Cell Biochemistry and Biophysics

, Volume 57, Issue 2–3, pp 67–76

MMP-9 Gene Ablation and TIMP-4 Mitigate PAR-1-Mediated Cardiomyocyte Dysfunction: A Plausible Role of Dicer and miRNA

  • Paras Kumar Mishra
  • Naira Metreveli
  • Suresh C. Tyagi
Original Paper


Although matrix metalloproteinase-9 (MMP‐9) is involved in cardiomyocytes contractility dysfunction, tissue inhibitor of metalloproteinase-4 (TIMP‐4) mitigates the effect of MMP‐9, and proteinase-activated receptor-1 (PAR‐1, a G-protein couple receptor, GPCR) is involved in the signaling cascade of MMP‐9-mediated cardiac dysfunction, the mechanism(s) are unclear. To test the hypothesis that induction of dicer and differential expression of microRNAs (miRNAs) contribute, in part, to the down regulation of sarcoplasmic reticulum calcium ATPase isoform 2a (serca-2a) in MMP-9 and PAR-1-mediated myocytes dysfunction, ventricular cardiomyocytes were isolated from C57BL/6J mice and treated with 3 ng/ml of MMP-9, 12 ng/ml of TIMP-4, and 10 and 100 μM of PAR-1 antagonist with MMP-9. Specific role of MMP-9 was determined by using MMP-9 knock out (MMP-9KO) and their corresponding control (FVB) mice. Ion Optics video-edge detection system and Fura 2-AM loading were used for determining the contractility and calcium release from cardiomyocytes. Quantitative and semi-quantitative PCR were used to determine the expression of dicer, TIMP-4 and serca-2a. miRNA microarrays were used for assessing the expression of different miRNAs between MMP-9KO and FVB cardiomyocytes. The results suggest that MMP‐9 treatment attenuates the voltage‐induced contraction of primary cardiomyocytes while TIMP‐4, an inhibitor of MMP‐9, reverses the inhibition. MMP‐9 treatment is also associated with reduced Ca2+ transients. This effect is blocked by a PAR‐1 antagonist, suggesting that PAR‐1 mediates this effect. The effect is not as great at high concentrations (100 μM) perhaps due to mild toxicity. The PAR‐1 antagonist effect did not affect calcium transients unlike TIMP‐4. Interestingly, we show that MMP‐KO myocytes contract more rapidly and release more Ca2+ than FVB. The relevant RNA species serca-2a is induced and dicer is inhibited. There is selective inhibition of miR-376b and over-expression of miR-1, miR-26a, miR-30d, and miR-181c in MMP‐9KO that are implicated in regulation of G-PCR and calcium handling.


MMP PAR-1 TIMP Cardiac dysfunction Dicer Serca-2a Calcium Cardiomyocytes Contractility 


  1. 1.
    Tyagi, S. C., & Hoit, B. D. (2002). Metalloproteinase in myocardial adaptation and maladaptation. Journal of Cardiovascular Pharmacology and Therapeutics, 7, 241–246.CrossRefPubMedGoogle Scholar
  2. 2.
    Ali, M. A., & Schulz, R. (2009). Activation of MMP-2 as a key event in oxidative stress injury to the heart. Frontiers in Bioscience, 14, 699–716.PubMedGoogle Scholar
  3. 3.
    Macfarlane, S. R., Seatter, M. J., Kanke, T., Hunter, G. D., & Plevin, R. (2001). Proteinase-activated receptors. Pharmacological Reviews, 53, 245–282.PubMedGoogle Scholar
  4. 4.
    Sabri, A., Muske, G., Zhang, H., Pak, E., Darrow, A., Andrade-Gordon, P., et al. (2000). Signaling properties and functions of two distinct cardiomyocyte protease-activated receptors. Circulation Research, 86, 1054–1061.PubMedGoogle Scholar
  5. 5.
    Elkington, P. T., & Friedland, J. S. (2006). Matrix metalloproteinases in destructive pulmonary pathology. Thorax, 61, 259–266.CrossRefPubMedGoogle Scholar
  6. 6.
    Graham, H. K., Horn, M., & Trafford, A. W. (2008). Extracellular matrix profiles in the progression to heart failure. European Young Physiologists Symposium Keynote Lecture-Bratislava 2007. Acta Physiol (Oxf), 194, 3–21.CrossRefGoogle Scholar
  7. 7.
    Kumar, S., Kain, V., & Sitasawad, S. L. (2009). Cardiotoxicity of calmidazolium chloride is attributed to calcium aggravation, oxidative and nitrosative stress, and apoptosis. Free Radical Biology and Medicine, 47, 699–709.CrossRefPubMedGoogle Scholar
  8. 8.
    Khan, H., Metra, M., Blair, J. E., Vogel, M., Harinstein, M. E., Filippatos, G. S., et al. (2009). Istaroxime, a first in class new chemical entity exhibiting SERCA-2 activation and Na-K-ATPase inhibition: A new promising treatment for acute heart failure syndromes? Heart Failure Reviews, 14, 277–287.CrossRefPubMedGoogle Scholar
  9. 9.
    Ide, J., Aoki, T., Ishivata, S., Glusa, E., & Strukova, S. M. (2007). Proteinase-activated receptor agonists stimulate the increase in intracellular Ca2+ in cardiomyocytes and proliferation of cardiac fibroblasts from chick embryos. Bulletin of Experimental Biology and Medicine, 144, 760–763.CrossRefPubMedGoogle Scholar
  10. 10.
    Mishra, P. K., Tyagi, N., Kumar, M., & Tyagi, S. C. (2009). MicroRNAs as a therapeutic target for cardiovascular diseases. Journal of Cellular and Molecular Medicine, 13, 778–789.CrossRefPubMedGoogle Scholar
  11. 11.
    Latronico, M. V., Catalucci, D., & Condorelli, G. (2007). Emerging role of microRNAs in cardiovascular biology. Circulation Research, 101, 1225–1236.CrossRefPubMedGoogle Scholar
  12. 12.
    Chen, J. F., Murchison, E. P., Tang, R., Callis, T. E., Tatsuguchi, M., Deng, Z., et al. (2008). Targeted deletion of dicer in the heart leads to dilated cardiomyopathy and heart failure. Proceedings of the National Academy of Sciences of the United States of America, 105, 2111–2116.CrossRefPubMedGoogle Scholar
  13. 13.
    Ye, G., Metreveli, N. S., Donthi, R. V., Xia, S., Xu, M., Carlson, E. C., et al. (2004). Catalase protects cardiomyocyte function in models of type 1 and type 2 diabetes. Diabetes, 53, 1336–1343.CrossRefPubMedGoogle Scholar
  14. 14.
    Mishra, P. K., Tyagi, N., Kundu, S., & Tyagi, S. C. (2009). MicroRNAs are involved in homocysteine-induced cardiac remodeling. Cell Biochemistry and Biophysics, 55, 153–162.CrossRefPubMedGoogle Scholar
  15. 15.
    Moshal, K. S., Tyagi, N., Henderson, B., Ovechkin, A. V., & Tyagi, S. C. (2005). Protease-activated receptor and endothelial-myocyte uncoupling in chronic heart failure. American Journal of Physiology. Heart and Circulatory Physiology, 288, H2770–H2777.CrossRefPubMedGoogle Scholar
  16. 16.
    Moshal, K. S., Tipparaju, S. M., Vacek, T. P., Kumar, M., Singh, M., Frank, I. E., et al. (2008). Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia. American Journal of Physiology. Heart and Circulatory Physiology, 295, H890–H897.CrossRefPubMedGoogle Scholar
  17. 17.
    Leon, H., Baczko, I., Sawicki, G., Light, P. E., & Schulz, R. (2008). Inhibition of matrix metalloproteinases prevents peroxynitrite-induced contractile dysfunction in the isolated cardiac myocyte. British Journal of Pharmacology, 153, 676–683.CrossRefPubMedGoogle Scholar
  18. 18.
    Schultz, J. J., Glascock, B. J., Witt, S. A., Nieman, M. L., Nattamai, K. J., Liu, L. H., et al. (2004). Accelerated onset of heart failure in mice during pressure overload with chronically decreased SERCA2 calcium pump activity. American Journal of Physiology. Heart and Circulatory Physiology, 286, H1146–H1153.CrossRefGoogle Scholar
  19. 19.
    Wisloff, U., Loennechen, J. P., Falck, G., Beisvag, V., Currie, S., Smith, G., et al. (2001). Increased contractility and calcium sensitivity in cardiac myocytes isolated from endurance trained rats. Cardiovascular Research, 50, 495–508.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Paras Kumar Mishra
    • 1
  • Naira Metreveli
    • 1
  • Suresh C. Tyagi
    • 1
  1. 1.Department of Physiology & Biophysics, School of MedicineUniversity of LouisvilleLouisvilleUSA

Personalised recommendations