Cyclophilin D, a target for counteracting skeletal muscle dysfunction in mitochondrial myopathy

Human Molecular Genetics, 2015, Vol. 24, No. 23 6580–6587 doi: 10.1093/hmg/ddv361 Advance Access Publication Date: 14 September 2015 Original Article ORIGINAL ARTICLE Cyclophilin D, a target for counteracting skeletal muscle dysfunction in mitochondrial myopathy 1 Department of Physiology and Pharmacology, 2Department of Laboratory Medicine, 3Department of Molecular Medicine and Surgery, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden and 4Center for Inherited Metabolic Disease (CMMS), Karolinska University Hospital, Stockholm, Sweden *To whom correspondence should be addressed. Email: (A.W.)/ (H.W.) Abstract Muscle weakness and exercise intolerance are hallmark symptoms in mitochondrial disorders. Little is known about the mechanisms leading to impaired skeletal muscle function and ultimately muscle weakness in these patients. In a mouse model of lethal mitochondrial myopathy, the muscle-specific Tfam knock-out (KO) mouse, we previously demonstrated an excessive mitochondrial Ca2+ uptake in isolated muscle fibers that could be inhibited by the cyclophilin D (CypD) inhibitor, cyclosporine A (CsA). Here we show that the Tfam KO mice have increased CypD levels, and we demonstrate that this increase is a common feature in patients with mitochondrial myopathy. We tested the effect of CsA treatment on Tfam KO mice during the transition from a mild to terminal myopathy. CsA treatment counteracted the development of muscle weakness and improved muscle fiber Ca2+ handling. Importantly, CsA treatment prolonged the lifespan of these muscle-specific Tfam KO mice. These results demonstrate that CsA treatment is an efficient therapeutic strategy to slow the development of severe mitochondrial myopathy. Introduction Mitochondrial myopathies are genetically heterogeneous metabolic disorders, originating from the dysfunction of one or more mitochondrial metabolic pathways. They can be caused by either mitochondrial (mt) or nuclear DNA mutations that alter expression or function of proteins in the mitochondrial respiratory chain, leading to defective oxidative phosphorylation (1,2). Mitochondrial Ca2+ homeostasis has been shown to be of critical importance for cell function and is finely tuned. Indeed, limited and transient mitochondrial Ca2+ uptake contributes to the elaborate control of cell metabolism, whereas prolonged and excessive uptake can activate cell death pathways (3–5). The recently identified mitochondrial Ca2+ uniporter (MCU) has been shown to be the main site of Ca2+ entry to the mitochondrial matrix (6,7), but the presence of Ca2+ in mitochondria from MCU −/− mice suggests that alternative mechanisms exist for Ca2+ entry (8). Mice with skeletal muscle-specific disruption of the mitochondrial transcription factor A [Tfam; Tfam knock-out (KO) mice] develop severe mtDNA depletion with many phenotypic features also observed in human pathology (9,10). Thus, similar to patients suffering from various forms of mitochondrial myopathy, Tfam KO mice have a respiratory chain enzyme complex deficiency, including abnormally shaped mitochondria, and † Charlotte Gineste and Andres Hernandez equally contributed to this work. Received and Revised: August 14, 2015. Accepted: September 1, 2015 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: 6580 Charlotte Gineste1,†, Andres Hernandez1,†, Niklas Ivarsson1, Arthur J. Cheng1, Karin Naess2, Rolf Wibom4, Nicole Lesko4, Helene Bruhn4, Anna Wedell3,4, Christoph Freyer2,4, Shi-Jin Zhang1, Mattias Carlström1, Johanna T. Lanner1, Daniel C. Andersson1, Joseph D. Bruton1, Anna Wredenberg2,4, * and Håkan Westerblad1, * Human Molecular Genetics, 2015, Vol. 24, No. 23 atrophic, ragged-red and a mosaic pattern of COX-deficient muscle fibers (2,11). Tfam KO muscle fibers demonstrated an excessive mitochondrial Ca2+ uptake during repeated contractions, which was partially inhibited by cyclosporine A (CsA) (12). CsA is widely used in clinical practice as an immunosuppressant to prevent the rejection of organ transplants and for treatment of various autoimmune diseases (13). CsA binds to the mitochondrial protein cyclophilin D (14,15) (CypD; encoded by the Ppif gene in the mouse, previously also called cyclophilin F) and has been shown to counteract the opening of the mitochondrial permeability transition pore (16–19). In this study, we assessed the involvement of CypD in the disease process of mitochondrial myopathies first by measuring the expression of CypD in muscles of mitochondrial myopathy patients and Tfam KO mice and second by testing whether CsA treatment counteracted the disease progression and muscle weakness in Tfam KO mice. Results To investigate whether CypD is important in mitochondrial muscle pathophysiology, we measured CypD mRNA and/or protein levels in skeletal muscle from Tfam KO mice as well as patients with diagnosed mitochondrial myopathy (Supplementary Material, Table S1). In skeletal muscle from the Tfam KO mice, CypD mRNA and protein levels were significantly increased (∼60 and ∼55%, respectively; Fig. 1A and B). The patients were selected based on OXPHOS dysfunction and were grouped according to their mtDNA integrity in skeletal muscle. These groups were as follows: patients with mtDNA deletions (n = 4); mtDNA depletion (n = 1); mt-tRNA mutations (n = 4) and mutations in mitochondrial encoded RC complex I subunits (n = 3). Figure 1C and D show that patients with mtDNA depletion or mtDNA mutations all have markedly higher (∼200%) CypD levels than the control group. The only patient group with predominantly unchanged CypD levels had mtDNA deletions (Fig. 1C and D). CsA treatment leads to extended lifespan and prevents muscle weakness in mitochondrial myopathy mice We next examined the effect of 4 weeks of CsA treatment (120 µg/ day) on the Tfam KO mice. The treatment spanned from 12 to 16 weeks of age, when the disease progresses from moderate to terminal. CsA treatment resulted in a significant increase in lifespan of the Tfam KO mice: all treated mice were still alive at the end of the treatment period at 16 weeks of age, whereas ∼40% of the untreated Tfam KO mice were dead (Fig. 2A). CsA treatment also resulted in an ∼15% higher hindlimb lean mass in treated than in untreated Tfam KO mice (Fig. 2B), while no difference was observed for hindlimb fat mass (Supplementary Material, Fig. S1). In 16-week-old Tfam KO mice, CsA treatment significantly increased extensor digitorum longus (EDL) specific force at all stimulation frequencies, whereas it did not affect EDL force production in control mice (Supplementary Material, Fig. S2). The increased contractile strength of CsA-treated Tfam KO mice was confirmed by force measurements in intact single flexor digitorum brevis (FDB) fibers (Fig. 3A and B). Preserved sarcoplasmic reticulum Ca2+ release in CsA-treated Tfam KO muscle fibers CsA treatment completely prevented the decrease in free cytosolic [C (...truncated)