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Skeletal Muscle Mitochondrial Function is Determined by Burn Severity, Sex, and Sepsis, and is Associated With Glucose Metabolism and Functional Capacity in Burned Children

Rontoyanni, Victoria G.*,†; Malagaris, Ioannis*,‡; Herndon, David N.*,†,§; Rivas, Eric; Capek, Karel D.*,†; Delgadillo, Anahi D.*,†; Bhattarai, Nisha*,†; Elizondo, Armando; Voigt, Charles D.*,†; Finnerty, Celeste C.*,†,§; Suman, Oscar E.*,†,‡; Porter, Craig*,†,‡

doi: 10.1097/SHK.0000000000001074
Clinical Science Aspects
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Background: Restoring normal mitochondrial function represents a new target for strategies aimed at mitigating the stress response to severe burn trauma and hastening recovery. Our objective was to investigate the determinants of skeletal muscle mitochondrial respiratory capacity and function and its association with glucose metabolism and functional capacity in burned children.

Methods: Data from burned children enrolled in the placebo arm of an ongoing prospective clinical trial were analyzed. Mitochondrial respiratory capacity was determined in permeabilized myofibers by high-resolution respirometry on at least one occasion per participant. In subsets of patients, glucose kinetics and cardiorespiratory fitness (VO2peak) were also determined. Mixed multiple regression models were used to identify the determinants of mitochondrial respiratory function and to assess the relationship between mitochondrial respiration and both glucose control and functional capacity (VO2peak).

Main results: Increasing full-thickness burn size was associated with greater adjusted coupled (ATP-producing) respiration, adjusted for age, sex, sepsis, and time of testing (P < 0.01; n = 55, obs = 97). Girls had on average 23.3% lower coupled respiration (adjusted mean and 95% confidence of interval [CI], −7.1; −12.6 to −1.7 pmol/s/mg; P < 0.025) and 29.8% lower respiratory control than boys (adjusted mean and 95% CI, −0.66; −1.07 to −0.25; P < 0.01; n = 55, obs = 97). The presence of sepsis was associated with lower respiration coupled to ATP production by an average of 25.5% compared with nonsepsis (adjusted mean and 95% CI, −6.9; −13.0 to −0.7 pmol/s/mg; P < 0.05; n = 55, obs = 97), after adjustment for age, sex, full-thickness burn size, and time of testing. During a hyperinsulinemic euglycemic clamp, hepatic glucose release was associated with greater coupled respiration and respiratory control (P < 0.05; n = 42, obs = 73), independent of age, sepsis, full-thickness burn size, and time postinjury testing. Coupled respiration was positively associated with VO2peak after adjustment for age, full-thickness burn size, and time of exercise testing (P < 0.025; n = 18, obs = 25).

Conclusions: Burn severity, sex, and sepsis influence skeletal muscle mitochondrial function in burned children. Glucose control and functional capacity are associated with altered mitochondrial respiratory function in muscle of burn survivors, highlighting the relationship of altered muscle bioenergetics with the clinical sequelae accompanying severe burn trauma.

*Metabolism Unit, Shriners Hospitals for Children, Galveston, Texas

Department of Surgery, University of Texas Medical Branch, Galveston, Texas

Rehabilitation Sciences, University of Texas Medical Branch, Galveston, Texas

§Institute for Translational Sciences, University of Texas Medical Branch, Galveston, Texas

School of Medicine, University of Texas Medical Branch, Galveston, Texas

Address reprint requests to Craig Porter, PhD, Department of Surgery, University of Texas Medical Branch, Shriners Hospitals for Children (Galveston), 815 Market Street, Room 617, Galveston, TX 77550. E-mail: cr2porte@utmb.edu

Received 11 October, 2017

Revised 30 October, 2017

Accepted 28 November, 2017

This work was supported by grants awarded from NIH (P50 GM060338, R01 GM056687, R01 HD049471, RO1 GM112936, 3R01HD049471-12S1, and T32 GM008256), NIDILRR (90DP00430100), and Shriners Hospitals for Children (80,100, 85,410, 84,080, 84,090, 71,000, 71,006, 71,008, and 71,009). This work was also supported by the Department of Surgery at UTMB, the Remembering the 15 Research Education Endowment Fund, and UTMB's Institute for Translational Sciences, which was supported in part by a Clinical and Translational Science Award (UL1TR001439) from the National Center for Advancing Translational Sciences (NIH).

The authors report no conflicts of interest to disclose.

Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com).

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INTRODUCTION

Burns are a leading cause of morbidity and the fifth most common cause of nonfatal pediatric injuries globally (1). In the United States, hospitalization costs for burned children exceed $211 million annually (2). Severe burns (>20% total body surface area [TBSA]) result in a profound adrenergic and hypermetabolic stress response that persists for over a year postinjury (3, 4). In the United States, individuals with burns covering up to half their body now have a mortality rate of less than 10%. Furthermore, one in every two individuals that suffer a burn on 90% of body surface can expect to survive (5). Improved mortality rates after severe burn trauma have meant that new strategies aimed at reducing morbidity and hastening recovery are needed.

Burn injuries are associated with long-term musculoskeletal morbidity (6). Skeletal muscle accounts for a significant portion (30%–40%) of total body mass and is endowed with a high mitochondrial volume density, which makes skeletal muscle a major determinant of resting metabolic rate in humans. Leg oxygen consumption can be doubled after burn trauma (7, 8), where altered skeletal muscle mitochondrial ATP production and thermogenesis has been observed (9, 10). Yet, how mitochondrial ATP production and thermogenesis relate to burn injury severity and burn complications, such as sepsis, remain unknown. Identification of clinically meaningful determinants of mitochondrial function may help identify patients that would benefit from targeted interventions. In addition, the acute and long-term clinical significance of altered mitochondrial function in response to burn trauma remains unclear.

Here, we set out to identify the determinants of skeletal muscle mitochondrial respiratory capacity and function in burn patients. Our hypothesis was that sepsis, injury severity, and time postinjury affect mitochondrial respiratory function and capacity in skeletal muscle after burns. Furthermore, we determined the relationship between skeletal muscle mitochondrial respiratory function and both glucose metabolism and cardiorespiratory fitness, to relate altered tissue bioenergetics with the clinical sequelae accompanying severe burn trauma.

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MATERIALS AND METHODS

Patients and standard burn care

This is a retrospective monocentric cohort study that was reviewed and approved by the Institutional Review Boards at the University of Texas Medical Branch and Shriners Hospitals for Children. Written informed consent was obtained from the parent or legal guardian of all participants before enrollment. To identify the determinants of skeletal muscle mitochondrial function in response to burns, we studied burned children (aged 0–18 years) admitted to Shriners Hospitals for Children—Galveston between July 2012 and September 2016 who were enrolled in the placebo arm of ongoing prospective clinical trials (NCT01957449, NCT00675714, NCT01574131, NCT02452255) and had at least one skeletal muscle biopsy collected for the determination of mitochondrial respiratory function during the acute hospitalization period before first hospital discharge. To determine the relationship between mitochondrial function and glucose metabolism, we studied burned children who had a skeletal muscle biopsy collected the same week as they underwent infusion of isotopically labeled glucose in the fasted state and during a hyperinsulinemic-euglycemic clamp. Finally, to determine the relationship between mitochondrial respiratory function and whole-body cardiorespiratory fitness, we studied burned children after their discharge from hospital who had undergone skeletal muscle biopsies and a cardiorespiratory fitness (VO2peak) test.

The inclusion criteria for all subanalyses included burns covering at least 20% of TBSA, age of 0 to 18 years, and more than 1 surgical procedure. Upon admission, patients received standard burn care, which included fluid resuscitation, full-thickness burn wound excision, and sequential skin grafting until burn wounds were closed. Patients rested in bed for 4 to 5 days after major surgical procedures. Patients received enteral nutrition upon admission and throughout acute hospitalization until they could consume sufficient nutrition ad libitum. Enteral nutrition consisted of 82% carbohydrates, 15% protein, and 3% fat (Vivonex T.E.N., Nestlé Health Science, Minneapolis, Minn) and was given at a rate of 1,500 kcal/m2 TBSA/d plus 1,500 kcal/m2 TBSA burned/d or at a caloric load equal to 1.4 times the measured resting energy expenditure.

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Patient demographics and clinical characteristics

Patient demographics and clinical characteristics were extracted from patient hospital charts. Age was defined as time from birth to study, and A2S was defined as time from admission to study. Burn-related characteristics and early comorbidities included indices of burn size, burn type, presence of sepsis, and presence of inhalation injury. TBSA-b was defined as the percent TBSA that was burned, serving as an index of burn size. TBSA-3rd was defined as the percent TBSA with full-thickness burns, serving as an index of full-thickness burn size. Burn type was categorized as flame (excluding electrical), electrical, or scald. Sepsis on a given day was defined as meeting at least three of the American Burn Association burn-specific 2007 sepsis criteria on at least 2 consecutive days as well as the presence of positive cultures (11). Inhalation injury was diagnosed by bronchoscopy.

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Muscle biopsy collection and preparation for mitochondrial bioenergetics

Skeletal muscle biopsy samples were obtained from the m. vastus lateralis under ketamine sedation and local anesthesia using a suction-adapted Bergström needle or from the lower limb muscles (thigh, calf, or in rare occasions, groin area) of patients during surgical procedures to excise and close burn wounds. The fresh muscle tissue sample (∼10–20 mg) was immediately immersed in an ice-cold preservation buffer (10 mM CaK2-EGTA, 0.1 μM free Ca2+, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM DTT, 6.56 mM MgCl2, 5.77 mM ATP, and 15 mM creatine phosphate; pH 7.1) and processed as previously described (9, 10, 12, 13). In short, muscle samples were gently separated into myofiber bundles using sharp forceps, and their sarcolemmal membranes were permeabilized in a sucrose buffer containing 5 μM saponin for 10 to 20 min at 4 °C. Next, 1 to 3 mg of muscle tissue was blotted on filter paper, weighed, and transferred to an Oxygraph-2K respirometer chamber (Oroboros Instruments, Innsbruck, Austria) containing 2 mL respiration buffer (0.5 mM EGTA, 3 mM MgCl2, 60 mM lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 10 mM sucrose, and 1 mg/mL bovine serum albumin) for high-resolution respirometry analysis.

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Mitochondrial respiratory function by high-resolution respirometry

Mitochondrial substrates and inhibitors were added sequentially to the respirometer chambers of the Oxygraph-2K to determine various respiratory states and mitochondrial coupling control, as previously described (9, 10).

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Mitochondrial respiratory states

State 2 (leak) respiration was determined after the addition of octanoyl-l-carnitine (1.5 mM), pyruvate (5 mM), malate (2 mM), and glutamate (10 mM) into the O2K chamber. State 3I respiration (coupled to ATP) supported by Complex I was assayed after the addition of adenosine diphosphate (ADP; 5 mM) into the O2K chamber. Maximal oxidative phosphorylation (State 3I+II respiration) with electron input through complexes I and II of the electron transport chain was assayed by adding 10 mM succinate into the O2K chamber. Finally, State 4O (leak) respiration was determined by adding 5 μM oligomycin, an ATP synthase inhibitor, into the O2K chamber.

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Calculated indices of mitochondrial function

The respiratory control ratio (RCR) for ADP, an index of mitochondrial coupling control and quality, was computed as the ratio of State-3I to State-2 respiration. The substrate control ratio (SCR) for succinate, an index of Complex II function, was computed as State 3I+II respiration/State 3I respiration. The coupling control ratio (CCR) for oligomycin was calculated as State 4O respiration/State 3I+II respiration. The CCR for oligomycin represents the proportion of respiration insensitive to oligomycin and thus uncoupled from ATP production.

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Stable isotope infusion of glucose at basal and hyperinsulinemic-euglycemic conditions

To assess glucose metabolism, we performed a hyperinsulinemic-euglycemic clamp, a well-characterized approach for determining glucose metabolism and insulin sensitivity (14). When combined with an isotopically labeled glucose infusion, the hyperinsulinemic-euglycemic clamp technique allows for determination of hepatic insulin sensitivity and whole-body (primarily skeletal muscle) glucose uptake (index of whole-body insulin sensitivity) (15). After a 6 to 8 h fast, a blood sample was collected to determine background glucose enrichment, followed by administration of a primed (20 μmol/kg), constant (0.44 μmol/kg/min) 4-h infusion of [6,6-2H2]-glucose (Cambridge Isotope Laboratories) through a catheter in a forearm vein to assess glucose kinetics. Blood samples were obtained every 10 min during the last 30 min of the first 2 h of the infusion period to determine glucose concentration and enrichment. During the next 2 h, the infusion of [6,6-2H2]-glucose was continued, and a hyperinsulinemic-euglycemic clamp (insulin infusion rate of 20 mU/m2/min) was performed as previously described (14). Insulin dissolved in sterile NaCl 0.9% (Lilly, Indianapolis, Ind) was administered at a rate of 1.5 mU/kg/min, which supresses hepatic glucose release in healthy humans but only marginally increases muscle glucose disposal (16). Glucose (20% dextrose) was infused to maintain blood glucose concentrations at fasting levels. Again, blood was sampled over the last 30 min of this 2-h period as described above. The rate of appearance (Ra) of glucose in the fasted state and during the clamp period was calculated from the tracer enrichment in blood and the tracer infusion rate. Glucose appearance rates were adjusted for unlabeled glucose infusion. In the fasted state, whole-body glucose Ra is an index of hepatic glucose release. In response to the clamp, hepatic glucose release can be calculated by subtracting the glucose infusion rate from the whole-body glucose Ra, where the suppression in glucose Ra with insulin and glucose infusion provides a measure of hepatic insulin sensitivity. In isotopic steady state conditions as those described here, whole-body Ra plus tracer infusion rate equals whole-body rate of disappearance (Rd; an index of whole-body glucose uptake) (17). Whole-body insulin-stimulated glucose uptake (i.e., the glucose metabolic clearance rate; an index of whole-body insulin sensitivity) was calculated as the ratio of Rd (in μmol/kg/min) to mean plasma glucose (in mg/dL) during the last 30 min of the 2-h clamp period (18).

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Cardiorespiratory fitness (VO2peak) test

Cardiorespiratory fitness (VO2peak), normalized to lean body mass (LBM), was examined using a modified Bruce protocol treadmill exercise test to volitional exhaustion. Briefly, this test started at a low walking intensity of 1.7 miles per hour (mph) at 0% elevation. During the following two 3-min stages, the grade was increased progressively (5% and 10% at 1.7 mph). Thereafter, the speed (2.5, 3.4, 4.2, and 5 mph) and elevation (2%) were increased every 3 min. Subjects were encouraged to complete each 3-min stage, and the test ended once peak volitional effort was achieved. Respiratory gasses were analyzed using breath-by-breath data on an automated MedGraphics Cardi O2 metabolic cart (St. Paul, Minn). Before each exercise test, O2 and CO2 gas and air-flow were calibrated using known gasses and a 3 L syringe. The test was determined to be maximal (or peak) once subjects signaled to stop exercise (volitional fatigue) and at least three of the following criteria were met and examined after the completion of the test: a respiratory exchange ratio (RER) of at least 1.05, a leveling off in VO2 with increasing workloads (less than 2 mL/kg/min), an exercise final HR of 190 bpm or greater, or a final test time from 8 to 15 min. All subjects met three of the aforementioned criteria. To control for growth and body composition differences between participants, VO2peak values were normalized to LBM. LBM was evaluated by the use of Dual-emission X-Ray Absorptiometry (Hologic QDR-4500W, Hologic Inc., Bedford, Mass) within 7 days of the VO2peak test.

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Statistical analysis

Descriptive statistics for continuous and categorical variables are presented as mean ± SD, and frequencies and proportions, respectively. The mitochondrial function outcomes tested were the mitochondrial respiratory capacities at different states (State 2 [leak], State 3I, maximal State 3I+II, State 4O) and the coupling control ratios (SCR, RCR, CCR). Mixed multiple regression models were used to assess the relationships between patient and injury characteristics, and mitochondrial respiration outcomes, and between mitochondrial respiratory states and VO2peak/LBM or glucose kinetics while blocking on participants to control for repeated observations. To adjust for varying time points of mitochondrial studies between burn patients, we included time to study in the models. Model selection was performed using the Bayesian Information Criteria (BIC), where a smaller BIC is indicative of a superior model. Likelihood ratio tests were used for pairwise model comparison. All analyses were conducted in SAS Studio (SAS University Edition, SAS Institute Inc., NC). Two-sided statistical significance was considered for alpha at the 0.05 level.

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RESULTS

Determinants of skeletal muscle mitochondrial function

A total of 97 biopsies obtained from 55 pediatric burn patients (69% male) were analyzed; patient descriptives are presented in Table 1. On average, biopsies were collected at 17 ± 11 days postburn, and patients were 8 ± 5 years old and had 49 ± 16% TBSA-b and 39 ± 21% TBSA-3rd. All muscle biopsies were collected from the lower limb area, with 88% from the thigh area, of which 96% were from m. vastus lateralis. Inhalation injury was diagnosed in 22% of the burn patients and presence of sepsis on 24% of the study days. Bioenergetics data from 3 of the 97 biopsies has been previously published in terms of absolute values and trends compared to a nonburn control group (10).

Table 1

Table 1

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Full-thickness burn size, sex, and sepsis: determinants of skeletal muscle mitochondrial function

Via an independent variable/model selection process (see Text Document, Supplemental Digital Content 1, http://links.lww.com/SHK/A682, which outlines the model selection process we conducted), the final models included five independent variables: age, sex, sepsis, TBSA-3rd, and A2S. The best-fit models for mitochondrial respiratory capacity and coupling control are presented in Tables 2 and 3.

Table 2

Table 2

Table 3

Table 3

Full-thickness burn size (indicated by TBSA-3rd) was significantly associated with State 3I respiration (P < 0.05) and maximal coupled respiration with electron provision supported by complex I and II of the electron transport chain (State 3I+II) (P < 0.01). As the full-thickness burn size increased by 1%, State 3I respiration and maximal phosphorylating State 3I+II respiration increased by 0.15 and 0.28 pmol/s/mg, respectively (P < 0.05 and P < 0.01; Fig. 1E), adjusted for age, sex, sepsis and A2S. Furthermore, increasing full-thickness burn size was met with lower contribution from uncoupled respiration as indicated by lower CCR for oligomycin (P < 0.01). Females exhibited lower State 3I respiration by an average of 23.3% (adjusted mean and 95% confidence interval [CI], −7.1; −12.6 to −1.7 pmol/s/mg; P < 0.025; Fig. 1C) and a lower mitochondrial respiratory control ratio (RCR) for ADP by 29.8% (adjusted mean and 95% CI, −0.66; −1.07 to −0.25; P < 0.01; Fig. 1D) compared with males, after adjusting for age, TBSA-3rd, sepsis and A2S. Finally, presence of sepsis was associated with lower respiration in the leak state (State 2) supported by substrates (in absence of ADP) and lower respiration coupled to ATP production supported by complex I of the electron transport chain (State 3I), adjusted for age, sex, TBSA-3rd and A2S. State 2 and State 3I respiration was lower on average by 21.1% (adjusted mean and 95% CI, −3.2; −5.9 to −0.5 pmol/s/mg) and 25.5% (adjusted mean and 95% CI, −6.9; −13.0 to −0.7 pmol/s/mg) in septic than nonseptic patients (P < 0.05, Fig. 1A). In addition, sepsis was associated with a 23.9% greater coupling control ratio (CCR) for oligomycin (adjusted mean and 95% CI, 0.09; 0.02 to 0.17; P < 0.025, Fig. 1B). There were no other significant relationships between patient and clinical characteristics and respiratory states.

Fig. 1

Fig. 1

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RELATIONSHIP BETWEEN SKELETAL MUSCLE MITOCHONDRIAL FUNCTION, GLUCOSE METABOLISM, AND FUNCTIONAL CAPACITY AFTER SEVERE BURN TRAUMA

Skeletal muscle mitochondrial function is associated with glucose metabolism

A total of 82 stable isotope infusion and skeletal muscle mitochondrial function studies (80% on same day) from 42 pediatric burn patients (84% male) were analyzed. Of those 82 studies, there were missing values for state 4O respiration data on 3 muscle biopsies, for sepsis on 2 study days, and data were unavailable for the hyperinsulinemic portion of the clamp due to technical difficulties on 7 occasions. On average, patients were 8 ± 5 years old and had 49 ± 16% TBSA-b and 42 ± 21% TBSA-3rd. Patients were identified as being septic on 16% of the biopsy days. Of the 82 muscle biopsies collected, 78 were from the thigh (m. vastus lateralis), 3 from the calf, and 1 from the groin area. Mean fasting glucose was 93 ± 14 mg/dL. During the hyperinsulinemic euglycemic experimental conditions, glucose was clamped on average at 94 ± 18 mg/dL, which was achieved by a mean glucose infusion rate of 8.6 ± 3.9 mg/kg/min. In the fasted state, endogenous glucose Ra (hepatic glucose release) averaged 29.1 ± 9.1 μmol/kg/min and whole-body glucose Rd (an index of glucose uptake) was 29.5 ± 9.1 μmol/kg/min. During hyperinsulinemia, endogenous glucose Ra fell to an average of 6.0 ± 7.0 μmol/kg/min via hepatic glucose suppression of 81 ± 20%. During the clamp, glucose Rd (an index of glucose uptake) was 54.1 ± 24.2 μmol/kg/min (or 9.7 ± 4.4 mg/kg/min).

The relationships between glucose kinetics and mitochondrial function were adjusted for the confounding effects of age, TBSA-3rd, sepsis and A2S (sex was not a significant predictor). When fasted, decreasing State 4O (leak) respiration was associated with greater hepatic glucose release (P < 0.025; 79 studies in 42 patients). During a hyperinsulinemic euglycemic clamp, both coupled State 3I respiration (Fig. 2A) and the RCR for ADP were positively associated with hepatic glucose release (P < 0.05; 73 studies in 42 patients). In both the fasted state and during the clamp, higher TBSA-3rd values (P < 0.05) and younger age (P < 0.01) were related to greater hepatic glucose release. Suppression of hepatic glucose release during the clamp was negatively associated with State 3I respiration (beta = −0.003%, P < 0.05). During the clamp, lower State 4O respiration (P < 0.05; 75 studies in 42 patients), higher RCR (P < 0.025; 76 studies in 42 patients), and younger age (P < 0.001) were associated with greater whole-body glucose uptake. By correcting whole-body glucose uptake rate for steady-state plasma glucose concentration (whole-body insulin sensitivity index), the positive association with RCR remained significant (beta = 0.05 μmol/kg/min (mg/dL), P = 0.025; 76 studies in 42 patients; Fig. 2B). The presence of sepsis was significantly associated with lower whole-body insulin sensitivity (P < 0.01) and was included in all the regression models. No other significant relationships were identified.

Fig. 2

Fig. 2

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Skeletal muscle mitochondrial function is associated with cardiorespiratory fitness

A total of 25 biopsies obtained from 18 pediatric burn patients (72% male) were analyzed. On average, patients were 13 ± 3 years old and had 45 ± 14% TBSA-b and 35 ± 19% TBSA-3rd. Over the 25 observations, VO2peak averaged 35.6 ± 9.1 mLO2/kg LBM/min. Of the 25 muscle biopsies, 23 were from the thigh area (m. vastus lateralis), one from the calf and one from the groin area.

VO2peak was positively associated with age and negatively associated with TBSA-3rd (P < 0.05). Coupled State 3I and State 3I+II respiration (ATP-producing states) were positively associated with VO2peak per kg of LBM after adjustment for age, TBSA-3rd and time of exercise testing (beta = 0.29 mlO2/kgLBM/min, P < 0.025; Fig. 2C). Sepsis was identified in 2/25 biopsy days and hence, was not accounted for in the aforementioned regression models.

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DISCUSSION

To our knowledge, this is the first time the determinants of skeletal muscle mitochondrial respiratory capacity and function have been identified in severely burned patients. Furthermore, we provide novel data underscoring the relevance of altered muscle bioenergetics with clinical markers in response to burn trauma. Prior research has implicated burn size in the severity of the hypermetabolic stress response to burn trauma (19, 20). Here, we extend these findings to describe associations between burn and patient characteristics with skeletal muscle physiology at the mitochondrial level. Our primary finding is that sepsis, full-thickness burn size and sex are significant predictors of skeletal muscle mitochondrial respiratory function in burned children. Furthermore, we demonstrate that mitochondrial respiration in skeletal muscle is associated with glucose metabolism and cardiorespiratory fitness (VO2peak), which supports the consideration of mitochondrial respiratory function as a therapeutic target in burn care.

Controlling burn-induced hypermetabolism is an important aspect of acute burn care. Skeletal muscle facilitates significant metabolic functions in response to severe burns, including wound healing and production of acute phase proteins, which partly explain the hypermetabolic stress response (3, 21). In addition, catabolism of skeletal muscle may supply the liver with substrates for gluconeogenesis, predominantly alanine. Indeed, the heightened rate of alanine release from protein breakdown in burned patients appears to return to approximately normal levels after infusion of high glucose dose in the post-absorptive state, underscoring a role for skeletal muscle catabolism in gluconeogenesis after burn trauma (22). Furthermore, evidence from our laboratory suggests a role for increased skeletal muscle mitochondrial thermogenesis in burn-induced hypermetabolism (9, 10). Thus, the mitochondrion may also represent a novel target for new therapeutic strategies aimed at blunting hypermetabolism after burn trauma.

In the present study, we identified the patient and clinical characteristics that influence skeletal muscle mitochondrial respiratory function in response to severe burn trauma. We demonstrated that full-thickness burn size is an independent predictor of maximal oxidative phosphorylation (ATP production), with greater full-thickness burn area being associated with higher coupled respiration supported by complexes I and II of the electron transport chain, after adjusting for age, sex, sepsis, and time postinjury. Given the positive association between total burn size and the degree of hypermetabolism (19, 20), our current findings provide mechanistic insight to this relationship, where greater burn size is associated with greater hypermetabolism at the level of skeletal muscle.

In addition to burn injury severity, we found that sex was an independent determinant of skeletal muscle mitochondrial respiratory function in burned children, after adjustment for age, sepsis, injury severity, and time post-injury. Girls exhibited a 23% to 30% lower capacity for respiration coupled to ATP production than boys. Although no surrogate markers of mitochondrial abundance were quantified in the present study, high-resolution respirometry allows for the sequential addition of saturating levels of substrates and inhibitors into a chamber containing the same myofiber preparation. This allows for determination of coupling control ratios, which reflect respiratory responses to substrates and inhibitors that are independent of mitochondrial density, therefore providing a measure of mitochondrial quality (23, 24). Girls exhibited lower coupling control in response to ADP, further suggesting a lower capacity for ATP production in females compared to males. Previous studies in healthy young adults suggest that skeletal muscle oxidative capacity is lower in females than males (as indicated by greater cytochrome c oxidase enzyme activity) (25). However, other studies of healthy young adults found no significant sex differences in mitochondrial respiration (26, 27). To the best of our knowledge, these current data provide the first evidence that sex may influence skeletal muscle bioenergetics in children. Attenuated respiratory control of muscle mitochondria may support lower ATP production in skeletal muscle protein turnover in girls, and thus may account for the observation that girls are less hypermetabolic than boys after severe burn trauma. This finding is in line with a previous study demonstrating that metabolic rate and muscle protein turnover are lower in severely burned girls compared to boys (28). Indeed, there is evidence implicating sex steroid hormones in the regulation of mitochondrial biogenesis and function. Estrogens appear to stimulate mitochondrial biogenesis and oxidative capacity in skeletal muscle of rodents (29, 30). Furthermore, testosterone may contribute to mitochondrial biogenesis in rodent skeletal muscle (31). Additionally, a significant correlation between testosterone levels and oxidative phosphorylation gene expression has been identified in older men (32). However, a small interventional study in aging men with subnormal testosterone levels that were treated with transdermal testosterone for 6 months did not confirm a role for testosterone in muscle bioenergetics (33).

Sepsis was identified as a significant predictor of mitochondrial respiratory function in burned children during their acute hospitalization. In prior studies, mitochondrial responses to sepsis vary depending on the research model used (animal, human endotoxin model, septic patients) and time-phase postinfection (34–36). At the very onset of sepsis, mitochondrial activity may be increased (34), whereas in the early phase of acute septic shock or later phase of sepsis-induced multiple organ failure a decrease in mitochondrial function has been reported (35). Here we show for the first time that sepsis in severely burned patients is associated with impaired skeletal muscle mitochondrial respiratory function.

We examined the association of altered mitochondrial function in severely burned patients with glucose metabolism in the fasting state and in response to a hyperinsulinemic euglycemic clamp. Severe burn injury is characterized by increased fasting hepatic glucose release, impaired suppression of hepatic glucose release in response to insulin (indicating blunted hepatic insulin sensitivity) and decreased insulin-stimulated whole-body glucose uptake (37–39). Here, we show that during hyperinsulinemia, a higher glucose clearance rate was associated with greater mitochondrial respiratory control for ADP, which suggests that greater whole-body glucose uptake is associated with larger capacity for ATP production in skeletal muscle of severely burned patients. Furthermore, as burn injury size increases, hepatic insulin sensitivity worsens as indicated by greater glucose production from the liver. Interestingly, during hyperinsulinemia, higher glucose release from the liver and lower hepatic insulin sensitivity were associated with greater mitochondrial respiration linked to ATP production in muscle, after adjusting for age, sepsis, injury severity and time postinjury. Whether this relationship is driven by the fact that both hepatic insulin resistance and muscle hypermetabolism are related to burn severity, or whether greater glucose release from the liver helps to fuel muscle ATP turnover, are interesting questions for future studies to address.

Cardiorespiratory fitness is recognized by the American Heart Association and the American College of Sport Medicine, among other health agencies, as an established determinant of cardiovascular health and all-cause mortality (40). Although peak oxygen consumption is predominantly determined by the oxygen transport capacity (41), it is also related to skeletal muscle mitochondrial function. In healthy young and older adults, there is a significant relationship between mitochondrial respiratory capacity and cardiorespiratory fitness (42, 43). Here, we extend these findings to patients with severe burns by showing a significant association between coupled (ATP producing) respiration and cardiorespiratory fitness, after adjusting for age, injury severity, and time postinjury. Thus, altered skeletal muscle bioenergetics are related to cardiorespiratory fitness in burn survivors, indication that skeletal muscle mitochondria are associated with functional capacity and thus long-term morbidity postburn. Subsequently, strategies that promote recovery of mitochondrial function in muscle of burned children may hasten restoration of cardiorespiratory fitness.

Our study has certain limitations. Given the observational nature of our data, no causality can be inferred. We also note that high-resolution respirometry, whereas a robust tool for in situ determination of mitochondrial bioenergetics in muscle fiber bundles (44, 45), uses supraphysiological O2 tensions and substrate concentrations and thus, quantifies maximal mitochondrial respiratory capacity. Yet, the associations presented here of skeletal muscle mitochondrial respiratory capacity with glucose control and cardiorespiratory fitness in burned children suggest that maximal coupled and uncoupled respiration is relevant to clinical markers of insulin sensitivity and functional capacity.

In summary, these data shed new light on the role of the mitochondrion in the pathophysiological response to severe burn trauma, where burn injury severity, sex, and sepsis are determinants of mitochondrial respiratory capacity and function in skeletal muscle of burned children. Therefore, injury severity, sex, and sepsis may need to be considered when devising treatment and rehabilitative strategies to address the hypermetabolic catabolic stress response to burn trauma. Furthermore, we provide novel evidence underscoring the clinical relevance of altered skeletal muscle mitochondrial function in response to severe burn injury. In particular, glucose control and functional capacity are associated with skeletal muscle mitochondrial function after severe burns, suggesting that strategies aimed at restoring skeletal muscle mitochondrial function after burn trauma may also improve the insulin sensitivity and functional capacity of burn survivors.

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Acknowledgments

The authors thank the clinical research staff at Shriners Hospitals for Children—Galveston for their assistance in recruiting and studying patients. We also thank the research staff in the Metabolism Unit at Shriners Hospitals for Children—Galveston for help in sample preparation and analysis. We thank Dr. Ronald Mlcak for providing information on the diagnosis of inhalation injury. Finally, we thank Dr. Kasie Cole for proofreading this manuscript.

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REFERENCES

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Keywords:

Burn injury; cardiorespiratory fitness; critically ill; insulin sensitivity; mitochondria; oxidative phosphorylation

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