Ruxolitinib: A new hope for ventilator-induced diaphragm dysfunction.

AIM: Mechanical ventilation (MV) results in diminished diaphragm size and strength, termed ventilator-induced diaphragm dysfunction (VIDD). VID increases dependence, prolongs weaning, and increases discharge mortality rates. The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway is implicated in VIDD, upregulated following MV. JAK/STAT inhibition alleviates chronic muscle wasting conditions. This study aimed to explore the therapeutic potential of Ruxolitinib, an FDA approved JAK1/2 inhibitor (JI) for the treatment of VIDD. METHODS: Rats were subjected to 5 days controlled MV (CMV) with and without daily Ruxolitinib gavage. Muscle fiber size and function were assessed. RNAseq, mitochondrial morphology, respirometry, and mass spectrometry were determined. RESULTS: CMV significantly reduced diaphragm size and specific force by 45% (p < 0.01), associated with a two-fold P-STAT3 upregulation (p < 0.001). CMV disrupted mitochondrial content and reduced the oxygen consumption rate (p < 0.01). Expression of the motor protein myosin was unaffected, however CMV alters myosin function via post-translational modifications (PTMs). Daily administration of JI increased animal survival (40% vs. 87%; p < 0.05), restricted P-STAT3 (p < 0.001), and preserved diaphragm size and specific force. JI was associated with preserved mitochondrial content and respiratory function (p < 0.01), and the reversal or augmentation of myosin deamidation PTMs of the rod and head region. CONCLUSION: JI preserved diaphragm function, leading to increased survival in an experimental model of VIDD. Functional enhancement was associated with maintenance of mitochondrial content and respiration and the reversal of ventilator-induced PTMs of myosin. These results demonstrate the potential of repurposing Ruxolitinib for treatment of VIDD.

Single fibre cytoarchitecture in ventilator-induced diaphragm dysfunction (VIDD) assessed by quantitative morphometry second harmonic generation imaging: Positive effects of BGP-15 chaperone co-inducer and VBP-15 dissociative corticosteroid treatment.

Ventilator-induced diaphragm dysfunction (VIDD) is a common sequela of intensive care unit (ICU) treatment requiring mechanical ventilation (MV) and neuromuscular blockade (NMBA). It is characterised by diaphragm weakness, prolonged respirator weaning and adverse outcomes. Dissociative glucocorticoids (e.g., vamorolone, VBP-15) and chaperone co-inducers (e.g., BGP-15) previously showed positive effects in an ICU-rat model. In limb muscle critical illness myopathy, preferential myosin loss prevails, while myofibrillar protein post-translational modifications are more dominant in VIDD. It is not known whether the marked decline in specific force (force normalised to cross-sectional area) is a pure consequence of altered contractility signaling or whether diaphragm weakness also has a structural correlate through sterical remodeling of myofibrillar cytoarchitecture, how quickly it develops, and to which extent VBP-15 or BGP-15 may specifically recover myofibrillar geometry. To address these questions, we performed label-free multiphoton Second Harmonic Generation (SHG) imaging followed by quantitative morphometry in single diaphragm muscle fibres from healthy rats subjected to five or 10 days of MV + NMBA to simulate ICU treatment without underlying confounding pathology (like sepsis). Rats received daily treatment of either Prednisolone, VBP-15, BGP-15 or none. Myosin-II SHG signal intensities, fibre diameters (FD) as well as the parameters of myofibrillar angular parallelism (cosine angle sum, CAS) and in-register of adjacent myofibrils (Vernier density, VD) were computed from SHG images. ICU treatment caused a decline in FD at day 10 as well as a significant decline in CAS and VD from day 5. Vamorolone effectively recovered FD at day 10, while BGP-15 was more effective at day 5. BGP-15 was more effective than VBP-15 in recovering CAS at day 10 although not to control levels. In-register VD levels were restored at day 10 by both compounds. Our study is the first to provide quantitative insights into VIDD-related myofibrillar remodeling unravelled by SHG imaging, suggesting that both VBP-15 and BGP-15 can effectively ameliorate the structure-related dysfunction in VIDD.

Direct electrical stimulation impacts on neuromuscular junction morphology on both stimulated and unstimulated contralateral soleus.

BACKGROUND: There is increasing evidence of crosstalk between organs. The neuromuscular junction (NMJ) is a peripheral chemical synapse whose function and morphology are sensitive to acetylcholine (ACh) release and muscle depolarization. In an attempt to improve our understanding of NMJ plasticity and muscle crosstalk, the effects of unilateral direct electrical stimulation of a hindlimb muscle on the NMJ were investigated in rats exposed long-term post-synaptic neuromuscular blockade. METHODS: Sprague Dawley rats were subjected to post-synaptic blockade of neuromuscular transmission by systemic administration of α-cobrotoxin and mechanically ventilated for up to 8 days and compared with untreated sham operated controls and animals exposed to unilateral chronic electrical stimulation 12 h/day for 5 or 8 days. RESULTS: NMJs produced axonal and glial sprouts (growth of processes that extend beyond the confines of the synapse defined by high-density aggregates of acetylcholine receptors [AChRs]) in response to post-synaptic neuromuscular blockade, but less than reported after peripheral denervation or pre-synaptic blockade. Direct electrical soleus muscle stimulation reduced the terminal Schwann cell (tSC) and axonal sprouting in both stimulated and non-stimulated contralateral soleus. Eight days chronic stimulation reduced (P < 0.001) the number of tSC sprouts on stimulated and non-stimulated soleus from 6.7 ± 0.5 and 6.9 ± 0.5 sprouts per NMJ, respectively, compared with 10.3 ± 0.9 tSC per NMJ (P < 0.001) in non-stimulated soleus from rats immobilized for 8 days. A similar reduction of axonal sprouts (P < 0.001) was observed in stimulated and non-stimulated contralateral soleus in response to chronic electrical stimulation. RNAseq-based gene expression analyses confirmed a restoring effect on both stimulated and unstimulated contralateral muscle. The cross-over effect was paralleled by increased cytokine/chemokine levels in stimulated and contralateral unstimulated muscle as well as in plasma. CONCLUSIONS: Motor axon terminals and terminal Schwann cells at NMJs of rats subjected to post-synaptic neuromuscular blockade exhibited sprouting responses. These axonal and glial responses were likely dampened by a muscle-derived myokines released in an activity-dependent manner with both local and systemic effects.

A prospective clinical study on the mechanisms underlying critical illness myopathy-A time-course approach.

BACKGROUND: Critical illness myopathy (CIM) is a consequence of modern critical care resulting in general muscle wasting and paralyses of all limb and trunk muscles, resulting in prolonged weaning from the ventilator, intensive care unit (ICU) treatment and rehabilitation. CIM is associated with severe morbidity/mortality and significant negative socioeconomic consequences, which has become increasingly evident during the current COVID-19 pandemic, but underlying mechanisms remain elusive. METHODS: Ten neuro-ICU patients exposed to long-term controlled mechanical ventilation were followed with repeated muscle biopsies, electrophysiology and plasma collection three times per week for up to 12 days. Single muscle fibre contractile recordings were conducted on the first and final biopsy, and a multiomics approach was taken to analyse gene and protein expression in muscle and plasma at all collection time points. RESULTS: (i) A progressive preferential myosin loss, the hallmark of CIM, was observed in all neuro-ICU patients during the observation period (myosin:actin ratio decreased from 2.0 in the first to 0.9 in the final biopsy, P < 0.001). The myosin loss was coupled to a general transcriptional downregulation of myofibrillar proteins (P < 0.05; absolute fold change >2) and activation of protein degradation pathways (false discovery rate [FDR] <0.1), resulting in significant muscle fibre atrophy and loss in force generation capacity, which declined >65% during the 12 day observation period (muscle fibre cross-sectional area [CSA] and maximum single muscle fibre force normalized to CSA [specific force] declined 30% [P < 0.007] and 50% [P < 0.0001], respectively). (ii) Membrane excitability was not affected as indicated by the maintained compound muscle action potential amplitude upon supramaximal stimulation of upper and lower extremity motor nerves. (iii) Analyses of plasma revealed early activation of inflammatory and proinflammatory pathways (FDR < 0.1), as well as a redistribution of zinc ions from plasma. CONCLUSIONS: The mechanical ventilation-induced lung injury with release of cytokines/chemokines and the complete mechanical silencing uniquely observed in immobilized ICU patients affecting skeletal muscle gene/protein expression are forwarded as the dominant factors triggering CIM.

Electrical stimulated GLUT4 signalling attenuates critical illness-associated muscle wasting.

BACKGROUND: Critical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a by-product of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood; however, loss of mechanical stimuli contributes to critical illness-associated muscle atrophy and weakness. Passive mechanical loading and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care. METHODS: Rats were subjected to 8 days of critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM. RESULTS: Following 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, myosin : actin ratio, and single muscle fibre maximum force normalized to cross-sectional area (CSA; specific force) were reduced by 40-50% (P < 0.0001). ES significantly reduced the loss of soleus muscle fibre CSA and myosin : actin ratio by approximately 30% (P < 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signalling in the soleus muscle following critical care, and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (P < 0.01). ES promoted phosphofructokinase and insulin signalling pathways to control levels (P < 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signalling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (P = 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately two-fold (P = 0.06). Reduction of muscle protein degradation rather than increased synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (P = 0.006) and MuRF1 (P = 0.08) by approximately 50%, downstream of AMPK-FoxO3. CONCLUSIONS: ES maintained GLUT4 translocation through increased AMPK-TBC1D4 signalling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signalling, and reduced muscle protein degradation in CIM.

Acute and severe trabecular bone loss in a rat model of critical illness myopathy.

Prolonged mechanical ventilation for critically ill patients with respiratory distress can result in severe muscle wasting with preferential loss of myosin. Systemic inflammation triggered by lung mechanical injury likely contributes to this myopathy, although the exact mechanisms are unknown. In this study, we hypothesized that muscle wasting following mechanical ventilation is accompanied by bone loss. The objective was to determine the rate, nature, and extent of bone loss in the femora of rats ventilated up to 10 days and to relate the bone changes to muscle deterioration. We have developed a rat model of ventilator-induced muscle wasting and established its feasibility and clinical validity. This model involves pharmacologic paralysis, parenteral nutrition, and continuous mechanical ventilation. We assessed the hindlimb muscle and bone of rats ventilated for 0, 2, 5, 8, and 10 days. Routine histology, microCT, and biomechanical evaluations were performed. Hindlimb muscles developed changes consistent with myopathy, whereas the femurs demonstrated a progressive decline in trabecular bone volume, mineral density, and microarchitecture beginning Day 8 of mechanical ventilation. Biomechanical testing showed a reduction in flexural strength and stiffness on Day 10. The bone changes correlated with the loss of muscle mass and myosin. These results demonstrate that mechanical ventilation leads to progressive trabecular bone loss parallel to muscle deterioration. The results of our study suggest that mechanically ventilated patients may be at risk of compromised bone integrity and muscle weakness, predisposing to post-ventilator falls and fractures, thereby warranting interventions to prevent progressive bone and muscle decline.

Multi-omics reveals age-related differences in the diaphragm response to mechanical ventilation: a pilot study.

BACKGROUND: Old age is associated with a significantly increased mortality in COVID-19 patients exposed to long-term controlled mechanical ventilation (CMV) and suggested to be due to the hyperinflammatory response associated with the viral infection. However, our understanding of age-related differences in the response to CMV in the absence of a viral infection remains insufficient. METHODS: Young (7-8 months) and old (28-32 months) F344 BN hybrid rats were exposed to the ICU condition for 5 days, i.e., complete immobilization, mechanical ventilation, and extensive monitoring. Transcriptomic (RNA-Seq) and proteomics (Proximity Extension Assay) analyses of the diaphragm and proteomics analysis of plasma were conducted to investigate the molecular differences between young and old rats exposed to the ICU condition. RESULTS: According to multi-omics analyses, significant differences were observed in the diaphragm between young and old rats in response to 5 days CMV and immobilization. In young rats, metabolic pathways were primarily downregulated in response to immobilization (post-synaptic blockade of neuromuscular transmission). In old rats, on the other hand, dramatic immune and inflammatory responses were observed, i.e., an upregulation of specific related pathways such as "IL-17 signaling pathway", along with a higher level of inflammatory factors and cytokine/chemokine in plasma. CONCLUSIONS: The dramatically increased mortality in old ICU patients with COVID-19-associated hyperinflammation and cytokine storm need not only reflect the viral infection but may also be associated with the ventilator induced diaphragm dysfunction (VIDD) and hyperinflammatory responses induced by long-term CMV per se. Although mechanical ventilation is a life-saving intervention in COVID-19 ICU patients, CMV should be cautiously used especially in old age and other means of respiratory support may be considered, such as negative pressure ventilation.

JAK/STAT inhibition augments soleus muscle function in a rat model of critical illness myopathy via regulation of complement C3/3R.

KEY POINTS: Critical illness myopathy (CIM) is a frequently observed negative consequence of modern critical care. Chronic Janus kinase (JAK)/signal transducer and activator of transcription activation impairs muscle size and function and is prominent following mechanical ventilation. We identify pSTAT-3 activation in tibialis anterior of CIM patients, before examining the potential benefits of JAK1/2 inhibition in an experimental model of CIM, where muscle mass and function are impaired. CIM activates complement cascade and increased monocyte infiltration in the soleus muscle, which was ameliorated by JAK1/2 inhibition, leading to reduced muscle degeneration and improved muscle force. Here, we demonstrate that JAK1/2 inhibition augments CIM muscle function through regulation of the complement cascade. ABSTRACT: Critical illness myopathy (CIM) is frequently observed in response to modern critical care with negative consequences for patient quality of life, morbidity, mortality and healthcare costs. Janus kinase (JAK)/signal transducer and activator of transcription (STAT) activation is observed in limb muscles following controlled mechanical ventilation. Chronic JAK/STAT activation promotes loss of muscle mass and function. Thus, we hypothesized that JAK1/2 inhibition would improve muscle outcomes for CIM. Following 12 days of intensive care unit conditions, pSTAT-3 levels increased in tibialis anterior muscle of CIM patients (P = 0.0489). The potential of JAK1/2 inhibition was assessed in an experimental model of CIM, where soleus muscle size and force are impaired. JAK1/2 inhibition restores soleus force (P < 0.0001). CIM activated muscle complement cascade, which was ameliorated by JAK1/2 inhibition (P < 0.05, respectively). Soleus macrophage number corresponded with complement activity, leading to reduced muscle degeneration and augmented muscle function (P < 0.05). Thus, JAK/STAT inhibition improves soleus function by modulating the complement cascade and muscle monocyte infiltration. Collectively, we demonstrate that JAK/STAT inhibition augments muscle function in CIM.

Maintenance of sarcomeric integrity in adult muscle cells crucially depends on Z-disc anchored titin.

The giant protein titin is thought to be required for sarcomeric integrity in mature myocytes, but direct evidence for this hypothesis is limited. Here, we describe a mouse model in which Z-disc-anchored TTN is depleted in adult skeletal muscles. Inactivation of TTN causes sarcomere disassembly and Z-disc deformations, force impairment, myocyte de-stiffening, upregulation of TTN-binding mechanosensitive proteins and activation of protein quality-control pathways, concomitant with preferential loss of thick-filament proteins. Interestingly, expression of the myosin-bound Cronos-isoform of TTN, generated from an alternative promoter not affected by the targeting strategy, does not prevent deterioration of sarcomere formation and maintenance. Finally, we demonstrate that loss of Z-disc-anchored TTN recapitulates muscle remodeling in critical illness 'myosinopathy' patients, characterized by TTN-depletion and loss of thick filaments. We conclude that full-length TTN is required to integrate Z-disc and A-band proteins into the mature sarcomere, a function that is lost when TTN expression is pathologically lowered.

Expression of SUMO enzymes is fiber type dependent in skeletal muscles and is dysregulated in muscle disuse.

SUMOylation is a dynamic, reversible, enzymatic drug-targetable post-translational modification (PTM) reaction where the Small Ubiquitin-like Modifier (SUMO) moieties are attached to proteins. This reaction regulates various biological functions like cell growth, differentiation, and it is crucial for maintaining organ homeostasis. However, the actions of SUMO in skeletal muscle pathophysiology are still not investigated. In this study, we quantified the abundance of the SUMO enzymes and determined the distribution of SUMOylated proteins along the fibers of nine different muscles. We find that skeletal muscles contain a distinctive group of SUMO enzymes and SUMOylated proteins in relation to their different metabolism, functions, and fiber type composition. In addition, before the activation of protein degradation pathways, this unique set is quickly altered in response to muscle sedentariness. Finally, we demonstrated that PAX6 acts as an upstream regulator of the SUMO conjugation reaction, which can become a potential therapeutic marker to prevent muscle diseases generated by inactivity.

Chaperone co-inducer BGP-15 mitigates early contractile dysfunction of the soleus muscle in a rat ICU model.

AIM: Critical illness myopathy (CIM) represents a common consequence of modern intensive care, negatively impacting patient health and significantly increasing health care costs; however, there is no treatment available apart from symptomatic and supportive interventions. The chaperone co-inducer BGP-15 has previously been shown to have a positive effect on the diaphragm in rats exposed to the intensive care unit (ICU) condition. In this study, we aim to explore the effects of BGP-15 on a limb muscle (soleus muscle) in response to the ICU condition. METHODS: Sprague-Dawley rats were subjected to the ICU condition for 5, 8 and 10 days and compared with untreated sham-operated controls. RESULTS: BGP-15 significantly improved soleus muscle fibre force after 5 days exposure to the ICU condition. This improvement was associated with the protection of myosin from post-translational myosin modifications, improved mitochondrial structure/biogenesis and reduced the expression of MuRF1 and Fbxo31 E3 ligases. At longer durations (8 and 10 days), BGP-15 had no protective effect when the hallmark of CIM had become manifest, that is, preferential loss of myosin. Unrelated to the effects on skeletal muscle, BGP-15 had a strong positive effect on survival compared with untreated animals. CONCLUSIONS: BGP-15 treatment improved soleus muscle fibre and motor protein function after 5 days exposure to the ICU condition, but not at longer durations (8 and 10 days) when the preferential loss of myosin was manifest. Thus, long-term CIM interventions targeting limb muscle fibre/myosin force generation capacity need to consider both the post-translational modifications and the loss of myosin.

Vamorolone treatment improves skeletal muscle outcome in a critical illness myopathy rat model.

AIM: Critical illness myopathy (CIM) is a consequence of modern critical care, leading to skeletal muscle atrophy/paralysis with negative consequences for mortality/morbidity and health care costs. Glucocorticoids (GCs) have been proposed to trigger CIM. Here, we compare outcomes of two GCs, the commonly used prednisolone and the newly developed dissociative vamorolone in response to the intensive care unit (ICU) condition for 5 days, ie, sedation, immobilization, and mechanical ventilation. METHODS: Rats were divided into a 0-day sham-operated control group, and three groups exposed to 5 days ICU condition during treatment with prednisolone (PRED) or vamorolone (VAM) or none of these GCs (ICU-group). Survival, body and muscle weights, cytokine concentrations, regulation of muscle contraction in single fast- and slow-twitch muscle fibres, myofibrillar protein expression and protein degradation pathways were studied. RESULTS: Critical illness myopathy geno- and pheno-types were confirmed in the ICU group. However, VAM and PRED groups showed reduced atrophy/weakness than the ICU group, and muscle specific differences with more severe negative effects on fast-twitch muscle fibres in the PRED than the other groups. CONCLUSION: These results show that vamorolone provides a GC intervention superior to typical GCs in improving CIM outcomes. Further, the findings do not support the notion that moderate-dose GC treatment represents a factor triggering CIM.

A Proteomic Approach to Identify Alterations in the Small Ubiquitin-like Modifier (SUMO) Network during Controlled Mechanical Ventilation in Rat Diaphragm Muscle.

The small ubiquitin-like modifier (SUMO) is as a regulator of many cellular functions by reversible conjugation to a broad number of substrates. Under endogenous or exogenous perturbations, the SUMO network becomes a fine sensor of stress conditions by alterations in the expression level of SUMO enzymes and consequently changing the status of SUMOylated proteins. The diaphragm is the major inspiratory muscle, which is continuously active under physiological conditions, but its structure and function is severely affected when passively displaced for long extents during mechanical ventilation (MV). An iatrogenic condition called Ventilator-Induced Diaphragm Dysfunction (VIDD) is a major cause of failure to wean patients from ventilator support but the molecular mechanisms underlying this dysfunction are not fully understood. Using a unique experimental Intensive Care Unit (ICU) rat model allowing long-term MV, diaphragm muscles were collected in rats control and exposed to controlled MV (CMV) for durations varying between 1 and 10 days. Endogenous SUMOylated diaphragm proteins were identified by mass spectrometry and validated with in vitro SUMOylation systems. Contractile, calcium regulator and mitochondrial proteins were of specific interest due to their putative involvement in VIDD. Differences were observed in the abundance of SUMOylated proteins between glycolytic and oxidative muscle fibers in control animals and high levels of SUMOylated proteins were present in all fibers during CMV. Finally, previously reported VIDD biomarkers and therapeutic targets were also identified in our datasets which may play an important role in response to muscle weakness seen in ICU patients. Data are available via ProteomeXchange with identifier PXD006085. Username: reviewer26663@ebi.ac.uk, Password: rwcP5W0o.

Targeting Heat Shock Proteins Mitigates Ventilator Induced Diaphragm Muscle Dysfunction in an Age-Dependent Manner.

Intensive care unit (ICU) patients are often overtly subjected to mechanical ventilation and immobilization, which leads to impaired limb and respiratory muscle function. The latter, termed ventilator-induced diaphragm dysfunction (VIDD) has recently been related to compromised heat shock protein (Hsp) activation. The administration of a pharmacological drug BGP-15 acting as a Hsp chaperone co-inducer has been found to partially alleviate VIDD in young rats. Considering that the mean age in the ICU is increasing, we aimed to explore whether the beneficial functional effects are also present in old rats. For that, we exposed young (7-8 months) and old (28-32 months) rats to 5-day controlled mechanical ventilation and immobilization with or without systemic BGP-15 administration. We then dissected diaphragm muscles, membrane-permeabilized bundles and evaluated the contractile function at single fiber level. Results confirmed that administration of BGP-15 restored the force-generating capacity of isolated muscle cells from young rats in conjunction with an increased expression of Hsp72. On the other hand, our results highlighted that old rats did not positively respond to the BGP-15 treatment. Therefore, it is of crucial importance to comprehend in more depth the effect of VIDD on diaphragm function and ascertain any further age-related differences.

The chaperone co-inducer BGP-15 alleviates ventilation-induced diaphragm dysfunction.

Ventilation-induced diaphragm dysfunction (VIDD) is a marked decline in diaphragm function in response to mechanical ventilation, which has negative consequences for individual patients' quality of life and for the health care system, but specific treatment strategies are still lacking. We used an experimental intensive care unit (ICU) model, allowing time-resolved studies of diaphragm structure and function in response to long-term mechanical ventilation and the effects of a pharmacological intervention (the chaperone co-inducer BGP-15). The marked loss of diaphragm muscle fiber function in response to mechanical ventilation was caused by posttranslational modifications (PTMs) of myosin. In a rat model, 10 days of BGP-15 treatment greatly improved diaphragm muscle fiber function (by about 100%), although it did not reverse diaphragm atrophy. The treatment also provided protection from myosin PTMs associated with HSP72 induction and PARP-1 inhibition, resulting in improvement of mitochondrial function and content. Thus, BGP-15 may offer an intervention strategy for reducing VIDD in mechanically ventilated ICU patients.

Impaired Ca(2+) release contributes to muscle weakness in a rat model of critical illness myopathy.

BACKGROUND: Critical illness myopathy is an acquired skeletal muscle disorder with severe myosin loss and muscle weakness frequently seen in intensive care unit (ICU) patients. It is unknown if impaired excitation-contraction coupling contributes to the muscle weakness. METHODS: We used a unique ICU model where rats were deeply sedated, post-synaptically pharmacologically paralyzed, mechanically ventilated and closely monitored for up to ten days. Single intact fibers from the flexor digitorum brevis muscle were isolated and used to measure force and free myoplasmic [Ca(2+)] ([Ca(2+)]i) during tetanic contractions. RESULTS: Fibers from ICU rats had 80 % lower tetanic [Ca(2+)]i and produced only 15 % of the force seen in fibers from sham-operated (SHAM) rats. In the presence of 5 mM caffeine, tetanic [Ca(2+)]i was similar in fibers from ICU and SHAM rats but force was 50 % lower in fibers from ICU rats than SHAM rats. Confocal imaging showed disrupted tetanic [Ca(2+)]i transients in fibers from ICU rats compared to SHAM rats. Western blots showed similar levels of Na(+) channel and dihydropyridine receptor (DHPR) protein expression, whereas ryanodine receptor (RyR) and sarco-endoplasmic reticulum Ca(2+) ATPase 1 (SERCA1) expression was markedly lower in muscle of ICU rats than in SHAM rats. Immunohistochemical analysis showed that distribution of Na(+) channel and DHPR protein on the sarcolemma was disrupted in fibers from ICU rats compared with SHAM rats. CONCLUSIONS: These results suggest that impaired SR Ca(2+) release contributes to the muscle weakness seen in patients in ICU.

Age related differences in diaphragm muscle fiber response to mid/long term controlled mechanical ventilation.

BACKGROUND: Critically ill intensive care patients are subjected to controlled mechanical ventilation (CMV) which has an important association in triggering the impaired muscle function and the consequent delayed weaning from the respirator. AIM: The main aim of this study was to measure the effects of age and CMV over a period up to 5days on rat diaphragm muscle fibers, more specifically focusing on the changes in fiber structure and function. METHODS: Diaphragm muscle fiber cross-sectional area (CSA) and force generating capacity were measured in young (6months) and old (28-32months) rats in response to five days of CMV. To investigate the biological age of the old rats in this rat strain (F344 BN hybrid), a second set of experiments comparing muscle fiber size and specific force (maximum force normalized to CSA) was investigated in fast- and slow-twitch distal hind limb muscles in 3 different age groups: young adults (6months), middle aged (18months) and old rats (28months). RESULTS: This study shows an unexpected response of the diaphragm fibers to 5days CMV, demonstrating an increased CSA (p<0.001) in both young and old animals. Furthermore, an observed decreased maximum force of 39.8-45.2% (p<0.001) in both young and old animals compared with controls resulted in a dramatic loss of specific force. We suggest that this increase in CSA and decrease in specific force observed in both the young and old diaphragm fibers is an ineffective compensatory hypertrophy in response to the CMV. These results demonstrate an important mechanism of significant importance for the weaning problems associated with mechanical ventilation.

Sparing of muscle mass and function by passive loading in an experimental intensive care unit model.

The response to mechanical stimuli, i.e., tensegrity, plays an important role in regulating cell physiological and pathophysiological function, and the mechanical silencing observed in intensive care unit (ICU) patients leads to a severe and specific muscle wasting condition. This study aims to unravel the underlying mechanisms and the effects of passive mechanical loading on skeletal muscle mass and function at the gene, protein and cellular levels. A unique experimental rat ICU model has been used allowing long-term (weeks) time-resolved analyses of the effects of standardized unilateral passive mechanical loading on skeletal muscle size and function and underlying mechanisms. Results show that passive mechanical loading alleviated the muscle wasting and the loss of force-generation associated with the ICU intervention, resulting in a doubling of the functional capacity of the loaded versus the unloaded muscles after a 2-week ICU intervention. We demonstrate that the improved maintenance of muscle mass and function is probably a consequence of a reduced oxidative stress revealed by lower levels of carbonylated proteins, and a reduced loss of the molecular motor protein myosin. A complex temporal gene expression pattern, delineated by microarray analysis, was observed with loading-induced changes in transcript levels of sarcomeric proteins, muscle developmental processes, stress response, extracellular matrix/cell adhesion proteins and metabolism. Thus, the results from this study show that passive mechanical loading alleviates the severe negative consequences on muscle size and function associated with the mechanical silencing in ICU patients, strongly supporting early and intense physical therapy in immobilized ICU patients.