225 Pharmacological Ameioration of Perturbed Mitophagy Response (Mitophagy Resistance) in Burns

Abstract

Mitochondrial dysfunctions in skeletal muscles accompanying muscle wasting leads to the poor prognosis of patients with burn injury (BI). The relationship among autophagy, mitochondrial dysfunction, and poor prognosis in BI was not fully determined. Recently, autophagy has been categorized largely into two subsets; non-selective autophagy and selective autophagy including mitophagy. In the latter system, damaged organelles are selectively cleared for their quality control purposes. We have previously discovered that despite the upregulation of basal level of non-selective autophagy, stimulated and selective autophagy (mitophagy) is significantly compromised in BI (state of ‘mitophagy resistance (MR)’). Trehalose, which some stress-resistant organisms including tardigrades utilize to survive under harsh conditions, is known to facilitate autophagy by mTOR-independent pathway. In this study, we investigated whether MR can be pharmacologically ameliorated by trehalose. To study the systemic effect of a whole body BI, a third-degree BI was administered onto the body trunk of wild type mice. Sham-burn (SB) mice served as controls. On post-burn day 3 (PBD3), in vivo microscopy (ivM) was performed to analyze the speed of stimulated mitophagy, mitochondrial membrane potential (MMP) and production of superoxide from mitochondria. For these quantitative ivM analyses, in vivo transfection of a reporter gene construct (mito-Kaede), or staining with MMP-sensitive dye (DiOC6) and/or with MitoSOX, was combined, respectively. For the stimulation of mitophagy, mitochondrial uncoupler, CCCP was intramuscularly injected. The result was compared between mice with BI in the presence or absence of trehalose treatment (2mg/gBW, ip). With mito-Kaede experiment, turnover rate of mitochondria specific to CCCP-induced mitophagy, was successfully monitored. BI mice showed perturbed mitophagy rate than SB (deltaFL/6hr; 18.1% vs. 52.5%), which was significantly ameliorated by trehalose treatment (48.2%, p<0.05). BI also showed decreased basal level of MMP (32% decrease) and increased SO production (243% increase) in response to CCCP as compared to SB, both of which were ameliorated by trehalose treatment (p<0.05). By using novel ivM techniques, we have documented the relationship among MR, MMP loss, and SO production. Successful treatment with trehalose against MR ameliorated MMP loss and SO production, suggesting that MR in BI was likely the cause of muscle dysfunction. Potential efficacy of trehalose against MR was documented for the first time. MR can be a therapeutic target in muscle dysfunctions in BI. It is possible many other organ dysfunctions in critical illnesses may have similar mechanisms.