In their letter to the editor, Månsson et al. cite studies showing that minocycline at concentrations as high as 200 μM causes respiratory inhibition in isolated mitochondria and sensitization to the mitochondrial permeability transition (MPT). Likewise in our recent study, minocycline at concentrations greater than about 50 μM caused mitochondrial dysfunction manifested by decreased adenosine diphosphate/oxygen ratio and increased respiratory control ratio.1 These adverse effects on mitochondrial function correlated with loss of efficacy of minocycline to prevent storage-reperfusion injury to transplanted liver grafts, because 18 μM minocycline in the preservation medium protected against injury, whereas higher doses up to 200 μM did not.
The dose dependency of minocycline cytoprotection is more clearly illustrated in a model of warm ischemia-reperfusion injury to cultured rat hepatocytes. Hepatocytes were subjected to 4 hours of anoxia in Krebs-Ringer-HEPES buffer at pH 6.2 to simulate ischemia, followed by reoxygenation at pH 7.4 to simulate reperfusion, as described.2, 3 Under these conditions, cell death occurred progressively after reperfusion (Fig. 1A). When ischemic hepatocytes were exposed to minocycline 15 minutes before and then continuously after reperfusion, protection against cell death occurred in a dose-dependent fashion with maximal protection at 20 μM minocycline. Protection was then lost as minocycline increased further to 50 μM.
Månsson et al. present new measurements of respiratory rate and extramitochondrial free Ca2+ during continuous infusion of CaCl2 into isolated rat and human liver mitochondria suspensions. Interpretation of these results is confounded by the several concurrent processes likely to occur during the incubation, including (1) respiratory stimulation due to electrogenic Ca2+ uptake, (2) respiratory stimulation due to Ca2+-dependent activation of oxidized nicotinamide adenine dinucleotide (NAD+)-linked dehydrogenases (e.g., pyruvate, isocitrate, and α-ketoglutamate dehydrogenase), (3) respiratory stimulation due to mitochondrial depolarization after opening of permeability transition (PT) pores, (4) reversal of Ca2+ activation of dehydrogenases due to Ca2+ release after PT pore opening, and (5) respiratory inhibition from cytochrome c and pyridine nucleotide release after MPT-induced mitochondrial swelling. However, noteworthy in the data is lack of any evidence of respiratory inhibition by 2.5-10 μM minocycline before Ca2+ infusion. Moreover, as minocycline increased, extramitochondrial Ca2+ rose more rapidly during Ca2+ infusion, which is consistent with inhibition by minocycline of the mitochondrial Ca2+ uniporter. Nonetheless, definitive interpretation of this data set cannot be made without simultaneous measurement of mitochondrial membrane potential and swelling.
Measurements in isolated mitochondria, including our own measurements, are typically unphysiological in many respects. For example, MPT assays are almost always conducted in Mg2+-free medium, because free Mg2+, a normal constituent of hepatic cytosol, inhibits MPT onset in in vitro assays. For our respiratory measurements, we included Mg2+ to prevent the confounding effects of MPT onset on oxygen uptake. Both with and without Mg2+, our results supported the conclusion that minocycline inhibits mitochondrial Ca2+ uptake. Although Månsson et al. suggest that minocycline may exert effects via Mg2+ chelation, for our respiratory measurements Mg2+ was in substantial molar excess (e.g., 5 mM Mg2+ versus no more than 100 μM minocycline), which would make consequences of such chelation negligible. Moreover, tetracycline also chelates Mg2+,4 but is not cytoprotective.
True physiological conditions exist when mitochondria are inside intact functioning cells, and our recent article in HEPATOLOGY showed by intravital multiphoton microscopy that treatment of stored livers with minocycline or the MPT inhibitor, NIM811, prevented mitochondrial depolarization, necrosis, and graft failure after transplantation.1 To show that protection against mitochondrial dysfunction by minocycline is specifically related to inner membrane permeabilization, the key event of the MPT, we recently examined the effect of minocycline on mitochondrial function during warm ischemia-reperfusion injury to cultured rat hepatocytes. After reperfusion with vehicle, most mitochondria inside hepatocytes depolarized within 20 minutes, as shown by decreased red fluorescence of potential-indicating tetramethylrhodamine methylester (TMRM) in three of four cells shown in Fig. 1B (asterisks, right panel). Simultaneously, inner membrane permeabilization occurred, as shown by uptake of green fluorescing calcein into the mitochondria matrix (Fig. 1B, left panel). As shown previously,1–3, 5 cyclosporin A and NIM811 block these changes, showing that depolarization and inner membrane permeabilization are a consequence of the MPT. Similarly, minocycline (20 μM) inhibited mitochondrial TMRM release and calcein uptake even after longer times of reperfusion, demonstrating directly that minocycline blocks the characteristic inner membrane permeabilization of the MPT (Fig. 1C). Thus, minocycline protects against mitochondrial dysfunction after both warm and cold ischemia-reperfusion by prevention of the MPT. These results under truly physiological conditions are simply incompatible with the proposal that the chief effects of minocycline are respiratory inhibition and MPT promotion. Rather, such mitochondrial dysfunction is likely the basis for loss of efficacy at higher nontherapeutic minocycline concentrations. Currently, we are screening a panel of tetracycline derivatives for compounds with a larger therapeutic window of efficacy.
Minocycline is a well-characterized and cost-effective anitbiotic of proven safety in a broad spectrum of indications.6 In rare instances, hepatotoxicity after chronic use occurs, which is attributed to the metabolite 4-epiminocycline.7 However, such toxicity would be unlikely after one-time use to minimize storage-reperfusion injury to transplanted livers. To decrease infection, antibiotics are a standard peritransplant regimen, and use of minocycline in donors, recipients, and during graft storage might have added benefit, especially in marginal livers from higher risk donors or in livers subjected to longer periods of storage. Overall, we agree with Månsson et al. that the safety and efficacy of minocycline for use in clinical liver transplantation deserve further exploration.