Mechanisms of Chronic Muscle Wasting and Dysfunction after an Intensive Care Unit Stay: A Pilot Study. Online Data Supplement

Transcription

1 Mechanisms of Chronic Muscle Wasting and Dysfunction after an Intensive Care Unit Stay: A Pilot Study C dos Santos, SNA Hussain, S Mathur, M Picard, M Herridge, J Correa, A Bain, Y Guo, A Advani, SL Advani, G Tomlinson, H Katzberg, CJ Streutker, JI Cameron, A Schols, H Gosker, and J Batt, for the MEND ICU group, the RECOVER Program investigators and the Canadian Critical Care Translational Biology Group Online Data Supplement

2 METHODS Participants We recruited our patient population from the RECOVER program (Phase 1; Toward RECOVER), a multi-centre prospective longitudinal study evaluating functional outcomes in critically ill patients following prolonged mechanical ventilation over a 1 year period after ICU discharge (1). were eligible for Towards RECOVER if they were: older than 16 years of age and mechanically ventilated for a minimum of one week in an ICU. They were excluded for any of the following: important current or pre-existing neurological injury that would preclude cognitive testing; a formal diagnosis of neuromuscular disease; lack of independence with activities of daily living or non-ambulatory prior to admission; anticipated death or withdrawal of life sustaining treatment within 48 hours; history of psychiatric illness or significant cognitive impairment with documented hospital admission; not fluent in English or French; living greater than 3 km from referral centre; physician refusal; patient or SDM (substitute decision maker) consent refusal; and no available next of kin or SDM available (if patient unable to provide consent). Additional exclusion criteria for this nested study included important current neurological injury that would preclude motor testing; known HIV, Hepatitis B or Hepatitis C infection; therapeutic anticoagulation and/or active cancer undergoing treatment. As we were interested in juxtaposing those patients at 6 months who continued to be weak with minimal improvement vs those who demonstrated substantial improvement, we based our sample size estimate on the clinical measure of weakness. We estimated, based on previous reports, the proportion of patients followed who would present with weakness and functional dependency compared to those who presented with minimal to no functional dependence and weakness at 6 months would be around 25%. With this estimate, we determined 12 patients E2

3 would be sufficient to provide the number of patients required to detect a significant difference between the two proportions (confidence level of 95%, power of 8%).[ For the purpose of the muscle molecular analyses outlined below previously banked muscle biopsy specimens collected from consenting healthy individuals at St Michaels Hospital and Toronto General Hospital Toronto (n = 9, Toronto, Canada), were used for comparison (Table E1). For the purpose of ultrastructure analyses, EM sections from previously banked muscle biopsy specimens collected from consenting healthy individuals at the University Hospital Maastricht, (n = 4, Maastricht, Netherlands) were used for comparison (Table E1). All volunteers had been screened by interview and self-reported the absence of malignancy, significant cardiac, pulmonary, hepatic, renal or endocrine disease. Targeted testing to exclude co-morbidities in the these individuals however, was not undertaken. Assessment of Baseline Physical Functional Capacity The Duke Activity Index The Duke Activity Status Index (DASI), which is a questionnaire that estimates an individual s peak oxygen uptake by assessing the ability to perform specific physical tasks (2), was used to estimate each patient s baseline physical functional capacity prior to their ICU admission. The questionnaire was administered after discharge from the ICU either at their 7 day or 6 month assessment, and thus was a retrospective recall of their physical capacity during health. In the DASI, each activity item has a specific weight based on its metabolic cost (MET). The final score ranges between zero and 58.2 points; the higher the score, the better the functional E3

4 capacity. The DASI score can be used to estimate the peak oxygen uptake (VO2 max = DASI * ) ; ie 58.2 estimating a peak oxygen uptake of 34.6mL/kg.min). Normal values in the population vary according to age, sex and level of fitness, but reported values for healthy, untrained males and females aged 2 to 7 years range from 34 47mL/kg.min and ml/kg.min respectively (3, 4). While the DASI has been validated to predict patients cardiovascular fitness for surgery, it has not been validated in the ICU patient population Indicators of Illness Severity APACHE II (Acute Physiology and Chronic Health Evaluation score II) APACHE II is a severity of disease classification score applied within 24 hours of ICU admission, that ranges from to 71 (5). A higher score is indicative of more severe disease and likelihood of death. Charlson Index Charlson Index predicts 1 year mortality based on a range of comorbid conditions with a higher score indicative of higher mortality (6). Outcome Measures of Physical Functioning, Strength and Muscle Mass 6 Minute Walk Distance The 6 minute walk distance was performed as directed by the American Thoracic Society established criteria with standardized instruction and encouragement as previously described (7). E4

5 It has been validated in survivors of critical illness. Absolute distances were measured, and the percent predicted 6 minute walk distance was then calculated for each patient. The predicted 6 minute walk distance is determined using reference equations previously described that normalize for gender, age, height and weight (7, 8), and specifies the distance a healthy individual of the same age, sex and body morphometric proportions would be able to walk. Functional Independence Measures (FIM) questionnaire Functional Independent Measures questionnaire (FIM) provides a numerical score for cognitive and motor function that has been validated and standardized across diverse patient populations. A higher FIM score (scale ) connotes better function in both domains (9). The motor subscore is based on the individual s ability to perform their activities of daily living (ADLs; toileting, dressing, walking, climbing stairs, eating). A healthy individual with complete and unassisted independence of ADL would achieve a maximal FIM motor subscore of 91. The measure is independent of an individual s gender or age. Medical Research Council (MRC) Sum Score Global muscle strength testing. Manual assessment of muscle strength was performed by a physiotherapist, grading muscle strength from to 5, as established by the Medical Research Council (1). The higher the score, the stronger the muscle group, with 5 representing what is deemed by the assessor to be normal strength, taking into account the individual s sex and age. The MRC sum score is calculated by tallying the total score for bilateral shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. The score for an individual with normal strength is 6 (11). E5

6 Determination of Quadriceps Isometric Peak Torque Quadriceps isometric peak torque (strength) was measured from maximal voluntary contractions of the knee extensors. Testing was undertaken at the University of Toronto Muscle Function & Performance Laboratory (Toronto, ON, Canada). The patient was seated on the Biodex dynamometer (Biodex System 4), with the hip at 85 degrees and knee at 6 degrees of flexion. The patient performed five maximal voluntary contractions with one minute rests, according to a previously described protocol (12). Peak torque was recorded in Newton-meters (Nm). peak torque was reported in absolute terms and as percentage of the patients predicted normals, determined using 2 different validated predictor equations derived by Gosselink et al (13) and Harbo et al (14) where sex, age, weight and height are taken into account, University of Toronto Muscle Function & Performance Laboratory based sex and age (year-specific) norms of isometric knee extensor peak torque for healthy individuals have been established (unpublished data), and were available for comparison to patients measured peak torque. These norms were not weight and height matched. Determination of Whole thigh and Quadriceps Cross Sectional Area The midthigh quadriceps femoris muscle cross-sectional area (CSA) was determined as previously described (15). Briefly, computed tomography imaging of the right thigh, halfway between the pubic symphysis and the inferior condyle of the femur, using a Light Speed QXi 4 slice helical scanner (General Electric, Milwaukee, WI), was performed with the subject in the supine position. Each image was 1 to 2 mm thick, and the muscle identified as tissue with a E6

7 density of 4 to 1 Hounsfield units. Images were analyzed and the CSA of thigh muscle determined by a single radiologist, blinded as to the categorization of each test subject. Quadriceps was then manually traced using ImageJ software (version 1.42q, NIH, USA) by a single investigator blinded to participant categorization and the CSA calculated. Determination of Quadriceps Voluntary Contractile Capacity As a measure of quadriceps contractility, we calculated the quadriceps muscle specific force, which normalizes the muscle power to the muscle size (quadriceps isometric peak torque Nm/quadriceps mid thigh cross sectional area cm 2 ) Electromyography and Nerve Conduction Studies Peripheral nerve conduction studies were performed to rule out a major neuropathic process that would distort interpretation of muscle pathology. The side selected for study was one with the least number of arterial/venous lines to minimize interference with recordings. Right or left peroneal motor, tibial motor and sural sensory tests were initially conducted. If these were normal, muscle electromyography was then performed. If these were abnormal, nerve conduction testing was conducted on the upper extremities; median motor and sensory (antidromic) studies with F wave, ulnar motor and sensory (antidromic) studies and radial sensory (antidromic) response, were assessed. Measurement of the upper limb nerves was undertaken because lower limb nerves are very vulnerable to technical effects from ICU related phenomenon (i.e. edema) and may falsely indicate a peripheral neuropathy that is not a truly systemic problem. Muscle needle EMG studies were performed on the deltoid, triceps, tensor fascia lata and vastus lateralis. These muscle were selected for study, since 1) distal muscles are more likely to E7

8 be affected by common compression palsies than proximal, and 2) the muscle selected are most commonly affected clinically by myopathic processes. Muscle Biopsy and Molecular Analyses Vastus Lateralis Percutaneous Biopsy Biopsy of the vastus lateralis muscle was performed under local anesthetic using a modified Bergstrom needle, as previously described (16). Briefly, under sterile conditions, the skin and subcutaneous tissue overlying the lateral aspect of the distal third of the muscle were anesthetized, and a small incision was made in the outer fascial layer with a scalpel blade. The Bergstrom needle was advanced through the incision roughly 1 cm into the muscle, suction was applied as the trochar was advanced, and several pieces of muscle tissue were obtained. The needle was withdrawn under counter pressure, the skin closed with a single suture, and a pressure dressing applied. Tissue (approx. 2mg in total) was immediately sectioned with a sterile scalpel blade and pieces were processed for electron and light microscopy or flash frozen in liquid N2. Protein extraction and Western blotting Total protein was extracted from flash frozen tissue by homogenizing (Polytron PT 12E, Kinematica, Lucerne, Switzerland) the muscle in Muscle Lysis Buffer [5 mm Tris-HCl ph 8., 1 mm EDTA, 1 mm EGTA, 1 mm b-mercaptoethanol, 1% glycerol, PMSF (1 mm), leupeptin, aprotinin (1 ug/ml each)] for 3 x 3 sec. and homogenates were centrifuged at 16xg for 1 min at 4ºC. The supernatant was cleared by centrifuging further for 1 min at 4uC, 1,6g. All fractions were quantified using the Pierce (Rockford, IL, USA) BCA Protein Assay Kit and E8

9 normalized for equal loading for SDS-PAGE and Western blot analysis. 25 or 5 µg of muscle lysates were separated by SDS-PAGE, transferred to nitrocellulose, Ponceau-S stained and immunoblotted. The commercial primary antibodies used included anti-ubiquitin, -LC3B, -Beclin1, - VSP 34, - Bnip3, -GAPDH and -tubulin. Secondary antibodies were Horseradish peroxidase (HRP)-linked anti-rabbit, anti-mouse or anti-rat and protein bands detected with Biorad ECL (Biorad Laboratories, Hercules, CA, USA) or Immun Star Western C (Biorad Laboratories). Quantification of the chemiluminescent signal from Western blots was performed with Bio-Rad Fluor S Max Acquisition System (Biorad Laboratories) and Image Lab software (Biorad Laboratories) (17) or alternatively densitometry as described (18). 2S Proteasome Measurements 2S proteasome activity (Chymotrypsin-like activity) was measured using the Chemicon 2S Proteasome Activity Kit (APT 28) as directed by the manufacturer. The 2S proteasome forms the catalytic core of the 26S proteasome. Briefly, muscle was lysed in muscle lysis buffer in the absence of protease inhibitors and stored in aliquots at -8C. 1ug of protein was incubated with the proteasome substrate LLVY-AMC, with or without the proteasome inhibitor lactacystin in the presence of SDS. Cleavage of LLVY-AMC by the proteasome generates the fluorophore 7- AMC, and the fluorescence is measured using a 38/46 filter set in a fluorimeter. Standard curves are generated using 2S proteasome positive control and AMC standards. The amount of 2S proteasome activity per sample is calculated by measuring the extent of fluorescence generated per sample in the absence of lactacystin minus the presence of lactacystin, and comparison to standard curves. E9

10 Electron Microscopy Samples for TEM were fixed in 2% glutaraldehyde in.1m sodium cacodylate buffer, or.1m phosphate buffer with 2.5% glutaraldehyde at ph7.4, rinsed in buffer, post-fixed in 1% osmium tetroxide in buffer, dehydrated in a graded ethanol series followed by propylene oxide, and embedded in Quetol-Spurr resin or epoxy resin (Glycid ether 1; Serva, Heidelberg, Germany). Sections (6 or 1nm thick) were cut on an RMC MT6 ultramicrotome, stained with uranyl acetate and lead citrate and viewed in an FEI Tecnai 2 TEM. Light Microscopy, and Immunohistochemistry Muscle biopsies were fixed in 1% buffered formalin phosphate for 24 hours at room temperature, rinsed in ethanol, paraffin embedded and sectioned (1 µm thickness) on cross section. The sections were rehydrated in a series of xylene and ethanol washes and then endogenous peroxidases were quenched with a 3 min. incubation in.3% H 2 O 2. Antigen retrieval was performed by microwaving the sections in 1 mm sodium citrate, ph 6., 3x5 min at 9% power (72 Watt oven). Sections were blocked with Protein Block Serum Free (Dako/Agilent, Mississauga, Canada). Sections were rinsed and fibres were immunostained using anti-pax-7 (Pax 7, Developmental Studies Hybridoma Bank, University of Iowa, USA), or anti-cd56 (123C3, Roche, Basel, Switzerland), markers of muscle satellite cells/myoblasts, followed by biotinylated secondary antibody and streptavidin-hrp/dab (Vectastain ABC Elite Peroxidase kit, Vector Laboratories Inc., Burlingame, CA, USA) or the Opti-view DAB IHC Amp kit with (Roche) respectively. For negative controls, the primary antibody was omitted E1

11 during immunostaining. Hematoxylin was used as a counterstain. The number of satellite cells, and myofibres were counted by 2 individuals blinded to sample identification on randomly selected areas of each histologic section such that a minimum of 5 myofibres per study participant were examined. Histologic cross sections were also prepared as noted above for immunostaining with antimyosin, fast twitch antibody (My-32, Sigma), which immunostains type II fast twitch fibres. Type I fibres remain unstained, and appear light purple with hematoxylin counterstaining. Fibre type specific cross sectional area was independently determined using ImageJ software by 4 individuals blinded to sample identification as previously described (19). To assess for inflammatory infiltrate, sections were immunostained with anti-cd45 (Abcam 1257, Abcam, Cambridge MA, USA), anti-cd68 (Abcam 12547) or anti-cd11c (Abcam 52632) antibodies, and detected with streptavidin/hrp-dab. The number of CD45, CD68 and CD11c positive cells and myofibres were counted by 2 individuals blinded to sample identification on randomly selected areas of each histologic section such that a minimum of 5 myofibres per study participant were examined. To determine muscle vascularity, histologic sections were immunostained for the endothelial cell marker, PECAM-1 (Abcam 28364). The number of capillaries and myofibres were counted by an individual blinded to sample identification and the vascularity described as number of capillaries per myofibre. A minimum of 5 myofibres were assessed. Mitochondria Analyses For electron microscopy analyses, samples were fixed and processed as above. Ultrathin sections were imaged in the longitudinal orientation at 13,5x magnification. Areas exhibiting E11

12 sarcomere hypercontraction, subsarcolemmal compartment or myonuclei were avoided. To quantify mitochondrial size and volume density, twelve randomly selected micrographs yielding on average 787 ± 312 (mean ± SD) intermyofibrillar mitochondria were analyzed for each subject. Mitochondrial size measurements were obtained as previously described (2), using Image J (version 1.42q, NIH, USA) by manually tracing discernable outlines of mitochondria. Mitochondrial surface area is reported in µm 2, and skeletal muscle mitochondrial volume density (mitochondrial content) was computed by dividing the sum of mitochondrial area and total surface area analyzed, thus generating % of muscle volume occupied by mitochondria. Computed values were imported into Microsoft Excel and Prism 6 (GraphPad Software) for data analysis. To produce frequency distributions of mitochondrial size, each mitochondrion was assigned to one of twenty bins of equal sizes and proportions were determined, yielding frequency histograms with overlaid fitted curves to facilitate comparison. Statistical analyses consisted of one-way ANOVA for between group differences, and paired student s T-test to compare 7 days and 6 months time points in patients. Reference List 1. Herridge MS, Chu LM, Matte A, Tomlinson G, Chan L, Thomas C, Friedrich JO, Mehta S, Lamontagne F, Levasseur M, et al. The RECOVER Program: Disability Risk Groups & One Year Outcome After >/= 7 Days of Mechanical Ventilation. Am J Respir Crit Care Med 216. Mar (epub ahead of print). E12

13 2. Hlatky MA, Boineau RE, Higginbotham MB, Lee KL, Mark DB, Califf RM, Cobb FR, Pryor DB. A Brief Self-Administered Questionnaire to Determine Functional Capacity (the Duke Activity Status Index). Am J Cardiol 1989;64: Heywood V. Advanced Fitness Assessment & Exercise Prescription., 3rd Edition ed. Champaigne, IL, USA Human Kinetics; Guyton A, Hall J. Textbook of Medical Physiology, 12th Ed. ed. Philadelphia, PA, USA WB Saunders Co., Knaus WA, Draper EA, Wagner DP, Zimmerman JE. APACHE II: a Severity of Disease Classification System. Crit Care Med 1985;13: Charlson ME, Pompei P, Ales KL, MacKenzie CR. A New Method of Classifying Prognostic Comorbidity in Longitudinal Studies: Development and Validation. J Chronic Dis 1987;4: Enright PL, Sherrill DL. Reference Equations for the Six-Minute Walk in Healthy Adults. Am J Respir Crit Care Med 1998;158: Cote CG, Casanova C, Marin JM, Lopez MV, Pinto-Plata V, de Oca MM, Dordelly LJ, Nekach H, Celli BR. Validation and Comparison of Reference Equations for the 6-Min Walk Distance Test. Eur Respir J 28;31: Oczkowski WJ, Barreca S. The Functional Independence Measure: Its Use to Identify Rehabilitation Needs in Stroke Survivors. Arch Phys Med Rehabil 1993;74: Medical Research Council. Aids to the Investigation of the Peripheral Nervous System. London, England: Her Majesty's Stationary office.; E13

14 11. Kleyweg RP, van der Meche FG, Schmitz PI. Interobserver Agreement in the Assessment of Muscle Strength and Functional Abilities in Guillain-Barre Syndrome. Muscle Nerve 1991;14: Mathur S, Makrides L, Hernandez P. Test-Retest Reliability of Isometric and Isokinetic Torque in With Chronic Obstructive Pulmonary Disease. Physiotherapy Canada 24;56: Gosselink R, Troosters T, Decramer M. Peripheral Muscle Weakness Contributes to Exercise Limitation in COPD. Am J Respir Crit Care Med 1996;153: Harbo T, Brincks J, Andersen H. Maximal Isokinetic and Isometric Muscle Strength of Major Muscle Groups Related to Age, Body Mass, Height, and Sex in 178 Healthy Subjects. Eur J Appl Physiol 212;112: Marquis K, Debigare R, Lacasse Y, LeBlanc P, Jobin J, Carrier G, Maltais F. Midthigh Muscle Cross- Sectional Area Is a Better Predictor of Mortality Than Body Mass Index in With Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med 22;166: Bourgeois JM, Tarnopolsky MA. Pathology of Skeletal Muscle in Mitochondrial Disorders. Mitochondrion 24;4: Plant PJ, Brooks D, Faughnan M, Bayley T, Bain J, Singer L, Correa J, Pearce D, Binnie M, Batt J. Cellular Markers of Muscle Atrophy in Chronic Obstructive Pulmonary Disease. Am J Respir Cell Mol Biol 21;42: Guo Y, Gosker HR, Schols AM, Kapchinsky S, Bourbeau J, Sandri M, Jagoe RT, Debigare R, Maltais F, Taivassalo T, et al. Autophagy in Locomotor Muscles of With Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med 213;188: E14

15 19. Nagpal P, Plant PJ, Correa J, Bain A, Takeda M, Kawabe H, Rotin D, Bain JR, Batt JA. The Ubiquitin Ligase Nedd4-1 Participates in Denervation-Induced Skeletal Muscle Atrophy in Mice. PLoS ONE 212;7:e Picard M, White K, Turnbull DM. Mitochondrial Morphology, Topology, and Membrane Interactions in Skeletal Muscle: a Quantitative Three-Dimensional Electron Microscopy Study. J Appl Physiol (1985 ) 213;114: E15

16 Control Age Gender Muscle Molecular and Cellular Analyses 1 43 F Myofibre CSA, UPS, Autophagy, Inflammatory infiltrate, Satellite cell, Vascularity 2 38 F Myofibre CSA, UPS, Autophagy, Inflammatory infiltrate, Satellite cell, Vascularity 3 58 F Myofibre CSA, UPS, Autophagy, Inflammatory infiltrate, Satellite cell 4 49 M Myofibre CSA, UPS, Autophagy, Inflammatory infiltrate, Satellite cell, Vascularity 5 22 M Myofibre CSA, UPS, Autophagy, Inflammatory infiltrate, Satellite cell, Vascularity 6 4 M Myofibre CSA, UPS, Autophagy, Inflammatory infiltrate, Satellite cell, Vascularity 7 31 F Myofibre CSA, UPS, Inflammatory infiltrate, Satellite cell, Vascularity 8 38 F Myofibre CSA, UPS, Inflammatory infiltrate, Vascularity 9 3 M Myofibre CSA, UPS, Inflammatory infiltrate, Satellite cell, Vascularity 1 62 M Mitochondrial analyses (EM) M Mitochondrial analyses (EM) F Mitochondrial analyses (EM) M Mitochondrial analyses (EM) Table E1 Banked muscle biopsy specimens from healthy individuals Demographics of the healthy individuals from whom banked quadriceps (vastus lateralis) biopsy specimens were obtained. The cellular and molecular analyses for which they were used are indicated.

17 13 Towards RECOVER participants 2 MSICU and 1 Trauma ICU Sept 21 April died 19 excluded 23 missed 32 refused 27 consented 15-7 day biopsy 11-6 month biopsy 2 - withdrew 1 repatriated 1 medical issues necessitated withdrawal 12-7 day no biopsy 2 withdrew 4 died ICU 2 medical issues necessitated withdrawal 1 attempted biopsy unsuccessful 1 - repatriated 2 withdrew reasons unknown 15 patients completed 7 day biopsy/assessment 11 completed 6 month biopsy/assessment Figure E1 Patient Recruitment

18 missed or excluded Median (25%,75%) N = 44 declined consent Median (25%,75%) N = 32 consented; no biopsy Median (25%,75%) N = 12 consented; biopsied Median (25%,75%) N = 15 P Value Healthy Individuals Median (25%,75%) N = 13 Gender 41% F 47% F 5% F 47% F 46% F Age 55 (47, 71) 55 (5, 69) 6 (42, 71) 53 (4, 62) NS 43 (35, 6) MV Duration (Days) Hospital Length of Stay (Days) 2 (14, 29) 14 (13, 23) 17 (13, 26) 14 (1, 29) NS 47 (34, 71) 38 (27, 55) 33 (26, 64) 38 (27, 58) NS Charlson 2 (, 3) 1 (, 2) 2 (1, 4) 1 (, 1) NS Apache II 19 (17, 23) 21 (15, 23) 19 (16, 23) 22 (14, 3) NS Table E2 Study patients are representative of the ICU population ICU patients participating in the trial were representative of the critically ill population undergoing prolonged mechanical ventilation (MV; > 1week) in 1 trauma and 2 medical/surgical ICUs in Toronto, Canada. Age, severity of illness (Apache II score), co-morbidities (Charlson index), duration of MV and hospital length of stay (LOS), are similar across patient groups. Healthy individuals from which the banked quadriceps biopsy specimens were obtained were slightly younger than the ICU population tested. (APACHE II - Acute Physiology and Chronic Health Evaluation score, NS = not significant).

19 A Quadriceps Isometric Peak Torque 6 Month (% pred) Quadriceps Isometric Peak Torque 6 month (% pred) FIM Motor Subscore B MRC Sum Score 6 Month MRC Sum score 6 Month 6 MW (% Pred) C p < 1 7 Day 6 Month D Day 6 Month p< r s =.76 p = 2 4 r s =.66 p = E FIM Motor Subscore F FIM Motor Subscore 1 8 r s =.71 p = r s =.53 p = Min Walk (% pred) Min Walk (% pred) Figure E2. Persistent muscle weakness correlates with sustained impairment of physical function. 7 days post ICU discharge patients demonstrated functional impairment as indicated by decreased (A) FIM motor subscores (maximum score is 91) and (B) percent predicted 6 minute walk (6MW) distance. At 6 months post ICU discharge, both measures improved significantly, but did not normalize in 82% (9/11) and 91% (1/11) of patients respectively. (Patient 8 did not undergo 7 day testing due to fractures). (C) Quadriceps strength (% predicted isometric peak torque, determined by Gosselink predictive equation 51 ) and (D) global strength (MRCSS) correlated with the FIM motor subscore at 6 months post ICU discharge. (E,F) Strength measures trended towards correlation with the % predicted 6MW distance, although this was not statistically significant.

20 Type I Fibre CSA ( m 2 ) Type II Fibre CSA ( m 2 ) Type I Fibre CSA ( m 2 ) Type II Fibre CSA ( m 2 ) 7D 6M 7D 6M F 5 5 Pt * Pt 15 * Pt * Pt 15 * Pt 13 Pt D 6M HI 7D 6M HI 7D 6M HI 7D 6M HI Female Male Female Male * * * * * * 1 1 7D 6M 7D 6M HI 7D 6M 7D 6M HI Pt 1,7,1, 15 3,5,6 13,14 11,12 8,9 Female Male 7D 6M 7D 6M HI 7D 6M 7D 6M HI Pt 1,7,1, 15 3,5,6 13,14 11,12 8,9 Female Male Figure E3 Muscle atrophy is sustained in the majority of patients 6 months following ICU discharge (A-E) Immunostaining of vastus lateralis histologic cross sections for fast twitch myosin heavy chain indicates type II (brown) and type I (mauve) myofibres. Representative sections are shown for patients 7 (A,B) and 15 (C,D) at 7 days (7D) and 6 months (6M) after ICU discharge, and (E) a sex matched healthy individual. (F) (blue circles females, purple circles males) demonstrate smaller CSA of both type I and type II fibres at 7 days (7D) post ICU discharge, compared to sex matched healthy individuals (HI, black circles, n = 9), which persists at 6 months (6M) in 7% of patients (5 to 11). In contrast, type 1 and type II myofibre CSAs in patients 13,14 and 15, who reconstitute muscle mass, are the same as sex matched healthy individuals at 6 months. Data are the mean +/- SD (*, ᶲ p < 5). (Insufficient sample was available from patients 2, 4 and 12 for analyses at 7D and/or 6M. Patient 8 underwent biopsy of the left vastus lateralis at both time points due to right femur fracture on ICU admission).

21 No. EMG 7 Day NCS 7 Day EMG 6 Month NCS 6 Month 5 Normal Normal Normal Normal 6 Normal Normal Normal Normal 7 Myopathy Normal Normal Normal 9 Myopathy?mild neuropathy Normal Isolated peroneal neuropathy 15 Myopathy Normal N/A (refused) N/A(refused) 8 Myopathy?isolated tensor fascia lata denervation Normal Isolated tensor fascia lata denervation 1 Myopathy Neuropathy N/A (refused) N/A (refused) 11 Myopathy Isolated peroneal neuropathy Myopathy Isolated peroneal neuropathy 13 Myopathy Normal Myopathy Normal 14 Myopathy Normal N/A (refused) N/A (refused) 12 Myopathy Neuropathy Myopathy Neuropathy 1 Normal Isolated peroneal involvement N/A (withdrew) N/A (withdrew) 2?Myopathy Axonal Neuropathy N/A (withdrew; active cancer) N/A ( withdrew: active cancer) 3 N/A (refused) N/A (refused) N/A (withdrew) N/A (withdrew) 4 Myopathy Axonal Sensory Polyneuropathy N/A (back to ICU) N/A (back to ICU) Table E3. EMG and NCS delineate electrophysiologic evidence of myopathy and peripheral neuropathy 79% (11/14) and 36% (5/14) of patients tested demonstrated myopathy and peripheral neuropathy respectively at 7 days post ICU discharge (1 patient refused testing). By 6 months neuropathy had resolved in all but 1 of the 8 patients who completed 6 month testing (3 patients refused testing). Electrophysiologic evidence of myopathy persisted in 38% (3/8 )patients. (N/A = not tested; EMG = electromyography, NCS = nerve conduction study)

22 CD45 +ve cells/myofibre CD68 +ve cells/myofibre CD11c +ve cells/myofibre CD45 +ve cells/myofibre CD68 +ve cells/myofibre Cd11c +ve cells/myofibre A 7 Day 6 Month HI CD45 CD68 CD11c B.6 * Day 6Month HI * Day 6 Month HI.4.2 * 7 Day 6 Month HI * D 6M 7D 6M 7D 6M 7D 6M 7D 6M 7D 6M Atrophic quads Normal quads Atrophic quads Normal quads Atrophic quads Normal quads Figure E4 Muscle inflammatory infiltrate is evident 7 days following ICU discharge (A) Quadriceps histologic cross sections were immunostained for the leukocyte markers CD45, CD68 and CD11c (inset shows higher magnification). Positive cells appear brown (black arrows). (B) Upper panels; CD45 (pan leukocytes) and CD11c (macrophages and dendritic cells) were increased at 7 days, but not at 6 months following ICU discharge. CD68 (macrophage marker) was not significantly increased at any time point. Lower panels; There was no difference in the influx of leukocytes between individuals with persistent muscle wasting (Atrophic quads), and those who reconstituted quadriceps CSA (Normal quads), at 6 months. (* p < 5). (HI = healthy individuals).

23 7 Day 6 Month Pt 11 P11 P1 Pt 13 P14 Figure E5 Sarcomere destruction is evident 7 days post-icu discharge Electron micrographs of vastus lateralis biopsies at 7 days post ICU discharge demonstrated myofibrillary disarray and Z band streaming in all patients. Sarcomeric structure is destroyed in localized areas (arrowheads). By 6 months post ICU discharge muscle sarcomere appears normal in all patients, regardless of the presence or absence of sustained muscle wasting. Representative micrographs from a patient with sustained atrophy (Patient 11) and one who normalized quadriceps size (Patient 13) are shown.

24 kda 135 P5 P6 P7 P8 P15 P9 7D 6M 7D 6M 7D 6M 7D 6M 7D 6M 7D 6M Ab Ub GAPDH kda 135 P1 P11 P13 P14 P12 7D 6M 7D 6M 7D 6M 7D 6M 7D 6M Ab Ub GAPDH kda 135 P1 P2 P3 7D 7D 7D HI1 HI2 HI3 HI4 HI5 HI6 Ab Ub GAPDH Figure E6 Increased vastus lateralis total protein ubiquitination is evident at 7 days, but not 6 months, post-icu discharge Muscle protein lysates of healthy individuals (HI) and study patients (P) at 7 days (7D) and 6 months (6M) post-icu discharge were immunoblotted for ubiquitin. GAPDH served as loading control. A constant reference standard was included on each blot to allow normalization across blots. Quantification of bands is shown in Figure 3 of the main manuscript. (Lysate from P13 on D7 was not available)

25 Beclin1 Protein Levels (arbitrary units) Beclin1 Protein Levels (arbitrary units) Beclin1 Protein Levels (arbitrary units) VPS34 Protein Levels (arbitrary units) VPS34 Protein Levels (arbitrary units) VPS34 Protein Levels (arbitrary units) Bnip3 Protein Levels (arbitrary units) Bnip3 Protein Levels (arbitrary units) Bnip3 Protein Levels (arbitrary units) LC3 II/LC3 1 LC3II/LC3I LC3II/LC3I A Day 6 Month HI 7 Day 6 Month HI 7 Day 6 Month 7 Day 6 Month B Atrophic Quads Normal Quads C 7 Day 6 month HI 7 Days 6 months HI 7 Day 6 Month 7 Day 6 Month Atrophic Quads Normal Quads 1.5 * ^ * ^ D 7 Day 6 month HI 7 Day 6 months HI 7 Day 6 Months 7 Day 6 Months Atrophic Quads Normal Quads * ^ * ^ Day 6 month HI 7 Days 6 months HI 7 Day 6 Month 7 Day 6 Month Atrophic Quads Normal Quads Figure E7 Altered autophagy is not evident in the quadriceps muscle of ICU survivors 6 months post-icu discharge (A) Autophagy was assessed in the quadriceps muscle of ICU survivors at 7 days and 6 months post ICU discharge by Western blotting and determining the extent of LC3B I conversion to LC3B- II. This serves as a functional readout of autophagy, since LC3B-1 is biochemically modified by lipidation to form LC3B-II at the initiation of autophagy. There was no difference between the LC3B-II/LC3B-I ratio between patients at 7 days and 6 months post ICU discharge, and healthy individuals (HI). (B,C,D) Protein lysates from 7 day and 6 month post ICU discharge and muscle biopsies from healthy normals were Western blotted for 3 component proteins of the autophagy pathway, Beclin1, VPS34 and Bnip3. Vps34 and Beclin1 were both increased at 7 days, but only Beclin 1 increases persisted at 6 months. (Western blots are shown on Figure E8. Bar graphs show mean +/- S.D. 5 to 12 = atrophic quads; 13 to 15 = normal quads, * and ^ p < 5).

26 P5 P6 P7 P8 P15 P9 P1 P11 kda 7D 6M 7D 6M 7D 6M 7D 6M Ab kda 7D 6M 7D 6M 7D 6M 7D 6M Ab 15 VPS34 15 VPS34 52 Beclin1 52 Beclin1 22 Bnip 3 22 Bnip LC3B-I LC3B-II Tubulin LC3B-I LC3B-II Tubulin 15 P13 P14 P12 P1 P2 kda 6M 7D 6M 7D 6M 7D 7D Ab VPS34 kda 15 P3 7D HI1 HI2 HI3 HI4 HI5 HI6 Ab VPS34 52 Beclin1 52 Beclin1 22 Bnip 3 22 Bnip LC3B-I LC3B-II LC3B-I LC3B-II Tubulin Tubulin Figure E8 Altered autophagy is not evident at 6 months post-icu discharge Muscle protein lysates of healthy individuals (HI) and study patients (P) at 7 days (7D) and 6 months (6M) post-icu discharge were Western blotted for component proteins of the autophagy pathway. Tubulin served as loading control. Bands were quantified and normalized to tubulin. A constant reference standard was included on each blot to allow for normalization across blots. Quantification of bands is shown in Figure E7. (Protein lysate from P13 was not available for all blots).

27 CD56 +ve cells/myofibre CD56 +ve cells/myofibre A P6 7D P6 6M B.4.3 ^.2 ^.1 Atrophic Normal Atrophic Normal HI Quads Quads Quads Quads P14 7D P14 6M C.4 7 Days 6 Months Day 6 Month 7 Day 6 Month HI Atrophic Quads Normal Quads Figure E9. Vastus lateralis satellite cell content is decreased in patients with persistent atrophy compared to those normalizing CSA at 6 months post ICU discharge. (A) Histologic cross sections were immunostained for CD56 (brown staining, indicated by arrows), a marker that is more sensitive, but less specific than Pax7, for satellite cells. Representative images are shown for patient 7 (P7) who demonstrated persistent quadriceps atrophy at 6 months post ICU discharge, and patient 14 (P14) who normalized quadriceps CSA. (B, C) Those patients with persistent quadriceps atrophy (atrophic quads, patients 5 to 12) at 6 months post ICU discharge demonstrate a decreased satellite cell population at both 7 days and 6 months post discharge compared to patients who normalized their quadriceps CSA (normal quads, patients 13,14,15) (Bar graphs show mean +/- S.D; ^ p < 5; HI = healthy individuals).

28 Capillaries/myofibre Capillaries/myofibre 7D 6M C 7D 6M E r 2 =.56 p < 1 F Pax 7+ve cells/myofibre 7D 6M 7D 6M HI Atrophic Quads Normal Quads Figure E1 Muscle vascularity (A-D) Quadriceps histologic cross sections were immunostained for PECAM-1 to indicate muscle capillaries (brown). Representative sections are shown for patients 7 (A,B) and 14 (C,D) at 7 days (7D) and 6 months (6M) following ICU discharge. (E) The capillary to myofibre ratio correlated with the muscle satellite cell content. (F) There was a trend towards a decrease in capillary to myofibre ratio in patients with persistent atrophy compared to those who reconstituted their quadriceps size at 6 months, but this was not significant. (Inadequate tissue was available from patients 5, 12 and 15 for 7D and/or 6M analyses; HI = healthy individuals).

29 Mitochondrial density (1 3 mitochondria/mm 2 ) Proportion of muscle occupied by mitochondria Percentage of muscle occupied by mitochondria Mitochondrial density (1 3 mitochondria/mm 2 ) Size of mitochondria (um 2 ) Percentage of muscle occupied by mitochondria Mitochondrial density (1 3 mitochondria/mm 2 ) Mitochondria Volume Density ^ * * ^ Number of Mitochondria ^ * * ^ Mitochondria Size (um 2) Size of Mitochondria 7 day 6 month HI 7Day 6 Month HI 7 Day 6 Month HI Mitochondria Volume Density Number of Mitochondria Size of Mitochondrial Day 6 Months HI 7 Day 6 Month HI 7 Day 6 Months HI Figure E11 - Vastus lateralis mitochondrial content is decreased at Day 7 post ICU discharge but normalizes at 6 months post ICU discharge. Mitochondrial Correlation number of Muscle and volume Specific density Force decreased in patients Correlation 7 Muscle days post Specific ICU Force discharge with Number of Mitochondria and Mitochondrial Volume Density compared to 6 months post discharge and healthy individuals (HI, n = 4). There was no 1. 1 difference in mitochondrial size at either time point. (Bar graphs show mean +/- S.D; * and ^ p.8 8 < 5) p = NS.6 p = NS Muscle Specific Force (Nm/cm 2 ) Muscle Specific Force (Nm/cm 2 )