Browsing by Author "Sehat, Alvand"
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Item Burn Serum Stimulated Mitochondrial Fission Was Decreased with IL-6 Antibody Treatment(2017-01-17) Sehat, Alvand; Song, Juquan; Kulangara, Rohan; Maredia, Navin; Maxwell, Christian; Panwar, Kunal; Huebinger, Ryan M.; Carlson, Deborah L.; Zang, Qun S.; Wolf, Steven E.INTRODUCTION: Burn patients suffer muscle mass loss associated with hyperinflammation and hypercatabolism. We previously observed that burn serum resulted in cell death with elevated mitochondrial fragmentation in C2C12 myoblast. IL-6 as the key cytokine response to thermal injury has been showed to increase mitochondrial fragmentation. We thus posit that inhibition of IL-6 expression in burn serum will alleviate mitochondrial fragmentation. The aim of this study is to investigate the neutralization effect of IL-6 antibody in burn serum stimulated myoblasts. METHODS: Murine myoblasts C2C12 cells were cultured with recombinant IL-6 protein from 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, and 100 ng/ml. Cells were labeled with MitoTracker Green dye and live cell images were captured with Confocal microscopy. Next, C2C12 cells were exposed to medium containing 1) 10% serum from control rat, 2) 10% serum from burn rat, and 3) 10% serum from burn rat and 0.5 ug/ml of IL-6 antibody. All cells were labeled as the first experiment and live cell images were recorded. The caspase 3 activity was further examined from cell protein lysate. RESULTS: The 1 ng/ml dose of r-IL6 showed a fourfold increase in mitochondrial volume (μm3) at 24 hours post challenge. The intensity signal of Mitotracker was significantly increased in the 1 ng/ml IL-6 dose group at 48 hours. The 1 ng/ml dose of r-IL6 showed an increase in mitochondrial fragmentation. In cells cultured with 10% serum from control rats, mitochondrial morphology maintained the elongated linear shape during the 48 hours. In cells cultured with 10% serum from burned rats, a reduction in mitochondrial sizes was significant. In cells cultured with 10% serum and IL-6 antibody treatment this effect was reversed. Further, the intensity signals increased in response to the burn serum challenge. However, with addition of IL-6 antibody treatment the intensity signals decreased. In addition, burn serum increased the total volume of mitochondria about 1.4 fold, while with IL-6 antibody treatment the total volume was decreased. Consistently, a significant decrease in the expression of caspase 3 in the IL-6 antibody treatment group was observed. CONCLUSION: IL-6 stimulates an increase in mitochondria fragmentation in myoblasts, while IL-6 antibody treatment decreases mitochondrial fragmentation and cell death in burn serum stimulated myoblasts. This project has shown that targeting cytokine levels may be an effective treatment strategy in the management of burn patients.Item Burn Serum Stimulates Mitochondrial Fission in C2C12 Myoblasts(2021-04-22) Sehat, Alvand; Wolf, Steven; Carlson, Deborah; Huebinger, RyanBACKGROUND: Burn patients suffer muscle mass loss associated with a hypercatabolic status. Impairment of mitochondrial function has been observed in the muscle of burn patient's. OBJECTIVE: We hypothesize that muscle atrophy due to burn injur y is associated with an alteration in mitochondrial dynamics. This study was designed to investigate changes in mitochondrial fission and fusion in response to a burn serum challenge in myoblasts in vitro. METHODS: Cultured murine myoblasts, C2C12 cells, w ere exposed to 10% rat serum isolated either from 40% total body surface area (TBSA) scald burn rats or control rats. Cells were then labeled with MitoTracker Green dye and live cell images were recorded by confocal microscopy. The expression of mitochondr ial fission/fusion factors was examined by Western blots. RESULTS: In cells cultured with 10% serum from control rats, mitochondrial morphology maintained the elongated linear shape during the 48 hours we observed. However, 24 hours of culturing in 10% scaled rat serum resulted in a significant reduction in mitochondrial size. .Further, the number of total mitochondria increased, indicating a stimulation of mitochondrial fragmentation in response to the burn serum challenge. In addition, burn serum increases the total volume of mitochondria about 1.4 fold. Consistently, Western blot analysis showed a significant decrease in the expression of mitochondrial fusion protein Mfn1. CONCLUSION: Burn serum stimulates an increase in mitochondria fission in myoblasts.Item Mitochondrial Fission with Function Impairment in Burn Serum Treated C2C12 Cells(2016-01-19) Sehat, Alvand; Song, Juquan; Kumar, Puneet; Cai, Anthony; Huebinger, Ryan M.; Carlson, Deborah, L.; Zang, Qun S.; Wolf, Steven E.BACKGROUND: Burn patients suffer muscle mass loss associated with a hypercatabolic status. Mitochondria dynamics cycle is affected by metabolic status, and mitochondrial fission mediated high glucose induced cell death. Mitochondria function impairment associated with muscle mass loss has been observed in severe burn patients. We hypothesize that severe burn impaired muscle mass loss is associated with increased mitochondria fission with function impairment. The study was to investigate mitochondrial dynamics in response to burn serum stimulation. METHODS: Murine myoblast C2C12 cells were treated with DMEM media containing 10% rat serum isolated either from 40% TBSA scald burn rats, or control rats. Mitochondria was labeled with 3nM of MitoTracker Green FM dye, and live cell images were taken sequentially under a Nikon Ti Eclipse Confocal microscope. Cell lysates were collected for molecular biological analysis. Mitochondrial function was evaluated with Enzo Mito-ID membrane potential cytotoxicity kit. Target protein signals from cell lysate were detected by SDS-PAGE and western blot analysis. RESULTS: Mitochondrial morphology maintained the elongated linear shape in C2C12 cells when treated with 10% control rat serum. In contrast, when cells were treated with 10% burn serum, mitochondria reduced the elongated linear shape at 24 to 48 hours, and the florescent dye diffused at 72 hours. The cell florescent images showed an increase in circularity and fragmentation of mitochondria in C2C12 cells with burn serum stimulation. Meanwhile mitochondrial membrane potential decreased with 6hr post-burn serum stimulation. Western blot data showed that mitofusion-1 (Mfn1) significantly decreased in C2C12 cells with burn serum stimulation, confirming the observation of mitochondrial fission in response to burn serum. Cell death marker caspase 3 increased its expression in C2C12 cells with burn serum stimulation, suggesting a superfluous cell death in skeletal muscle after burn. CONCLUSION: Our results show an increase in the mitochondria fission/fusion ratio in C2C12 cells stimulated with burn serum isolated 6 hours after burn. The mechanism of mitochondrial fission with function impairment leading to muscle death is under investigation.Item Myokine Musclin Expression Is Elevated in Rats after Burn(2017-01-17) Maredia, Navin; Song, Juquan; Sehat, Alvand; Maxwell, Christian; Panwar, Kunal; Kulangara, Rohan; Carlson, Deborah; Huebinger, Ryan; Wolf, StevenINTRODUCTION: Annually, over 2 million people in the US experience severe burns, a condition marked by a hypercatabolic state with significant muscle loss. Muscle is necessary for glucose and lipid metabolism. Previous studies have shown detrimental effects of insulin resistance and hyperglycemia associated with muscle loss due to burns. Recently, the novel skeletal muscle myokine musclin has been found to regulate glucose in vitro. Thus, we attempted to better understand the effects of burns on musclin levels. We aimed to investigate the effects of burns on 1.) musclin levels systemically and 2.) musclin mRNA expression in vitro. METHODS: Thirty-one adult male Sprague-Dawley rats received 40% total body surface area (TBSA) burns. Rat serum was collected from 6 hours to 14 days after burn. Nine animals without injury served as control. Musclin levels in serum were measured by ELISA. Mouse C2C12 myoblasts were stimulated with 10% rat burn serum for 24 hours. Cells were stimulated with non-burn serum, 6-hour post burn serum, 72-hour post burn serum, and 14-day post burn serum. Following stimulation for 24 hours, C2C12 cells were collected and musclin expression was quantified by real time PCR analysis. RESULTS: Circulating musclin levels were 59.3 ± 3.3 ng/mL in non burned control rats. Musclin levels in the serum significantly increased to 76.7 ± 6.0 ng/mL at 6 hours and 76.7 ± 1.9 ng/mL at 24 hours after burn (p<0.05). Musclin levels in the serum returned to baseline until 14 days. Normalized to GAPDH mRNA level, musclin mRNA expression was 7.87 ± 0.56 fold in C2C12 myoblasts with 10% non-burn serum stimulation. Musclin mRNA expression significantly increased with the addition of the following burn rat serums: 22.66 ± 5.18 fold with 6-hour post-burn serum, 15.00 ± 1.93 fold with 72-hour post-burn serum, and 12.17 ± 0.82 fold with 14-day post-burn serum (p<0.05). CONCLUSIONS: Musclin levels increase in rat serum following 40% TBSA burn injury. In vitro stimulation of muscle cells with burn serum increases musclin expression.