Leigh Ann Callahan

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.

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.

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.

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.

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.