A randomized controlled trial to determine whether beta-hydroxy-beta-methylbutyrate and/or eicosapentaenoic acid improves diaphragm and quadriceps strength in critically Ill mechanically ventilated patients.

BACKGROUND: Intensive care unit acquired weakness is a serious problem, contributing to respiratory failure and reductions in ambulation. Currently, there is no pharmacological therapy for this condition. Studies indicate, however, that both beta-hydroxy-beta-methylbutyrate (HMB) and eicosapentaenoic acid (EPA) increase muscle function in patients with cancer and in older adults. The purpose of this study was to determine whether HMB and/or EPA administration would increase diaphragm and quadriceps strength in mechanically ventilated patients. METHODS: Studies were performed on 83 mechanically ventilated patients who were recruited from the Medical Intensive Care Units at the University of Kentucky. Diaphragm strength was assessed as the trans-diaphragmatic pressure generated by supramaximal magnetic phrenic nerve stimulation (PdiTw). Quadriceps strength was assessed as leg force generated by supramaximal magnetic femoral nerve stimulation (QuadTw). Diaphragm and quadriceps thickness were assessed by ultrasound. Baseline measurements of muscle strength and size were performed, and patients were then randomized to one of four treatment groups (placebo, HMB 3 gm/day, EPA 2 gm/day and HMB plus EPA). Strength and size measurements were repeated 11 days after study entry. ANCOVA statistical testing was used to compare variables across the four experimental groups. RESULTS: Treatments failed to increase the strength and thickness of either the diaphragm or quadriceps when compared to placebo. In addition, treatments also failed to decrease the duration of mechanical ventilation after study entry. CONCLUSIONS: These results indicate that a 10-day course of HMB and/or EPA does not improve skeletal muscle strength in critically ill mechanically ventilated patients. These findings also confirm previous reports that diaphragm and leg strength in these patients are profoundly low. Additional studies will be needed to examine the effects of other anabolic agents and innovative forms of physical therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01270516. Registered 5 January 2011, https://clinicaltrials.gov/ct2/show/NCT01270516?term=Supinski&draw=2&rank=4 .

MitoTEMPOL, a mitochondrial targeted antioxidant, prevents sepsis-induced diaphragm dysfunction.

Clinical studies indicate that sepsis-induced diaphragm dysfunction is a major contributor to respiratory failure in mechanically ventilated patients. Currently there is no drug to treat this form of diaphragm weakness. Sepsis-induced muscle dysfunction is thought to be triggered by excessive mitochondrial free radical generation; we therefore hypothesized that therapies that target mitochondrial free radical production may prevent sepsis-induced diaphragm weakness. The present study determined whether MitoTEMPOL, a mitochondrially targeted free radical scavenger, could reduce sepsis-induced diaphragm dysfunction. Using an animal model of sepsis, we compared four groups of mice: 1) sham-operated controls, 2) animals with sepsis induced by cecal ligation puncture (CLP), 3) sham controls given MitoTEMPOL (10 mg·kg-1·day-1 ip), and 4) CLP animals given MitoTEMPOL. At 48 h after surgery, we measured diaphragm force generation, mitochondrial function, proteolytic enzyme activities, and myosin heavy chain (MHC) content. We also examined the effects of delayed administration of MitoTEMPOL (by 6 h) on CLP-induced diaphragm weakness. The effects of MitoTEMPOL on cytokine-mediated alterations on muscle cell superoxide generation and cell size in vitro were also assessed. Sepsis markedly reduced diaphragm force generation. Both immediate and delayed MitoTEMPOL administration prevented sepsis-induced diaphragm weakness. MitoTEMPOL reversed sepsis-mediated reductions in mitochondrial function, activation of proteolytic pathways, and decreases in MHC content. Cytokines increased muscle cell superoxide generation and decreased cell size, effects that were ablated by MitoTEMPOL. MitoTEMPOL and other compounds that target mitochondrial free radical generation may be useful therapies for sepsis-induced diaphragm weakness.

Does Standard Physical Therapy Increase Quadriceps Strength in Chronically Ventilated Patients? A Pilot Study.

OBJECTIVES: Physical therapy is standard care for mechanically ventilated patients, but there is no evidence, using nonvolitional, objective measurements, that physical therapy increases muscle strength in this population. The present study tested the hypothesis that 2 weeks of standard, conventional physical therapy provided at a ventilator weaning facility would increase quadriceps strength in mechanically ventilated patients. DESIGN: Prospective observational study. SETTING: Ventilator weaning unit. PATIENTS: Patients who were transferred from an acute care hospital because of failure to wean from mechanical ventilation and who were receiving physical therapy as prescribed by facility staff. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We employed a novel, nonvolitional objective technique, quadriceps twitch force generation in response to femoral nerve magnetic stimulation, to assess leg strength before and after 2 weeks of conventional physical therapy. The duration and specific exercises provided to patients were also recorded. In a subset of patients, we measured muscle activation intensity using wireless electromyogram recordings. Indices of respiratory function (maximum inspiratory pressure generation and the rapid shallow breathing index) were also assessed. Patients' responses to 2 weeks of physical therapy were poor; on average, quadriceps twitch fell by -1.02 ± 0.71 Newtons. Neither physical therapy duration nor specific forms of exercise were identified to positively impact quadriceps twitch. Electromyogram recordings indicated that during training, muscle activation was poor. Consequently, therapists spent substantial time performing exercises that elicited little muscle activation. Physical therapy did not improve respiratory function. CONCLUSIONS: Standard physical therapy delivered in a ventilator weaning facility failed to improve quadriceps leg strength in a majority of mechanically ventilated patients. The fact that mechanically ventilated patients fail to achieve high levels of muscle activation during physical therapy provides a potential explanation as to why physical therapy may often be ineffective. We speculate that use of novel methods which increase muscle activation during exercise may improve responses of mechanically ventilated patients to physical therapy.

Increased myocardial short-range forces in a rodent model of diabetes reflect elevated content of ß myosin heavy chain.

Diastolic dysfunction is a clinically significant problem for patients with diabetes and often reflects increased ventricular stiffness. Attached cross-bridges contribute to myocardial stiffness and produce short-range forces, but it is not yet known whether these forces are altered in diabetes. In this study, we tested the hypothesis that cross-bridge-based short-range forces are increased in the streptozotocin (STZ) induced rat model of type 1 diabetes. Chemically permeabilized myocardial preparations were obtained from 12week old rats that had been injected with STZ or vehicle 4weeks earlier, and activated in solutions with pCa (=-log10[Ca(2+)]) values ranging from 9.0 to 4.5. The short-range forces elicited by controlled length changes were ∼67% greater in the samples from the diabetic rats than in the control preparations. This change was mostly due to an increased elastic limit (the length change at the peak short-range force) as opposed to increased passive muscle stiffness. The STZ-induced increase in short-ranges forces is thus unlikely to reflect changes to titin and/or collagen filaments. Gel electrophoresis showed that STZ increased the relative expression of ß myosin heavy chain. This molecular mechanism can explain the increased short-ranges forces observed in the diabetic tissue if ß myosin molecules remain bound between the filaments for longer durations than α molecules during imposed movements. These results suggest that interventions that decrease myosin attachment times may be useful treatments for diastolic dysfunction associated with diabetes.

Diaphragm weakness in mechanically ventilated critically ill patients.

INTRODUCTION: Studies indicate that mechanically ventilated patients develop significant diaphragm muscle weakness, but the etiology of weakness and its clinical impact remain incompletely understood. We assessed diaphragm strength in mechanically ventilated medical ICU patients, correlated the development of diaphragm weakness with multiple clinical parameters, and examined the relationship between the level of diaphragm weakness and patient outcomes. METHODS: Transdiaphragmatic twitch pressure (PdiTw) in response to bilateral magnetic stimulation of the phrenic nerves was measured. Diaphragm weakness was correlated with the presence of infection, blood urea nitrogen, albumin, and glucose levels. The relationship of diaphragm strength to patient outcomes, including mortality and the duration of mechanical ventilation for successfully weaned patients, was also assessed. RESULTS: We found that infection is a major risk factor for diaphragm weakness in mechanically ventilated medical ICU patients. Outcomes for patients with severe diaphragm weakness (PdiTw<10 cmH2O) were poor, with a markedly increased mortality (49%) compared to patients with PdiTw≥10 cmH2O (7% mortality, P=0.022). In addition, survivors with PdiTw<10 cmH2O required a significantly longer duration of mechanical ventilation (12.3±1.7 days) than those with PdiTw≥10 cmH2O (5.5±2.0 days, P=0.016). CONCLUSIONS: Infection is a major cause of severe diaphragm weakness in mechanically ventilated patients. Moreover, diaphragm weakness is an important determinant of poor outcomes in this patient population.

Magnetic twitch assessment of diaphragm and quadriceps weakness in critically ill mechanically ventilated patients.

Critically ill mechanically ventilated (MV) patients develop significant muscle weakness, which has major clinical consequences. There remains uncertainty, however, regarding the severity of leg weakness, the precise relationship between muscle strength and thickness, and the risk factors for weakness in MV patients. We therefore measured both diaphragm (PdiTw) and quadriceps (QuadTw) strength in MV patients using magnetic stimulation and compared strength to muscle thickness. Both PdiTw and QuadTw were profoundly reduced for MV patients, with PdiTw 19 % of normal and QuadTw 6% of normal values. There was a poor correlation between strength and thickness for both muscles, with thickness often remaining in the normal range when strength was severely reduced. Regression analysis revealed reductions in PdiTw correlated with presence of infection (p = 0.006) and age (p = 0.007). QuadTw best correlated with duration of MV (p = 0.036). Limb muscles are profoundly weak in critically ill patients, with a severity that mirrors the level of weakness observed in the diaphragm.

Doxorubicin causes diaphragm weakness in murine models of cancer chemotherapy.

Doxorubicin is a chemotherapeutic agent prescribed for a variety of tumors. While undergoing treatment, patients exhibit frequent symptoms that suggest respiratory muscle weakness. Cancer patients can receive doxorubicin chemotherapy through either intravenous (IV) or intraperitoneal (IP) injections. We hypothesized that respiratory muscle function would be depressed in a murine model of chemotherapy. We tested this hypothesis by treating C57BL/6 mice with a clinical dose of doxorubicin (20 mg/kg) via IV or IP injection. Three days later we measured contractile properties of muscle fiber bundles isolated from the diaphragm. Doxorubicin consistently depressed diaphragm force with both methods of administration (P < 0.01). Doxorubicin IP exaggerated the depression in diaphragm force and stimulated tissue inflammation and muscle fiber injury. These results suggest that clinically relevant doses of doxorubicin cause respiratory muscle weakness and that the loss of function depends, in part, on the route of administration.

Skeletal muscle-specific calpastatin overexpression mitigates muscle weakness in aging and extends life span.

Calpain activation has been postulated as a potential contributor to the loss of muscle mass and function associated with both aging and disease, but limitations of previous experimental approaches have failed to completely examine this issue. We hypothesized that mice overexpressing calpastatin (CalpOX), an endogenous inhibitor of calpain, solely in skeletal muscle would show an amelioration of the aging muscle phenotype. We assessed four groups of mice (age in months): 1) young wild type (WT; 5.71 ± 0.43), 2) young CalpOX (5.6 ± 0.5), 3) old WT (25.81 ± 0.56), and 4) old CalpOX (25.91 ± 0.60) for diaphragm and limb muscle (extensor digitorum longus, EDL) force frequency relations. Aging significantly reduced diaphragm and EDL peak force in old WT mice, and decreased the force-time integral during a fatiguing protocol by 48% and 23% in aged WT diaphragm and EDL, respectively. In contrast, we found that CalpOX mice had significantly increased diaphragm and EDL peak force in old mice, similar to that observed in young mice. The impact of aging on the force-time integral during a fatiguing protocol was abolished in the diaphragm and EDL of old CalpOX animals. Surprisingly, we found that CalpOX had a significant impact on longevity, increasing median survival from 20.55 mo in WT mice to 24 mo in CalpOX mice (P = 0.0006).NEW & NOTEWORTHY This is the first study to investigate the role of calpastatin overexpression on skeletal muscle specific force in aging rodents. Muscle-specific overexpression of calpastatin, the endogenous calpain inhibitor, prevented aging-induced reductions in both EDL and diaphragm specific force and, remarkably, increased life span. These data suggest that diaphragm dysfunction in aging may be a major factor in determining longevity. Targeting the calpain/calpastatin pathway may elucidate novel therapies to combat skeletal muscle weakness in aging.

Mitoquinone mesylate (MitoQ) prevents sepsis-induced diaphragm dysfunction.

Sepsis-induced diaphragm dysfunction is a major contributor to respiratory failure in mechanically ventilated patients. There are no pharmacological treatments for this syndrome, but studies suggest that diaphragm weakness is linked to mitochondrial free radical generation. We hypothesized that administration of mitoquinone mesylate (MitoQ), a mitochondrially targeted free radical scavenger, would prevent sepsis-induced diaphragm dysfunction. We compared diaphragm function in 4 groups of male mice: 1) sham-operated controls treated with saline (0.3 mL ip), 2) sham-operated treated with MitoQ (3.5 mg/kg/day given intraperitoneally in saline), 3) cecal ligation puncture (CLP) mice treated with saline, and 4) CLP mice treated with MitoQ. Forty-eight hours after surgery, we assessed diaphragm force generation, myosin heavy chain content, state 3 mitochondrial oxygen consumption (OCR), and aconitase activity. We also determined effects of MitoQ in female mice with CLP sepsis and in mice with endotoxin-induced sepsis. CLP decreased diaphragm specific force generation and MitoQ prevented these decrements (e.g. maximal force averaged 30.2 ± 1.3, 28.0 ± 1.3, 12.8 ± 1.9, and 30.0 ± 1.0 N/cm2 for sham, sham + MitoQ, CLP, and CLP + MitoQ groups, respectively, P < 0.001). CLP also reduced diaphragm mitochondrial OCR and aconitase activity; MitoQ blocked both effects. Similar responses were observed in female mice and in endotoxin-induced sepsis. Moreover, delayed MitoQ treatment (by 6 h) was as effective as immediate treatment. These data indicate that MitoQ prevents sepsis-induced diaphragm dysfunction, preserving force generation. MitoQ may be a useful therapeutic agent to preserve diaphragm function in critically ill patients with sepsis.NEW & NOTEWORTHY This is the first study to show that mitoquinone mesylate (MitoQ), a mitochondrially targeted antioxidant, treats sepsis-induced skeletal muscle dysfunction. This biopharmaceutical agent is without known side effects and is currently being used by healthy individuals and in clinical trials in patients with various diseases. When taken together, our results suggest that MitoQ has the potential to be immediately translated into treatment for sepsis-induced skeletal muscle dysfunction.

Calpain activation contributes to endotoxin-induced diaphragmatic dysfunction.

Calpain activation occurs in skeletal muscle in response to infection, but it is unknown if calpain inhibition improves muscle functional capacity. We hypothesized that infection induces diaphragm calpain activation, that calpain activation results in cleavage of important diaphragm cytoskeletal proteins, and that inhibition of calpain attenuates infection-induced diaphragm dysfunction. Mice (n = 4-6/group) were given: (1) saline (intraperitoneal); (2) endotoxin (12 mg/kg intraperitoneal); (3) calpain inhibitor peptide III (12 mg/kg intraperitoneal); and (4) endotoxin (12 mg/kg) plus calpain inhibitor peptide III (12 mg/kg). At 24 hours, diaphragms were removed and the following determined: (1) calpain activity by fluorogenic assay; (2) calpain I and II protein levels; (3) talin protein levels; and (4) the force-frequency relationship. Endotoxin significantly increased diaphragm calpain activity (P < 0.001), active calpain I protein (P < 0.001), active calpain II protein (P < 0.01), levels of a calpain-specific cleavage talin degradation product (P < 0.003), and reduced diaphragm force (P < 0.001). Calpain inhibitor III administration prevented endotoxin-induced increases in calpain activity, reduced talin degradation, and attenuated reductions in diaphragm force. Diaphragm-specific force at 150 Hz stimulation was significantly higher in control, endotoxin plus calpain inhibitor III, and calpain inhibitor III alone groups (23 +/- 1, 20 +/- 1 and 23 +/- 1 N/cm(2), respectively) than in the endotoxin alone group (15 +/- 1 N/cm(2)) (P < 0.01). This model of sepsis results in significant diaphragm calpain activation and calpain-dependent diaphragm cytoskeletal protein cleavage. Moreover, calpain inhibition attenuates endotoxin-induced diaphragm weakness, suggesting that such inhibitors may be a potential treatment to improve respiratory muscle function in infected patients.

p38 Mitogen-activated protein kinase modulates endotoxin-induced diaphragm caspase activation.

We postulated that the p38 pathway is activated in the diaphragm during sepsis and contributes to sepsis-induced diaphragm caspase activation and contractile dysfunction. This study determined whether: (1) endotoxin administration elicits p38 activation in the diaphragm; (2) cytokines activate p38 in isolated muscle cells; (3) activation of p38 is accompanied by caspase 8 activation; (4) inhibition of p38 prevents caspase 8 activation and; (5) inhibition of p38 prevents diaphragm dysfunction in endotoxin-treated animals. We first evaluated the time course of diaphragm p38 activation after endotoxin in mice. We then determined if p38 inhibitor administration could prevent caspase 8 activation in endotoxin-treated mice. We also assessed p38 and caspase 8 activation in C2C12 muscle cells treated with control media or a cytokine mixture, with or without concomitant chemical inhibition of p38 (using SB203580, 25 muM) or loss of p38 function due to cell transfection with a dominant negative p38 genetic construct. Endotoxin administration activated diaphragm p38 (P < 0.001), and cytokines activated p38 in C2C12 cells (P < 0.05). In both the diaphragm and cells, p38 activation was accompanied by increases in active caspase 8 (P < 0.01). Inhibition of p38 with either SB203580 or with a dominant negative p38 construct prevented caspase activation (P < 0.001). p38 inhibitors also prevented endotoxin-induced diaphragm weakness (P < 0.001). p38 modulates cytokine-induced skeletal muscle caspase activation.

Diaphragm weakness and mechanical ventilation--what's the critical issue?

While animal studies indicate that controlled mechanical ventilation (MV) induces diaphragm weakness and myofiber atrophy, there are no data in humans that confirm MV per se produces diaphragm weakness. Whether or not diaphragm weakness results from MV, sepsis, corticosteroids, hyperglycemia, or a combination of these factors, however, is not the most important issue raised by the recent study from Hermans and colleagues. This study makes an important contribution by providing additional evidence that many critically ill patients have profound diaphragm weakness. If diaphragm weakness of this magnitude is present in most mechanically ventilated patients, a strong argument can be made that respiratory muscle weakness is a major contributor to respiratory failure.

Eicosapentaenoic acid preserves diaphragm force generation following endotoxin administration.

INTRODUCTION: Infections produce severe respiratory muscle weakness, which contributes to the development of respiratory failure. An effective, safe therapy to prevent respiratory muscle dysfunction in infected patients has not been defined. This study examined the effect of eicosapentaenoic acid (EPA), an immunomodulator that can be safely administered to patients, on diaphragm force generation following endotoxin administration. METHODS: Rats were administered the following (n = 5/group): (a) saline, (b) endotoxin, 12 mg/kg IP, (c) endotoxin + EPA (1.0 g/kg/d), and (d) EPA alone. Diaphragms were removed and measurements made of the diaphragm force-frequency curve, calpain activation, caspase activation, and protein carbonyl levels. RESULTS: Endotoxin elicited large reductions in diaphragm specific force generation (P < 0.001), and increased diaphragm caspase activation (P < 0.01), calpain activation (P < 0.001) and protein carbonyl levels (P < 0.01). EPA administration attenuated endotoxin-induced reductions in diaphragm specific force, with maximum specific force levels of 27 +/- 1, 14 +/- 1, 23 +/- 1, and 24 +/- 1 N/cm2, respectively, for control, endotoxin, endotoxin + EPA, and EPA treated groups (P < 0.001). EPA did not prevent endotoxin induced caspase activation or protein carbonyl formation but significantly reduced calpain activation (P < 0.02). CONCLUSIONS: These data indicate that endotoxin-induced reductions in diaphragm specific force generation can be partially prevented by administration of EPA, a nontoxic biopharmaceutical that can be safely given to patients. We speculate that it may be possible to reduce infection-induced skeletal muscle weakness in critically ill patients by administration of EPA.

Mitochondria and Critical Illness.

Classically, mitochondria have largely been believed to influence the development of illness by modulating cell metabolism and determining the rate of production of high-energy phosphate compounds (eg, adenosine triphosphate). It is now recognized that this view is simplistic and that mitochondria play key roles in many other processes, including cell signaling, regulating gene expression, modulating cellular calcium levels, and influencing the activation of cell death pathways (eg, caspase activation). Moreover, these multiple mitochondrial functional characteristics are now known to influence the evolution of cellular and organ function in many disease states, including sepsis, ICU-acquired skeletal muscle dysfunction, acute lung injury, acute renal failure, and critical illness-related immune function dysregulation. In addition, diseased mitochondria generate toxic compounds, most notably released mitochondrial DNA, which can act as danger-associated molecular patterns to induce systemic toxicity and damage multiple organs throughout the body. This article reviews these evolving concepts relating mitochondrial function and acute illness. The discussion is organized into four sections: (1) basics of mitochondrial physiology; (2) cellular mechanisms of mitochondrial pathophysiology; (3) critical care disease processes whose initiation and evolution are shaped by mitochondrial pathophysiology; and (4) emerging treatments for mitochondrial dysfunction in critical illness.

SS31, a mitochondrially targeted antioxidant, prevents sepsis-induced reductions in diaphragm strength and endurance.

Sepsis-induced diaphragm dysfunction contributes to respiratory failure and mortality in critical illness. There are no treatments for this form of diaphragm weakness. Studies show that sepsis-induced muscle dysfunction is triggered by enhanced mitochondrial free radical generation. We tested the hypothesis that SS31, a mitochondrially targeted antioxidant, would attenuate sepsis-induced diaphragm dysfunction. Four groups of mice were studied: 1) sham-operated controls, 2) sham-operated+SS31 (10 mg·kg-1·day-1), 3) cecal ligation puncture (CLP), and 4) CLP+SS31. Forty-eight hours postoperatively, diaphragm strips with attached phrenic nerves were isolated, and the following were assessed: muscle-field-stimulated force-frequency curves, nerve-stimulated force-frequency curves, and muscle fatigue. We also measured calpain activity, 20S proteasomal activity, myosin heavy chain (MHC) levels, mitochondrial function, and aconitase activity, an index of mitochondrial superoxide generation. Sepsis markedly reduced diaphragm force generation; SS31 prevented these decrements. Diaphragm-specific force generation averaged 30.2 ± 1.4, 9.4 ± 1.8, 25.5 ± 2.3, and 27.9 ± 0.6 N/cm2 for sham, CLP, sham+SS31, and CLP+SS31 groups (P < 0.001). Similarly, with phrenic nerve stimulation, CLP depressed diaphragm force generation, effects prevented by SS31. During endurance trials, force was significantly reduced with CLP, and SS31 prevented these reductions (P < 0.001). Sepsis also increased diaphragm calpain activity, increased 20S proteasomal activity, decreased MHC levels, reduced mitochondrial function (state 3 rates and ATP generation), and reduced aconitase activity; SS31 prevented each of these sepsis-induced alterations (P ≤ 0.017 for all indices). SS31 prevents sepsis-induced diaphragm dysfunction, preserving force generation, endurance, and mitochondrial function. Compounds with similar mechanisms of action may be useful therapeutically to preserve diaphragm function in patients who are septic and critically ill.NEW & NOTEWORTHY Sepsis-induced diaphragm dysfunction is a major contributor to mortality and morbidity in patients with critical illness in intensive care units. Currently, there is no proven pharmacological treatment for this problem. This study provides the novel finding that administration of SS31, a mitochondrially targeted antioxidant, preserves diaphragm myosin heavy chain content and mitochondrial function, thereby preventing diaphragm weakness and fatigue in sepsis.

Taurine administration ablates sepsis induced diaphragm weakness.

Infection induced diaphragm weakness is a major contributor to death and prolonged mechanical ventilation in critically ill patients. Infection induced muscle dysfunction is associated with activation of muscle proteolytic enzymes, and taurine is known to suppress proteolysis. We therefore postulated that taurine administration may prevent infection induced diaphragm dysfunction. The purpose of this study was to test this hypothesis using a clinically relevant animal model of infection, i.e. cecal ligation puncture induced sepsis (CLP). Studies were performed on (n = 5-7 mice/group): (a) sham operated controls, (b) animals with sepsis induced by CLP, (c) sham operated animals given taurine (75 mg/kg/d, intraperitoneally), and (d) CLP animals given taurine. At intervals after surgery animals were euthanized, diaphragm force generation measured in vitro, and diaphragm calpain, caspase and proteasomal activity determined. CLP elicited a large reduction in diaphragm specific force generation at 24 h (1-150 Hz, p < 0.001) and taurine significantly attenuated CLP induced diaphragm weakness at all stimulation frequencies (p < 0.001). CLP induced significant increases in diaphragm calpain, caspase and proteasomal activity; taurine administration prevented increases in the activity of all three pathways. In additional time course experiments, diaphragm force generation remained at control levels over 72 h in CLP animals treated with daily taurine administration, while CLP animals demonstrated severe, sustained reductions in diaphragm strength (p < 0.01 for all time points). Our results indicate that taurine administration prevents infection induced diaphragm weakness and reduces activation of three major proteolytic pathways. Because this agent is has been shown to be safe, non-toxic when administered to humans, taurine may have a role in treating infection induced diaphragm weakness. Future clinical studies will be needed to assess this possibility.

Sepsis-induced myopathy.

Sepsis is a major cause of morbidity and mortality in critically ill patients, and despite advances in management, mortality remains high. In survivors, sepsis increases the risk for the development of persistent acquired weakness syndromes affecting both the respiratory muscles and the limb muscles. This acquired weakness results in prolonged duration of mechanical ventilation, difficulty weaning, functional impairment, exercise limitation, and poor health-related quality of life. Abundant evidence indicates that sepsis induces a myopathy characterized by reductions in muscle force-generating capacity, atrophy (loss of muscle mass), and altered bioenergetics. Sepsis elicits derangements at multiple subcellular sites involved in excitation contraction coupling, such as decreasing membrane excitability, injuring sarcolemmal membranes, altering calcium homeostasis due to effects on the sarcoplasmic reticulum, and disrupting contractile protein interactions. Muscle wasting occurs later and results from increased proteolytic degradation as well as decreased protein synthesis. In addition, sepsis produces marked abnormalities in muscle mitochondrial functional capacity and when severe, these alterations correlate with increased death. The mechanisms leading to sepsis-induced changes in skeletal muscle are linked to excessive localized elaboration of proinflammatory cytokines, marked increases in free-radical generation, and activation of proteolytic pathways that are upstream of the proteasome including caspase and calpain. Emerging data suggest that targeted inhibition of these pathways may alter the evolution and progression of sepsis-induced myopathy and potentially reduce the occurrence of sepsis-mediated acquired weakness syndromes.

The JNK MAP kinase pathway contributes to the development of endotoxin-induced diaphragm caspase activation.

We previously demonstrated that endotoxin-induced sepsis results in caspase 8-mediated diaphragmatic dysfunction. The upstream signaling pathways modulating diaphragm caspase 8 activation in response to endotoxin administration are, however, unknown. The purpose of the present study was to test the hypothesis that the JNK (Jun N-terminal Kinase) pathway is activated in the diaphragm during sepsis and contributes to sepsis-induced diaphragm caspase 8 activation. Endotoxin was administered to intact animals to model the effects of sepsis. We first assessed the time course of JNK activation after endotoxin (12 mg/kg i.p.) administration to mice. We then determined whether JNK inhibitor administration (30 microm/kg i.p. SP600125) could prevent caspase 8 activation and diaphragm weakness in endotoxin-treated mice. Experiments were then repeated comparing the effects of endotoxin on control and transgenic JNK knockout mice. We finally determined whether cytomix (LPS, TNFalpha, IL1beta, and IFN-gamma) exposure activated caspase 8 in C2C12 muscle cells and whether caspase 8 activation was attenuated by either chemical inhibition of JNK (30 microM SP600125) or transfection with a dominant negative JNK construct. We found that endotoxin activated diaphragm JNK (P < 0.001) and increased active caspase 8 (P < 0.01). Inhibition of JNK with SP600125 or by use of JNK-deficient animals prevented diaphragm caspase 8 activation (P < 0.01) and prevented diaphragm weakness (P < 0.05). JNK inhibition also prevented caspase 8 activation in cytokine-treated muscle cells (P < 0.001). These data implicate JNK activation as a major factor mediating inflammation-induced skeletal muscle caspase 8 activation and weakness.

Hyperglycemia and acquired weakness in critically ill patients: potential mechanisms.

Critical illness polyneuropathy/critical illness myopathy (CIP/CIM) is a major cause of mortality and long-term morbidity in critically ill patients, but the true incidence and prevalence of these syndromes are not known. Hermans and colleagues show that when intensive insulin therapy is used as part of routine clinical practice in the intensive care unit, the incidence of CIP/CIM as determined by electrophysiologic testing is reduced. Our understanding of the mechanisms responsible for inducing prolonged weakness in intensive care unit patients is limited, and the role of hyperglycemia in modulating these processes is unknown. Intensive insulin therapy currently remains the only effective therapeutic intervention that has been shown to reduce the incidence of CIP/CIM.