Musculoskeletal
Melatonin Attenuates I/R-Induced Mitochondrial Dysfunction in Skeletal Muscle

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Background

Our recent studies have shown that ischemia/reperfusion (I/R) produces significant necrosis and apoptosis in the cells of skeletal muscle. Our previous studies also demonstrated that melatonin provides significant protection against superoxide generation, endothelial dysfunction, and cell death in the skeletal muscle after I/R. Mitochondria are essential for cell survival, because of their roles as ATP producers as well as regulators of cell death. However, the efficacy of melatonin on I/R-induced mitochondrial dysfunction in the skeletal muscle in vivo has not been demonstrated in the literature.

Materials and Methods

Vascular pedicle isolated rat gracilis muscle model was used. After 4 h of ischemia followed by 24 h of reperfusion, gracilis muscle was harvested, and mitochondrial as well as cytosolic fractions were isolated. Mitochondrial dysfunction was determined by the alteration of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c. Three groups were designed; sham I/R, I/R-V (I/R with vehicle), and I/R-Mel (I/R with melatonin). Melatonin or vehicle was given intravenously 10 min prior to reperfusion and 10 min after reperfusion.

Results

We found that the capability of uptake of fluorescent JC-1 dye in skeletal muscle cells was substantially improved in I/R-Mel group compared with I/R-V group. Melatonin significantly inhibited the outflow of cytochrome c from mitochondria to cytoplasm, which was demonstrated in the I/R-V group.

Conclusions

Melatonin significantly attenuates I/R-induced mitochondrial dysfunction, such as the depolarization of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c, from the mitochondria.

Introduction

Our previous studies 1, 2 have shown that prolonged ischemia followed by reperfusion not only induces necrosis but also accelerates apoptosis in the cells of skeletal muscle. Mitochondria are essential for cell survival, because of their roles as generators of energy as well as regulators of cell death [3]. Mitochondria are a major source of reactive oxygen species (ROS) that could be the toxic stimuli produced during reperfusion and the key components of I/R injury 4, 5. Our previous study [6] found that ischemia/reperfusion (I/R) causes a significant mitochondrial dysfunction, which could be an underlying mechanism of I/R-induced cell death. The present study is the continuation of our long-term project focused on I/R injury in skeletal muscle.

Melatonin (N-acetyl-5-methoxytryptamine), released from the pineal gland, is a highly efficient scavenger of ROS [7]. The highest melatonin concentrations are found in the mitochondria 8, 9, 10. Mitochondria have been identified as a target for melatonin actions 11, 12. In the present study, we hypothesized that melatonin might be able to attenuate I/R-induced mitochondrial dysfunction in skeletal muscle. Mitochondria dysfunction was determined by the depolarization of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c, from the mitochondria. The mitochondrial membrane potential was measured by JC-1 assay in the mitochondrial fractions. The concentrations of cytochrome c were determined by cytochrome c immunoassay in both mitochondrial and cytosolic fractions.

Section snippets

Methods

All experimental procedures and care of the animals were approved by the Institutional Animal Care and Use committee. Male Sprague-Dawley rats weighing 400 to 450 g were used. Anesthesia was accomplished using intraperitoneal sodium pentobarbital (50 mg/kg).

Results

The result of mitochondrial membrane potential (Fig. 1): The average uptake of the fluorescent JC-1 dye (FLU ± SEM/mg protein) in the mitochondrial fraction of muscle cells was significantly decreased in I/R-V group (80697 ± 5355/mg protein) compared with sham I/R group (141303 ± 10986/mg protein) with P < 0.001. This 43% reduction of uptake of JC-1 dye in I/R-V group is an indicator of the depolarization of mitochondrial membrane potential. However, the treatment of melatonin (I/R-Mel)

Discussion

Melatonin, released from the pineal gland, is a great ROS scavenger 7, 8. Melatonin has shown the protective effect on I/R injury in many different organs and tissues (such as in the heart, lung, brain, spinal cord, peripheral nerve, kidney, liver, stomach, intestine, random pattern skin flap, hamster cheek pouch microcirculation, and skeletal muscle), in multiple animal species and in both in vivo and in vitro models 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. Melatonin has higher

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