Reactive oxygen species in skeletal muscle injury, fatigue, regeneration and ageing: In memory of John Faulkner The role of zinc and matrix metalloproteinases in myofibrillar protein degradation in critical illness myopathy.

Due to an unexpected activation of different zinc (Zn) transporters in a recent prospective clinical study, we have revisited the role of Zn homeostasis and the activation of matrix metalloproteinases (MMPs) in skeletal muscle exposed to the intensive care unit (ICU) condition (immobilization and mechanical ventilation). ICU patients exposed to 12 days ICU condition were followed longitudinally with six repeated muscle biopsies while they showed a progressive preferential myosin loss, i.e., the hallmark of Critical Illness Myopathy (CIM), in parallel with the activation of Zn-transporters. In this study, we have revisited the expression of Zn-transporters and the activation of MMPs in clinical as well as in experimental studies using an established ICU model. MMPs are a group Zn-dependent endopeptidases which do not only target and cleave extracellular proteins but also intracellular proteins including multiple sarcomeric proteins. MMP-9 is of specific interest since the hallmark of CIM, the preferential myosin loss, has also been reported in dilated cardiomyopathy and coupled to MMP-9 activation. Transcriptional activation of Zn-transporters was observed in both clinical and experimental studies as well as the activation of MMPs, in particular MMP-9, in various limb and respiratory muscles in response to long-term exposure to the ICU condition. The activation of Zn-transporters was paralleled by increased Zn levels in skeletal muscle which in turn showed a negative linear correlation with the preferential myosin loss associated with CIM, offering a potential intervention strategy. Thus, activation of Zn-transporters, increased intramuscular Zn levels, and activation of the Zn-dependent MMPs are forwarded as a probable mechanism involved in CIM pathophysiology. These effects were confirmed in different rat strains subjected to a model of CIM and exacerbated by old age. This is of specific interest since old age and muscle wasting are the two factors most strongly associated with ICU mortality.

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.

Neurogenic vs. Myogenic Origin of Acquired Muscle Paralysis in Intensive Care Unit (ICU) Patients: Evaluation of Different Diagnostic Methods.

Introduction. The acquired muscle paralysis associated with modern critical care can be of neurogenic or myogenic origin, yet the distinction between these origins is hampered by the precision of current diagnostic methods. This has resulted in the pooling of all acquired muscle paralyses, independent of their origin, into the term Intensive Care Unit Acquired Muscle Weakness (ICUAW). This is unfortunate since the acquired neuropathy (critical illness polyneuropathy, CIP) has a slower recovery than the myopathy (critical illness myopathy, CIM); therapies need to target underlying mechanisms and every patient deserves as accurate a diagnosis as possible. This study aims at evaluating different diagnostic methods in the diagnosis of CIP and CIM in critically ill, immobilized and mechanically ventilated intensive care unit (ICU) patients. Methods. ICU patients with acquired quadriplegia in response to critical care were included in the study. A total of 142 patients were examined with routine electrophysiological methods, together with biochemical analyses of myosin:actin (M:A) ratios of muscle biopsies. In addition, comparisons of evoked electromyographic (EMG) responses in direct vs. indirect muscle stimulation and histopathological analyses of muscle biopsies were performed in a subset of the patients. Results. ICU patients with quadriplegia were stratified into five groups based on the hallmark of CIM, i.e., preferential myosin loss (myosin:actin ratio, M:A) and classified as severe (M:A < 0.5; n = 12), moderate (0.5 ≤ M:A < 1; n = 40), mildly moderate (1 ≤ M:A < 1.5; n = 49), mild (1.5 ≤ M:A < 1.7; n = 24) and normal (1.7 ≤ M:A; n = 19). Identical M:A ratios were obtained in the small (4-15 mg) muscle samples, using a disposable semiautomatic microbiopsy needle instrument, and the larger (>80 mg) samples, obtained with a conchotome instrument. Compound muscle action potential (CMAP) duration was increased and amplitude decreased in patients with preferential myosin loss, but deviations from this relationship were observed in numerous patients, resulting in only weak correlations between CMAP properties and M:A. Advanced electrophysiological methods measuring refractoriness and comparing CMAP amplitude after indirect nerve vs. direct muscle stimulation are time consuming and did not increase precision compared with conventional electrophysiological measurements in the diagnosis of CIM. Low CMAP amplitude upon indirect vs. direct stimulation strongly suggest a neurogenic lesion, i.e., CIP, but this was rarely observed among the patients in this study. Histopathological diagnosis of CIM/CIP based on enzyme histochemical mATPase stainings were hampered by poor quantitative precision of myosin loss and the impact of pathological findings unrelated to acute quadriplegia. Conclusion. Conventional electrophysiological methods are valuable in identifying the peripheral origin of quadriplegia in ICU patients, but do not reliably separate between neurogenic vs. myogenic origins of paralysis. The hallmark of CIM, preferential myosin loss, can be reliably evaluated in the small samples obtained with the microbiopsy instrument. The major advantage of this method is that it is less invasive than conventional muscle biopsies, reducing the risk of bleeding in ICU patients, who are frequently receiving anticoagulant treatment, and it can be repeated multiple times during follow up for monitoring purposes.

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.

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.

Hormone replacement therapy improves contractile function and myonuclear organization of single muscle fibres from postmenopausal monozygotic female twin pairs.

Ageing is associated with a decline in muscle mass and strength leading to increased physical dependency in old age. Postmenopausal women experience a greater decline than men of similar age in parallel with the decrease in female sex steroid hormone production. We recruited six monozygous female twin pairs (55-59 years old) where only one twin pair was on hormone replacement therapy (HRT use = 7.8 ± 4.3 years) to investigate the association of HRT with the cytoplasmic volume supported by individual myonuclei (myonuclear domain (MND) size,) together with specific force at the single fibre level. HRT use was associated with a significantly smaller (∼27%; P < 0.05) mean MND size in muscle fibres expressing the type I but not the IIa myosin heavy chain (MyHC) isoform. In comparison to non-users, higher specific force was recorded in HRT users both in muscle fibres expressing type I (∼27%; P < 0.05) and type IIa (∼23%; P < 0.05) MyHC isoforms. These differences were fibre-type dependent, i.e. the higher specific force in fast-twitch muscle fibres was primarily caused by higher force per cross-bridge while slow-twitch fibres relied on both a higher number and force per cross-bridge. HRT use had no effect on fibre cross-sectional area (CSA), velocity of unloaded shortening (V0) and relative proportion of MyHC isoforms. In conclusion, HRT appears to have significant positive effects on both regulation of muscle contraction and myonuclei organization in postmenopausal women.

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.

Muscle atrophy and preferential loss of myosin in prolonged critically ill patients.

OBJECTIVE: Muscle weakness contributes to prolonged rehabilitation and adverse outcome of critically ill patients. Distinction between a neurogenic and/or myogenic underlying problem is difficult using routine diagnostic tools. Preferential loss of myosin has been suggested to point to a myogenic component. We evaluated markers of muscle atrophy and denervation, and the myosin/actin ratio in limb and abdominal wall skeletal muscle of prolonged critically ill patients and matched controls in relation to insulin therapy and known risk factors for intensive care unit-acquired weakness. DESIGN: Secondary analysis of two large, prospective, single-center randomized clinical studies. SETTING: University hospital surgical and medical intensive care unit. PATIENTS: Critically ill patients and matched controls. INTERVENTIONS: Intensive care unit patients had been randomized to blood glucose control to 80-110 mg/dL with insulin infusion or conventional glucose management, where insulin was only administered when glucose levels rose above 215 mg/dL. MEASUREMENTS AND MAIN RESULTS: As compared with controls, rectus abdominis and vastus lateralis muscle of critically ill patients showed smaller myofiber size, decreased mRNA levels for myofibrillar proteins, increased proteolytic enzyme activities, and a lower myosin/actin ratio, virtually irrespective of insulin therapy. Increased forkhead box O1 action may have played a role. Most alterations were more severe in patients treated with corticosteroids. Duration of corticosteroid treatment, independent of duration of intensive care unit stay or other risk factors, was a dominant risk factor for a low myosin/actin ratio. The immature acetylcholine receptor subunit γ messenger RNA expression was elevated in vastus lateralis, independent of the myosin/actin ratio. CONCLUSIONS: Both limb and abdominal wall skeletal muscles of prolonged critically ill patients showed downregulation of protein synthesis at the gene expression level as well as increased proteolysis. This affected myosin to a greater extent than actin, resulting in a decreased myosin/actin ratio. Muscle atrophy was not ameliorated by intensive insulin therapy, but possibly aggravated by corticosteroids.

Higher expression of myosin heavy chain IIx in wrist flexors in cerebral palsy.

BACKGROUND: Children with cerebral palsy (CP) use their paretic arm less than normal but have a relative overactivity of wrist flexors, causing an impairing flexed position of the wrist. Voluntary use of a muscle downregulates myosin heavy chain (MyHC) IIx, but it is unclear whether the relative overactivity of wrist flexors and extensors in children with CP affects MyHC expression compared to normal subjects. QUESTIONS/PURPOSES: We therefore asked whether MyHC expression composition differs in wrist flexors compared to extensors in children with CP and in controls and whether it is related to clinical findings. METHODS: We took muscle biopsies from wrist flexors and extensors during hand surgery in children with CP (n = 9) and during open reduction of forearm fractures in control children (n = 5). The expression of the MyHC I, IIa, and IIx isoforms were determined on silver-stained 6% SDS-PAGE. RESULTS: CP flexors showed a higher proportion of MyHC IIx (40%) than control flexors (16%) and CP extensors (20%). MyHC IIa isoform proportion was lower in CP flexors (27%) than in control flexors (46%) and in CP extensors (45%). MyHC I expression was lower in CP (36%) than in controls (46%) for wrist extensors only. CONCLUSIONS: Both the brain injury in CP and the different demands on flexors and extensors affect the expression of MyHCs. The higher amount of MyHC IIx in CP could be caused by a decreased voluntary use of the hemiplegic arm. CLINICAL RELEVANCE: More information on the structural difference between flexors and extensors in normal and spastic muscle could improve the understanding of strain of wrist extensors and possibly the development of flexion contractures in CP.

Preferential skeletal muscle myosin loss in response to mechanical silencing in a novel rat intensive care unit model: underlying mechanisms.

The muscle wasting and impaired muscle function in critically ill intensive care unit (ICU) patients delay recovery from the primary disease, and have debilitating consequences that can persist for years after hospital discharge. It is likely that, in addition to pernicious effects of the primary disease, the basic life support procedures of long-term ICU treatment contribute directly to the progressive impairment of muscle function. This study aims at improving our understanding of the mechanisms underlying muscle wasting in ICU patients by using a unique experimental rat ICU model where animals are mechanically ventilated, sedated and pharmacologically paralysed for duration varying between 6 h and 14 days. Results show that the ICU intervention induces a phenotype resembling the severe muscle wasting and paralysis associated with the acute quadriplegic myopathy (AQM) observed in ICU patients, i.e. a preferential loss of myosin, transcriptional down-regulation of myosin synthesis, muscle atrophy and a dramatic decrease in muscle fibre force generation capacity. Detailed analyses of protein degradation pathways show that the ubiquitin proteasome pathway is highly involved in this process. A sequential change in localisation of muscle-specific RING finger proteins 1/2 (MuRF1/2) observed during the experimental period is suggested to play an instrumental role in both transcriptional regulation and protein degradation. We propose that, for those critically ill patients who develop AQM, complete mechanical silencing, due to pharmacological paralysis or sedation, is a critical factor underlying the preferential loss of the molecular motor protein myosin that leads to impaired muscle function or persisting paralysis.

Novel transcriptional profile in wrist muscles from cerebral palsy patients.

BACKGROUND: Cerebral palsy (CP) is an upper motor neuron disease that results in a progressive movement disorder. Secondary to the neurological insult, muscles from CP patients often become spastic. Spastic muscle is characterized by an increased resistance to stretch, but often develops the further complication of contracture which represents a prominent disability in children with CP. This study's purpose is to characterize alterations of spastic muscle on the transcriptional level. Increased knowledge of spastic muscle may lead to novel therapies to improve the quality of life for children with CP. METHOD: The transcriptional profile of spastic muscles were defined in children with cerebral palsy and compared to control patients using Affymetrix U133A chips. Expression data were verified using quantitative-PCR (QPCR) and validated with SDS-PAGE for select genes. Significant genes were determined using a 2 x 2 ANOVA and results required congruence between 3 preprocessing algorithms. RESULTS: CP patients clustered independently and 205 genes were significantly altered, covering a range of cellular processes. Placing gene expression in the context of physiological pathways, the results demonstrated that spastic muscle in CP adapts transcriptionally by altering extracellular matrix, fiber type, and myogenic potential. Extracellular matrix adaptations occur primarily in the basal lamina although there is increase in fibrillar collagen components. Fiber type is predominately fast compared to normal muscle as evidenced by contractile gene isoforms and decrease in oxidative metabolic gene transcription, despite a paradoxical increased transcription of slow fiber pathway genes. We also found competing pathways of fiber hypertrophy with an increase in the anabolic IGF1 gene in parallel with a paradoxical increase in myostatin, a gene responsible for stopping muscle growth. We found evidence that excitation-contraction coupling genes are altered in muscles from patients with CP and may be a significant component of disease. CONCLUSION: This is the first transcriptional profile performed on spastic muscle of CP patients and these adaptations were not characteristic of those observed in other disease states such as Duchenne muscular dystrophy and immobilization-induced muscle atrophy. Further research is required to understand the mechanism of muscle adaptation to this upper motor neuron lesion that could lead to the development of innovative therapies.

Myofibrillar protein and gene expression in acute quadriplegic myopathy.

The dramatic muscle wasting, preferential loss of myosin and impaired muscle function in intensive care unit (ICU) patients with acute quadriplegic myopathy (AQM) have traditionally been suggested to be the result of proteolysis via specific proteolytic pathways. In this study we aim to investigate the mechanisms underlying the preferential loss of thick vs. thin filament proteins and the reassembly of the sarcomere during the recovery process in muscle samples from ICU patients with AQM. Quantitative and qualitative analyses of myofibrillar protein and mRNA expression were analyzed using SDS-PAGE, confocal microscopy, histochemistry and real-time PCR. The present results demonstrate that the transcriptional regulation of myofibrillar protein synthesis plays an important role in the loss of contractile proteins, as well as the recovery of protein levels during clinical improvement, myosin in particular, presumably in concert with proteolytic pathways, but the mechanisms are specific to the different thick and thin filament proteins studied.