Biochemistry (Moscow)

, Volume 82, Issue 2, pp 168–175 | Cite as

Chronic alcohol intoxication is not accompanied by an increase in calpain proteolytic activity in cardiac muscle of rats

  • Yu. V. Gritsyna
  • N. N. Salmov
  • A. G. Bobylev
  • I. S. Fadeeva
  • N. I. Fesenko
  • D. G. Sadikova
  • N. I. Kukushkin
  • Z. A. Podlubnaya
  • I. M. VikhlyantsevEmail author


Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression of μ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the “heart weight/body weight” parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents of μ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis of μ-calpain was also not verified, which could indicate that proteolytic activity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months. A decrease in μ-calpain and calpastatin mRNA content (~1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (~1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.


cardiac muscle μ-calpain calpastatin titin chronically alcoholized rats 



myosin heavy chains


intact titin molecule located between the M-band and Z-disk of a sarcomere


proteolytic fragment of the intact T1 linked to myosin filaments in A-disk of a sarcomere


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  1. 1.
    Suzuki, K. (1990) The structure of the calpains and the calpain gene, in Intracellular Calcium-Dependent Proteolysis (Mellgren, R. L., and Murachi, T., eds.) Boca Raton, CRC Press, FL,pp. 25–35.Google Scholar
  2. 2.
    Goll, D. E., Thompson, V. F., Li, H., Wei, W., and Cong, J. (2003) The calpain system, Physiol. Rev., 83, 731–801.CrossRefPubMedGoogle Scholar
  3. 3.
    Goll, D. E., Neti, G., Mares, S. W., and Thompson, V. F. (2008) Myofibrillar protein turnover: the proteasome and the calpains, J. Anim. Sci., 86, e19-35.CrossRefGoogle Scholar
  4. 4.
    Baki, A., Tompa, P., Alexa, A., Molnar, O., and Friedrich, P. (1996) Autolysis parallels activation of mu-calpain, Biochem. J., 318, 897–901.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Grishina, D. A., Suponeva, N. A., Shvedkov, V. V., and Belopasova, A. V. (2015) Inherited progressive limb-girdle muscular dystrophy type 2A (calpainopathy): a review of literature, Nerv. Mysh. Bol., 1, 25–36.Google Scholar
  6. 6.
    Campbell, R. L., and Davies, P. L. (2012) Structure-function relationships in calpains, Biochem. J., 447, 335–351.CrossRefPubMedGoogle Scholar
  7. 7.
    Mohrhauser, D. A., Underwood, K. R., and Weaver, A. D. (2011) In vitro degradation of bovine myofibrils is caused by µ-calpain, not caspase-3, J. Anim. Sci., 89, 798–808.Google Scholar
  8. 8.
    Enns, D. L., Raastad, T., Ugelstad, I., and Belcastro, A. N. (2007) Calpain/calpastatin activities and substrate depletion patterns during hindlimb unweighting and reweighting in skeletal muscle, Eur. J. Appl. Physiol., 100, 445–455.CrossRefPubMedGoogle Scholar
  9. 9.
    Shenkman, B. S., Podlubnaya, Z. A., Vikhlyantsev, I. M., Litvinova, K. S., Udal’tsov, S. N., Nemirovskaya, T. L., Lemesheva, Yu. S., Mukhina, A. M., and Kozlovskaya, I. B. (2004) Contractile characteristics and proteins of sarcomeric cytoskeleton of human m. soleus fibers under conditions of gravitational unloading. Role of support stimulus, Biofizika, 49, 881–890.PubMedGoogle Scholar
  10. 10.
    Udaka, J., Ohmori, S., Terui, T., Ohtsuki, I., Ishiwata, S., Kurihara, S., and Fukuda, N. (2008) Disuse-induced preferential loss of the giant protein titin depresses muscle performance via abnormal sarcomeric organization, J. Gen. Physiol., 131, 33–41.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Vikhlyantsev, I. M., and Podlubnaya, Z. A. (2012) New titin (connectin) isoforms and their functional role in striated muscles of mammals: facts and suppositions, Biochemistry (Moscow), 77, 1515–1535.CrossRefGoogle Scholar
  12. 12.
    Okuneva, A. D., Vikhlyantsev, I. M., Shpagina, M. D., Rogachevskii, V. V., Khutzyan, S. S., Podlubnaya, Z. A., and Grigoriev, A. I. (2012) Changes in titin and myosin heavy chain isoform composition in skeletal muscles of Mongolian gerbil (Meriones unguiculatus) after 12-day spaceflight, Biophysics, 57, 581–586.CrossRefGoogle Scholar
  13. 13.
    Ulanova, A., Gritsyna, Y., Vikhlyantsev, I., Salmov, N., Bobylev, A., Abdusalamova, Z., Rogachevsky, V., Shenkman, B., and Podlubnaya, Z. (2015) Isoform composition and gene expression of thick and thin filament proteins in striated muscles of mice after 30-day space flight, Biomed Res. Int., doi: 10.1155/2015/104735.Google Scholar
  14. 14.
    Gritsyna, Y. V., Vikhlyantsev, I. M., Salmov, N. N., Bobylev, A. G., Kukushkin, N. I., and Podlubnaya, Z. A. (2016) The role of calpain system in atrophy of skeletal muscles of alcohol-fed rats, in Biological Motility, pp. 79-85.Google Scholar
  15. 15.
    Warren, C. M., Jordan, M. C., Roos, K. P., Krzesinski, P. R., and Greaser, M. L. (2003) Titin isoform expression in normal and hypertensive myocardium, Cardiovasc. Res., 59, 86–94.CrossRefPubMedGoogle Scholar
  16. 16.
    Karaduleva, E. V., Vikhlyantsev, I. M., and Podlubnaya, Z. A. (2010) Expression of titin in the myocardium of spontaneously hypertensive rats during development of hypertrophy, Biophysics, 55, 550–554.CrossRefGoogle Scholar
  17. 17.
    Lang, C. N., Wu, D., Frost, R. A., Jefferson, L. S., Kimball, S. R., and Vary, T. C. (1999) Inhibition of muscle protein synthesis by alcohol is associated with modulation of eIF2B and eIF4E, Am. J. Physiol. Endocrinol. Metab., 277, e268-E276.Google Scholar
  18. 18.
    Lieber, C. S., and DeCarli, L. M. (1989) Liquid diet technique of ethanol administration: 1989 update, Alcohol Alcohol., 24, 197–211.PubMedGoogle Scholar
  19. 19.
    Murphy, R. M., Snow, R. J., and Lamb, G. D. (2006) muCalpain and calpain-3 are not autolyzed with exhaustive exercise in humans, Am. J. Physiol. Cell. Physiol., 290, C116-C122.Google Scholar
  20. 20.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680–685.CrossRefPubMedGoogle Scholar
  21. 21.
    Tatsumi, R., and Hattori, A. (1995) Detection of giant myofibrillar proteins connectin and nebulin by electrophoresis in 2% polyacrylamide slab gels strengthened with agarose, Anal. Biochem., 224, 28–31.CrossRefPubMedGoogle Scholar
  22. 22.
    Borbely, A., Falcao-Pires, I., van Heerebeek, L., Hamdani, N., Edes, I., Gavina, C., Leite- Moreira, A. F., Bronzwaer, J. G., Papp, Z., van der Velden, J., Stienen, G. J., and Paulus, W. J. (2009) Hypophosphorylation of the Stiff N2B-titin isoform raises cardiomyocyte resting tension in failing human myocardium, Circ. Res., 104, 780–786.CrossRefPubMedGoogle Scholar
  23. 23.
    Opitz, C. A., Leake, M. C., Makarenko, I., Benes, V., and Linke, W. A. (2004) Developmentally regulated switching of titin size alters myofibrillar stiffness in the perinatal heart, Circ. Res., 94, 967–975.CrossRefPubMedGoogle Scholar
  24. 24.
    Livak, K. J., and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method, Methods, 25, 402–408.CrossRefPubMedGoogle Scholar
  25. 25.
    Murphy, R. M., Verburg, E., and Lamb, G. D. (2006) Ca2+ activation of diffusible and bound pools of mu-calpain in rat skeletal muscle, J. Physiol., 576, 595–612.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Sandmann, S., Yu, M., and Unger, T. (2001) Transcriptional and translational regulation of calpain in the rat heart after myocardial infarction-effects of AT(1) and AT(2) receptor antagonists and ACE inhibitor, Br. J. Pharmacol., 132, 767–777.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Salmov, N. N., Gritsyna, Yu. V., Ulanova, A. D., Vikhlyantsev, I. M., and Podlubnaya, Z. A. (2015) On the role of titin phosphorylation in the development of muscular atrophy, Biophysics, 60, 684–686.CrossRefGoogle Scholar
  28. 28.
    Reilly, M. E., McKoy, G., Mantle, D., Peters, T. J., Goldspink, G., and Preedy, V. R. (2000) Protein and mRNA levels of the myosin heavy chain isoforms Ibeta, IIa, IIx and IIb in type I and type IIfibre-predominant rat skeletal muscles in response to chronic alcohol feeding, J. Muscle Res. Cell Motil., 21, 763–773.PubMedGoogle Scholar
  29. 29.
    Vary, T. C., and Deiter, G. (2005) Long-term alcohol administration inhibits synthesis of both myofibrillar and sarcoplasmic proteins in heart, Metabolism, 54, 212–219.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2017

Authors and Affiliations

  • Yu. V. Gritsyna
    • 1
  • N. N. Salmov
    • 1
  • A. G. Bobylev
    • 1
  • I. S. Fadeeva
    • 1
  • N. I. Fesenko
    • 1
  • D. G. Sadikova
    • 2
  • N. I. Kukushkin
    • 2
  • Z. A. Podlubnaya
    • 1
    • 3
  • I. M. Vikhlyantsev
    • 1
    • 3
    Email author
  1. 1.Institute of Theoretical and Experimental BiophysicsRussian Academy of SciencesPushchino, Moscow RegionRussia
  2. 2.Institute of Cell BiophysicsPushchino, Moscow RegionRussia
  3. 3.Pushchino State Institute of Natural SciencesPushchino, Moscow RegionRussia

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