Skeletal Muscle Regeneration in Survivors of Critical Illness: How to Prevent Satellite Cell Failure?

Study Overview
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Brief Summary
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Modern intensive care enables patients to survive insults that in the past would have been supralethal. Nonetheless, increased number of survivors suffer from failed functional outcomes associated with prolonged muscle weakness and fatiguability. Whilst alterations of skeletal muscle biology that occur during critical illness slowly disappear over the period of months, muscle weakness remains. Recent pilot studies have shown that muscle weakness is associated with loss and alteration of satellite skeletal muscle cells, which are supposed to proliferate and repair damaged muscle tissue. The pathogenesis of this phenomenon has not been fully understood. In this grant project, we will study function and structure of satellite cells and their organelles (particularly mitochondria) using both classical bioenergetics and advanced microscopic techniques. Satellite cells will be isolated from biopsies taken from critically ill patients with developed muscle weakness in the acute and protracted phase of a disease and after 6 months. In time points, an ultrasound examination of muscle mass will be performed, and metabolism will be assessed using insulin clamps. In an in vitro experiments, we will test also effect of nutritional and anabolic factors and drugs, commonly used in ICU, on satellite cells. In a control branch, cells will be isolated from skeletal muscle of volunteers undergoing elective hip replacement surgery. Results of this study could significantly contribute to understanding of mechanisms leading to ICU acquired muscle weakness and to identify therapeutic strategy in future.

Detailed Description
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Background: Muscle weakness is a common complication in patients who survived a serious critical illness or trauma. Altered muscle strength and functional ability significantly worsens the patients' performance and quality of life. Specific treatment does not exist and the pathogenesis is not yet fully understood. Acute sepsis or extensive inflammatory response are the main risk factors for the muscle weakness development in critically ill patients. Recent studies have shown that muscle weakness can be caused by the loss of ability of skeletal muscle cells to react to the injury and regenerate. Skeletal muscle satellite cells, which are localized beneath the basal lamina of individual muscle fibers, are responsible for muscle regeneration. After the muscle damage, satellite cells are activated from quiescent state (G0 phase) and enter the cell cycle (G1 phase). Subsequently, they proliferate and differentiate into the myoblasts which then fuse and form multinucleated cylindrical myotubes. The cells then merge into the myofibrils and join the muscle fibers that were not damaged. Some satellite cells return to the G0 state to replenish the pool of quiescent skeletal muscle cells. In response to satellite cell damage, mitochondrial biogenesis and synthesis of new myofibrillar proteins are activated to build the new muscle mass containing new intracellular content. Thus, satellite cells have a crucial role for muscle fiber regeneration. Pilot studies performed on animal models (e. g. laboratory mice that developed the acute sepsis) demonstrated a reduction in mitochondrial content and DNA, increased production of reactive oxygen species and changes in oxidative phosphorylation. The abnormalities in the bioenergetic profile of satellite cells are considered a cause of their reduced ability to regenerate. However, the exact mechanism has not yet been fully elucidated. Changes in the mitochondrial structure and dynamics of satellite cells in critically ill patients are also unknown. In last years, the association of mitochondrial functions with mitochondrial dynamics has been investigated in various pathological conditions and diseases. Depending on external insults and metabolic demands, mitochondria undergo dramatic shape changes that can have a very significant impact on cellular metabolism. The balanced process of mitochondrial fission and fusion plays a key role in the mitochondrial biogenesis and removal of damaged mitochondria. The process is absolutely necessary for the proper growth and function of the muscle tissue. Mitochondrial dynamics and morphology can be altered under pathological circumstances: under the mild stress and starvation, mitochondrial morphology can change from small spheres or short rods to long tubules with an increased capacity for oxidative phosphorylation. On the other hand, acute severe stress leads to a mitochondrial fission and defective oxidative phosphorylation. Several studies performed on animal models demonstrated that proteins responsible for the process of mitochondrial fusion and fission are absolutely crucial for the proper growth and function of skeletal muscle cells. Therefore, alterations in mitochondrial dynamics often play a crucial role in skeletal muscle dysfunction and have been recently extensively studied in several myopathies. In this project, the investigators would like to investigate the causal relationships between mitochondrial function and their shape in satellite cells obtained from critically ill patients in the acute, protracted and post-ICU phase of critical illness. Hypotheses and aims of the project: In light of this, the investigators hypothesize that critical illness induces damage to satellite cells in skeletal muscle that later impairs skeletal muscle structural and functional recovery and contributes to persistent weakness and failed functional outcome. First, structural and functional (incl. bioenergetic) characteristics of satellite cells will be compared in acute, protracted and post-ICU phase in critically ill patients vs. control subjects. Second, the investigators will test the hypothesis that structural and/or functional alteration of satellite cells corelate with gross muscle mass and power in survivors of critical illness. Third, the factors that influence bioenergetics and mitochondrial morphology of satellite cells will be studied (such as extracellular inflammatory milieu, drugs common in ICU as well as nutritional and anabolic factors). Design: Prospective cohort study with exploratory physiological end-points. Study subjects: Critically ill patients: receiving mechanical ventilation to be enrolled within 72 hours of admission, who are likely to need 7 days or more of ICU stay; sudden onset of disease, which can be determined in time (such as trauma, stroke, sudden cardiac arrest etc.). Control subjects: orthopedic patients undergoing elective hip replacement surgery with a very good to excellent performance status, only limited to joint pain (ECOG 0). Informed consent procedure: All patients with capacity will be asked to provide a prospective written informed consent. For patients without capacity, a deferred consent procedure will take a place. In this case, an independent clinician will review and sign that the patient is lacking capacity and he/she fulfills all criteria to be enrolled to the study. The patient's next of kin will be informed about the study as soon as practical with the aid of information leaflet. The patient will be asked to provide and sign informed consent as soon as he/she regains the capacity to do so. In case of consent refusal, patient's data will not be used in per-protocol analysis. Methods: General methodological approaches to be used are as follows. Time points: Eligible patients will be assessed at the baseline, after 7 days and after 6 months by clinical tests, metabolic tests and muscle biopsies. Briefly, on day 0 muscle mass will be assessed using diagnostic ultrasound (a measurement of rectus muscle cross-sectional area) and biopsy of musculus vastus lateralis will be performed using Bergström needle. Baseline blood samples will be taken, plasma will be separated and frozen at -80° C for the later analysis of cytokines and hormone levels. Urine samples will be collected daily, surfaced with toluene and stored in a deep freeze facility for later determination of nitrogen content and 3-methyl histidine levels (to calculate muscle catabolism rate and nitrogen balance). On day 7, all above mentioned procedures will be performed again. In addition, whenever the patient regains consciousness, also muscle power by Medical Research Council (MRC) score will be assessed (standardized testing of muscle power [0-5] on 12 muscle groups on all 4 limbs, giving the score 0-60 (60 suggesting normal muscle power)). At ICU discharge, the patients and relatives will be asked to provide contact details for 6 months follow up. On day 180, all the procedures will be repeated. Furthermore, insulin sensitivity will be measured after overnight fasting by hyperinsulinaemic euglycemic clamp on days 7 and 180. Clinical outcomes: will be assessed by objective validated tests such as SF-36 questionnaire for the quality of life assessment, Medical Research Council Score of muscle power and 6-minutes walking test to measure aerobic performance. Metabolic characteristics: will be assessed at the whole body level by hyperinsulinaemic euglycaemic clamps and at the tissue level by vastus lateralis biopsies. Insulin sensitivity and substrate oxidation will be measured after overnight fasting by hyperinsulinaemic euglycemic clamp. Laboratory procedures: mitochondrial research: muscle tissue biopsies will be performed from vastus lateralis muscle by Bergström needle biopsy technique. The sample from each biopsy will be separated into three parts (from 25-100mg per each). One part will be immediately frozen in liquid nitrogen-cooled isopentane for later analysis of muscle fibres typing and immunohistochemistry analysis. The second part will be placed into the respiration medium on ice for the preparation of homogenates and measurement on high-resolution respirometry which enables to determine the function of individual respiratory complexes in the cytosolic context and measure basic functional metabolic indices. Mainly, ATP production, mitochondrial uncoupling, electron transport chain capacity and respiration linked to individual complexes will be assessed. The third part will be used for isolation and culture of skeletal muscle cells. Firstly, satellite cells from biopsies using FACS or magnetic beads will be isolated. The satellite cells will be cultivated and global mitochondrial indices will be assessed by measurement of oxygen consumption rate. This will be processed with extracellular flux analyzer or high resolution respirometry which enables measurement of oxygen consumption rate and lactate production in a real time. This enables to determine ATP production in living cells, uncoupling of inner mitochondrial membrane, maximal respiratory capacity of respiratory chain, glycolytic capacity and fatty acid oxidation or respiration linked to individual complexes of respiratory chain. Simultaneously, reactive oxygen species and mitochondrial membrane potential will be measured. Additionally, shape and size of mitochondria, density of mitochondrial network and dynamic arrangement of these interconnected organelles will be analyzed using confocal laser scanning microscopy in live-cell imaging. All the parameters will be studied in the acute, protracted and post-ICU phase of critical illness.

Official Title
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Skeletal Muscle Regeneration in Survivors of Critical Illness: How to Prevent Satellite Cell Failure?

Conditions
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Intensive Care Unit-acquired Weakness

Participation Criteria
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Eligibility Criteria
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Eligibility Criteria for group of Critically ill patients: Inclusion Criteria: Critically ill patients receiving mechanical ventilation, to be enrolled within 72 hours of admission, who are likely to need 7 days or more of ICU stay Sudden onset of disease, which can be determined in time (such as trauma, stroke, sudden cardiac arrest etc.) Informed consent signed by patient or patient's representative Exclusion Criteria: Unlikely to survive 6 months Premorbid downslope functional trajectory or poor performance status (ECOG Gr. 3 or worse) or baseline functional status unknown Bleeding disorder (INR≥1.5 or PLT< that would preclude muscle biopsies) Known mitochondrial disease Endocrine crisis as a reason for admission Pregnant women Eligibility Criteria for a control group: Elective hip surgery patients with a very good to excellent performance status, only limited to joint pain (ECOG 0)

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Critically ill patients<br>Critically ill patients with sudden onset of disease receiving mechanical ventilation, to be enrolled within 72 hours of admission, who are likely to need 7 days or more of ICU stay. | |
| Volunteers with a very good to excellent performance status<br>Elective hip surgery patients with a very good to excellent performance status, only limited to joint pain (ECOG 0) | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Quality of life as per 36-Item Short Form Health Survey (SF-36) | A set of quality-of-life measures. A questionnaire includes questions about: physical functioning, bodily pain, role limitations due to physical health problems, role limitations due to personal or emotional problems, emotional well-being, social functioning, energy/fatigue, and general health perceptions. The minimum and maximum scores are 0 and 100. A higher score defines a more favorable health state. | on day 180 | 
| Changes of muscle mass between baseline, day 7 and day 180 | By measurement of musculus rectus femoris cross-sectional area by diagnostic ultrasound. | Changes between days 0, 7 and 180 | 
| A measurement of muscle power by Medical Research Council Score | A standardized testing of muscle power [0-5] on 12 muscle groups on all 4 limbs using Medical Research Council Score, giving the score from minimum 0 to maximum 60. A higher score defines a more favorable health state, 60 points suggest a normal muscle power. | on day 180 (eventually on day 7 if the patient is conscious) | 
| Changes of mitochondrial function of satellite cells between baseline, day 7 and day 180 | Mitochondrial functional parameters will be assessed by Extracellular XF24 Seahorse Analyzer or high-resolution respirometry which enables continous real-time measurement of oxygen consumption in living cells at the baseline and after addition of various substrates, uncouplers and inhibitors of the respiratory chain. This allow to estimate parameters as ATP production, maximal respiratory capacity, respiration in baseline etc. The techniques measure oxygen consumption rate of living cells in pmol/min. | Changes between days 0, 7 and 180 | 
| Changes of mitochondrial structure of satellite cells between baseline, day 7 and day 180 | A mitochondrial structure and architecture of its network (mitochondrial density and a length of its branches etc.) will be assessed after staining of mitochondria by fluorescent probes and imaging on confocal laser scanning microscopy. The length/density will be measured in microns/microns 2. | Changes between days 0, 7 and 180 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Nitrogen balance measured in g/m2 of body surface area | From collected urine samples, a determination of nitrogen content and 3-methyl histidine levels will be performed (to calculate muscle catabolism rate and nitrogen balance). | on first 7 days | 
| 6-minutes walking test to measure aerobic performance | 6-minutes of exercise (walking) test used to assess aerobic capacity and endurance. | on day 180 | 
| Changes of insulin sensitivity between days 7 and 180 | This parameter will be measured after overnight fasting by hyperinsulinaemic euglycemic clamp. | Changes between days 7 and 180 | 
| Length of ICU stay in days | A number of days spent on ICU. | on day 28 | 
| Number of ventilator-free days | A number of ventilator-free days spent on ICU. | on day 28 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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intensive care unit-acquired weakness, critically ill, skeletal muscle regeneration, satellite cells