Long-term in vitro Treatment of Metformin Altered Biological and  Biochemical Properties of Cardiac Cells

Pornthanate Seenak; Nitchawat Paiyabhroma; Phatiwat Chotimon; Titiporn Mekrungruangwong; Sittiruk Roytrakul; Sarawut Kumphune

Authors

Pornthanate Seenak Biomedical Research Unit in Cardiovascular Sciences (BRUCS)
Nitchawat Paiyabhroma Biomedical Research Unit in Cardiovascular Sciences (BRUCS)
Phatiwat Chotimon Biomedical Research Unit in Cardiovascular Sciences (BRUCS)
Titiporn Mekrungruangwong Department of Cardio-Thoracic Technology
Sittiruk Roytrakul Genome Institute, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, 12120
Sarawut Kumphune Department of Medical Technology Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, 65000

Keywords:

diabetes, metformin, longterm effects of metformin, cardiac cell, proteomics

Abstract

A cardiovascular complication from diabetes, especially ischemic heart disease (IHD), is a major cause of death in diabetic patients. Metformin is an effective anti-diabetic drug that has been used worldwide. Several studies have reported the cardioprotective effect of Metformin in an in vivo and ex vivo model. However, information on the biological alterations of long-duration treatment with Metformin at the cellular and molecular levels is still unclear. The purpose of this study was to demonstrate the effects of long-duration cardioprotective doses of Metformin treatment on the biological, biochemical and proteomes characteristics of cardiac cells. Rat cardiac myoblast cell lines (H9c2) were cultured for 30 days in Dulbecco’s modified Eagle’s medium (DMEM) in both the presence and absence of 3mM Metformin, which was optimized as a cardioprotective dose, in a simulated ischemia/reperfusion model. The changes in cell proliferation were determined by the characteristic of the growth curve and calculated populations doubling time (PDT). The cellular morphology was measured by F-actin cytoskeleton staining with phalloidin conjugated with TRITC and visualized under a fluorescence microscope. The phosphorylation of p38 MAPK was demonstrated by western blotting. The alteration of the expressed cellular protein was determined by a proteomics technique. The results showed that the long term effects of 3 mM Metformin was an increase of cell proliferation, decreased populations’ doubling time, slight disorganized actin, and decreased phosphorylation of the p38 MAPK level. In conclusion, the data suggested that the in vitro effect of long-duration Metformin treatment altered the biological, biochemical and proteome characteristics of the cardiac cells.

References

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular Biology of the Cell, 4th edition. Signaling through Enzyme-Linked Cell-Surface Receptors. New York: Garland Science.

Capano, M., & Crompton, M. (2006). Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases. Biochem J, 395(1), 57-64.

Chen, Z., Raman, M., Chen, L., Lee, S. F., Gilman, A. G., & Cobb, M. H. (2003). TAO (thousand-and-one amino acid) protein kinases mediate signaling from carbachol to p38 mitogen-activated protein kinase and ternary complex factors. J Biol Chem, 278(25), 22278-22283.

Costa, Y., Speed, R., Ollinger, R., Alsheimer, M., Semple, C. A., Gautier, P., … Cooke, H. J. (2005). Two novel proteins recruited by synaptonemal complex protein 1 (SYCP1) are at the centre of meiosis. J Cell Sci, 118(Pt 12), 2755-2762.

Hauton, D. (2011). Does long-term metformin treatment increase cardiac lipoprotein lipase? Metabolism, 60(1), 32-42.

Hu, F. B. (2011). Globalization of diabetes: the role of diet, lifestyle, and genes. Diabetes Care, 34(6), 1249-1257.

Jermsri, P., & Kumphune, S. (2012). Ethylacetate extract of Aquilaria crassna preserve actin cytoskeleton on simulated ischemia induced cardiac cell death. Journal of medicinal plants research, 6(23), 4057-4062.

Kravchuk, E., Grineva, E., Bairamov, A., Galagudza, M., & Vlasov, T. (2011). The effect of metformin on the myocardial tolerance to ischemia-reperfusion injury in the rat model of diabetes mellitus type II. Exp Diabetes Res, 2011(2011), 907496. Retrieved from http://dx.doi.10.1155/2011/907496

Kucharczyka, R., Zickb, M., Bietenhadera, M., Raka, M., Couplanc, E., Blondelc, M., … Rago, J. P. (2009). Mitochondrial ATP synthase disorders: Molecular mechanisms and the quest for curative therapeutic approaches. Biochimica et Biophysica Acta, 1739(1), 186–199.

Kumphune, S., Jermsri, P., & Paiyabhroma, N. (2012). An in vitro anti-ischemic effect of Aquilaria crassna in isolated adult rat ventricular myocytes subjected to simulated ischemia. Journal of Phytotherapy and Pharmacology, 1(3), 47-54.

Lowry, O. H., Rosenbrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin Phenol Reagent. J Bio Chem, 193, 265-275.

Mager, H. W., Boer, H. A., Siderius, H. M., & Voss, P. H. (2005). Cellular responses to oxidative and osmotic stress. Cell Stress & Chaperones, 5 (2), 73–75.

Morrish, N. J., Wang, S. L., Stevens, L. K., Fuller, J. H., & Keen, H. (2001). Mortality and causes of death in the WHO Multinational Study of Vascular Disease in Diabetes. Diabetologia, 44(2), S14-21.

Neckers, L. (2007). Heat shock protein 90: the cancer chaperone. J Biosci, 32(3), 517-530.

Ng, S. S., Cheung, Y. T., An, X. M., Chen, Y. C., Li, M., Li, G. H., … Lin, M. C. (2007). Cell Cycle–Related Kinase: A Novel Candidate Oncogene in Human Glioblastoma. J Natl Cancer Inst, 99, 936–948.

Rahbar, S., Natarajan, R., Yerneni, K., Scott, S., Gonzales, N., & Nadler, J. L. (2000). Evidence that pioglitazone, metformin and pentoxifylline are inhibitors of glycation. Clin Chim Acta, 301(1-2), 65-77.

Rossi, G. (2010). Diagnosis and classification of diabetes mellitus. Recenti Prog Med, 101(7-8), 274-276.

Wang, X. F., Zhang, J. Y., Li, L., & Zhao, X. Y. (2011). Beneficial effects of metformin on primary cardiomyocytes via activation of adenosine monophosphate-activated protein kinase. Chin Med J, 124(12), 1876-1884.

Yin, M., van der Horst, I. C., van Melle, J. P., Qian, C., van Gilst, W. H., Sillje, H. H., & de Boer, R. A. (2011). Metformin improves cardiac function in a nondiabetic rat model of post-MI heart failure. Am J Physiol Heart Circ Physiol, 301(2), H459-468.