translocation of AP1 and CREB only in the CM cultures; whereas, the CM/FB co-cultures and the FB cultures did not respond to isoprenaline. Similarly, if CM and FB were separated by a microporous membrane (Transwell® system) the isoprenaline-induced increase in CM Cx43 was completely suppressed, suggesting the existence of a soluble factor responsible for the suppressant effect of FB. Angiotensin II determination in types 1 and 2 cell culture supernatants revealed that the CM/FB co-cultures exhibited a significant higher angiotensin II release than the CM cultures. Furthermore, we aimed to inhibit angiotensin II signal transduction pathway: blockade of AT1 receptors or PKC inhibition restored the responsiveness of CM/FB co-cultures to isoprenaline. Moreover, external addition of angiotensin II to CM cultures also resulted in suppression of isoprenaline-stimulated Cx43 expression in an AT1-receptor- and PKC-dependent manner. Thus, our study indicates that cardiac fibroblasts inhibit β-adrenoceptor-dependent Cx43 signalling in CM involving angiotensin II.

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

Abbreviations

ACE:

Angiotensin-converting enzyme

AP1:

Activator protein 1

AT1-R:

Angiotensin II receptor type 1

ATII:

Angiotensin II

Cx43:

Connexin43

CREB:

CRE-binding protein

Ct:

Threshold cycle

EMSA:

Electrophoretic mobility shift assay

ERK1/2:

Extracellular signal-regulated kinase 1/2 (p42/44)

PCR:

Polymerase chain reaction

PKC:

Protein kinase C

References

  • Aggeli IKS, Gaitanaki C, Beis I (2006) Involvement of JNKs and p38-MAPK/MSK1 pathways in H2O2-induced upregulation of heme oxygenase-1 mRNA in H9c2 cells. Cell Signal 18:1801–1812

    Article  PubMed  CAS  Google Scholar 

  • Andersson-Sjöland A, de Alba CG, Nihlberg K, Becerril C, Ramírez R, Pardo A, Westergren-Thorsson G, Selman M (2008) Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis. Int J Biochem Cell Biol 40:2129–2140

    Article  PubMed  Google Scholar 

  • Arino T, Tanonaka K, Kawahara Y, Maki T, Takagi N, Yagi A, Takeo S (2008) Effects of tanshinone VI on phosphorylation of ERK and Akt in isolated cardiomyocytes and cardiac fibroblasts. Eur J Pharmacol 580:298–305

    Article  PubMed  CAS  Google Scholar 

  • Bailey J, Phillips RJ, Pollard AJ, Gilmore K, Robson SC, Europe-Finner GN (2002) Characterization and functional analysis of cAMP response element modulator protein and activating transcription factor 2 (ATF2) isoforms in the human myometrium during pregnancy and labor: identification of a novel ATF2 species with potent transactivation properties. J Clin Endocrinol Metab 87:1717–1728

    Article  PubMed  CAS  Google Scholar 

  • Beardslee MA, Laing JG, Beyer EC, Saffitz JE (1998) Rapid turnover of connexin43 in the adult rat heart. Circ Res 83:629–635

    Article  PubMed  CAS  Google Scholar 

  • Brodde OE, Bruck H, Leineweber K (2006) Cardiac adrenoceptors: physiological and pathophysiological relevance. J Pharmacol Sci 100:323–337

    Article  PubMed  CAS  Google Scholar 

  • Brown MA, Iyer RK, Radisic M (2008) Pulsatile perfusion bioreactor for cardiac tissue engineering. Biotechnol Prog 24:907–920

    Article  PubMed  CAS  Google Scholar 

  • Darrow BJ, Fast VG, Kléber AG, Beyer EC, Saffitz JE (1996) Functional and structural assessment of intercellular communication. Increased conduction velocity and enhanced connexin expression in dibutyryl cAMP-treated cultured cardiac myocytes. Circ Res 79:174–183

    Article  PubMed  CAS  Google Scholar 

  • Davis LM, Rodefeld ME, Green K, Beyer EC, Saffitz JE (1995) Gap junction protein phenotypes of the human heart and conduction system. J Cardiovasc Electrophysiol 6:813–822

    Article  PubMed  CAS  Google Scholar 

  • de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T (2000) International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 52:415–472

    PubMed  Google Scholar 

  • Doble BW, Kardami E (1995) Basic fibroblast growth factor stimulates connexin-43 expression and intercellular communication of cardiac fibroblasts. Mol Cell Biochem 143:81–87

    Article  PubMed  CAS  Google Scholar 

  • Dodge SM, Beardslee MA, Darrow BJ, Green KG, Beyer EC, Saffitz JE (1998) Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes. J Am Coll Cardiol 32:800–807

    Article  PubMed  CAS  Google Scholar 

  • Dostal DE, Baker KM (1999) The cardiac renin–angiotensin system: conceptual, or a regulator of cardiac function? Circ Res 85:643–650

    Article  PubMed  CAS  Google Scholar 

  • Dostal DE, Rothblum KN, Conrad KM, Cooper GR, Baker KM (1992) Detection of angiotensin I and II in cultured rat cardiac myocytes and fibroblasts. Am J Physiol 263:C851–C863

    PubMed  CAS  Google Scholar 

  • Dostal DE, Booz GW, Baker KM (2000) Regulation of angiotensinogen gene expression and protein in neonatal rat cardiac fibroblasts by glucocorticoid and beta-adrenergic stimulation. Basic Res Cardiol 95:485–490

    Article  PubMed  CAS  Google Scholar 

  • Echetebu CO, Ali M, Izban MG, MacKay L, Garfield RE (1999) Localization of regulatory protein binding sites in the proximal region of human myometrial connexin43 gene. Mol Hum Reprod 5:757–766

    Article  PubMed  CAS  Google Scholar 

  • Gilbert EM, Sandoval A, Larrabee P, Renlund DG, O'Connell JB, Bristow MR (1993) Lisinopril lowers cardiac adrenergic drive and increases beta-receptor density in the failing human heart. Circulation 88:472–480

    Article  PubMed  CAS  Google Scholar 

  • Goldsmith EC, Hoffman A, Morales MO, Potts JD, Price RL, McFadden A, Rice M, Borg TK (2004) Organization of fibroblasts in the heart. Dev Dyn 230:787–794

    Article  PubMed  CAS  Google Scholar 

  • Henegar JR, Schwartz DD, Janicki JS (1998) ANG II-related myocardial damage: role of cardiac sympathetic catecholamines and beta-receptor regulation. Am J Physiol 275:H534–541

    PubMed  CAS  Google Scholar 

  • Huang D, Wang Y, Yang C, Liao Y, Huang K (2009) Angiotensin II promotes poly(ADP-ribosyl)ation of c-Jun/c-Fos in cardiac fibroblasts. J Mol Cell Cardiol 46:25–32

    Article  PubMed  CAS  Google Scholar 

  • Huang J, Wang D, Zheng J, Huang X, Jin H (2012) Hydrogen sulfide attenuates cardiac hypertrophy and fibrosis induced by abdominal aortic coarctation in rats. Mol Med Report 5:923–928

    CAS  Google Scholar 

  • Hussain W, Patel PM, Chowdhury RA, Cabo C, Ciaccio EJ, Lab MJ, Duffy HS, Wit AL, Peters NS (2012) The renin–angiotensin system mediates the effects of stretch on conduction velocity, connexin43 expression, and redistribution in intact ventricle. J Cardiovasc Electrophysiol 21:1276–1283

    Article  Google Scholar 

  • Inoue N, Ohkusa T, Nao T, Lee JK, Matsumoto T, Hisamatsu Y, Satoh T, Yano M, Yasui K, Kodama I, Matsuzaki M (2004) Rapid electrical stimulation of contraction modulates gap junction protein in neonatal rat cultured cardiomyocytes: involvement of mitogen-activated protein kinases and effects of angiotensin II-receptor antagonist. J Am Coll Cardiol 44:914–922

    PubMed  CAS  Google Scholar 

  • Isakson BE, Kronke G, Kadl A, Leitinger N, Duling BR (2006) Oxidized phospholipids alter vascular connexin expression, phosphorylation, and heterocellular communication. Arterioscler Thromb Vasc Biol 26:2216–2221

    Article  PubMed  CAS  Google Scholar 

  • Ishida J, Kai M, Ohkura Y (1986) High-performance liquid chromatography of tyrosine-containing peptides by pre-column derivatization involving formylation followed by fluorescence reaction with 1,2-diamino-4,5-dimethoxybenzene. J Chromatogr 356:171–177

    Article  PubMed  CAS  Google Scholar 

  • Jaffré F, Bonnin P, Callebert J, Debbabi H, SetolaV DS, Monassier L, Mettauer B, Blaxall BC, Launay JM, Maroteaux L (2009) Serotonin and angiotensin receptors in cardiac fibroblasts coregulate adrenergic-dependent cardiac hypertrophy. Circ Res 104:113–123

    Article  PubMed  Google Scholar 

  • Jugdutt BI (2003) Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough? Circulation 108:1395–1403

    Article  PubMed  Google Scholar 

  • Kohl P, Camelliti P, Burton FL, Smith GL (2005) Electrical coupling of fibroblasts and myocytes: relevance for cardiac propagation. J Electrocardiol 38:45–50

    Article  PubMed  Google Scholar 

  • Kostin S (2007) Zonula occludens-1 and connexin43 expression in the failing human heart. J Cell Mol Med 11:892–895

    Article  PubMed  Google Scholar 

  • Kostin S, Rieger M, Dammer S, Hein S, Richter M, Klövekorn WP, Bauer EP, Schaper J (2003) Gap junction remodeling and altered connexin43 expression in the failing human heart. Mol Cell Biochem 242:135–144

    Article  PubMed  CAS  Google Scholar 

  • Li L, Fan D, Wang C, Wang JY, Cui XB, Wu D, Zhou Y, Wu LL (2011) Angiotensin II increases periostin expression via Ras/p38 MAPK/CREB and ERK1/2/TGF-β1 pathways in cardiac fibroblasts. Cardiovasc Res 91:80–89

    Article  PubMed  CAS  Google Scholar 

  • Makino T, Hattori Y, Matsuda N, Onozuka H, Sakuma I, Kitabatake A (2003) Effects of angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on beta-adrenoceptor signaling in heart failure produced by myocardial infarction in rabbits: reversal of altered expression of beta-adrenoceptor kinase and G i alpha. J Pharmacol Exp Ther 304:370–379

    Article  PubMed  CAS  Google Scholar 

  • Markou T, Hadzopoulou-Cladaras M, Lazou A (2004) Phenylephrine induces activation of CREB in adult rat cardiac myocytes through MSK1 and PKA signaling pathways. J Mol Cell Cardiol 37:1001–1011

    Article  PubMed  CAS  Google Scholar 

  • Mazzolai L, Nussberger J, Aubert JF, Brunner DB, Gabbiani G, Brunner HR, Pedrazzini T (1998) Blood pressure-independent cardiac hypertrophy induced by locally activated renin–angiotensin system. Hypertension 31:1324–1330

    Article  PubMed  CAS  Google Scholar 

  • Peng HJ, Dai DZ, Ji H, Dai Y (2010) The separate roles of endothelin receptors participate in remodeling of matrix metalloproteinase and connexin43 of cardiac fibroblasts in maladaptive response to isoproterenol. Eur J Pharmacol 634:101–106

    Article  PubMed  CAS  Google Scholar 

  • Polontchouk L, Ebelt B, Jackels M, Dhein S (2002) Chronic effects of endothelin 1 and angiotensin II on gap junctions and intercellular communication in cardiac cells. FASEB J 16:87–89

    PubMed  CAS  Google Scholar 

  • Porter KE, Turner NA (2009) Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol Ther 123:255–278

    Article  PubMed  CAS  Google Scholar 

  • Radisic M, Park H, Martens TP, Salazar-Lazaro JE, Geng W, Wang Y, Langer R, Freed LE, Vunjak-Novakovic G (2008) Pre-treatment of synthetic elastomeric scaffolds by cardiac fibroblasts improves engineered heart tissue. J Biomed Mater Res A 86:713–724

    PubMed  Google Scholar 

  • Salameh A, Frenzel C, Boldt A, Rassler B, Glawe I, SchulteJ MK, Zimmer HG, Pfeiffer D, Dhein S (2006) Subchronic alpha- and beta-adrenergic regulation of cardiac gap junction protein expression. FASEB J 20:365–367

    PubMed  CAS  Google Scholar 

  • Salameh A, Krautblatter S, Baessler S, Karl S, Rojas Gomez D, Dhein S, Pfeiffer D (2008) Signal transduction and transcriptional control of cardiac connexin43 up-regulation after alpha 1-adrenoceptor stimulation. J Pharmacol Exp Ther 326:315–322

    Article  PubMed  CAS  Google Scholar 

  • Salameh A, Krautblatter S, Karl S, Blanke K, Rojas-Gomez D, Dhein S, Pfeiffer D, Janousek J (2009) The signal transduction cascade regulating the expression of the gap junction protein connexin43 by β-adrenoceptors. Brit J Pharmacol 158:198–208

    Article  CAS  Google Scholar 

  • Salameh A, Karl S, Djilali H, Dhein S, Janousek J, Daehnert I (2010a) Opposing and synergistic effects of cyclic mechanical stretch and α- or β-adrenergic stimulation on the cardiac gap junction protein Cx43. Pharmacol Res 62:506–513

    Article  PubMed  CAS  Google Scholar 

  • Salameh A, Wustmann A, Karl S, Blanke K, Apel D, Rojas-Gomez D, Franke H, Mohr FW, Janousek J, Dhein S (2010b) Cyclic mechanical stretch induces cardiomyocyte orientation and polarisation of the gap junction protein connexin43. Circ Res 106:1592–1602

    Article  PubMed  CAS  Google Scholar 

  • Santiago JJ, Dangerfield AL, Rattan SG, Bathe KL, Cunnington RH, Raizman JE, Bedosky KM, Freed DH, Kardami E, Dixon IM (2010) Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts. Dev Dyn 239:1573–1584

    Article  PubMed  CAS  Google Scholar 

  • Schwartz DD, Naff BP (1997) Activation of protein kinase C by angiotensin II decreases beta 1-adrenergic receptor responsiveness in the rat heart. J Cardiovasc Pharmacol 29:257–264

    Article  PubMed  CAS  Google Scholar 

  • Teunissen BE, Jansen AT, van Amersfoorth SC, O'Brien TX, Jongsma HJ, Bierhuizen MF (2003) Analysis of the rat connexin43 proximal promoter in neonatal cardiomyocytes. Gene 322:123–136

    Article  PubMed  CAS  Google Scholar 

  • Teunissen BE, Smeets PJ, Willemsen PH, De Windt LJ, Van der Vusse GJ, Van Bilsen M (2007) Activation of PPARdelta inhibits cardiac fibroblast proliferation and the transdifferentiation into myofibroblasts. Cardiovasc Res 75:519–529

    Article  PubMed  CAS  Google Scholar 

  • Tilley DG, Rockman HA (2006) Role of β-adrenergic receptor signaling and desensitization in heart failure: new concepts and prospects for treatment. Expert Rev Cardiovasc Ther 4:417–432

    Article  PubMed  CAS  Google Scholar 

  • Turner NA, Porter KE, Smith WH, White HL, Ball SG, Balmforth AJ (2003) Chronic beta2-adrenergic receptor stimulation increases proliferation of human cardiac fibroblasts via an autocrine mechanism. Cardiovasc Res 57:784–792

    Article  PubMed  CAS  Google Scholar 

  • Wang J, Seth A, McCulloch CA (2000) Force regulates smooth muscle actin in cardiac fibroblasts. Am J Physiol Heart Circ Physiol 279:H2776–H2785

    PubMed  CAS  Google Scholar 

  • Yoshida H, Takahashi M, Tanonaka K, Maki T, Nasa Y, Takeo S (2001) Effects of ACE inhibition and angiotensin II type 1 receptor blockade on cardiac function and G proteins in rats with chronic heart failure. Br J Pharmacol 134:150–160

    Article  PubMed  CAS  Google Scholar 

  • Yoshida M, Ohkusa T, Nakashima T, Takanari H, Yano M, Takemura G, Honjo H, Kodama I, Mizukami Y, Matsuzaki M (2011) Alterations in adhesion junction precede gap junction remodelling during the development of heart failure in cardiomyopathic hamsters. Cardiovasc Res 92:95–105

    Article  PubMed  CAS  Google Scholar 

  • Zabrouskov V, Ge Y, Schwartz J, Walker JW (2008) Unravelling molecular complexity of phosphorylated human cardiac troponin I by top down electron capture dissociation/electron transfer dissociation mass spectrometry. Mol Cell Proteomics 7:1838–1849

    Article  PubMed  CAS  Google Scholar 

  • Zhang GX, Kimura S, Nishiyama A, Shokoji T, Rahman M, Yao L, Nagai Y, Fujisawa Y, Miyatake A, Abe Y (2005) Cardiac oxidative stress in acute and chronic isoproterenol-infused rats. Cardiovasc Res 65:230–238

    Article  PubMed  CAS  Google Scholar 

Download references

Disclosure statement

None

Author information

Authors and Affiliations

  1. Clinic for Paediatric Cardiology, University of Leipzig, Heart Centre, Struempellstr. 39, 04289, Leipzig, Germany

    A. Salameh, H. Djilali, K. Blanke, J. Gonzalez Casanova & I. Dähnert

  2. Clinic for Cardiac Surgery, Heart Centre, University of Leipzig, Leipzig, Germany

    S. von Salisch, A. Savtschenko & S. Dhein

Authors
  1. A. Salameh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. Djilali
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. Blanke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Gonzalez Casanova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S. von Salisch
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. Savtschenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S. Dhein
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. I. Dähnert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. Salameh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Salameh, A., Djilali, H., Blanke, K. et al. Cardiac fibroblasts inhibit β-adrenoceptor-dependent connexin43 expression in neonatal rat cardiomyocytes. Naunyn-Schmiedeberg's Arch Pharmacol 386, 421–433 (2013). https://doi.org/10.1007/s00210-013-0843-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00210-013-0843-6

Keywords

Search

Navigation