Intensive Care Med DOI 10.1007/s00134-014-3224-9
Tobias Wollersheim Janine Woehlecke Martin Krebs Jida Hamati Doerte Lodka Anja Luther-Schroeder Claudia Langhans Kurt Haas Theresa Radtke Christian Kleber Claudia Spies Siegfried Labeit Markus Schuelke Simone Spuler Joachim Spranger Steffen Weber-Carstens Jens Fielitz
ORIGINAL
Dynamics of myosin degradation in intensive care unit-acquired weakness during severe critical illness
J. Woehlecke J. Hamati D. Lodka C. Langhans S. Spuler J. Spranger J. Fielitz ()) Ó Springer-Verlag Berlin Heidelberg and Experimental and Clinical Research ESICM 2014 Center (ECRC), A Cooperation Between Max-Delbru¨ck-Centrum and T. Wollersheim and J. Woehlecke contributed equally to this work. Charite´–Universita¨tsmedizin Berlin, Campus Buch, S. Weber-Carstens and J. Fielitz contributed Lindenberger Weg 80, equally to this work. 13125 Berlin, Germany e-mail: [email protected] Take-home message: Intensive care unit Tel.: ?49-30-450540424 (ICU)-acquired muscle wasting is a Fax: ?49-30-450540928 devastating complication leading to persistent weakness and functional C. Kleber disability. We demonstrate that decreased synthesis and increased degradation of Center for Musculoskeletal Surgery, myosin heavy chains contribute to ICUCharite´–Universita¨tsmedizin Berlin, Berlin, acquired muscle wasting, and conclude that Germany therapeutic interventions must be initiated very early during critical illness. S. Labeit Universita¨tsmedizin Mannheim, Mannheim, Trial registration The study was registered at http://www.controlled-trials.com Germany as ISRCTN77569430. M. Schuelke Electronic supplementary material NeuroCure Clinical Research Center, The online version of this article Charite´–Universita¨tsmedizin Berlin, Berlin, (doi:10.1007/s00134-014-3224-9) contains Germany supplementary material, which is available to authorized users. J. Spranger Department of Endocrinology, Diabetes and Nutrition, T. Wollersheim M. Krebs Charite´–Universita¨tsmedizin Berlin, A. Luther-Schroeder K. Haas Berlin, Germany T. Radtke C. Spies S. Weber-Carstens Anesthesiology and Operative Intensive J. Spranger Care Medicine, Charite´– Center for Cardiovascular Research, Universita¨tsmedizin Berlin, Campus Charite´–Universita¨tsmedizin Berlin, Berlin, Virchow and Campus Mitte, Berlin, Germany Germany Received: 9 September 2013 Accepted: 18 January 2014
J. Fielitz Department of Cardiology, Charite´– Universita¨tsmedizin Berlin, Campus Virchow, Berlin, Germany
Abstract Importance: Intensive care unit (ICU)-acquired muscle wasting is a devastating complication leading to persistent weakness and functional disability. The mechanisms of this myopathy are unclear, but a disturbed balance of myosin heavy chain (MyHC) is implicated. Objective: To investigate pathways of myosin turnover in severe critically ill patients at high risk of ICUacquired weakness. Design: Prospective, mechanistic, observational study. Setting: Interdisciplinary ICUs of a university hospital. Participants: Twenty-nine patients with Sequential Organ Failure Assessment (SOFA) scores of at least 8 on three consecutive days within the first 5 days in ICU underwent two consecutive open skeletal muscle biopsies from the vastus lateralis at median days 5 and 15. Control biopsy specimens were from healthy subjects undergoing hip-replacement surgery. Interventions: None. Main outcome(s) and measure(s): Timedependent changes in myofiber architecture, MyHC synthesis, and
degradation were determined and correlated with clinical data. Results: ICU-acquired muscle wasting was characterized by early, disrupted myofiber ultrastructure followed by atrophy of slow- and fasttwitch myofibers at later time points. A rapid decrease in MyHC mRNA and protein expression occurred by day 5 and persisted at day 15 (P \ 0.05). Expression of the atrophy
Introduction
genes MuRF-1 and Atrogin1 was increased at day 5 (P \ 0.05). Early MuRF-1 protein content was closely associated with late myofiber atrophy and the severity of weakness. Conclusions and relevance: Decreased synthesis and increased degradation of MyHCs contribute to ICUacquired muscle wasting. The rates and time frames suggest that pathogenesis of muscle failure is initiated
very early during critical illness. The persisting reduction of MyHC suggests that sustained treatment is required. Keywords ICUAW MuRF-1 Myosin degradation Inflammation Muscle atrophy dmCMAP
Failure Assessment (SOFA) scores at different time points of critical illness. Our results were recently pubIntensive care unit (ICU)-acquired muscle wasting and lished in part as abstracts [25, 26]. weakness are devastating complications of critical illness [1]. The condition is common in patients with sepsis, systemic inflammatory response syndrome, and those who are mechanically ventilated [2, 3]. The resultant myopathy Methods leads to persistent functional disability that compromises patients long after discharge from hospital [4, 5]. The The institutional review board of the Charite´ approved the pathophysiology of ICU-acquired muscle wasting and study and written informed consent was obtained from weakness is poorly understood and there are no specific legal proxy or from the study subject (Charite´ EA2/061/ therapies. However, skeletal muscle wasting, particularly 06). We specifically included patients at high risk of atrophy of fast-twitch (type-II) myofibers and reduced developing ICU-acquired muscle wasting and weakness myosin heavy chain (MyHC), is consistently observed [6, [12]. Accordingly, critically ill, mechanically ventilated 7]. Early pathways leading to MyHC loss and its kinetics ICU patients were eligible for inclusion once they showed are poorly defined because most investigators reported later SOFA scores of at least 8 on three consecutive days time points when ICU-acquired weakness was already within the first 5 days after ICU admission. Not included established [7–9]. MyHC loss could be caused by a dis- by prior definition were patients less than 18 years of age, turbed balance between its synthesis and degradation [10]. pretreatment longer than 7 days, a body mass index Several risk factors predispose to ICU-acquired weakness greater than 35 kg/m2, pre-existing neuromuscular disand skeletal muscle wasting, such as systemic inflamma- eases, insulin-dependent diabetes mellitus, and moribund tion, sepsis, immobilization, sedation, hyperglycemia, patients. ICU patients received treatment according to exposure to neuromuscular blocking agents, and cortico- standard procedures including goal-directed sedation steroids [11, 12], leading to reduced muscle mass [13] and treatment and sepsis guidelines. In addition, physiotherstrength by increasing protein degradation and/or apy was performed by an experienced physiotherapist decreasing protein synthesis [14]. starting from day 1 in ICU, including passive range of The major protein-degrading system in muscle is the motion, positioning, and active exercises as soon as posubiquitin–proteasome system (UPS) that targets MyHC sible. A physician unaware of the histological findings for breakdown [15, 16]. In critically ill patients, the UPS assessed the patients’ muscle strength according to the is activated and mediates muscle atrophy [17–19]. The crucial Medical Research Council (MRC) score. The muscle-enriched E3 ubiquitin ligase (MuRF)-1 and the MRC score was evaluated when patients were alert F-box adaptor protein FBXO32/Atrogin1 are implicated. (defined as Richmond Agitation Sedation Scale scores of Early upregulation of MuRF-1 and Atrogin1 expression -1, 0, or ?1) and responded adequately to three or more and increased protein production are sensitive markers of five verbal commands for the first time [1, 27]. Since for muscle atrophy [20]. MuRF-1 and Atrogin1 are MRC values have their limitations in critically ill patients induced in skeletal muscle of critically ill patients [13, because of patient cooperation issues and cognitive dys14, 19, 21, 22]. Because MuRF-1 mediates MyHC function we reassessed MRC values at the consecutive degradation [23], MuRF-1 could play a role in MyHC day. Sequential open muscle biopsy from the vastus loss in ICU-acquired muscle wasting and weakness [13, lateralis was performed in the ICU patients to analyze 24]. We investigated MyHC synthesis and degradation myocyte cross-sectional area (MCSA), mRNA, protein in the skeletal muscle of patients at high risk of ICU- content, and ultrastructure by electron microscopy. Five acquired weakness identified by increased Sepsis Organ age- and gender-matched patients undergoing elective
orthopedic surgery, but otherwise healthy, permitted a biopsy from the vastus lateralis at the time point of elective surgery; this biopsy was assumed to be representative of muscular integrity just before the onset of critical illness in terms of MCSA, mRNA, and protein analysis. The study was registered at http://www. controlled-trials.com as ISRCTN77569430. Open muscle biopsy from the vastus lateralis was performed at median day 5 from 29 ICU patients, referred to as the early time point of critical illness. Of these 29 patients 22 remained in the ICU at least to median day 15, referred to as the late time point of critical illness, when a second biopsy specimen from the vastus lateralis was obtained. Hematoxylin–eosin and Gomori trichrome stainings on histological cryosections were performed to assess overall muscle pathology as described earlier [23]. Metachromatic ATPase staining was performed for fiber type analysis and measurements of fiber-type-specific MCSA [23, 28]. MCSA was determined by an investigator blinded to the kind of samples using ImageJ software version 1.46 (fiber count [100). From those 22 patients who underwent sequential biopsies and the five control subjects, MCSA measurements were only possible in 16 patients at both time points and in 4 controls owing to freezing artifacts. For detailed methods on Western blot, RNA analyses, and electron microscopy refer to the electronic supplementary material. Biostatistics Non-parametric tests, namely the Mann–Whitney test to analyze group differences and the Wilcoxon test for dependent samples, were performed. Spearman’s rank correlation coefficients were calculated. Statistical tests were computed using SPSS (version 19.0.0.1); box plots were made by Sigma Plot software (version 12.0). Data are provided as interquartile range (IQR) unless otherwise indicated. P \ 0.05 was accepted as significant.
Results The patients’ characteristics are shown in Table 1. Muscle strength according to MRC score was measured in 20 ICU patients when they emerged from analgesics and sedation at median day 13 (IQR 11–20). The median MRC score at this time point was 3.3 (IQR 3.0–3.9). Muscle biopsy specimens were obtained from 29 patients on median day 5 (IQR 4–7) after ICU admission referred to as first-biopsy and early time point. Twenty-two patients were still in the ICU at 2 weeks and allowed a second muscle biopsy at median day 15 (IQR 14–19), referred to as the second biopsy and late time point (Fig. 1 in the electronic supplementary material). Data during clinical course on organ failure, glucose metabolism and nutrition, as well as analgesics, sedation, and awareness are shown in Table 1 in the electronic supplementary material. Neuromuscular blocking agents were sparingly administered and their cumulative dosage was unremarkably low. Patients did not receive high-dose corticosteroid treatment. Hydrocortisone (200 mg/24 h, ‘‘low dose infusion’’) was given to patients with septic shock. No differences in MCSA of fast-twitch (type-IIa and -IIb) fibers were detected between controls and the first biopsy specimens of ICU patients. MCSAs of slow-twitch (type-I) fibers were smaller in controls compared to ICU patients in the first biopsy (Fig. 1a). In contrast, MCSAs of type-I, -IIa, and -IIb fibers decreased significantly between the first and second biopsy (Fig. 1a, b; Fig. 2 in the electronic supplementary material). The decrease in MCSA was predominant in type-II fibers. Comparing the median MCSA values of individual fiber types, we found that the strongest decrease of MCSA was for type-IIa fibers reaching 42 % (P \ 0.005), followed by type-IIb (32 %, P \ 0.005) and type-I (28 %, P \ 0.01) fibers. In contrast, no change in fiber type composition was observed between the first and second biopsy specimens
Table 1 Patients characteristics Patients’ characteristics
All patients enrolled
Controls
Number Age (years) Gender (m/f) BMI (kg/m2) MRC score Diagnosis [n (%)]
29 54 (41/68) 22/7 (76 %/24 %) 28 (24/31) 3.3 (3.0/3.9) ALI/ARDS: n = 11 (37.9 %) Sepsis: n = 7 (24.1 %) Trauma: n = 6 (20.7 %) CNS: n = 5 (17.2 %) 23 (79.3 %) ICU scores SOFA: 12 (10/14) SAPS-II: 42 (36/53)
5 69 (67/71) 3/2 (60 %/40 %) ND 5.0
Survivors [n (%)] Severity of illness (at ICU admission)
5 (100 %) NA
ALI/ARDS acute lung injury/acute respiratory distress syndrome, BMI body mass index, CNS central nervous system, NA not applicable, ND not determined, SOFA Sequential Organ Failure Assessment score, SAPS-II Simplified Acute Physiology Score-II
A
Type-I 8000
Myocyte cross sectional area [µm2 ]
Fig. 1 Fast- and slow-twitch fibers atrophied during critical illness. a Myocyte crosssectional area (MCSA, lm2) from controls (white) and ICU patients’ first (gray) and second (black) biopsy specimens in type-I (left), type-IIa (middle), and type-IIb (right) fibers. **P \ 0.01; *P \ 0.05; n.s. not significant. b ATPase stained fiber-type analyses from early (left) and late (right) time points (type-I, -IIa, and -IIb fibers are indicated; scale bar 100 lm). c Representative electron micrographs from the early (left) and late (right) time points revealed early destruction of myofiber ultrastructure (scale bar 2 lm). Myosin loss and mitochondrial ballooning occurred early during critical illness (left). At the later time point myosin became squeezed and distorted, Z-lines were deformed and H-zone shapes were blurred (right). Myosin loss (thick black arrow), Z-lines (white arrow), H-zone (small black arrow), mitochondria (white star)
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and was comparable to the fiber type composition in control subjects (Fig. 3 and Table 2 in the electronic supplementary material). There was no evidence of necrosis, regeneration, inflammatory infiltrates, or mitochondrial abnormalities, such as ragged red fibers or cytochrome C oxidase-negative fibers (data not shown). These data suggest that histological fiber atrophy can only be detected in the later phase of critical illness. The data also showed that all fiber types atrophy during the disease course.
To investigate if ultrastructural changes occurred early during critical illness we performed electron microscopy on biopsy specimens of ICU patients. Early biopsy specimens were characterized by sarcomeres devoid of MyHC fibrils, swollen mitochondria, edema, and increased spacing between sarcomeres (Fig. 1c, left). In the later biopsy specimens, not only was MyHC gone from the sarcomeres but also the sarcomeric ultrastructure was disrupted. Swollen mitochondria, edema, and increased spacing between sarcomeres were
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Fig. 2 Myosin heavy chain (MyHC) decreases in slow- and fasttwitch fibers. a Representative Western blots for slow (type-I) and fast (type-II) MyHC of four control subjects and four ICU patients (left), a-actin was used as loading control. Western blots of slow and fast MyHC of four ICU patients in the first (I) and second (II)
biopsy (right). b Quantification of Western blot signals are depicted as box plots showing median (IQR) and documents the marked decrease in MyHC. c Correlation between slow and fast MyHC protein contents in ICU patients (black circles and line). Controls are shown as white circles and dashed lines
found to a lesser degree (Fig. 1c, right). These changes paralleled the reduction of MCSA that was only detected in the biopsy specimens of the later time point. Thus, fiber ultrastructure and MyHC-containing fibrils were already perturbed at the early biopsy specimen, a time point where no changes were apparent by light microscopy. Western blotting showed a substantial reduction of MyHC in the first biopsy specimen (Fig. 2a, b). This reduction was consistent with the myosin loss seen by electron microscopy even before a decrease in MCSA was observed. Moreover, comparing the first and second biopsy specimens, we note that the slow and fast MyHC protein contents remained significantly low and did not differ between the two time points (Fig. 2a, b). Although slow and fast MyHC protein contents decreased simultaneously, the reduction of fast MyHC was more pronounced compared to slow MyHC. The findings are
consistent with a more pronounced atrophy of the fasttwitch fibers (Fig. 2c). These data showed an unexpected early decrease in both fast and slow MyHC proteins in severe critically ill patients. Decreased gene expression of the MyHC isoforms MyHC-1, -2, and -7 was found in the first biopsy specimens compared to controls. MyHC-7 expression encoding for slow-twitch type-I MyHC was significantly decreased in the early and later biopsy specimens. MyHC-1 and -2 expression, encoding for fast-twitch type-IIx and -IIa MyHC, were decreased in the early biopsy specimen, whereas MyHC-4 expression, encoding for fast-twitch type-IIb MyHC, remained unchanged. MyHC-2, -4, and -7 expression was significantly higher in the second biopsy specimen, compared to the first, suggesting some degree of recovery (Fig. 3). These data suggested that reduced MyHC synthesis contributed to early MyHC loss during severe critical illness. However, these results did
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Fig. 3 Myosin heavy chain (MyHC) gene expression is regulated early in muscle of ICU patients. Gene expression analyses of slow (MyHC-7) and fast (MyHC-1, -2, and -4) MyHC by real-time RT-PCR. ***P \ 0.001; **P \ 0.01; *P \ 0.05; n.s. not significant
not exclude increased MyHC degradation as a contributing factor for decreased muscular MyHC contents in critical illness. In fact, UPS-dependent protein degradation has been implicated as the major pathway responsible for muscular protein degradation in skeletal muscle atrophy [15, 16]. Gene expression of MuRF-1, Atrogin1, Calpain-1, Caspase-3, and FOXO-1, which are all involved in skeletal muscle protein degradation, was quantitated by RTPCR (Fig. 4a). MuRF-1, Atrogin1, and FOXO-1 mRNA expression significantly increased in the first biopsy specimen and returned to control levels in the second. Caspase-3 expression was significantly increased in the first biopsy specimen and remained at this level in the second. Calpain-1 expression was unchanged in the first biopsy specimen, but increased in the second. These data showed that atrophy-related genes were activated at different time points during the disease course. MuRF-1 protein was significantly increased in the first biopsy by Western blotting, followed by a significant reduction in the second biopsy specimen (Fig. 4b). These data showed that changes in MuRF-1 protein content paralleled its mRNA expression in skeletal muscle in early critical illness. We next relied on RT-PCR to investigate the proteasome beta subunits (PSMB) of the 20S core particle of the proteasome. The three PSMB types-1, -2, and -5 encoding peptidyl-glutamyl peptide hydrolyzing, trypsin-like, and chymotrypsin-like activity, respectively, were significantly increased at the time of the first biopsy, as were PSMB-3, -4, and -7, which are catalytically inactive. Gene
expression of PSMB-6 remained unchanged. Gene expression of the interferon gamma-inducible PSMB subunits was either decreased (PSMB-9 and -10) or increased (PSMB-8). No significant differences in gene expression were found for any of the PSMB genes between the first and second biopsy specimens (Fig. 5). These data indicate that the proteasome was activated early and remained at this level during the disease course. We observed a close and inverse correlation between MuRF-1 protein contents in the first biopsy specimens and the MCSA of all three fiber types measured in the second biopsy specimens (Fig. 4a in the electronic supplementary material). In contrast, such a correlation was not found for the myosin protein (data not shown). Furthermore, higher MuRF-1 protein contents at the early stage of critical illness were significantly associated with the crucial MRC score during the disease course when patients were regaining consciousness (Fig. 4b in the electronic supplementary material) linking myosin loss and weakness. By univariate regression analyses we investigated if patient characteristics, severity of illness, medication (e.g., hydrocortisone) as well as non-excitable muscle membrane (Table 3 in the electronic supplementary material) were associated with the molecular findings. We found that increased age was associated with early increased MuRF1 protein content, and a higher SAPS-II score at admission was associated with greater MCSA reduction of type-IIb muscle fibers between the first and second biopsy. None of the other clinical parameters were associated with our experimental data.
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Fig. 4 Activation of protein degradation in muscle. a Values from controls are open boxes. Gene expression of MuRF-1, Atrogin1, Caspase-3, Calpain-1, and FOXO-1 exceeded normal subjects and decreased variably thereafter. b MuRF-1 quantification of Western blot signals are depicted as box plots showing median (IQR).
c Representative Western blots of MuRF-1 from four controls and four ICU patients document marked upregulation. a-Actin was used as loading control (left). By the second biopsy MuRF-1 upregulation had subsided (right)
Discussion
is particularly important for primary-care physicians who are generally responsible for these patients once they survive their acute illness. We draw attention to mechanisms contributing to ICU-acquired muscle wasting and weakness which often lead to persistent disabilities compromising patients long after hospital discharge [4, 5, 29]. It is unclear if neuropathic changes were involved in myosin loss. However, this seems to be unlikely for our study, because molecular changes in muscle occurred before electrophysiological signs of critical illness polyneuropathy were detectable.
The important finding in our study is the unexpected early and persistent loss of MyHC in skeletal muscle of severe critically ill patients occurring 5 days after ICU admission. Our data suggest that although MyHC is lost rapidly, the capacity of muscle from critically ill patients to regenerate is limited. Furthermore massive myosin loss implicates a need for long-term and persistent treatment strategies to strengthen muscle function once patients are diagnosed with ICU-acquired weakness. The latter point
PSMB1
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Fig. 5 Activation of proteasomal subunits in skeletal muscle. persistent induction of the ubiquitin proteasome system (UPS). Gene expression of proteasome beta subunits (PSMB) 1–10 was ***P \ 0.001; **P \ 0.01; *P \ 0.05; n.s. not significant quantitated by real-time RT-PCR and revealed an early and
MyHC loss was accompanied by decreased MyHC mRNA expression and MyHC protein contents during the early phase of critical illness. The dynamics of MyHC gene expression and protein turnover implicate pathways responsible for MyHC loss that are possibly deregulated during the first days, if not hours, of severe critical illness. Our data are supported by other findings showing decreased protein synthesis in the skeletal muscle of critically ill patients [24]. In contrast, others report that protein synthesis is unchanged in critically ill patients [13]. Discrepancies between our study and the latter could be explained by time-dependent changes in gene and protein expression during the disease course.
MyHC loss was paralleled by an activation of proteins involved in myosin degradation. Proteasomal degradation of myofibrillar proteins requires initial proteolysis of actomyosin complexes by caspase-3 [30] and calpain-1 [31]. Since both proteases were increased in our study, access to the UPS could be alleviated, thereby possibly easing its degradation. In addition, MuRF-1 and Atrogin1 [20], key proteins for UPS-dependent MyHC degradation, were activated during early critical illness. The rapid loss of MyHC during the early disease phase may reflect effective cooperation between these components of UPS-mediated protein degradation. Our results are in line with findings in animal models, where various atrophy stimuli, such as
denervation, immobilization, and cancer [21] increased MuRF-1 and Atrogin1 expression in a typical time course with a maximum of expression 3–5 days after the insult and a subsequent decrease to control levels [20]. This time course might explain differences with other studies in which no changes in MuRF-1 expression in critically ill patients were found in late muscle biopsy specimens [13]. Increased MuRF-1 expression in critical illness is of significant clinical importance for ICU-acquired muscle wasting and weakness because MuRF-1 was directly correlated with myofiber atrophy and muscle weakness. We think that systemic inflammation—notably severe sepsis, a hallmark of the early disease phase in our study—caused enhanced muscular protein degradation by increasing MuRF-1 expression. This view is supported by the induction of FOXO-1. FOXO-1 is activated by inflammation and sepsis [32] and increases MuRF-1 and Atrogin1 expression during atrophy [33]. The decline in MuRF-1 and Atrogin1 expression, observed at the later disease phase when inflammation declined, supports this interpretation. Following ubiquitin-chain formation, myofibrillar proteins are recognized and degraded by the UPS. The activity and substrate specificity of the UPS are regulated by the composition of its catalytic subunits [34]. Recently, an increase in UPS activity was found in the diaphragms of patients with diaphragm disuse [18] and in ICUacquired weakness [13]. Our findings extend these data showing that muscular protein degradation involves different components of the UPS and that UPS-mediated protein degradation remains increased during the disease course of critical illness. Skeletal muscle atrophy during critical illness was mainly observed in fast-twitch fibers, which strongly depend on glycolytic metabolism. Earlier, we observed disturbed glucose utilization in skeletal muscle of patients with critical illness myopathy [35]. We reason that disturbed glucose utilization aggravates atrophy of fasttwitch fibers in critically ill patients. In addition, differences in the time course of fast- and slow-twitch fiber atrophy argue for distinct fiber-type-specific pathways involved favoring early atrophy of fast-twitch fibers. Indeed, this hypothesis is supported by our recent work showing preferential expression of MuRF-1 in fast-twitch fibers [36]. We believe that systemic inflammation and sepsis are key factors not only to increase protein degradation but also to impair glucose utilization in muscle during early critical illness. This view is supported by data showing that sepsis activates FOXO-1 leading to increased expression of its proteolytic target genes and to impaired carbohydrate oxidation in muscle [37]. High-dose corticosteroids can induce UPS-dependent protein degradation in muscle and lead to myopathy [38].
High-dose corticosteroids were an independent predictor of ICU-acquired paresis [1, 39]. In contrast, low-dose corticosteroids have not been shown to induce myopathy [12]. We have no reason to believe that low-dose hydrocortisone had an effect on skeletal muscle atrophy or ICU-acquired weakness in our patients. First, no highdose corticosteroid treatment was applied. Second, only patients with septic shock refractory to fluid resuscitation and vasopressor therapy received low-dose hydrocortisone treatment. Third, glucocorticosteroids were only applied sporadically and were unlikely to affect the skeletal muscle. Fourth, no correlation between the cumulative dose of hydrocortisone and any of the molecular, clinical, or morphological data obtained here were found by regression analyses. Although the number of patients in our study is small, sequential muscle biopsies strengthen our results. We bewail the absence of muscle biopsies at the time point of ICU admission; however, the first muscular biopsy only became possible once a legal proxy was named by the court, which was at day 3 after ICU admission at the earliest. General internists are likely to be faced with survivors of muscle wasting and ICU-acquired weakness [40, 41]. Survivors can reckon with sustained physical impairment [42]. One year after hospital discharge, half of the patients are unable to work and have substantially reduced health-related quality of life [4]. The economic impact of ICU-acquired weakness is staggering; the costs in post-discharge care are estimated at US$300,000/patient/year [43]. Even a small advance to avoid or ameliorate the course of ICU-acquired weakness would not only be helpful to our patients but also a major contribution to health-care providers. Our study shows that pathophysiological mechanisms resulting in ICU-acquired weakness are activated early during critical illness. Our hope is that our study can be helpful to develop strategies to treat these patients. Further clinical studies are needed to investigate therapeutic options to maintain muscle integrity and function during critical illness. Acknowledgments We are thankful for the patience and courage of our patients and their consenting relatives. We thank Anika Lindner and Josefine Russ for technical assistance. We thank Friedrich C. Luft, MD, FACP for his continued support and editorial assistance. The Deutsche Forschungsgemeinschaft (FI 965/21, FI 965/4-1 and La668/14-1, KFO 192—WE 4386/1-2), Muscular Dystrophy Association, Marie Curie International Reintegration grant (FP7-PEOPLE-2007-4-3-IRG), and the Deutsche Gesellschaft fu¨r Muskelkranke supported this work. Conflicts of interest interest.
The authors are not aware of any conflicts of
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Tobias Wollersheim Janine Woehlecke Martin Krebs Jida Hamati Doerte Lodka Anja Luther-Schroeder Claudia Langhans Kurt Haas Theresa Radtke Christian Kleber Claudia Spies Siegfried Labeit Markus Schuelke Simone Spuler Joachim Spranger Steffen Weber-Carstens Jens Fielitz
ORIGINAL
Dynamics of myosin degradation in intensive care unit-acquired weakness during severe critical illness
J. Woehlecke J. Hamati D. Lodka C. Langhans S. Spuler J. Spranger J. Fielitz ()) Ó Springer-Verlag Berlin Heidelberg and Experimental and Clinical Research ESICM 2014 Center (ECRC), A Cooperation Between Max-Delbru¨ck-Centrum and T. Wollersheim and J. Woehlecke contributed equally to this work. Charite´–Universita¨tsmedizin Berlin, Campus Buch, S. Weber-Carstens and J. Fielitz contributed Lindenberger Weg 80, equally to this work. 13125 Berlin, Germany e-mail: [email protected] Take-home message: Intensive care unit Tel.: ?49-30-450540424 (ICU)-acquired muscle wasting is a Fax: ?49-30-450540928 devastating complication leading to persistent weakness and functional C. Kleber disability. We demonstrate that decreased synthesis and increased degradation of Center for Musculoskeletal Surgery, myosin heavy chains contribute to ICUCharite´–Universita¨tsmedizin Berlin, Berlin, acquired muscle wasting, and conclude that Germany therapeutic interventions must be initiated very early during critical illness. S. Labeit Universita¨tsmedizin Mannheim, Mannheim, Trial registration The study was registered at http://www.controlled-trials.com Germany as ISRCTN77569430. M. Schuelke Electronic supplementary material NeuroCure Clinical Research Center, The online version of this article Charite´–Universita¨tsmedizin Berlin, Berlin, (doi:10.1007/s00134-014-3224-9) contains Germany supplementary material, which is available to authorized users. J. Spranger Department of Endocrinology, Diabetes and Nutrition, T. Wollersheim M. Krebs Charite´–Universita¨tsmedizin Berlin, A. Luther-Schroeder K. Haas Berlin, Germany T. Radtke C. Spies S. Weber-Carstens Anesthesiology and Operative Intensive J. Spranger Care Medicine, Charite´– Center for Cardiovascular Research, Universita¨tsmedizin Berlin, Campus Charite´–Universita¨tsmedizin Berlin, Berlin, Virchow and Campus Mitte, Berlin, Germany Germany Received: 9 September 2013 Accepted: 18 January 2014
J. Fielitz Department of Cardiology, Charite´– Universita¨tsmedizin Berlin, Campus Virchow, Berlin, Germany
Abstract Importance: Intensive care unit (ICU)-acquired muscle wasting is a devastating complication leading to persistent weakness and functional disability. The mechanisms of this myopathy are unclear, but a disturbed balance of myosin heavy chain (MyHC) is implicated. Objective: To investigate pathways of myosin turnover in severe critically ill patients at high risk of ICUacquired weakness. Design: Prospective, mechanistic, observational study. Setting: Interdisciplinary ICUs of a university hospital. Participants: Twenty-nine patients with Sequential Organ Failure Assessment (SOFA) scores of at least 8 on three consecutive days within the first 5 days in ICU underwent two consecutive open skeletal muscle biopsies from the vastus lateralis at median days 5 and 15. Control biopsy specimens were from healthy subjects undergoing hip-replacement surgery. Interventions: None. Main outcome(s) and measure(s): Timedependent changes in myofiber architecture, MyHC synthesis, and
degradation were determined and correlated with clinical data. Results: ICU-acquired muscle wasting was characterized by early, disrupted myofiber ultrastructure followed by atrophy of slow- and fasttwitch myofibers at later time points. A rapid decrease in MyHC mRNA and protein expression occurred by day 5 and persisted at day 15 (P \ 0.05). Expression of the atrophy
Introduction
genes MuRF-1 and Atrogin1 was increased at day 5 (P \ 0.05). Early MuRF-1 protein content was closely associated with late myofiber atrophy and the severity of weakness. Conclusions and relevance: Decreased synthesis and increased degradation of MyHCs contribute to ICUacquired muscle wasting. The rates and time frames suggest that pathogenesis of muscle failure is initiated
very early during critical illness. The persisting reduction of MyHC suggests that sustained treatment is required. Keywords ICUAW MuRF-1 Myosin degradation Inflammation Muscle atrophy dmCMAP
Failure Assessment (SOFA) scores at different time points of critical illness. Our results were recently pubIntensive care unit (ICU)-acquired muscle wasting and lished in part as abstracts [25, 26]. weakness are devastating complications of critical illness [1]. The condition is common in patients with sepsis, systemic inflammatory response syndrome, and those who are mechanically ventilated [2, 3]. The resultant myopathy Methods leads to persistent functional disability that compromises patients long after discharge from hospital [4, 5]. The The institutional review board of the Charite´ approved the pathophysiology of ICU-acquired muscle wasting and study and written informed consent was obtained from weakness is poorly understood and there are no specific legal proxy or from the study subject (Charite´ EA2/061/ therapies. However, skeletal muscle wasting, particularly 06). We specifically included patients at high risk of atrophy of fast-twitch (type-II) myofibers and reduced developing ICU-acquired muscle wasting and weakness myosin heavy chain (MyHC), is consistently observed [6, [12]. Accordingly, critically ill, mechanically ventilated 7]. Early pathways leading to MyHC loss and its kinetics ICU patients were eligible for inclusion once they showed are poorly defined because most investigators reported later SOFA scores of at least 8 on three consecutive days time points when ICU-acquired weakness was already within the first 5 days after ICU admission. Not included established [7–9]. MyHC loss could be caused by a dis- by prior definition were patients less than 18 years of age, turbed balance between its synthesis and degradation [10]. pretreatment longer than 7 days, a body mass index Several risk factors predispose to ICU-acquired weakness greater than 35 kg/m2, pre-existing neuromuscular disand skeletal muscle wasting, such as systemic inflamma- eases, insulin-dependent diabetes mellitus, and moribund tion, sepsis, immobilization, sedation, hyperglycemia, patients. ICU patients received treatment according to exposure to neuromuscular blocking agents, and cortico- standard procedures including goal-directed sedation steroids [11, 12], leading to reduced muscle mass [13] and treatment and sepsis guidelines. In addition, physiotherstrength by increasing protein degradation and/or apy was performed by an experienced physiotherapist decreasing protein synthesis [14]. starting from day 1 in ICU, including passive range of The major protein-degrading system in muscle is the motion, positioning, and active exercises as soon as posubiquitin–proteasome system (UPS) that targets MyHC sible. A physician unaware of the histological findings for breakdown [15, 16]. In critically ill patients, the UPS assessed the patients’ muscle strength according to the is activated and mediates muscle atrophy [17–19]. The crucial Medical Research Council (MRC) score. The muscle-enriched E3 ubiquitin ligase (MuRF)-1 and the MRC score was evaluated when patients were alert F-box adaptor protein FBXO32/Atrogin1 are implicated. (defined as Richmond Agitation Sedation Scale scores of Early upregulation of MuRF-1 and Atrogin1 expression -1, 0, or ?1) and responded adequately to three or more and increased protein production are sensitive markers of five verbal commands for the first time [1, 27]. Since for muscle atrophy [20]. MuRF-1 and Atrogin1 are MRC values have their limitations in critically ill patients induced in skeletal muscle of critically ill patients [13, because of patient cooperation issues and cognitive dys14, 19, 21, 22]. Because MuRF-1 mediates MyHC function we reassessed MRC values at the consecutive degradation [23], MuRF-1 could play a role in MyHC day. Sequential open muscle biopsy from the vastus loss in ICU-acquired muscle wasting and weakness [13, lateralis was performed in the ICU patients to analyze 24]. We investigated MyHC synthesis and degradation myocyte cross-sectional area (MCSA), mRNA, protein in the skeletal muscle of patients at high risk of ICU- content, and ultrastructure by electron microscopy. Five acquired weakness identified by increased Sepsis Organ age- and gender-matched patients undergoing elective
orthopedic surgery, but otherwise healthy, permitted a biopsy from the vastus lateralis at the time point of elective surgery; this biopsy was assumed to be representative of muscular integrity just before the onset of critical illness in terms of MCSA, mRNA, and protein analysis. The study was registered at http://www. controlled-trials.com as ISRCTN77569430. Open muscle biopsy from the vastus lateralis was performed at median day 5 from 29 ICU patients, referred to as the early time point of critical illness. Of these 29 patients 22 remained in the ICU at least to median day 15, referred to as the late time point of critical illness, when a second biopsy specimen from the vastus lateralis was obtained. Hematoxylin–eosin and Gomori trichrome stainings on histological cryosections were performed to assess overall muscle pathology as described earlier [23]. Metachromatic ATPase staining was performed for fiber type analysis and measurements of fiber-type-specific MCSA [23, 28]. MCSA was determined by an investigator blinded to the kind of samples using ImageJ software version 1.46 (fiber count [100). From those 22 patients who underwent sequential biopsies and the five control subjects, MCSA measurements were only possible in 16 patients at both time points and in 4 controls owing to freezing artifacts. For detailed methods on Western blot, RNA analyses, and electron microscopy refer to the electronic supplementary material. Biostatistics Non-parametric tests, namely the Mann–Whitney test to analyze group differences and the Wilcoxon test for dependent samples, were performed. Spearman’s rank correlation coefficients were calculated. Statistical tests were computed using SPSS (version 19.0.0.1); box plots were made by Sigma Plot software (version 12.0). Data are provided as interquartile range (IQR) unless otherwise indicated. P \ 0.05 was accepted as significant.
Results The patients’ characteristics are shown in Table 1. Muscle strength according to MRC score was measured in 20 ICU patients when they emerged from analgesics and sedation at median day 13 (IQR 11–20). The median MRC score at this time point was 3.3 (IQR 3.0–3.9). Muscle biopsy specimens were obtained from 29 patients on median day 5 (IQR 4–7) after ICU admission referred to as first-biopsy and early time point. Twenty-two patients were still in the ICU at 2 weeks and allowed a second muscle biopsy at median day 15 (IQR 14–19), referred to as the second biopsy and late time point (Fig. 1 in the electronic supplementary material). Data during clinical course on organ failure, glucose metabolism and nutrition, as well as analgesics, sedation, and awareness are shown in Table 1 in the electronic supplementary material. Neuromuscular blocking agents were sparingly administered and their cumulative dosage was unremarkably low. Patients did not receive high-dose corticosteroid treatment. Hydrocortisone (200 mg/24 h, ‘‘low dose infusion’’) was given to patients with septic shock. No differences in MCSA of fast-twitch (type-IIa and -IIb) fibers were detected between controls and the first biopsy specimens of ICU patients. MCSAs of slow-twitch (type-I) fibers were smaller in controls compared to ICU patients in the first biopsy (Fig. 1a). In contrast, MCSAs of type-I, -IIa, and -IIb fibers decreased significantly between the first and second biopsy (Fig. 1a, b; Fig. 2 in the electronic supplementary material). The decrease in MCSA was predominant in type-II fibers. Comparing the median MCSA values of individual fiber types, we found that the strongest decrease of MCSA was for type-IIa fibers reaching 42 % (P \ 0.005), followed by type-IIb (32 %, P \ 0.005) and type-I (28 %, P \ 0.01) fibers. In contrast, no change in fiber type composition was observed between the first and second biopsy specimens
Table 1 Patients characteristics Patients’ characteristics
All patients enrolled
Controls
Number Age (years) Gender (m/f) BMI (kg/m2) MRC score Diagnosis [n (%)]
29 54 (41/68) 22/7 (76 %/24 %) 28 (24/31) 3.3 (3.0/3.9) ALI/ARDS: n = 11 (37.9 %) Sepsis: n = 7 (24.1 %) Trauma: n = 6 (20.7 %) CNS: n = 5 (17.2 %) 23 (79.3 %) ICU scores SOFA: 12 (10/14) SAPS-II: 42 (36/53)
5 69 (67/71) 3/2 (60 %/40 %) ND 5.0
Survivors [n (%)] Severity of illness (at ICU admission)
5 (100 %) NA
ALI/ARDS acute lung injury/acute respiratory distress syndrome, BMI body mass index, CNS central nervous system, NA not applicable, ND not determined, SOFA Sequential Organ Failure Assessment score, SAPS-II Simplified Acute Physiology Score-II
A
Type-I 8000
Myocyte cross sectional area [µm2 ]
Fig. 1 Fast- and slow-twitch fibers atrophied during critical illness. a Myocyte crosssectional area (MCSA, lm2) from controls (white) and ICU patients’ first (gray) and second (black) biopsy specimens in type-I (left), type-IIa (middle), and type-IIb (right) fibers. **P \ 0.01; *P \ 0.05; n.s. not significant. b ATPase stained fiber-type analyses from early (left) and late (right) time points (type-I, -IIa, and -IIb fibers are indicated; scale bar 100 lm). c Representative electron micrographs from the early (left) and late (right) time points revealed early destruction of myofiber ultrastructure (scale bar 2 lm). Myosin loss and mitochondrial ballooning occurred early during critical illness (left). At the later time point myosin became squeezed and distorted, Z-lines were deformed and H-zone shapes were blurred (right). Myosin loss (thick black arrow), Z-lines (white arrow), H-zone (small black arrow), mitochondria (white star)
*
Type-IIa
Type-IIb n.s.
n.s. n.s.
8000
n.s.
8000
**
**
**
6000
6000
6000
4000
4000
4000
2000
2000
2000
0
0 control day 5 day 15
n.s.
0 control day 5 day 15
ICU-patients
ICU-patients
B Early
control day 5 day 15 ICU-patients
Late
IIa
I IIb
C Early
Late
*
and was comparable to the fiber type composition in control subjects (Fig. 3 and Table 2 in the electronic supplementary material). There was no evidence of necrosis, regeneration, inflammatory infiltrates, or mitochondrial abnormalities, such as ragged red fibers or cytochrome C oxidase-negative fibers (data not shown). These data suggest that histological fiber atrophy can only be detected in the later phase of critical illness. The data also showed that all fiber types atrophy during the disease course.
To investigate if ultrastructural changes occurred early during critical illness we performed electron microscopy on biopsy specimens of ICU patients. Early biopsy specimens were characterized by sarcomeres devoid of MyHC fibrils, swollen mitochondria, edema, and increased spacing between sarcomeres (Fig. 1c, left). In the later biopsy specimens, not only was MyHC gone from the sarcomeres but also the sarcomeric ultrastructure was disrupted. Swollen mitochondria, edema, and increased spacing between sarcomeres were
A
Controls
ICU-patients
I
biopsy
II
I
II
I
II
I
MyHC slow
MyHC slow
170 kDa
170 kDa MyHC fast
MyHC fast 170 kDa
α-actin
B
II
170 kDa α-actin
43 kDa
MyHC slow
43 kDa
C
MyHC fast
* ** n.s.
0.5
0
Relative MyHC slow protein content
1.0
1.5
1.5
Relative protein content
Relative protein content
1.5
*** *** n.s.
1.0
0.5
0 control day 5 day 15
ICU-patients
control day 5 day 15
ICU-patients
1.0
0.5
k=0.414 R2 =0.211 P=0.005 0 0
0.5
1.0
1.5
Relative MyHC fast protein content
Fig. 2 Myosin heavy chain (MyHC) decreases in slow- and fasttwitch fibers. a Representative Western blots for slow (type-I) and fast (type-II) MyHC of four control subjects and four ICU patients (left), a-actin was used as loading control. Western blots of slow and fast MyHC of four ICU patients in the first (I) and second (II)
biopsy (right). b Quantification of Western blot signals are depicted as box plots showing median (IQR) and documents the marked decrease in MyHC. c Correlation between slow and fast MyHC protein contents in ICU patients (black circles and line). Controls are shown as white circles and dashed lines
found to a lesser degree (Fig. 1c, right). These changes paralleled the reduction of MCSA that was only detected in the biopsy specimens of the later time point. Thus, fiber ultrastructure and MyHC-containing fibrils were already perturbed at the early biopsy specimen, a time point where no changes were apparent by light microscopy. Western blotting showed a substantial reduction of MyHC in the first biopsy specimen (Fig. 2a, b). This reduction was consistent with the myosin loss seen by electron microscopy even before a decrease in MCSA was observed. Moreover, comparing the first and second biopsy specimens, we note that the slow and fast MyHC protein contents remained significantly low and did not differ between the two time points (Fig. 2a, b). Although slow and fast MyHC protein contents decreased simultaneously, the reduction of fast MyHC was more pronounced compared to slow MyHC. The findings are
consistent with a more pronounced atrophy of the fasttwitch fibers (Fig. 2c). These data showed an unexpected early decrease in both fast and slow MyHC proteins in severe critically ill patients. Decreased gene expression of the MyHC isoforms MyHC-1, -2, and -7 was found in the first biopsy specimens compared to controls. MyHC-7 expression encoding for slow-twitch type-I MyHC was significantly decreased in the early and later biopsy specimens. MyHC-1 and -2 expression, encoding for fast-twitch type-IIx and -IIa MyHC, were decreased in the early biopsy specimen, whereas MyHC-4 expression, encoding for fast-twitch type-IIb MyHC, remained unchanged. MyHC-2, -4, and -7 expression was significantly higher in the second biopsy specimen, compared to the first, suggesting some degree of recovery (Fig. 3). These data suggested that reduced MyHC synthesis contributed to early MyHC loss during severe critical illness. However, these results did
MyHC-7
MyHC-2
** Relative mRNA expression
n.s.
*
2.5
MyHC-1
n.s.
**
*** 2.5
MyHC-4
*
n.s.
**
15
*
2.5
2.0
2.0
12
2.0
1.5
1.5
9
1.5
1.0
1.0
6
1.0
0.5
0.5
3
0.5
0
0 control day 5 day 15 ICU-patients
0 control day 5 day 15 ICU-patients
n.s. n.s.
0 control day 5 day 15 ICU-patients
control day 5 day 15 ICU-patients
Fig. 3 Myosin heavy chain (MyHC) gene expression is regulated early in muscle of ICU patients. Gene expression analyses of slow (MyHC-7) and fast (MyHC-1, -2, and -4) MyHC by real-time RT-PCR. ***P \ 0.001; **P \ 0.01; *P \ 0.05; n.s. not significant
not exclude increased MyHC degradation as a contributing factor for decreased muscular MyHC contents in critical illness. In fact, UPS-dependent protein degradation has been implicated as the major pathway responsible for muscular protein degradation in skeletal muscle atrophy [15, 16]. Gene expression of MuRF-1, Atrogin1, Calpain-1, Caspase-3, and FOXO-1, which are all involved in skeletal muscle protein degradation, was quantitated by RTPCR (Fig. 4a). MuRF-1, Atrogin1, and FOXO-1 mRNA expression significantly increased in the first biopsy specimen and returned to control levels in the second. Caspase-3 expression was significantly increased in the first biopsy specimen and remained at this level in the second. Calpain-1 expression was unchanged in the first biopsy specimen, but increased in the second. These data showed that atrophy-related genes were activated at different time points during the disease course. MuRF-1 protein was significantly increased in the first biopsy by Western blotting, followed by a significant reduction in the second biopsy specimen (Fig. 4b). These data showed that changes in MuRF-1 protein content paralleled its mRNA expression in skeletal muscle in early critical illness. We next relied on RT-PCR to investigate the proteasome beta subunits (PSMB) of the 20S core particle of the proteasome. The three PSMB types-1, -2, and -5 encoding peptidyl-glutamyl peptide hydrolyzing, trypsin-like, and chymotrypsin-like activity, respectively, were significantly increased at the time of the first biopsy, as were PSMB-3, -4, and -7, which are catalytically inactive. Gene
expression of PSMB-6 remained unchanged. Gene expression of the interferon gamma-inducible PSMB subunits was either decreased (PSMB-9 and -10) or increased (PSMB-8). No significant differences in gene expression were found for any of the PSMB genes between the first and second biopsy specimens (Fig. 5). These data indicate that the proteasome was activated early and remained at this level during the disease course. We observed a close and inverse correlation between MuRF-1 protein contents in the first biopsy specimens and the MCSA of all three fiber types measured in the second biopsy specimens (Fig. 4a in the electronic supplementary material). In contrast, such a correlation was not found for the myosin protein (data not shown). Furthermore, higher MuRF-1 protein contents at the early stage of critical illness were significantly associated with the crucial MRC score during the disease course when patients were regaining consciousness (Fig. 4b in the electronic supplementary material) linking myosin loss and weakness. By univariate regression analyses we investigated if patient characteristics, severity of illness, medication (e.g., hydrocortisone) as well as non-excitable muscle membrane (Table 3 in the electronic supplementary material) were associated with the molecular findings. We found that increased age was associated with early increased MuRF1 protein content, and a higher SAPS-II score at admission was associated with greater MCSA reduction of type-IIb muscle fibers between the first and second biopsy. None of the other clinical parameters were associated with our experimental data.
A
MuRF-1
Atrogin1
**
** n.s.
Relative mRNA expression
8
***
*
n.s.
8
Calpain-1
**
16
*
FOXO-1
**
n.s.
* n.s.
16
n.s.
8
n.s.
6
6
12
12
6
4
4
8
8
4
2
2
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B
MuRF-1 30
***
0 control day 5 day 15
*
0
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control day 5 day 15
control day 5 day 15
ICU-patients
ICU-patients
ICU-patients
C
** ***
Relative protein content
Caspase-3
Controls
ICU-patients
ICU-patients biopsy
MuRF-1
20
α-actin
— 43kDa
— 43kDa
I
II
I
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I
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10
0 control day 5 day 15 ICU-patients
Fig. 4 Activation of protein degradation in muscle. a Values from controls are open boxes. Gene expression of MuRF-1, Atrogin1, Caspase-3, Calpain-1, and FOXO-1 exceeded normal subjects and decreased variably thereafter. b MuRF-1 quantification of Western blot signals are depicted as box plots showing median (IQR).
c Representative Western blots of MuRF-1 from four controls and four ICU patients document marked upregulation. a-Actin was used as loading control (left). By the second biopsy MuRF-1 upregulation had subsided (right)
Discussion
is particularly important for primary-care physicians who are generally responsible for these patients once they survive their acute illness. We draw attention to mechanisms contributing to ICU-acquired muscle wasting and weakness which often lead to persistent disabilities compromising patients long after hospital discharge [4, 5, 29]. It is unclear if neuropathic changes were involved in myosin loss. However, this seems to be unlikely for our study, because molecular changes in muscle occurred before electrophysiological signs of critical illness polyneuropathy were detectable.
The important finding in our study is the unexpected early and persistent loss of MyHC in skeletal muscle of severe critically ill patients occurring 5 days after ICU admission. Our data suggest that although MyHC is lost rapidly, the capacity of muscle from critically ill patients to regenerate is limited. Furthermore massive myosin loss implicates a need for long-term and persistent treatment strategies to strengthen muscle function once patients are diagnosed with ICU-acquired weakness. The latter point
PSMB1
PSMB2
*** 15
*** ***
Relative mRNA expression
PSMB4
*** **
15 n.s.
*** **
15
PSMB5
n.s.
** **
15 n.s.
n.s.
n.s.
12
12
12
12
9
9
9
9
9
6
6
6
6
6
3
3
3
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0 control day 5 day 15 ICU-patients
PSMB6
PSMB7
**
n.s. n.s.
control day 5 day 15
control day 5 day 15
ICU-patients
ICU-patients
PSMB8
PSMB9
ICU-patients
PSMB10
* **
30
0
0 control day 5 day 15
ICU-patients
n.s.
15
0 control day 5 day 15
** *
n.s.
30
n.s.
12
8
8
6 20
**
15
12
0
Relative mRNA expression
PSMB3
* n.s.
6
***
20
**
9
n.s.
4
4
2
2
6 10
10
3
0
0 control day 5 day 15 ICU-patients
0
0 control day 5 day 15
control day 5 day 15
ICU-patients
ICU-patients
0 control day 5 day 15 ICU-patients
control day 5 day 15 ICU-patients
Fig. 5 Activation of proteasomal subunits in skeletal muscle. persistent induction of the ubiquitin proteasome system (UPS). Gene expression of proteasome beta subunits (PSMB) 1–10 was ***P \ 0.001; **P \ 0.01; *P \ 0.05; n.s. not significant quantitated by real-time RT-PCR and revealed an early and
MyHC loss was accompanied by decreased MyHC mRNA expression and MyHC protein contents during the early phase of critical illness. The dynamics of MyHC gene expression and protein turnover implicate pathways responsible for MyHC loss that are possibly deregulated during the first days, if not hours, of severe critical illness. Our data are supported by other findings showing decreased protein synthesis in the skeletal muscle of critically ill patients [24]. In contrast, others report that protein synthesis is unchanged in critically ill patients [13]. Discrepancies between our study and the latter could be explained by time-dependent changes in gene and protein expression during the disease course.
MyHC loss was paralleled by an activation of proteins involved in myosin degradation. Proteasomal degradation of myofibrillar proteins requires initial proteolysis of actomyosin complexes by caspase-3 [30] and calpain-1 [31]. Since both proteases were increased in our study, access to the UPS could be alleviated, thereby possibly easing its degradation. In addition, MuRF-1 and Atrogin1 [20], key proteins for UPS-dependent MyHC degradation, were activated during early critical illness. The rapid loss of MyHC during the early disease phase may reflect effective cooperation between these components of UPS-mediated protein degradation. Our results are in line with findings in animal models, where various atrophy stimuli, such as
denervation, immobilization, and cancer [21] increased MuRF-1 and Atrogin1 expression in a typical time course with a maximum of expression 3–5 days after the insult and a subsequent decrease to control levels [20]. This time course might explain differences with other studies in which no changes in MuRF-1 expression in critically ill patients were found in late muscle biopsy specimens [13]. Increased MuRF-1 expression in critical illness is of significant clinical importance for ICU-acquired muscle wasting and weakness because MuRF-1 was directly correlated with myofiber atrophy and muscle weakness. We think that systemic inflammation—notably severe sepsis, a hallmark of the early disease phase in our study—caused enhanced muscular protein degradation by increasing MuRF-1 expression. This view is supported by the induction of FOXO-1. FOXO-1 is activated by inflammation and sepsis [32] and increases MuRF-1 and Atrogin1 expression during atrophy [33]. The decline in MuRF-1 and Atrogin1 expression, observed at the later disease phase when inflammation declined, supports this interpretation. Following ubiquitin-chain formation, myofibrillar proteins are recognized and degraded by the UPS. The activity and substrate specificity of the UPS are regulated by the composition of its catalytic subunits [34]. Recently, an increase in UPS activity was found in the diaphragms of patients with diaphragm disuse [18] and in ICUacquired weakness [13]. Our findings extend these data showing that muscular protein degradation involves different components of the UPS and that UPS-mediated protein degradation remains increased during the disease course of critical illness. Skeletal muscle atrophy during critical illness was mainly observed in fast-twitch fibers, which strongly depend on glycolytic metabolism. Earlier, we observed disturbed glucose utilization in skeletal muscle of patients with critical illness myopathy [35]. We reason that disturbed glucose utilization aggravates atrophy of fasttwitch fibers in critically ill patients. In addition, differences in the time course of fast- and slow-twitch fiber atrophy argue for distinct fiber-type-specific pathways involved favoring early atrophy of fast-twitch fibers. Indeed, this hypothesis is supported by our recent work showing preferential expression of MuRF-1 in fast-twitch fibers [36]. We believe that systemic inflammation and sepsis are key factors not only to increase protein degradation but also to impair glucose utilization in muscle during early critical illness. This view is supported by data showing that sepsis activates FOXO-1 leading to increased expression of its proteolytic target genes and to impaired carbohydrate oxidation in muscle [37]. High-dose corticosteroids can induce UPS-dependent protein degradation in muscle and lead to myopathy [38].
High-dose corticosteroids were an independent predictor of ICU-acquired paresis [1, 39]. In contrast, low-dose corticosteroids have not been shown to induce myopathy [12]. We have no reason to believe that low-dose hydrocortisone had an effect on skeletal muscle atrophy or ICU-acquired weakness in our patients. First, no highdose corticosteroid treatment was applied. Second, only patients with septic shock refractory to fluid resuscitation and vasopressor therapy received low-dose hydrocortisone treatment. Third, glucocorticosteroids were only applied sporadically and were unlikely to affect the skeletal muscle. Fourth, no correlation between the cumulative dose of hydrocortisone and any of the molecular, clinical, or morphological data obtained here were found by regression analyses. Although the number of patients in our study is small, sequential muscle biopsies strengthen our results. We bewail the absence of muscle biopsies at the time point of ICU admission; however, the first muscular biopsy only became possible once a legal proxy was named by the court, which was at day 3 after ICU admission at the earliest. General internists are likely to be faced with survivors of muscle wasting and ICU-acquired weakness [40, 41]. Survivors can reckon with sustained physical impairment [42]. One year after hospital discharge, half of the patients are unable to work and have substantially reduced health-related quality of life [4]. The economic impact of ICU-acquired weakness is staggering; the costs in post-discharge care are estimated at US$300,000/patient/year [43]. Even a small advance to avoid or ameliorate the course of ICU-acquired weakness would not only be helpful to our patients but also a major contribution to health-care providers. Our study shows that pathophysiological mechanisms resulting in ICU-acquired weakness are activated early during critical illness. Our hope is that our study can be helpful to develop strategies to treat these patients. Further clinical studies are needed to investigate therapeutic options to maintain muscle integrity and function during critical illness. Acknowledgments We are thankful for the patience and courage of our patients and their consenting relatives. We thank Anika Lindner and Josefine Russ for technical assistance. We thank Friedrich C. Luft, MD, FACP for his continued support and editorial assistance. The Deutsche Forschungsgemeinschaft (FI 965/21, FI 965/4-1 and La668/14-1, KFO 192—WE 4386/1-2), Muscular Dystrophy Association, Marie Curie International Reintegration grant (FP7-PEOPLE-2007-4-3-IRG), and the Deutsche Gesellschaft fu¨r Muskelkranke supported this work. Conflicts of interest interest.
The authors are not aware of any conflicts of
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