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Mouse Genetics pp 385-400 | Cite as

Generating Double Knockout Mice to Model Genetic Intervention for Diabetic Cardiomyopathy in Humans

  • Vishalakshi Chavali
  • Shyam Sundar Nandi
  • Shree Ram Singh
  • Paras Kumar MishraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1194)

Abstract

Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/−) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3–4 weeks and show robust hyperglycemia at the age of 10–12 weeks. It is a chronic model of T1D. In Ins2+/− Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/−/Mmp9−/− (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy.

Key words

Ins2+/− Akita Mmp9 Diabetes Heart failure Cardiomyopathy 

Notes

Acknowledgement

This work is supported by National Institute of Health grants HL-113281 and HL116205 to Paras Kumar Mishra.

References

  1. 1.
    Wild S, Roglic G, Green A, Sicree R, King H (2004) Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27:1047–1053PubMedCrossRefGoogle Scholar
  2. 2.
    Chavali V, Tyagi SC, Mishra PK (2013) Predictors and prevention of diabetic cardiomyopathy. Diabetes Metab Syndr Obes 6:151–160PubMedCentralPubMedGoogle Scholar
  3. 3.
    Mathew V, Gersh BJ, Williams BA, Laskey WK, Willerson JT, Tilbury RT, Davis BR, Holmes DR Jr (2004) Outcomes in patients with diabetes mellitus undergoing percutaneous coronary intervention in the current era: a report from the Prevention of REStenosis with Tranilast and its Outcomes (PRESTO) trial. Circulation 109:476–480PubMedCrossRefGoogle Scholar
  4. 4.
    Pignone M, Alberts MJ, Colwell JA, Cushman M, Inzucchi SE, Mukherjee D, Rosenson RS, Williams CD, Wilson PW, Kirkman MS (2010) Aspirin for primary prevention of cardiovascular events in people with diabetes: a position statement of the American Diabetes Association, a scientific statement of the American Heart Association, and an expert consensus document of the American College of Cardiology Foundation. Diabetes Care 33:1395–1402PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Rota M, LeCapitaine N, Hosoda T, Boni A, De AA, Padin-Iruegas ME, Esposito G, Vitale S, Urbanek K, Casarsa C, Giorgio M, Luscher TF, Pelicci PG, Anversa P, Leri A, Kajstura J (2006) Diabetes promotes cardiac stem cell aging and heart failure, which are prevented by deletion of the p66shc gene. Circ Res 99:42–52PubMedCrossRefGoogle Scholar
  6. 6.
    Sarwar N, Gao P, Seshasai SR, Gobin R, Kaptoge S, Di AE, Ingelsson E, Lawlor DA, Selvin E, Stampfer M, Stehouwer CD, Lewington S, Pennells L, Thompson A, Sattar N, White IR, Ray KK, Danesh J (2010) Diabetes mellitus, fasting blood glucose concentration, and risk of vascular disease: a collaborative meta-analysis of 102 prospective studies. Lancet 375:2215–2222PubMedCrossRefGoogle Scholar
  7. 7.
    Schramm TK, Gislason GH, Kober L, Rasmussen S, Rasmussen JN, Abildstrom SZ, Hansen ML, Folke F, Buch P, Madsen M, Vaag A, Torp-Pedersen C (2008) Diabetes patients requiring glucose-lowering therapy and nondiabetics with a prior myocardial infarction carry the same cardiovascular risk: a population study of 3.3 million people. Circulation 117:1945–1954PubMedCrossRefGoogle Scholar
  8. 8.
    Spencer EA, Pirie KL, Stevens RJ, Beral V, Brown A, Liu B, Green J, Reeves GK (2008) Diabetes and modifiable risk factors for cardiovascular disease: the prospective Million Women Study. Eur J Epidemiol 23:793–799PubMedCrossRefGoogle Scholar
  9. 9.
    Herman WH, Zimmet P (2012) Type 2 diabetes: an epidemic requiring global attention and urgent action. Diabetes Care 35:943–944PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    King H, Aubert RE, Herman WH (1998) Global burden of diabetes, 1995-2025: prevalence, numerical estimates, and projections. Diabetes Care 21:1414–1431PubMedCrossRefGoogle Scholar
  11. 11.
    Stoy J, Edghill EL, Flanagan SE, Ye H, Paz VP, Pluzhnikov A, Below JE, Hayes MG, Cox NJ, Lipkind GM, Lipton RB, Greeley SA, Patch AM, Ellard S, Steiner DF, Hattersley AT, Philipson LH, Bell GI (2007) Insulin gene mutations as a cause of permanent neonatal diabetes. Proc Natl Acad Sci U S A 104:15040–15044PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Chang CL, Chen YC, Chen HM, Yang NS, Yang WC (2013) Natural cures for type 1 diabetes: a review of phytochemicals, biological actions, and clinical potential. Curr Med Chem 20:899–907PubMedGoogle Scholar
  13. 13.
    Simsek DG, Aycan Z, Ozen S, Cetinkaya S, Kara C, Abali S, Demir K, Tunc O, Ucakturk A, Asar G, Bas F, Cetinkaya E, Aydin M, Karaguzel G, Orbak Z, Siklar Z, Altincik A, Okten A, Ozkan B, Ocal G, Semiz S, Arslanoglu I, Evliyaoglu O, Bundak R, Darcan S (2013) Diabetes care, glycemic control, complications, and concomitant autoimmune diseases in children with type 1 diabetes in Turkey: a multicenter study. J Clin Res Pediatr Endocrinol 5:20–26PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Mishra PK, Singh SR, Joshua IG, Tyagi SC (2010) Stem cells as a therapeutic target for diabetes. Front Biosci 15:461–477CrossRefGoogle Scholar
  15. 15.
    Nokoff NJ, Rewers M, Cree GM (2012) The interplay of autoimmunity and insulin resistance in type 1 diabetes. Discov Med 13:115–122PubMedGoogle Scholar
  16. 16.
    Masoudi FA, Inzucchi SE (2007) Diabetes mellitus and heart failure: epidemiology, mechanisms, and pharmacotherapy. Am J Cardiol 99:113B–132BPubMedCrossRefGoogle Scholar
  17. 17.
    Mishra PK, Tyagi N, Kumar M, Tyagi SC (2009) MicroRNAs as a therapeutic target for cardiovascular diseases. J Cell Mol Med 13:778–789PubMedCrossRefGoogle Scholar
  18. 18.
    Agyemang C, Kunst AE, Bhopal R, Zaninotto P, Nazroo J, Unwin N, van Valkengoed I, Redekop WK, Stronks K (2013) A cross-national comparative study of metabolic syndrome among non-diabetic Dutch and English ethnic groups. Eur J Public Health 23:447–452PubMedCrossRefGoogle Scholar
  19. 19.
    Barengo NC, Trejo R, Sposetti G (2013) Prevalence of type 2 diabetes in Argentina 1979-2012. Diabetes Metab Res Rev, Epub. July, 16Google Scholar
  20. 20.
    Gable D, Sanderson SC, Humphries SE (2007) Genotypes, obesity and type 2 diabetes–can genetic information motivate weight loss? A review. Clin Chem Lab Med 45:301–308PubMedCrossRefGoogle Scholar
  21. 21.
    Zhang Q, Wang Y, Huang ES (2009) Changes in racial/ethnic disparities in the prevalence of Type 2 diabetes by obesity level among US adults. Ethn Health 14:439–457PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Mishra PK, Tyagi N, Kundu S, Tyagi SC (2009) MicroRNAs are involved in homocysteine-induced cardiac remodeling. Cell Biochem Biophys 55:153–162PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Mishra PK, Tyagi N, Sen U, Joshua IG, Tyagi SC (2010) Synergism in hyperhomocysteinemia and diabetes: role of PPAR gamma and tempol. Cardiovasc Diabetol 9:49–61PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Mishra PK, Metreveli N, Tyagi SC (2010) MMP-9 gene ablation and TIMP-4 mitigate PAR-1-mediated cardiomyocyte dysfunction: a plausible role of dicer and miRNA. Cell Biochem Biophys 57:67–76PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Mishra PK, Chavali V, Metreveli N, Tyagi SC (2012) Ablation of MMP9 induces survival and differentiation of cardiac stem cells into cardiomyocytes in the heart of diabetics: a role of extracellular matrix. Can J Physiol Pharmacol 90:353–360PubMedCrossRefGoogle Scholar
  26. 26.
    Mishra PK, Kuypers NJ, Singh SR, Leiberh ND, Chavali V, Tyagi SC (2013) Cardiac Stem Cell Niche, MMP9, and Culture and Differentiation of Embryonic Stem Cells. Methods Mol Biol 1035:153–163PubMedCrossRefGoogle Scholar
  27. 27.
    Buralli S, Dini FL, Ballo P, Conti U, Fontanive P, Duranti E, Metelli MR, Marzilli M, Taddei S (2010) Circulating matrix metalloproteinase-3 and metalloproteinase-9 and tissue Doppler measures of diastolic dysfunction to risk stratify patients with systolic heart failure. Am J Cardiol 105:853–856PubMedCrossRefGoogle Scholar
  28. 28.
    Bradham WS, Moe G, Wendt KA, Scott AA, Konig A, Romanova M, Naik G, Spinale FG (2002) TNF-alpha and myocardial matrix metalloproteinases in heart failure: relationship to LV remodeling. Am J Physiol Heart Circ Physiol 282:H1288–H1295PubMedGoogle Scholar
  29. 29.
    Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT (2000) Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 106:55–62PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Lindsey ML, Escobar GP, Dobrucki LW, Goshorn DK, Bouges S, Mingoia JT, McClister DM Jr, Su H, Gannon J, MacGillivray C, Lee RT, Sinusas AJ, Spinale FG (2006) Matrix metalloproteinase-9 gene deletion facilitates angiogenesis after myocardial infarction. Am J Physiol Heart Circ Physiol 290:H232–H239PubMedCrossRefGoogle Scholar
  31. 31.
    Spinale FG (2002) Matrix metalloproteinases: regulation and dysregulation in the failing heart. Circ Res 90:520–530PubMedCrossRefGoogle Scholar
  32. 32.
    Romanic AM, Harrison SM, Bao W, Burns-Kurtis CL, Pickering S, Gu J, Grau E, Mao J, Sathe GM, Ohlstein EH, Yue TL (2002) Myocardial protection from ischemia/reperfusion injury by targeted deletion of matrix metalloproteinase-9. Cardiovasc Res 54:549–558PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Vishalakshi Chavali
    • 1
  • Shyam Sundar Nandi
    • 1
  • Shree Ram Singh
    • 2
  • Paras Kumar Mishra
    • 1
    • 3
    Email author
  1. 1.Department of Cellular and Integrative PhysiologyUniversity of Nebraska Medical CenterOmahaUSA
  2. 2.Stem Cell Regulation and Animal Aging Section, Basic Research LaboratoryNational Cancer Institute, National Institutes of HealthFrederickUSA
  3. 3.Department of AnesthesiologyUniversity of Nebraska Medical CenterOmahaUSA

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