• Noorul Izzati Hanafi Institute of Medical Molecular Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA (UiTM), Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Siti Hamimah Sheikh Abdul Kadir Institute of Medical Molecular Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA (UiTM), Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Anis Syamimi Mohamed Institute of Medical Molecular Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA (UiTM), Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Julina Md Noor Faculty of Medicine, Universiti Teknologi MARA (UiTM), Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Nora Julianna Osman Faculty of Medicine, Universiti Teknologi MARA (UiTM), Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Rosfaiizah Siran Faculty of Medicine, Universiti Teknologi MARA (UiTM), Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Sharaniza Ab Rahim Faculty of Medicine, Universiti Teknologi MARA (UiTM), Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Narimah Abdul Hamid Hasani Faculty of Medicine, Universiti Teknologi MARA (UiTM), Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia




UDCA, hypoxia, cardiomyocytes, caspase, ROS


Ursodeoxycholic acid (UDCA) is known as a therapeutic agent in treating cholestasis and liver diseases. Recently, UDCA has been suggested as a therapeutic drug for heart related diseases. Cardioprotective effect of UDCA against the development of ischemia has been studied. Yet, the mechanism of UDCA-cardioprotection is not clearly understood. Therefore, this study aimed to elucidate the mechanisms of UDCA cardioprotection against hypoxia by investigating the expression of caspase -3/-9 and ROS generation using an in vitro hypoxic heart model. A newborn (0-2 days old) rat heart was isolated for primary cell culture of cardiomyocytes. Hypoxia was chemically induced by using CoCl2. Cardiomyocytes were then incubated with UDCA. The treated cardiomyocytes were subjected for ROS generation detection assay, QuantiGene Plex assay for caspase-3/-9 gene expression and ELISA for caspase-3/-9 protein expression. The data were analyzed by using sample paired t-test and One-way ANOVA. Our results showed that UDCA abolishes the effects on CoCl2 in ROS production and UDCA downregulates caspase-9 protein  expression in CoCl2 treated cardiomyocytes. This study provides an insight of UDCA in protecting cardiomyocytes against hypoxia mediated by anti-apoptosis mechanism.

Author Biography

  • Noorul Izzati Hanafi, Institute of Medical Molecular Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA (UiTM), Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
    Postgraduate Student (MSc)


M. Trauner, P. Claudel T FAU - Fickert, T. Fickert P FAU - Moustafa, M. Moustafa T FAU - Wagner, and M. Wagner. 2010. Bile Acids As Regulators Of Hepatic Lipid And Glucose Metabolism. 1421-9875 (Electronic). Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria. michael.trauner@meduni-graz.at FAU - Claudel, Thierry. 220-224,

T. T. Lu, M. Makishima, J. J. Repa, K. Schoonjans, T. A. Kerr, J. Auwerx, and D. J. Mangelsdorf. 2000. Molecular Basis for Feedback Regulation of Bile Acid Synthesis by Nuclear Receptors. Mol. Cell. 6(3): 507-515.

P. B. Hylemon, H. Zhou, W. M. Pandak, S. Ren, G. Gil, and P. Dent. 2009. Bile Acids As Regulatory Molecules. J. Lipid Res. 50: 1509-1520.

E. C. Von Rosenvinge and J.-P. Raufman. 2011. Muscarinic Receptor Signaling In Colon Cancer. Cancers (Basel). 3(1): 971-81.

J. D. Amaral, R. J. S. Viana, R. M. Ramalho, C. J. Steer, and C. M. P. Rodrigues. 2009. Bile Acids: Regulation Of Apoptosis By Ursodeoxycholic Acid. J. Lipid Res. 50(9): 1721-34.

S. H. Sheikh Abdul Kadir, M. Miragoli, S. Abu-Hayyeh, A. V Moshkov, Q. Xie, V. Keitel, V. O. Nikolaev, C. Williamson, and J. Gorelik. 2010. Bile Acid-Induced Arrhythmia Is Mediated By Muscarinic M2 Receptors In Neonatal Rat Cardiomyocytes. PLoS One. 5(3): e9689.

M. J. Perez. 2009. Bile-Acid-Induced Cell Injury And Protection. World J. Gastroenterol. 15(14): 1677.

G. Paumgartner and U. Beuers. 2002. Ursodeoxycholic Acid In Cholestatic Liver Disease: Mechanisms Of Action And Therapeutic Use Revisited. Hepatology. 36(3): 525-31.

E. Im and J. D. Martinez. 2004. Ursodeoxycholic Acid (UDCA) Can Inhibit Deoxycholic Acid (DCA)-induced apoptosis Via Modulation Of EGFR/Raf-1/ERK Signaling In Human Colon Cancer Cells. J. Nutr. 134(2): 483-6.

J. D. Amaral, R. E. Castro, S. Solá, C. J. Steer, and C. M. P. Rodrigues. 2007. P53 Is a Key Molecular Target of Ursodeoxycholic Acid in Regulating Apoptosis. J. Biol. Chem. 282(47): 34250-9.

J. H. Boatright, J. M. Nickerson, A. G. Moring, and M. T. Pardue. 2009. Bile Acids In Treatment Of Ocular Disease. J. Ocul. Biol. Dis. Infor. 2(3): 149-159.

A. A. Goldberg, V. I. Titorenko, A. Beach, and J. T. Sanderson. 2013. Bile Acids Induce Apoptosis Selectively In Androgen-Dependent And -Independent Prostate Cancer Cells. PeerJ. 1(e122).

M. Dubreuil, S. Ruiz-Gaspà , N. Guañabens, P. Peris, L. Alvarez, A. Monegal, A. Combalia, and A. Parés. 2013. Ursodeoxycholic Acid Increases Differentiation And Mineralization And Neutralizes The Damaging Effects Of Bilirubin On Osteoblastic Cells. Liver Int. 33(7): 1029-38.

M. Miragoli, S. H. S. A. Kadir, M. N. Sheppard, N. Salvarani, M. Virta, S. Wells, M. J. Lab, V. O. Nikolaev, A. Moshkov, W. M. Hague, S. Rohr, C. Williamson, and J. Gorelik. 2011. A Protective Antiarrhythmic Role Of Ursodeoxycholic Acid In An In Vitro Rat Model Of The Cholestatic Fetal Heart. Hepatology. 54(4): 1282-92.

S. von Haehling, J. C. Schefold, E. a Jankowska, J. Springer, A. Vazir, P. R. Kalra, A. Sandek, G. Fauler, T. Stojakovic, M. Trauner, P. Ponikowski, H.-D. Volk, W. Doehner, A. J. S. Coats, P. a Poole-Wilson, and S. D. Anker. 2012. Ursodeoxycholic Acid In Patients With Chronic Heart Failure: A Double-Blind, Randomized, Placebo-Controlled, Crossover Trial. J. Am. Coll. Cardiol. 59(6): 585-92.

J. C. Sluimer, J. M. Gasc, J. L. van Wanroij, N. Kisters, M. Groeneweg, M. D. Sollewijn Gelpke, J. P. Cleutjens, L. H. van den Akker, P. Corvol, B. G. Wouters, M. J. Daemen, and A. P. J. Bijnens. 2008. Hypoxia, Hypoxia-Inducible Transcription Factor, and Macrophages in Human Atherosclerotic Plaques Are Correlated With Intraplaque Angiogenesis. J. Am. Coll. Cardiol. 51(13): 1258-1265.

J. E. Ziello, I. S. Jovin, and Y. Huang. 2007. Hypoxia-Inducible Factor (HIF) -1 Regulatory Pathway and its Potential for Therapeutic Intervention in Malignancy and Ischemia. Yale J. Biol. Med. Med. 80: 51-60.

M. C. Brahimi-Horn, J. Chiche, and J. Pouyss. 2007. Hypoxia Signalling Controls Metabolic Demand. Curr. Opin. Cell Biol. 19: 223-229.

R. Clark, I. Christlieb, M. Sanmarco, R. Diaz-perez, and J. F. Dammann. 1963. Relationship of Hypoxia to Arrhythmia and Cardiac Conduction Hemorrhage. Circulation. XXVII(April): 724-47.

K. Sarkar, Z. Cai, R. Gupta, N. Parajuli, K. Fox-talbot, and M. S. Darshan. 2012. Hypoxia-inducible Factor 1 Transcriptional Activity In Endothelial Cells Is Required For Acute Phase Cardioprotection Induced By Ischemic Preconditioning. Pnas.

X. Wang, H. Fang, Z. Huang, W. Shang, T. Hou, A. Cheng, and H. Cheng. 2013. Imaging ROS Signaling In Cells And Animals. J. Mol. Med. (Berl). 91(8): 917-27.

J. Cui, Z. Li, L. Qian, Q. Gao, J. Wang, M. Xue, X. Lou, I. C. Bruce, Q. Xia, and H. Wang. 2013. Reducing The Oxidative Stress Mediates The Cardioprotection Of Bicyclol Against Ischemia-Reperfusion Injury In Rats. J. Zhejiang Univ. Sci. B. 14(6): 487-95.

J.-Y. Kim and J.-H. Park. 2003. ROS-dependent Caspase-9 Activation In Hypoxic Cell Death. FEBS Lett. 549(1-3): 94-98.

J. Neckář, O. Szárszoi, J. Herget, B. Ošťádal, and F. Kolář. 2003. Cardioprotective Effect Of Chronic Hypoxia Is Blunted By Concomitant Hypercapnia. Physiol. Res. 52: 171-175.

S. N. Sclair, E. Little, and C. Levy. 2015. Current Concepts in Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis. Clin Transl Gastroenterol. 6(8): e109.

S. C. Lim, H. Q. Duong, K. R. Parajuli, and S. I. Han. 2012. Pro-apoptotic Role Of The MEK/ERK Pathway In Ursodeoxycholic Acid-Induced Apoptosis In SNU601 Gastric Cancer Cells. Oncol. Rep. 28(4): 1429-1434.

M. J. D. E. F. Smith 111., A. J. Nichols., T. S. Sellers., S. R. O’Brien., D. E. Griswold., J. W. Egan., L. M. Hillegass., J. A. Vasko., M. J. Slivjak, P. A. Davis., C. E, Wolff. 1991. Reduction in Myocardial Ischemic/Reperfusion Injury and Neutrophil Accumulation After Therapeutis Administration of Streptokinase. 72-738.

L. Bonello, P. Sbragia, N. Amabile, O. Com, S. V Pierre, S. Levy, and F. Paganelli. 2007. Protective Effect Of An Acute Oral Loading Dose Of Trimetazidine On Myocardial Injury Following Percutaneous Coronary Intervention. Heart. 93(6): 703-7.

M. U. Khan, Y. Cheema, A. U. Shahbaz, R. A. Ahokas, Y. Sun, I. C. Gerling, S. K. Bhattacharya, and K. T. Weber. 2012. Mitochondria Play A Central Role In Nonischemic Cardiomyocyte Necrosis: Common To Acute And Chronic Stressor States. Pflügers Arch. 464(1): 123-131.

J. D. Luo, F. Xie, W. W. Zhang, X. D. Ma, J. X. Guan, and X. Chen. 2001. Simvastatin Inhibits Noradrenaline-Induced Hypertrophy Of Cultured Neonatal Rat Cardiomyocytes. Br. J. Pharmacol. 132(1): 159-64.

Y. Liu, G. Fiskum, and D. Schubert. 2002. Generation of Reactive Oxygen Species By The Mitochondrial Electron Transport Chain. J. Neurochem. 80(5): 780-7.

D. de Moissac, R. M. Gurevich, H. Zheng, P. K. Singal, and L. A. Kirshenbaum. 2000. Caspase Activation And Mitochondrial Cytochrome C Release During Hypoxia-Mediated Apoptosis Of Adult Ventricular Myocytes. J. Mol. Cell. Cardiol. 32(1): 53-63.

Y. Qin, T. L. Vanden Hoek, K. Wojcik, T. Anderson, C.-Q. Li, Z.-H. Shao, L. B. Becker, and K. J. Hamann. 2004. Caspase-Dependent Cytochrome C Release And Cell Death In Chick Cardiomyocytes After Simulated Ischemia-Reperfusion. Am. J. Physiol. Heart Circ. Physiol. 286(6): H2280-6.

X. Borenstein, G. L. Fiszman, A. Blidner, S. I. Vanzulli, and M. a. Jasnis. 2010. Functional Changes In Murine Mammary Cancer Cells Elicited By Cocl2-Induced Hypoxia. Nitric Oxide - Biol. Chem. 23(3): 234-241.

J. Piret, D. Mottet, M. Raes, and C. Michiels. 2002. Is HIF-1a A Pro- Or An Anti-Apoptotic Protein? 64(January): 889-892,

L. Xi, M. Taher, C. Yin, F. Salloum, and R. C. Kukreja. 2004. Cobalt Chloride Induces Delayed Cardiac Preconditioning In Mice Through Selective Activation Of HIF-1alpha And AP-1 And Inos Signaling. Am. J. Physiol. Heart Circ. Physiol. 287: H2369-75.

Y. Saini, K. K. Greenwood, C. Merrill, K. Y. Kim, S. Patial, N. Parameswaran, J. R. Harkema, and J. J. LaPres. 2010. Acute Cobalt-Induced Lung Injury And The Role Of Hypoxia-Inducible Factor 1?? In Modulating Inflammation. Toxicol. Sci. 116(2): 673-681.

G. Chachami, G. Simos, A. Hatziefthimiou, S. Bonanou, P. A. Molyvdas, and E. Paraskeva. 2004. Cobalt Induces Hypoxia-Inducible Factor-1?? Expression In Airway Smooth Muscle Cells By A Reactive Oxygen Species- And PI3K-Dependent Mechanism. Am. J. Respir. Cell Mol. Biol. 31: 544-551.

a Linna, P. Oksa, K. Groundstroem, M. Halkosaari, P. Palmroos, S. Huikko, and J. Uitti. 2004. Exposure To Cobalt In The Production Of Cobalt And Cobalt Compounds And Its Effect On The Heart. Occup. Environ. Med. 61: 877-885.

Y. Kakinuma, R. G. Katare, M. Arikawa, K. Muramoto, F. Yamasaki, and T. Sato, 2008. A HIF-1alpha-Related Gene Involved In Cell Protection From Hypoxia By Suppression Of Mitochondrial Function. FEBS Lett. 582(2): 332-340.

R. Malhotra, D. W. Tyson, H. M. Rosevear, and F. C. B. Iii. 2008. Hypoxia-inducible Factor-1alpha Is A Critical Mediator Of Hypoxia Induced Apoptosis In Cardiac H9c2 And Kidney Epithelial HK-2 Cells. 11: 1-11.

A. Kulisz, N. Chen, N. S. Chandel, Z. Shao, and P. T. Schumacker. 2002. Mitochondrial ROS Initiate Phosphorylation of p38 MAP Kinase During Hypoxia In Cardiomyocytes. Am. J. Physiol. Lung Cell. Mol. Physiol. 282: L1324-9.

R. Malhotra, V. Valuckaite, M. L. Staron, T. Theccanat, K. M. D’Souza, J. C. Alverdy, and S. a Akhter. 2011. High-molecular-weight Polyethylene Glycol Protects Cardiac Myocytes From Hypoxia- and Reoxygenation-Induced Cell Death And Preserves Ventricular Function. Am. J. Physiol. Heart Circ. Physiol. 300: H1733-42.

F. J. Giordano. 2005. Review Series Oxygen, Oxidative Stress, Hypoxia, And Heart Failure. 115(3): 500-508.

J. Duranteau, N. S. Chandel, A. Kulisz, Z. Shao, and P. T. Schumacker. 1998. Intracellular Signaling by Reactive Oxygen Species during Hypoxia in Cardiomyocytes TL - 273. J. Biol. Chem. 273(19): 11619-11624.

J. Duranteau. 1998. Intracellular Signaling by Reactive Oxygen Species during Hypoxia in Cardiomyocytes. J. Biol. Chem. 273(19): 11619-11624.

C. M. Rodrigues, X. Ma, C. Linehan-Stieers, G. Fan, B. T. Kren, and C. J. Steer. 1999. Ursodeoxycholic Acid Prevents Cytochrome C Release In Apoptosis By Inhibiting Mitochondrial Membrane Depolarization And Channel Formation. Cell Death Differ. 6: 842-54.

Y. Yuan, G. Hilliard, T. Ferguson, and D. E. Millhorn. 2003. Cobalt Inhibits The Interaction Between Hypoxia-Inducible Factor-?? And Von Hippel-Lindau Protein By Direct Binding To Hypoxia-Inducible Factor-?? J. Biol. Chem. 278(18): 15911-15916.

W. Zou, J. Zeng, M. Zhuo, W. Xu, L. Sun, J. Wang, and X. Liu. 2002. Involvement Of Caspase-3 And P38 Mitogen-Activated Protein Kinase In Cobalt Chloride-Induced Apoptosis In PC12 Cells. J. Neurosci. Res. 67(6): 837-843.

K. Kuida, T. F. Haydar, C. Y. Kuan, Y. Gu, C. Taya, H. Karasuyama, M. S. S. Su, P. Rakic, and R. a. Flavell. 1998. Reduced apoptosis and cytochrome C-mediated caspase activation in mice lacking Caspase 9. Cell. 94: 325-337.

P. M. Kang, a Haunstetter, H. Aoki, a Usheva, and S. Izumo. 2000. Morphological And Molecular Characterization Of Adult Cardiomyocyte Apoptosis During Hypoxia And Reoxygenation. Circ. Res. 87: 118-125.

B. M. Kim and H. W. Chung. 2007. Hypoxia/reoxygenation Induces Apoptosis Through A ROS-Mediated Caspase-8/Bid/Bax Pathway In Human Lymphocytes. Biochem. Biophys. Res. Commun. 363(3): 745-50.

J. Moungjaroen, U. Nimmannit, P. S. Callery, L. Wang, N. Azad, V. Lipipun, P. Chanvorachote, and Y. Rojanasakul. 2006. Reactive Oxygen Species Mediate Caspase Activation And Apoptosis Induced By Lipoic Acid In Human Lung Epithelial Cancer Cells Through Bcl-2 Down-Regulation. J. Pharmacol. Exp. Ther. 319(3): 1062-1069.

V. Viswanath, Y. Wu, R. Boonplueang, S. Chen, F. F. Stevenson, F. Yantiri, L. Yang, M. F. Beal, and J. K. Andersen. 2001. Caspase-9 Activation Results in Downstream Caspase-8 Activation and Bid Cleavage in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s Disease. J. Neurosci. 21(24): 9519-9528.

and H. M. Marieke H. Schoemaker, Laura Conde de la Rosa, Manon Buist-Homan, Titia E. Vrenken, Rick Havingga, Klaas Poelstra, Hidde J. haisma, Peter L. M. Jansen. 2004. Tauroursodeoxycholic Acid Protects Rat Hepatocytes from Bile-Induced Apoptosis via Activation of survival Pathways. 1563-1573.

K. Å kemiene, G. Jablonskiene, J. Liobikas, and V. Borutaite. 2013. Protecting The Heart Against Ischemia/Reperfusion-Induced Necrosis And Apoptosis: The Effect Of Anthocyanins. Med. 49(2): 84-88.

H. Zhu, S. McElwee-Witmer, M. Perrone, K. L. Clark, and a Zilberstein. 2000. Phenylephrine protects neonatal rat cardiomyocytes from hypoxia and serum deprivation-induced apoptosis. Cell Death Differ. 7: 773-784.

N. I. Hanafi, A. S. Mohamed, J. Noor, and N. A. H. Hasani. 2016. Ursodeoxycholic Acid Upregulates ERK and Akt in The Protection Of Cardiomyocytes Against CoCl 2. 15(2): 1-12.

Carvour, C., Song, C., Kaul, S., Anantharam, A., Kandasamy, A. & Kandasamy, A. 2008. Chronic Low Dose Oxidative Stress Induces Caspase-3 Dependent Pkcδ Proteolytic Activation And Apoptosis In A Cell Culture Model Of Dopaminergic Neurodegeneration. Ann N.Y Acad Sci. 1139(515): 197-205.






Science and Engineering

How to Cite