Screening for Novel Calcium-Binding Proteins that Regulate Cardiac Hypertrophy: CIB1 as an Example

  • Joerg HeinekeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 963)


Calcium-binding proteins have a crucial function in the regulation of cardiac contractility as well as in the regulation of cardiac signal-transduction. Because they sense calcium concentrations and at the same time bind specific signaling molecules, some of these proteins are critically involved in the establishment of signaling microdomains, which are insulated from the large cytosolic calcium fluctuations involved in cardiac excitation–contraction coupling. In this regard, we have recently identified the calcium-binding protein CIB1 as an important regulator of pathological cardiac hypertrophy and transition to heart failure. It is almost certain that more, currently unknown calcium-binding proteins with similar regulatory function in cardiac signaling exist. Here, I suggest screening strategies to identify these calcium-binding proteins with impact on cardiac hypertrophy and provide a detailed protocol for the identification of protein interaction partners. I also describe cell culture-based models for cardiomyocyte hypertrophy as well as mouse models for pathological or physiological hypertrophy and strategies to analyze the impact of candidate genes on the development of hypertrophy.

Key words

Calcium Heart Hypertrophy Screening CIB1 Calcium-binding proteins, EF-hand 


  1. 1.
    Heineke J, Molkentin JD (2006) Regulation of cardiac hypertrophy by intracellular signaling pathways. Nat Rev Mol Cell Biol 7:589–600PubMedCrossRefGoogle Scholar
  2. 2.
    Bers DM (2002) Cardiac excitation-contraction coupling. Nature 415:198–205PubMedCrossRefGoogle Scholar
  3. 3.
    Kiriazis H, Sato Y, Kadambi VJ, Schmidt AG, Gerst MJ, Hoit BD, Kranias EG (2002) Hypertrophy and functional alterations in hyperdynamic phospholamban-knockout mouse hearts under chronic aortic stenosis. Cardiovasc Res 53:372–381PubMedCrossRefGoogle Scholar
  4. 4.
    Molkentin JD (2006) Dichotomy of Ca2+ in the heart: contraction versus intracellular signaling. J Clin Invest 116:623–626PubMedCrossRefGoogle Scholar
  5. 5.
    Schaub MC, Heizmann CW (2008) Calcium, troponin, calmodulin, S100 proteins: from myocardial basics to new therapeutic strategies. Biochem Biophys Res Commun 369:247–264PubMedCrossRefGoogle Scholar
  6. 6.
    Wu X, Zhang T, Bossuyt J, Li X, McKinsey TA, Dedman JR, Olson EN, Chen J, Brown JH, Bers DM (2006) Local InsP(3)-dependent perinuclear Ca signaling in cardiac myocyte excitation-transcription coupling. J Clin Invest 116:675–682PubMedCrossRefGoogle Scholar
  7. 7.
    Backs J, Backs T, Neef S, Kreusser MM, Lehmann LH, Patrick DM, Grueter CE, Qi X, Richardson JA, Hill JA, Katus HA, Bassel-Duby R, Maier LS, Olson EN (2009) The delta isoform of CaM kinase II is required for pathological cardiac hypertrophy and remodeling after pressure overload. Proc Natl Acad Sci USA 106:2342–2347PubMedCrossRefGoogle Scholar
  8. 8.
    Ling H, Zhang T, Pereira L, Means CK, Cheng H, Gu Y, Dalton ND, Peterson KL, Chen J, Bers D, Heller Brown J (2009) Requirement for Ca2+/calmodulin-dependent kinase II in the transition from pressure overload-induced cardiac hypertrophy to heart failure in mice. J Clin Invest 119:1230–1240PubMedCrossRefGoogle Scholar
  9. 9.
    Zhang T, Johnson EN, Gu Y, Morissette MR, Sah VP, Gigena MS, Belke DD, Dillmann WH, Rogers TB, Schulman H, Ross J Jr, Brown JH (2002) The cardiac-specific nuclear delta(B) isoform of Ca2+/calmodulin-dependent protein kinase II induces hypertrophy and dilated cardiomyopathy associated with increased ­protein phosphatase 2A activity. J Biol Chem 277:1261–1267PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang T, Maier LS, Dalton ND, Miyamoto S, Ross J Jr, Bers DM, Brown JH (2003) The deltaC isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. Circ Res 92:912–919PubMedCrossRefGoogle Scholar
  11. 11.
    Song K, Backs J, McAnally J, Qi X, Gerard RD, Richardson JA, Hill JA, Bassel-Duby R, Olson EN (2006) The transcriptional coactivator CAMTA2 stimulates cardiac growth by opposing class II histone deacetylases. Cell 125:453–466PubMedCrossRefGoogle Scholar
  12. 12.
    Bueno OF, van Rooij E, Molkentin JD, Doevendans PA, De Windt LJ (2002) Calcineurin and hypertrophic heart disease: novel insights and remaining questions. Cardiovasc Res 53:806–821PubMedCrossRefGoogle Scholar
  13. 13.
    Bueno OF, Wilkins BJ, Tymitz KM, Glascock BJ, Kimball TF, Lorenz JN, Molkentin JD (2002) Impaired cardiac hypertrophic response in calcineurin Abeta -deficient mice. Proc Natl Acad Sci USA 99:4586–4591PubMedCrossRefGoogle Scholar
  14. 14.
    Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN (1998) A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93:215–228PubMedCrossRefGoogle Scholar
  15. 15.
    Yang SA, Klee CB (2000) Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A. Biochemistry 39:16147–16154PubMedCrossRefGoogle Scholar
  16. 16.
    Heineke J, Auger-Messier M, Correll RN, Xu J, Benard MJ, Yuan W, Drexler H, Parise LV, Molkentin JD (2010) CIB1 is a regulator of pathological cardiac hypertrophy. Nat Med 16:872–879PubMedCrossRefGoogle Scholar
  17. 17.
    Naik UP, Patel PM, Parise LV (1997) Identification of a novel calcium-binding ­protein that interacts with the integrin alphaIIb cytoplasmic domain. J Biol Chem 272:4651–4654PubMedCrossRefGoogle Scholar
  18. 18.
    Gentry HR, Singer AU, Betts L, Yang C, Ferrara JD, Sondek J, Parise LV (2005) Structural and biochemical characterization of CIB1 delineates a new family of EF-hand-containing proteins. J Biol Chem 280:8407–8415PubMedCrossRefGoogle Scholar
  19. 19.
    Naik MU, Naik UP (2003) Calcium-and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen. Blood 102:3629–3636PubMedCrossRefGoogle Scholar
  20. 20.
    Stabler SM, Ostrowski LL, Janicki SM, Monteiro MJ (1999) A myristoylated calcium-binding protein that preferentially interacts with the Alzheimer’s disease presenilin 2 protein. J Cell Biol 145:1277–1292PubMedCrossRefGoogle Scholar
  21. 21.
    Haataja L, Kaartinen V, Groffen J, Heisterkamp N (2002) The small GTPase Rac3 interacts with the integrin-binding protein CIB and promotes integrin alpha(IIb)beta(3)-mediated adhesion and spreading. J Biol Chem 277:8321–8328PubMedCrossRefGoogle Scholar
  22. 22.
    Jarman KE, Moretti PA, Zebol JR, Pitson SM (2010) Translocation of sphingosine kinase 1 to the plasma membrane is mediated by calcium and integrin binding protein 1. J Biol Chem 285:483–492PubMedCrossRefGoogle Scholar
  23. 23.
    Leisner TM, Liu M, Jaffer ZM, Chernoff J, Parise LV (2005) Essential role of CIB1 in regulating PAK1 activation and cell migration. J Cell Biol 170:465–476PubMedCrossRefGoogle Scholar
  24. 24.
    Rockman HA, Ross RS, Harris AN, Knowlton KU, Steinhelper ME, Field LJ, Ross J Jr, Chien KR (1991) Segregation of atrial-specific and inducible expression of an atrial natriuretic ­factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci USA 88:8277–8281PubMedCrossRefGoogle Scholar
  25. 25.
    Heineke J, Ruetten H, Willenbockel C, Gross SC, Naguib M, Schaefer A, Kempf T, Hilfiker-Kleiner D, Caroni P, Kraft T, Kaiser RA, Molkentin JD, Drexler H, Wollert KC (2005) Attenuation of cardiac remodeling after myocardial infarction by muscle LIM protein-calcineurin signaling at the sarcomeric Z-disc. Proc Natl Acad Sci USA 102:1655–1660PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Klinik für Kardiologie und Angiologie, Rebirth-Cluster of ExcellenceMedizinische Hochschule HannoverHannoverGermany

Personalised recommendations