Critical illness myopathy : mechanisms and pharmacological interventions

The human body typically contains over 600 skeletal muscles that make up about 40% of total body weight. These muscles work together to perform functions such as locomotion, breathing, mastication, heat regulation and speech. Skeletal muscles can change their size and protein content in response to endogenous or exogenous signals, including physical, neural or chemical ones. Critically ill patients, usually treated intensively, are prone to developing a condition of muscle wasting and paralysis, called critical illness myopathy (CIM), where limb and truck muscles suffer severe atrophy and loss of force production capacity coupled with a preferential myosin loss, but craniofacial muscles remain less affected. Triggers of CIM are thought to be the exposure to the intensive care unit (ICU) interventions per se, such as unloading, mechanical ventilation (MV) and high doses of certain drugs such as muscle relaxants and glucocorticoids (GCs). The rapidly compromised diaphragm function due to the impact of the ventilator has been given a specific name, ventilator induced diaphragm dysfunction (VIDD). CIM and VIDD have dire consequences and research into their underlying mechanisms is urgently needed. This research is inherently difficult in patients and thus suitable animal models mimicking the ICU condition must be implemented. In this thesis, we used a pig and a rat ICU models with extended periods of immobilization, deep sedation and MV, in which we used different analyses to understand the muscle specific mechanistic differences of the masticatory, limb and the diaphragm muscles. In addition, we explored the effects of two new drugs on skeletal muscles: BGP-15, a chaperone co-inducer and vamorolone, a first-in-class dissociative GC. In paper I, we report that a 5-day GC treatment in the pig ICU model induces numerous transcriptional changes that affect myofiber function in a limb muscle. In paper II, we conclude that the masseter, the main masticatory muscle, is partially protected from CIM effects by several mechanisms that reduce proteolysis, including early heat shock protein (HSPs) activation. In paper III, we report that treatment with BGP-15 activates HSP70 and improves the diaphragm muscle fiber function in young but not old rats. In the last paper, we report differences between the new GC, vamorolone and the traditional GC, prednisolone, in the rat ICU model where the former drug shows less negative effects on fast twitch EDL muscle and both show positive effects on the slow twitch soleus muscle. These results emphasize the uniqueness of each muscle response to ICU interventions and also shed some light on a couple of promising pharmacological interventions that may counteract CIM deleterious effects

Masseter Muscle Myofibrillar Protein Synthesis and Degradation in an Experimental Critical Illness Myopathy Model

Critical illness myopathy (CIM) is a debilitating common consequence of modern intensive care, characterized by severe muscle wasting, weakness and a decreased myosin/actin (M/A) ratio. Limb/trunk muscles are primarily affected by this myopathy while cranial nerve innervated muscles are spared or less affected, but the mechanisms underlying these muscle-specific differences remain unknown. In this time-resolved study, the cranial nerve innervated masseter muscle was studied in a unique experimental rat intensive care unit (ICU) model, where animals were exposed to sedation, neuromuscular blockade (NMB), mechanical ventilation, and immobilization for durations varying between 6 h and 14d. Gel electrophoresis, immunoblotting, RT-PCR and morphological staining techniques were used to analyze M/A ratios, myofiber size, synthesis and degradation of myofibrillar proteins, and levels of heat shock proteins (HSPs). Results obtained in the masseter muscle were compared with previous observations in experimental and clinical studies of limb muscles. Significant muscle-specific differences were observed, i.e., in the masseter, the decline in M/A ratio and muscle fiber size was small and delayed. Furthermore, transcriptional regulation of myosin and actin synthesis was maintained, and Akt phosphorylation was only briefly reduced. In studied degradation pathways, only mRNA, but not protein levels of MuRF1, atrogin-1 and the autophagy marker LC3b were activated by the ICU condition. The matrix metalloproteinase MMP-2 was inhibited and protective HSPs were up-regulated early. These results confirm that the cranial nerve innervated masticatory muscles is less affected by the ICU-stress response than limb muscles, in accordance with clinical observation in ICU patients with CIM, supporting the model' credibility as a valid CIM model

Primers and probes used in real time PCR analyses.

<p>Primers and probes used in real time PCR analyses.</p

Hematoxylin and eosin (H&E) staining and cross-sectional area (CSA).

<p><b>A, B, C, D</b>) H&E staining for the measurement of CSA of 0.25–4, 5–8 and 9–14 day groups, respectively, <b>E</b>) CSA (μm²) of the masseter muscle fibers, and <b>F</b>) the lesser fiber diameter (μm). Asterisks (**p<0.01) denote significant differences compared with controls. Values are means + SEM.</p

MuRF1, atrogin-1 and LC3b mRNA expression.

<p><b>A</b>) MuRF1 (black bars) and atrogin-1 (grey bars), <b>B</b>) LC3b mRNA expression, according to real-time PCR in the masseter muscle. Asterisks (*p<0.05) denote significant differences compared with controls. Values are means of starting quantities + SEM.</p

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 five-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 Effect of Nutritional Status in the Pathogenesis of Critical Illness Myopathy (CIM)

The muscle wasting and loss of specific force associated with Critical Illness Myopathy (CIM) is, at least in part, due to a preferential loss of the molecular motor protein myosin. This acquired myopathy is common in critically ill immobilized and mechanically ventilated intensive care patients (ICU). There is a growing understanding of the mechanisms underlying CIM, but the role of nutritional factors triggering this serious complication of modern intensive care remains unknown. This study aims at establishing the effect of nutritional status in the pathogenesis of CIM. An experimental ICU model was used where animals are mechanically ventilated, pharmacologically paralysed post-synaptically and extensively monitored for up to 14 days. Due to the complexity of the experimental model, the number of animals included is small. After exposure to this ICU condition, animals develop a phenotype similar to patients with CIM. The results from this study show that the preferential myosin loss, decline in specific force and muscle fiber atrophy did not differ between low vs. eucaloric animals. In both experimental groups, passive mechanical loading had a sparing effect of muscle weight independent on nutritional status. Thus, this study confirms the strong impact of the mechanical silencing associated with the ICU condition in triggering CIM, overriding any potential effects of caloric intake in triggering CIM. In addition, the positive effects of passive mechanical loading on muscle fiber size and force generating capacity was not affected by the nutritional status in this study. However, due to the small sample size these pilot results need to be validated in a larger cohort

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