Mitochondrial Permeability Transition Induces Skeletal Muscle Atrophy in Single Living Myofibers

Abstract

Introduction In advanced age (≥75 y), skeletal muscle atrophy leads to physical frailty, impaired mobility, and increased mortality. Mitochondrial dysfunction has been linked to aging skeletal muscle atrophy, although the specific mechanisms are unclear. Mitochondrial permeability transition (MPT) has been implicated in aging biology, which involves formation of a pore across the inner mitochondrial membrane that causes dissipation of mitochondrial membrane potential and cytoplasmic release of reactive oxygen species (ROS) and mitochondrial proteins. Although we find mitochondria are sensitized to MPT in aging skeletal muscle, the role of MPT in aging muscle biology has not been explored. To gain insight to the impact of MPT in aging muscle, we recently examined the cross-sectional area (CSA) of muscle fibers with myonuclei that contained apoptosis inducing factor (AIF), a protein released from mitochondria during MPT. On average, fibers containing AIF-positive myonuclei had smaller CSA, suggesting a link between MPT and muscle atrophy in advanced age. To more directly test the involvement of MPT in muscle atrophy, we used cultured mouse skeletal muscle fibers to determine if acute chemical induction of MPT would cause fiber atrophy that would be prevented by MPT inhibition. We also hypothesized that treatment with an MPT inhibitor would prevent atrophy in a model of muscle fiber ‘disuse’ where isolated single mouse myofibers were maintained in culture conditions for 5 d. Methods Single fibers were isolated from mouse flexor digitorum brevis (FDB) muscles, plated, and treated with DMSO (vehicle control), doxorubicin (DOX; 1µM), a chemotherapy agent known to induce MPT, or DOX plus the MPT inhibitor cyclosporine A (CsA; 1 µM). Fibers were imaged at 20x upon plating and after 24h treatment. Myofiber diameter was assessed in ImageJ (average of 10 diameter measurements per fiber). Individual fibers were matched pre- to post-treatment for determination of percent change in myofiber diameter. Previous studies have established that maintaining single isolated muscle fibers in culture conditions is associated with fiber atrophy and can therefore be used as a model of disuse/denervation atrophy. On this basis, we isolated single mouse FDB myofibers and treated them with vehicle (DMSO) or 1 µM CsA every 24h under standard culture conditions for 5 d. Myofiber diameter was determined at baseline and day 5, as described above. Results Whereas 24h treatment of myofibers with DOX induced significant atrophy compared to baseline (percent change in myofiber diameter; DOX: -16.64 ± 7.78%, P<0.05), the addition of CsA prevented atrophy over this brief period (DOX+CsA: 0.22 ± 15.38%, NS). Similarly, in a model of muscle disuse, MPT inhibition using CsA prevented myofiber atrophy (baseline: 38.11 ± 5.87 µm vs. day 5: 36.49 ± 7.66 µm, NS) compared with DMSO (baseline: 35.65 ± 7.30 µm vs. day 5: 31.15 ± 7.01 µm, P<0.05). Discussion Consistent with our hypotheses, both inducing MPT using DOX and 5 days of muscle disuse led to atrophy in single living myofibers that was prevented by MPT inhibition. Therefore, our results identify MPT as a novel mechanism of skeletal muscle atrophy and a mechanism that appears relevant to causing the atrophy seen in aging skeletal muscle.