Cardiovascular Drugs and Therapy

, Volume 31, Issue 3, pp 255–267 | Cite as

Argatroban Attenuates Diabetic Cardiomyopathy in Rats by Reducing Fibrosis, Inflammation, Apoptosis, and Protease-Activated Receptor Expression

  • Yogesh Bulani
  • Shyam Sunder SharmaEmail author



Chronic diabetes is associated with cardiovascular dysfunctions. Diabetic cardiomyopathy (DCM) is one of the serious cardiovascular complications associated with diabetes. Despite significant efforts in understanding the pathophysiology of DCM, management of DCM is not adequate due to its complex pathophysiology. Recently, involvement of protease-activated receptors (PARs) has been postulated in cardiovascular diseases. These receptors are activated by thrombin, trypsin, or other serine proteases. Expression of PAR has been shown to be increased in cardiac diseases such as myocardial infarction, viral myocarditis, and pulmonary arterial hypertension. However, the role of PAR in DCM has not been elucidated yet. Therefore, in the present study, we have investigated the role of PAR in the condition of DCM using a pharmacological approach. We used argatroban, a direct thrombin inhibitor for targeting PAR.


Type-2 diabetes mellitus (T2DM) was induced by high-fat feeding along with low dose streptozotocin (STZ 35 mg/kg, i.p. single dose) in male Sprague-Dawley rats. After 16 weeks of diabetes induction, animals were treated with argatroban at 0.3 and 1 mg/kg dose daily for 4 weeks. After 20 weeks, ventricular functions were measured using ventricular catheterization. Cardiac histology, TUNEL staining, and immunoblotting were performed to evaluate cardiac fibrosis, DNA fragmentation, and expression level of different proteins, respectively.


T2DM was associated with cardiac structural and functional disturbances as evidenced from impaired cardiac functional parameters and increased fibrosis. There was a significant increase in PAR expression after 20 weeks of diabetes induction. Four weeks argatroban treatment ameliorated metabolic alterations (reduced plasma glucose and cholesterol), ventricular dysfunctions (improved systolic and diastolic functions), cardiac fibrosis (reduced percentage area of collagen in picro-sirius red staining), and apoptosis (reduced TUNEL positive nuclei). Reduced expression of PAR1 and PAR4 in the argatroban-treated group indicates a response towards inhibition of thrombin. In addition, AKT (Ser-473), GSK-3β (Ser-9), p-65 NFĸB phosphorylation, TGF-β, COX-2, and caspase-3 expression were reduced significantly along with an increase in SERCA expression in argatroban-treated diabetic rats which indicated the anti-fibrotic, anti-inflammatory, and anti-apoptotic potential of argatroban in DCM.


This study suggests the ameliorative effects of argatroban in diabetic cardiomyopathy by improving ventricular functions and reducing fibrosis, inflammation, apoptosis, and PAR expression.


Diabetic cardiomyopathy Ventricular functions Fibrosis Protease activated receptors Argatroban 



The authors acknowledge the financial assistance from the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India (Project ID: NPLC-SSSharma) for this work.

Compliance with Ethical Standard

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

All the guidelines (national and institutional) for the care and use of animals were followed for conducting studies for this manuscript. Experimental protocol was approved (Protocol approval no. IAEC/14/63) by Institutional Animal Ethics Committee, NIPER, S.A.S. Nagar, Punjab, India.

Informed Consent

Not required.


  1. 1.
    Rubler S, Dlugash J, Yuceoglu YZ, Kumral T, Branwood AW, Grishman A. New type of cardiomyopathy associated with diabetic glomerulosclerosis. Am J Cardiol. 1972;30(6):595–602.CrossRefPubMedGoogle Scholar
  2. 2.
    Kannel WB, McGee DL. Diabetes and cardiovascular disease. The Framingham study JAMA. 1979;241(19):2035–8.PubMedGoogle Scholar
  3. 3.
    Boudina S, Abel ED. Diabetic cardiomyopathy revisited. Circulation. 2007;115(25):3213–23.CrossRefPubMedGoogle Scholar
  4. 4.
    Ernande L, Derumeaux G. Diabetic cardiomyopathy: myth or reality? Arch Cardiovasc Dis. 2012;105(4):218–25.CrossRefPubMedGoogle Scholar
  5. 5.
    Poornima IG, Parikh P, Shannon RP. Diabetic cardiomyopathy: the search for a unifying hypothesis. Circ Res. 2006;98(5):596–605.CrossRefPubMedGoogle Scholar
  6. 6.
    Bloomgarden ZT. Glycemic control in diabetes: a tale of three studies. Diabetes Care. 2008;31(9):1913–9.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Cesario DA, Brar R, Shivkumar K. Alterations in ion channel physiology in diabetic cardiomyopathy. Endocrinol Metab Clin N Am. 2006;35(3):601–10. ix-x CrossRefGoogle Scholar
  8. 8.
    Aoki I, Shimoyama K, Aoki N, Homori M, Yanagisawa A, Nakahara K, et al. Platelet-dependent thrombin generation in patients with diabetes mellitus: effects of glycemic control on coagulability in diabetes. J Am Coll Cardiol. 1996;27(3):560–6.CrossRefPubMedGoogle Scholar
  9. 9.
    Ersoz G, Yakaryilmaz A, Turan B. Effect of sodium selenite treatment on platelet aggregation of streptozotocin-induced diabetic rats. Thromb Res. 2003;111(6):363–7.CrossRefPubMedGoogle Scholar
  10. 10.
    Romano M, Guagnano MT, Pacini G, Vigneri S, Falco A, Marinopiccoli M, et al. Association of inflammation markers with impaired insulin sensitivity and coagulative activation in obese healthy women. J Clin Endocrinol Metab. 2003;88(11):5321–6.CrossRefPubMedGoogle Scholar
  11. 11.
    Antoniak S, Owens AP 3rd, Baunacke M, Williams JC, Lee RD, Weithauser A, et al. PAR-1 contributes to the innate immune response during viral infection. J Clin Invest. 2013;123(3):1310–22.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Nickel KF, Laux V, Heumann R, von Degenfeld G. Thrombin has biphasic effects on the nitric oxide-cGMP pathway in endothelial cells and contributes to experimental pulmonary hypertension. PLoS One. 2013;8(6):e63504.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Strande JL, Hsu A, Su J, Fu X, Gross GJ, Baker JE. SCH 79797, a selective PAR1 antagonist, limits myocardial ischemia/reperfusion injury in rat hearts. Basic Res Cardiol. 2007;102(4):350–8.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Sabri A, Muske G, Zhang H, Pak E, Darrow A, Andrade-Gordon P, et al. Signaling properties and functions of two distinct cardiomyocyte protease-activated receptors. Circ Res. 2000;86(10):1054–61.CrossRefPubMedGoogle Scholar
  15. 15.
    Strande JL, Hsu A, Su J, Fu X, Gross GJ, Baker JE. Inhibiting protease-activated receptor 4 limits myocardial ischemia/reperfusion injury in rat hearts by unmasking adenosine signaling. J Pharmacol Exp Ther. 2008;324(3):1045–54.CrossRefPubMedGoogle Scholar
  16. 16.
    Bulani Y, Sharma SS. Therapeutic potential of targeting protease activated receptors in cardiovascular diseases. Curr Pharm Des. 2015;21(30):4392–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Sonin DL, Wakatsuki T, Routhu KV, Harmann LM, Petersen M, Meyer J, et al. Protease-activated receptor 1 inhibition by SCH79797 attenuates left ventricular remodeling and profibrotic activities of cardiac fibroblasts. J Cardiovasc Pharmacol Ther. 2013;18(5):460–75.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Sakai T, Nambu T, Katoh M, Uehara S, Fukuroda T, Nishikibe M. Up-regulation of protease-activated receptor-1 in diabetic glomerulosclerosis. Biochem Biophys Res Commun. 2009;384(2):173–9.CrossRefPubMedGoogle Scholar
  19. 19.
    Waasdorp M, Duitman J, Florquin S, Spek CA. Protease-activated receptor-1 deficiency protects against streptozotocin-induced diabetic nephropathy in mice. Sci Rep. 2016;6:33030.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Srinivasan K, Viswanad B, Asrat L, Kaul CL, Ramarao P. Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol Res. 2005;52(4):313–20.CrossRefPubMedGoogle Scholar
  21. 21.
    Mihara M, Aihara K, Ikeda Y, Yoshida S, Kinouchi M, Kurahashi K, et al. Inhibition of thrombin action ameliorates insulin resistance in type 2 diabetic db/db mice. Endocrinology. 2010;151(2):513–9.CrossRefPubMedGoogle Scholar
  22. 22.
    Pacher P, Nagayama T, Mukhopadhyay P, Batkai S, Kass DA. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc. 2008;3(9):1422–34.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–75.PubMedGoogle Scholar
  24. 24.
    Ti Y, Xie GL, Wang ZH, Bi XL, Ding WY, Wang J, et al. TRB3 gene silencing alleviates diabetic cardiomyopathy in a type 2 diabetic rat model. Diabetes. 2011;60(11):2963–74.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Marsh SA, Dell'italia LJ, Chatham JC. Interaction of diet and diabetes on cardiovascular function in rats. Am J Physiol Heart Circ Physiol. 2009;296(2):H282–92.CrossRefPubMedGoogle Scholar
  26. 26.
    Zhao SM, Wang YL, Guo CY, Chen JL, Wu YQ. Progressive decay of Ca2+ homeostasis in the development of diabetic cardiomyopathy. Cardiovasc Diabetol. 2014;13:75.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Bers DM. Cardiac excitation-contraction coupling. Nature. 2002;415(6868):198–205.CrossRefPubMedGoogle Scholar
  28. 28.
    Trost SU, Belke DD, Bluhm WF, Meyer M, Swanson E, Dillmann WH. Overexpression of the sarcoplasmic reticulum Ca (2+)-ATPase improves myocardial contractility in diabetic cardiomyopathy. Diabetes. 2002;51(4):1166–71.CrossRefPubMedGoogle Scholar
  29. 29.
    Vetter R, Rehfeld U, Reissfelder C, Weiss W, Wagner KD, Gunther J, et al. Transgenic overexpression of the sarcoplasmic reticulum Ca2+ATPase improves reticular Ca2+ handling in normal and diabetic rat hearts. FASEB J. 2002;16(12):1657–9.PubMedGoogle Scholar
  30. 30.
    Suarez J, Scott B, Dillmann WH. Conditional increase in SERCA2a protein is able to reverse contractile dysfunction and abnormal calcium flux in established diabetic cardiomyopathy. Am J Physiol Regul Integr Comp Physiol. 2008;295(5):R1439–45.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Mishra PK, Metreveli N, Tyagi SC. MMP-9 gene ablation and TIMP-4 mitigate PAR-1-mediated cardiomyocyte dysfunction: a plausible role of dicer and miRNA. Cell Biochem Biophys. 2010;57(2–3):67–76.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Martin J, Kelly DJ, Mifsud SA, Zhang Y, Cox AJ, See F, et al. Tranilast attenuates cardiac matrix deposition in experimental diabetes: role of transforming growth factor-β. Cardiovasc Res. 2005;65(3):694–701.CrossRefPubMedGoogle Scholar
  33. 33.
    Miric G, Dallemagne C, Endre Z, Margolin S, Taylor SM, Brown L. Reversal of cardiac and renal fibrosis by pirfenidone and spironolactone in streptozotocin-diabetic rats. Br J Pharmacol. 2001;133(5):687–94.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Border WA, Noble NA. Transforming growth factor beta in tissue fibrosis. N Engl J Med. 1994;331(19):1286–92.CrossRefPubMedGoogle Scholar
  35. 35.
    Way KJ, Isshiki K, Suzuma K, Yokota T, Zvagelsky D, Schoen FJ, et al. Expression of connective tissue growth factor is increased in injured myocardium associated with protein kinase C beta2 activation and diabetes. Diabetes. 2002;51(9):2709–18.CrossRefPubMedGoogle Scholar
  36. 36.
    Kassel KM, Sullivan BP, Cui W, Copple BL, Luyendyk JP. Therapeutic administration of the direct thrombin inhibitor argatroban reduces hepatic inflammation in mice with established fatty liver disease. Am J Pathol. 2012;181(4):1287–95.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Huisamen B. Protein kinase B in the diabetic heart. Mol Cell Biochem. 2003;249(1–2):31–8.CrossRefPubMedGoogle Scholar
  38. 38.
    Bagul PK, Dinda AK, Banerjee SK. Effect of resveratrol on sirtuins expression and cardiac complications in diabetes. Biochem Biophys Res Commun. 2015;468(1–2):221–7.CrossRefGoogle Scholar
  39. 39.
    Tian R. Another role for the celebrity: Akt and insulin resistance. Circ Res. 2005;96(2):139–40.CrossRefPubMedGoogle Scholar
  40. 40.
    Shamhart PE, Luther DJ, Hodson BR, Koshy JC, Ohanyan V, Meszaros JG. Impact of type 1 diabetes on cardiac fibroblast activation: enhanced cell cycle progression and reduced myofibroblast content in diabetic myocardium. Am J Physiol Endocrinol Metab. 2009;297(5):E1147–53.CrossRefPubMedGoogle Scholar
  41. 41.
    Pawlinski R, Tencati M, Hampton CR, Shishido T, Bullard TA, Casey LM, et al. Protease-activated receptor-1 contributes to cardiac remodeling and hypertrophy. Circulation. 2007;116(20):2298–306.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Sabri A, Short J, Guo J, Steinberg SF. Protease-activated receptor-1-mediated DNA synthesis in cardiac fibroblast is via epidermal growth factor receptor transactivation: distinct PAR-1 signaling pathways in cardiac fibroblasts and cardiomyocytes. Circ Res. 2002;91(6):532–9.CrossRefPubMedGoogle Scholar
  43. 43.
    Spronk HM, De Jong AM, Verheule S, De Boer HC, Maass AH, Lau DH, et al. Hypercoagulability causes atrial fibrosis and promotes atrial fibrillation. Eur Heart J. 2017;38(1):38–50.CrossRefPubMedGoogle Scholar
  44. 44.
    Sugden PH, Fuller SJ, Weiss SC, Clerk A. Glycogen synthase kinase 3 (GSK3) in the heart: a point of integration in hypertrophic signalling and a therapeutic target? A critical analysis. Br J Pharmacol. 2008;153(Suppl 1):S137–53.PubMedPubMedCentralGoogle Scholar
  45. 45.
    Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ. Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway. Diabetes. 2002;51(6):1938–48.CrossRefPubMedGoogle Scholar
  46. 46.
    Cai L, Wang Y, Zhou G, Chen T, Song Y, Li X, et al. Attenuation by metallothionein of early cardiac cell death via suppression of mitochondrial oxidative stress results in a prevention of diabetic cardiomyopathy. J Am Coll Cardiol. 2006;48(8):1688–97.CrossRefPubMedGoogle Scholar
  47. 47.
    Bhandari U, Kumar V, Kumar P, Tripathi CD, Khanna G. Protective effect of pioglitazone on cardiomyocyte apoptosis in low-dose streptozotocin & high-fat diet-induced type-2 diabetes in rats. Indian J Med Res. 2015;142(5):598–605.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Guo Z, Xia Z, Jiang J, McNeill JH. Downregulation of NADPH oxidase, antioxidant enzymes, and inflammatory markers in the heart of streptozotocin-induced diabetic rats by N-acetyl-L-cysteine. Am J Physiol Heart Circ Physiol. 2007;292(4):H1728–36.CrossRefPubMedGoogle Scholar
  49. 49.
    Suzuki H, Kayama Y, Sakamoto M, Iuchi H, Shimizu I, Yoshino T, et al. Arachidonate 12/15-lipoxygenase-induced inflammation and oxidative stress are involved in the development of diabetic cardiomyopathy. Diabetes. 2015;64(2):618–30.CrossRefPubMedGoogle Scholar
  50. 50.
    Kellogg AP, Converso K, Wiggin T, Stevens M, Pop-Busui R. Effects of cyclooxygenase-2 gene inactivation on cardiac autonomic and left ventricular function in experimental diabetes. Am J Physiol Heart Circ Physiol. 2009;296(2):H453–61.CrossRefPubMedGoogle Scholar
  51. 51.
    Kassel KM, Owens AP 3rd, Rockwell CE, Sullivan BP, Wang R, Tawfik O, et al. Protease-activated receptor 1 and hematopoietic cell tissue factor are required for hepatic steatosis in mice fed a western diet. Am J Pathol. 2011;179(5):2278–89.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Pharmacology and ToxicologyNational Institute of Pharmaceutical Education and Research (NIPER)Nagar (Mohali)India

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