Burn Serum Stimulates Myoblast Cell Death Associated with IL-6-Induced Mitochondrial Fragmentation

Abstract

Background: Burn patients suffer muscle mass loss associated with hyperinflammation and hypercatabolism. The mitochondria are affected by this metabolic alteration. Mitochondrial fission activates a caspase cascade that ultimately leads to cell death. We postulate that burn-induced muscle loss is associated with increased mitochondrial fission and subsequent functional impairment. Further, we investigated whether the cytokine IL-6 plays a major role in mitochondrial fission-associated cell death after burn. Methods: Murine myoblast C2C12 cells were treated with 10% serum isolated either from control rats or 40% total body surface area burned rats. Mitochondria were labeled with MitoTracker Green for live cell images. Mitochondrial function was assessed with an Enzo Mito-ID membrane potential cytotoxicity kit. Protein signals were detected by Western blot analysis. Moreover, recombinant IL-6 was applied to stimulate C2C12 to differentiate the role of cytokine IL-6; lastly, we treated burn serum-stimulated cells with IL-6 antibodies. Results: Caspase 3 activity increased in C2C12 cells with burn serum stimulation, suggesting increased cell death in skeletal muscle after burn. Mitochondrial morphology shortened and mitochondrial membrane potential decreased in cells treated with burn serum. Western blot data showed that mitofusion-1 expression significantly decreased in burn serum-treated cells, supporting the morpho-logic observation of mitochondrial fission. Mitochondrial fragmentation increased with IL-6 stimulation, and IL-6 antibody decreased caspase 3 activity and mitochondrial membrane potential improved in burn serum-stimulated cells. Conclusion: Burn serum caused muscle cell death associated with increased mitochondrial fission and functional impairment. This alteration was alleviated with IL-6 antibody treatment, suggesting the cytokine plays a role in mitochondrial changes in muscle after systemic injury.

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“…Moreover, the previous study also implicated cardiac apoptosis and inflammation, which has also shown to be time dependent (9,10). As one of the cytokines implicated in the acute inflammatory response to burn, IL-6 has also been shown to induce mitochondrial fragmentation of myoblasts in vitro (41) and may contribute to mitochondrial dysfunction in this model. Additionally, although hyperglycemia has been shown to induce cardiac mitochondrial damage in other burn models (49), we did not see any difference in circulating glucose levels in the two groups (data not shown).…”

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“…Furthermore, previous studies have proposed skeletal muscle cell apoptosis was increased following burn and involved in classical caspase-dependent pathways [19][20][21][22][23]. As Yasuhara et al reported increased apoptosis and caspase-3 activity in skeletal muscle within a few hours after burn in a rat model [20].…”

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“…The pathways by which IL-6 might act in the nervous system are not completely understood yet. 2 , 16 , 19 , 51 , 98 Interestingly, some very recent studies show that upon brain injury, a reduction of IL-6 prevented neuronal cell death, therefore suggesting a clear link between IL-6 and neurodegeneration. 2 , 19 …”