Cardiovascular Drugs and Therapy

, Volume 21, Issue 6, pp 415–422 | Cite as

Effect of Long-term Monotherapy with the Aldosterone Receptor Blocker Eplerenone on Cytoskeletal Proteins and Matrix Metalloproteinases in Dogs with Heart Failure

  • Sharad Rastogi
  • Sudhish Mishra
  • Valerio Zacà
  • Issa Alesh
  • Ramesh C. Gupta
  • Sidney Goldstein
  • Hani N. Sabbah



Long-term monotherapy with the aldosterone receptor blocker eplerenone in dogs with HF was previously shown to improve LV systolic and diastolic function. This study examined the effects of long-term monotherapy with the aldosterone receptor blocker eplerenone on mRNA and protein expression of the cytoskeletal proteins titin, tubulin, fibronectin and vimentin, the matrix metalloproteinases (MMPs)-1, -2 and -9, and the tissue inhibitors of MMPs (TIMPs)-1 and -2 in left ventricular (LV) myocardium of dogs with heart failure (HF).


HF was produced in 12 dogs by intracoronary microembolizations. Dogs were randomized to 3 months oral therapy with eplerenone (10 mg/kg twice daily, n = 6) or to no therapy at all (HF-control, n = 6). LV tissue from six normal dogs was used for comparison. mRNA expression was measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and protein expression using Western blots.


Compared to NL dogs, control dogs showed upregulation of mRNA and protein expression for tubulin, fibronectin, MMP-1, -2 and -9, and down-regulation of mRNA and protein expression for total titin. Normalization of mRNA and protein expression for all these genes was seen after treatment with eplerenone. N2BA/N2B-titin mRNA expression ratio increased significantly in dogs with HF treated with eplerenone. No differences in expression for vimentin, TIMP-1 and -2 were observed among groups.


In dogs with HF, long-term eplerenone therapy normalizes mRNA and protein expression of key cytoskeletal proteins and MMPs. Reversal of these molecular maladaptations may partly explain the improvement in LV diastolic function seen after long-term therapy with eplerenone.

Key words

titin heart failure cytoskeletal proteins reverse remodeling 



This study was supported, in part, by grants from the National Heart, Lung, and Blood Institute PO1 HL074237-04.


  1. 1.
    Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium: fibrosis and renin-angiotensin-adosterone system. Circulation.. 1991;83:1849–65.PubMedGoogle Scholar
  2. 2.
    Wilke A, Funck R, Rupp H. Effects of the renin-angiotensin-aldosterone system on the cardiac interstitium in heart failure. Basic Res Cardiol.. 1996;91:79–84.PubMedCrossRefGoogle Scholar
  3. 3.
    Dieterich HA, Wendt C, Saborowski F. Cardioprotection by aldosterone receptor antagonism in heart failure. Part I. The role of aldosterone in heart failure. Fiziol Cheloveka.. 2005;31:97–105.PubMedGoogle Scholar
  4. 4.
    Wu Y, Bell SP, Trombitas K, et al. Changes in titin isoform expression in pacing-induced cardiac failure give rise to increased passive muscle stiffness. Circulation.. 2002;106:1384–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Suzuki G, Morita H, Mishima T, et al. Effects of long-term monotherapy with eplerenone, a novel aldosterone blocker, on progression of left ventricular dysfunction and remodeling in dogs with heart failure. Circulation.. 2002;106:2967–72.PubMedCrossRefGoogle Scholar
  6. 6.
    Sabbah HN, Sharov VG, Lesch M, Goldstein S. Progression of heart failure: a role for interstitial fibrosis. Mol Cell Biochem.. 1995;147:29–34.PubMedCrossRefGoogle Scholar
  7. 7.
    Morita H, Khanal S, Rastogi S, et al. Selective matrix metalloproteinase inhibition attenuates progression of left ventricular dysfunction and remodeling in dogs with chronic heart failure. Am J Physiol.. 2006;290:H25–7.Google Scholar
  8. 8.
    Funck RC, Wilke A, Rupp H, Brilla CG. Regulation and role of myocardial collagen matrix remodeling in hypertensive heart disease. Adv Exp Med Biol.. 1997;432:35–44. Review.PubMedGoogle Scholar
  9. 9.
    Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med.. 1999;341:709–17. Sep 2.PubMedCrossRefGoogle Scholar
  10. 10.
    Pitt B, Williams G, Remme W, Martinez F, Lopez-Sendon J, Zannad F, et al. The EPHESUS trial: eplerenone in patients with heart failure due to systolic dysfunction complicating acute myocardial infarction. Eplerenone Post-AMI Heart Failure Efficacy and Survival Study. Cardiovasc Drugs Ther.. 2001;15:79–87.PubMedCrossRefGoogle Scholar
  11. 11.
    Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, et al. Eplerenone Post-acute Myocardial Infarction Heart Failure Efficacy and Survival Study Investigators. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med.. 2003;348:1309–21.PubMedCrossRefGoogle Scholar
  12. 12.
    Sabbah HN, Stein PD, Kono T, et al. A canine model of chronic heart failure produced by multiple sequential coronary microembolizations. Am J Physiol.. 1991;260:H1379–84.PubMedGoogle Scholar
  13. 13.
    Sabbah HN, Shimoyama H, Kono T, et al. Effects of long-term monotherapy with enalapril, metoprolol, and digoxin on the progression of left ventricular dysfunction and dilation in dogs with reduced ejection fraction. Circulation.. 1994;89:2852–9.PubMedGoogle Scholar
  14. 14.
    Spencer WE, Christensen MJ. Multiplex relative RT-PCR method for verification of differential gene expression. Biotechniques. 1999;27:1044–6, 1048–50, 1052.Google Scholar
  15. 15.
    Feldman AM, Ray PE, Silan CM, Mercer JA, Minobe W, Bristow MR. Selective gene expression in failing human heart. Quantification of steady-state levels of messenger RNA in endomyocardial biopsies using the polymerase chain reaction. Circulation. 1991;83:1866–72.PubMedGoogle Scholar
  16. 16.
    Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc.. 2006;1:581–5.PubMedCrossRefGoogle Scholar
  17. 17.
    Gupta RC, Mishra S, Mishima T, et al. Reduced sarcoplasmic reticulum Ca(2+)-uptake and expression of phospholamban in left ventricular myocardium of dogs with heart failure. J Mol Cell Cardiol.. 1999;7:1381–9.CrossRefGoogle Scholar
  18. 18.
    Mishra S, Gupta RC, Tiwari N, et al. Molecular mechanisms of reduced sarcoplasmic reticulum Ca(2+) uptake in human failing left ventricular myocardium. J Heart Lung Transplant.. 2002;21:366–73.PubMedCrossRefGoogle Scholar
  19. 19.
    Lowry OH, Rosebrough NJ, Farr AL, et al. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature.. 1951;227:680–85.Google Scholar
  20. 20.
    Barnes BJ, Howard PA. Eplerenone: a selective aldosterone receptor antagonist for patients with heart failure. Ann Pharmacother.. 2005 Jan;39:68–76.Google Scholar
  21. 21.
    Staessen J, Lijnen P, Fagard R, Verschueren LJ, Amery A. Rise in plasma concentration of aldosterone during long-term angiotensin II suppression. J Endocrinol. 1981;91:457–65.PubMedCrossRefGoogle Scholar
  22. 22.
    McKelvie RS, Yusuf S, Pericak D, Avezum A, Burns RJ, Probstfield J, et al. Comparison of candesartan, enalapril, and their combination in congestive heart failure: Randomized Evaluation of Strategies for Left Ventricular Dysfunction (RESOLVD) pilot study. The RESOLVD Pilot Study Investigators. Circulation. 1999;100:1056–64.PubMedGoogle Scholar
  23. 23.
    Brown NJ. Eplerenone: cardiovascular protection. Circulation.. 2003;107:2512–8. Review.PubMedCrossRefGoogle Scholar
  24. 24.
    Keller TCS. Structure and function of titin and nebulin. Curr Opin Cell Biol.. 1995;7:32–8.PubMedCrossRefGoogle Scholar
  25. 25.
    Granzier H, Wu Y, Siegfried L, et al. Titin: physiological function and role in cardiomyopathy and failure. Heart Fail Rev.. 2005;10:211–23.PubMedCrossRefGoogle Scholar
  26. 26.
    Wu Y, Cazorla O, Labeit D, Labeit S, Granzier H. Changes in titin and collagen underlie diastolic stiffness diversity of cardiac muscle. J Mol Cell Cardiol.. 2000;32:2151–62.PubMedCrossRefGoogle Scholar
  27. 27.
    Neagoe C, Kulke M, del Monte F, et al. Titin isoform switch in ischemic human heart disease. Circulation.. 2002;106:1333–41.PubMedCrossRefGoogle Scholar
  28. 28.
    Wu Y, Bell SP, Trombitas K, et al. Changes in titin isoform expression in pacing-induced cardiac failure give rise to increased passive muscle stiffness. Circulation.. 2002;106:1384–89.PubMedCrossRefGoogle Scholar
  29. 29.
    Cazorla O, Freiburg A, Helmes M, Centner T, McNabb M, Wu Y. Differential expression of cardiac titin isoforms and modulation of cellular stiffness. Circ Res.. 2000;86:59–67.PubMedGoogle Scholar
  30. 30.
    Miller KM, Granzier H, Ehler E, Gregorio CC. The sensitive giant: the role of titin-based stretch sensing complexes in the heart. Trends Cell Biol.. 2004;14:119–26.PubMedCrossRefGoogle Scholar
  31. 31.
    Warren CM, Jordan MC, Roos KP, Krzesinski PR, Greaser ML. Titin isoform expression in normal and hypertensive myocardium. Cardiovasc Res.. 2003;59:86–94.PubMedCrossRefGoogle Scholar
  32. 32.
    Aquila-Pastir LA, Dipaola NR, Matteo RG, Smedira NG, McCarthy PM, Moravec CS. Quantification and distribution of beta-tubulin in human cardiac myocytes. J Mol Cell Cardiol.. 2002;34:1513–23.PubMedCrossRefGoogle Scholar
  33. 33.
    Sharov VG, Kostin S, Todor A, Schaper J, Sabbah HN. Expression of cytoskeletal, linkage and extracellular proteins in failing dog myocardium. Heart Fail Rev.. 2005;10:297–303.PubMedCrossRefGoogle Scholar
  34. 34.
    Hynes RO. Fibronectins. Berlin, Germany: Springer; 1989.Google Scholar
  35. 35.
    Heling A, Zimmermann R, Kostin S, et al. Increased expression of cytoskeletal, linkage, and extracellular proteins in failing human myocardium. Circ Res.. 2000;86:846–53.PubMedGoogle Scholar
  36. 36.
    Kossmehl P, Schonberger J, Shakibaei M, et al. Increase of fibronectin and osteopontin in porcine hearts following ischemia reperfusion. J Mol Med.. 2005;83:626–37.PubMedCrossRefGoogle Scholar
  37. 37.
    Rastogi S, Mishra S, Gupta RC, Sabbah HN. Reversal of maladaptive gene program in left ventricular myocardium of dogs with heart failure following long-term therapy with the acorn cardiac support device. Heart Fail Rev.. 2005;10:157–63.PubMedCrossRefGoogle Scholar
  38. 38.
    Tsutsui H, Ishihara K, Cooper G. Cytoskeletal role for contractile dysfunction of hypertrophied myocardium. Science.. 1993;260:682–7.PubMedCrossRefGoogle Scholar
  39. 39.
    Tsutsui H, Tagawa H, Kent RL, et al. Role of microtubules in contractile dysfunction of hypertrophied cardiocytes. Circulation.. 1994;90:533–55.PubMedGoogle Scholar
  40. 40.
    Tagawa H, Koide M, Sato I, Cooper G. Cytoskeletal role in the contractile dysfunction of cardiomyocytes from hypertrophied and failing right ventricular myocardium. Proc Assoc Am Physicians.. 1996;108:218–29.PubMedGoogle Scholar
  41. 41.
    Tagawa H, Rozich JD, Tsutsui H, et al. Basis of increased microtubules in pressure hypertrophied cardiocytes. Circulation.. 1996;93:1230–43.PubMedGoogle Scholar
  42. 42.
    Tagawa H, Wang N, Narishige T, Ingber DE, Zile MR, Cooper G. Cytoskeletal mechanics in pressure-overload cardiac hypertrophy. Circ Res.. 1997;80:281–9.PubMedGoogle Scholar
  43. 43.
    Hein S, Kostin S, Heling A, Maeno Y, Schaper J. The role of the cytoskeleton in heart failure. Cardiovasc Res.. 2000;45:273–8.PubMedCrossRefGoogle Scholar
  44. 44.
    Wang X, Li F, Campbell SE, Gerdes M. Chronic pressure overload hypertrophy and failure in guinea pigs. J Mol Cell Cardiol.. 1999;31:319–31.PubMedCrossRefGoogle Scholar
  45. 45.
    Lemler MS, Bies RD, Frid MG, et al. Myocyte cytoskeletal disorganization and right heart failure in hypoxia-induced neonatal pulmonary hypertension. Am J Physiol.. 2000;279:H136–76.Google Scholar
  46. 46.
    Thomas CV, Coker MI, Zellner JL, Handy JR, Crumbley AJ, Spinale FG. Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation.. 1998;97:1708–15.PubMedGoogle Scholar
  47. 47.
    Rastogi S, Gupta RC, Mishra S, Morita H, Tanhehco EJ, Sabbah HN. Long-term therapy with acorn cardiac support device normalizes gene expression of growth factors and gelatinases in dogs with heart failure. J Heart Lung Transplant.. 2005;10:1619–25.CrossRefGoogle Scholar
  48. 48.
    Li YY, McTiernan CF, Feldman AM. Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling. Cardiovasc Res.. 2000;40:214–24.CrossRefGoogle Scholar
  49. 49.
    Lu L, Gunja-Smith Z, Woessner JF. Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo. Am J Physiol.. 2000;279:H601–09.Google Scholar
  50. 50.
    Tyagi SC, Kumar S, Cassatt S, Parker JL. Temporal expression of extracellular matrix metalloproteinases and tissue plasminogen activator in the development of collateral vessels in the canine model of coronary occlusion. Can J Physiol Pharmacol.. 1996;74:983–95.PubMedCrossRefGoogle Scholar
  51. 51.
    Lindsey ML, Gannon J, Aikawa M, et al. Selective matrix metalloproteinase inhibition reduces left ventricular remodeling but does not inhibit angiogenesis after myocardial infarction. Circulation.. 2002;105:753–58.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Sharad Rastogi
    • 1
  • Sudhish Mishra
    • 1
  • Valerio Zacà
    • 1
  • Issa Alesh
    • 1
  • Ramesh C. Gupta
    • 1
  • Sidney Goldstein
    • 1
  • Hani N. Sabbah
    • 1
    • 2
  1. 1.Department of Medicine, Division of Cardiovascular MedicineHenry Ford Health SystemDetroitUSA
  2. 2.Cardiovascular ResearchHenry Ford HospitalDetroitUSA

Personalised recommendations