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To the Editor:

The antibiotic minocycline exerts cytoprotection in animal disease models. One proposed mechanism is modulation of the mitochondrial permeability transition (mPT), a Ca2+-dependent pathogenic event leading to necrotic and/or apoptotic cell death.1–5 A recent study in HEPATOLOGY by Theruvath et al.,6 investigating storage/reperfusion injury following rat liver transplantation, concluded that minocycline prevented mPT and mitigated liver injury by decreasing mitochondrial Ca2+ uptake without affecting mitochondrial respiration. Further, the authors argue that it could be consistent with clinical practice to (pre)treat stored livers and graft recipients with minocycline. The driving force for mitochondrial Ca2+ transport is the mitochondrial membrane potential and the amount of Ca2+ retained is dependent on the proton gradient and the matrix pH.7 Respiratory inhibition will decrease Ca2+ retention capacity and sensitize mitochondria toward mPT.5, 7 Further, endogenous inhibitors of mPT such as adenine nucleotides and Mg2+ will influence the amount of Ca2+ sequestered prior to mPT. In Theruvath et al., the effect of minocyline on mPT was determined in two classical assays, both using bolus additions of calcium chloride: (1) the swelling assay and (2) the Ca2+ retention capacity assay. In both assays, the endpoint is Ca2+ overload and induction of mPT. The authors found that minocycline prevented Ca2+-induced swelling and decreased Ca2+ retention and interpreted this as a specific inhibitory effect on Ca2+ uptake. They excluded respiratory inhibition as the explanation to their findings by determining the respiration of mitochondria exposed to minocycline with and without Ca2+ addition. However, the buffer used in the respiration assay was different from the one used in the Ca2+ bolus assays, with high Mg2+ concentration (Mg2+ is a known endogenous inhibitor of mPT) and with the presence of the potent pharmacological mPT inhibitor cyclosporin A during Ca2+ addition. We argue that minocycline at moderate to high dosing, similar to what we have shown in brain mitochondria, prevents Ca2+-uptake and mPT-induced swelling by respiratory inhibition.1, 5 Further, depending on the buffer system used, the decreased Ca2+ retention can be explained by minocycline-induced increase of mPT sensitivity related to (1) inhibited respiration1, 5 and (2) chelating of Mg2+,8 or (3) direct activation of mPT (even during concurrent cyclosporin A treatment) by adding Ca2+ or in Ca2+ loaded mitochondria, as recently shown by Kupsch et al.8

To stringently evaluate effects of minocycline during the process of Ca2+ uptake, retention, and mPT, mitochondrial oxygen consumption can be monitored during a continuous Ca2+ infusion (Fig. 1A,B). This assay provides information of the bioenergetic demand on mitochondria caused by Ca2+ uptake as well as the respiratory inhibition triggered by mitochondrial Ca2+ overload and mPT.5, 7 Alternatively, the effect of minocycline on isolated mitochondria can be displayed by following changes of extramitochondrial Ca2+ during a slow infusion of the cation.

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Figure 1. Reduction of calcium retention capacity in rat and human liver mitochondria by minocycline. Calcium retention capacity (CRC) was determined in mitochondria exposed to a continuous Ca2+ infusion by monitoring changes in (A, B) oxygen consumption or (C, D) extramitochondrial calcium concentration ([Ca2+]). Experimental conditions were as described in Morota et al.5 All experiments were replicated in 3–4 separate mitochondrial preparations. Representative traces of (A) oxygen consumption and (C) Fura 6F fluorescence ratio during continuous Ca2+ infusion in isolated rat liver mitochondria (100 μg/mL, 200 nmol Ca2+/mg/minute) and human liver mitochondria (150 μg/mL, 50 nmol Ca2+/mg/minute), oxidizing complex I-linked substrates in presence of indicated concentrations of minocycline (Mino). During mitochondrial calcium uptake, oxygen consumption increased and extramitochondrial [Ca2+] was kept at a plateau level until activation of permeability transition (mPT) when oxygen consumption rapidly decreased and the retained Ca2+ in mitochondria was released. Calculations of CRC are displayed in (B) and (D) and minocycline dose-dependently decreased the ability of rat (P = 0.0002, r2 = 0.69) and human liver mitochondria (P = 0.0046, r2 = 0.6086) to hold Ca2+.

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In these more physiologically relevant models, minocycline dose-dependently reduces Ca2+ retention capacity and sensitizes rat and, importantly, human liver mitochondria to the mPT in the dose range used by Theruvath et al. (0–100 nmol/mg mitochondria; Fig. 1).

In conclusion, minocycline may be a promising agent for cytoprotection at relevant dosing through mechanisms other than mPT inhibition. In the clinical setting, prevention of mitochondrial Ca2+ uptake via respiratory inhibition is likely not beneficial to the organism. Further, to sensitize mitochondria to mPT by chelating Mg2+ is not a viable strategy for cytoprotection. This must be kept in mind when considering the use of minocycline, even at moderate dosing, to mitigate storage/reperfusion injury during liver transplantation.

Acknowledgements

Human Subjects: The study of mitochondria derived from human liver tissue was carried out in compliance with national legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (Declaration of Helsinki) and approved by the Ethical Committee of Hachioji Medical Center, Tokyo Medical University, Tokyo, Japan with permit number 12–0.

Animal Experimentation: All animal procedures were approved by the Malmö/Lund (Sweden) Ethical Committee for Animal Research (permit numbers M230-03, M44-07). Adequate measures were taken to minimize pain or discomfort, and all experiments were conducted in accordance with U.S. and international standards on animal welfare.

References

  1. Top of page
  • 1
    Mansson R, Hansson MJ, Morota S, Uchino H, Ekdahl CT, Elmer E. Re-evaluation of mitochondrial permeability transition as a primary neuroprotective target of minocycline. Neurobiol Dis 2007; 25: 198205.
  • 2
    Teng YD, Choi H, Onario RC, Zhu S, Desilets FC, Lan S, et al. Minocycline inhibits contusion-triggered mitochondrial cytochrome c release and mitigates functional deficits after spinal cord injury. Proc Natl Acad Sci U S A 2004; 101: 30713076.
  • 3
    Wang X, Zhu S, Drozda M, Zhang W, Stavrovskaya IG, Cattaneo E, et al. Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease. Proc Natl Acad Sci U S A 2003; 100: 1048310487.
  • 4
    Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M, et al. Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature 2002; 417: 7478.
  • 5
    Morota S, Mansson R, Hansson MJ, Kasuya K, Shimazu M, Hasegawa E, et al. Evaluation of putative inhibitors of mitochondrial permeability transition for brain disorders–specificity vs. toxicity. Exp Neurol 2009; 218: 353362.
  • 6
    Theruvath TP, Zhong Z, Pediaditakis P, Ramshesh VK, Currin RT, Tikunov A, et al. Minocycline and N-methyl-4-isoleucine cyclosporin (NIM811) mitigate storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial permeability transition. HEPATOLOGY 2008; 47: 236246.
  • 7
    Chalmers S, Nicholls DG. The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria. J Biol Chem 2003; 278: 1906219070.
  • 8
    Kupsch K, Hertel S, Kreutzmann P, Wolf G, Wallesch CW, Siemen D, et al. Impairment of mitochondrial function by minocycline. FEBS J 2009; 276: 17291738.

Roland Månsson* †, Saori Morota*, Magnus J. Hansson* ‡, Ichiro Sonoda¶, Yoshihiro Yasuda¶, Motohide Shimazu¶, Ayumu Sugiura††, Shigeru Yanagi††, Hitoshi Miura‡‡, Hiroyuki Uchino**, Eskil Elmér* §, * Mitochondrial Pathophysiology Unit, Laboratory for Experimental Brain Research, Department of Clinical Sciences, Lund University, Lund, Sweden, † Department of Neurology, Malmö University Hospital, Malmö, Sweden, ‡ Department of Clinical Physiology, Center for Medical Imaging and Physiology, Lund University Hospital, Lund, Sweden, § Department of Clinical Neurophysiology, Lund University Hospital, Lund, Sweden, ¶ Department of Gastroenterological Surgery, Tokyo Medical University, Hachioji Medical Center, Hachioji, Tokyo, Japan, ** Department of Anesthesiology, Tokyo Medical University, Hachioji Medical Center, Hachioji, Tokyo, Japan, †† Laboratory of Molecular Biochemistry, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan, ‡‡ Division of Anesthesiology and Critical Care Medicine, Ohyachi Hospital, Sapporo City, Sapporo, Hokkaido, Japan.