Relief of HCV-Induced Oxidative Stress by the Ca++-Uniporter Inhibitor Ruthenium Red

F Agriesti1, R Scrima2, A D\'Aprile2, M Ripoli3, T Tataranni1, V Ruggieri1, D Moradpour4, N Capitanio2, C Piccoli1,2. 1Laboratory of Pre-Clinical and Translational Research, IRCCS, Centro di Riferimento Oncologico della Basilicata, Rionero in Vulture (Pz), Italy, 2Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Fg), Italy, 3Liver Unit, IRCCS Casa Sollievo della Sofferenza Hospital, viale Cappuccini n1, 71013, San Giovanni Rotondo (Fg), Italy, 4Division of Gastroenterlogy and Hepatology, Center Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland

Recent findings of co-localization of a proportion of hepatitis C virus (HCV) core protein, expressed in cultured cell lines, at physical contact sites between ER and mitochondria have put forward the possibility that Ca++ and/or oxidative stress may be involved in HCV infection.

We have extended these observations by investigating the mitochondrial oxidative metabolism and intracellular calcium distribution in U2-OS human osteosarcoma-derived cell lines inducibily expressing the entire HCV open reading frame.

A comparative analysis of the OXPHOS complexes activity and related ROS production along with mitochondrial morpho-functional and intracellular calcium confocal microscopy [1, 2] imaging was carried out in U2-OS cells inducibly expressing the HCV polyprotein under the control of a tetracycline-regulated gene expression system. In these cells viral protein expression is repressed in the presence of tetracycline whereas viral proteins become detectable 4-6 h and reach a steady state 24-48 h following tetracycline withdrawal [3].

Confocal microscopy analysis using specific probes revealed that cell expressing HCV proteins exhibited: i) marked mtΔΨ depolarisation (25±10 vs 50±10 A.U.); ii) increase of mitochondrial ROS production (125±15 vs 25±4 A.U.); iii) enhanced mitochondrial Ca++ load (140±12 vs 70±10 A.U.); iv) an 80% increase of ATP level as compared to controls. Moreover complex I activity as well as the endogenous respiration was significantly reduced (65±5 vs 100±6 activity % of non induced and 1,8±0,4 vs 2,9±0,6 nmoles/min/106 cells respectively) whereas complexes III and IV activities were not affected (121±24 vs 99±21 and 119±21 vs 99±23 activity % of non induced respectively). Of note, all the alterations observed were rescued to the control levels upon treatment of the infected cells with ruthenium red (Fig. 1).

Our study shows a detrimental effect of HCV proteins on the cell oxidative metabolism with inhibition of respiratory chain activity and increased production of reactive oxygen species. These alteration come together with de-regulation of the calcium recycling between cytoplasm and intracellular Ca++ stores. The link appears to be causative since all the modifications observed in U2-OS cells expressing the HCV were completely reversed by the Ca++ uniporter inhibitor ruthenium red. These results, provide new insight into a possible involvement of the mitochondrial dysfunction in the pathogenesis of hepatitis C and its potential role in the development of hepatocellular carcinoma, thus suggesting alternative approaches for therapeutic strategy.

Reference

[1] Piccoli C, Scrima R et al, Transformation by retroviral vectors of bone marrow-derived mesenchymal cells induces mitochondria-dependent cAMP-sensitive reactive oxygen species production. Stem Cells. 2008 Nov; 26(11):2843-54.

[2] Papa S, Sardanelli AM et al, Mitochondrial respiratory dysfunction and mutations in mitochondrial DNA in PINK1 familial parkinsonism. J Bioenerg Biomembr. 2009 Dec;41(6):509-16.

[3] Moradpour D, Heim MK et al, Cell lines that allow regulated expression of HCV proteins: principles and application, in: R.F. Schinazi, J.-P. Sommadossi, C.M. Rice (Eds.), Frontiers in Viral Hepatisis, Elsevier B.V., Amsterdam, 2003, pp. 175–186.