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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.

The thoracic diaphragm is the muscle that drives the respiratory cycle of a human being. Using a system of partial differential equations (PDEs) that models linear elasticity we compute displacements and stresses in a two-dimensional cross section of the diaphragm in its contracted state. The boundary data consists of a mix of displacement and traction conditions. If these are imposed as they are, and the conditions are not compatible, this leads to reduced smoothness of the solution. Therefore, the boundary data is first smoothed using the least-squares radial basis function generated finite difference (RBF-FD) framework. Then the boundary conditions are reformulated as a Robin boundary condition with smooth coefficients. The same framework is also used to approximate the boundary curve of the diaphragm cross section based on data obtained from a slice of a computed tomography (CT) scan. To solve the PDE we employ the unfitted least-squares RBF-FD method. This makes it easier to handle the geometry of the diaphragm, which is thin and non-convex. We show numerically that our solution converges with high-order towards a finite element solution evaluated on a fine grid. Through this simplified numerical model we also gain an insight into the challenges associated with the diaphragm geometry and the boundary conditions before approaching a more complex three-dimensional model.

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 (

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 wa

Mechanical ventilation (MV), however brief results in the loss of diaphragm muscle mass and strength, termed ventilator induced diaphragm dysfunction (VIDD). VIDD increases dependence, complicates and prolongs weaning and significantly increases discharge mortality rate and health care costs worldwide. The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway was recently identified as an important signalling pathway implicated in VIDD, upregulated in the diaphragm following MV and limb muslces during critical care. Regulation of STAT3 is imperritve to skeletal muscle mass and function, as STAT3 is required in proper muscle growth and regeneration, while chronic activation of STAT3 is implicated in muscle dysfunction. As JAK/STAT pathway inhibition can restrict the development of chronic muscle wasting conditons, this study aimed to explore the therapeutic potential of Ruxolitinib, an approved JAK1/2 inhibitor for myelofibrosis, for treatment of CIM. We hypothesised Ruxolitinib would reduce loss of muscle mass and function associated with VIDD. Here, rats were subjected to five days controlled MV (CMV) with and without daily Ruxolitinib gavage. Five-days CMV significantly reduced diaphragm muscle size and impaired specific force, which was associated with 2-fold upregulation of P-STAT3, disrupted mitochondrial structure and respiratory function. Expression of the motor protein myosin was not affected, however CMV may alter myosin function through deamidation post translational modification. Ruxolitinib increases five-day survival rate, restored P-STAT3 expression and preserved diaphragm muscle size and specific force. These functional improvements were associated with improved mitochondrial structure, augmented mitochondrial respiratory function and reversal or augmentation of myosin deamidations. These results provide evidence of the preclinical potential of repurposing Ruxolitinib for the treatment of VIDD.

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 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.

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 post-translational 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.