Inflammation-induced acute phase response in skeletal muscle and critical illness myopathy.

OBJECTIVES: Systemic inflammation is a major risk factor for critical-illness myopathy (CIM) but its pathogenic role in muscle is uncertain. We observed that interleukin 6 (IL-6) and serum amyloid A1 (SAA1) expression was upregulated in muscle of critically ill patients. To test the relevance of these responses we assessed inflammation and acute-phase response at early and late time points in muscle of patients at risk for CIM. DESIGN: Prospective observational clinical study and prospective animal trial. SETTING: Two intensive care units (ICU) and research laboratory. PATIENTS/SUBJECTS: 33 patients with Sequential Organ Failure Assessment scores ≥ 8 on 3 consecutive days within 5 days in ICU were investigated. A subgroup analysis of 12 patients with, and 18 patients without CIM (non-CIM) was performed. Two consecutive biopsies from vastus lateralis were obtained at median days 5 and 15, early and late time points. Controls were 5 healthy subjects undergoing elective orthopedic surgery. A septic mouse model and cultured myoblasts were used for mechanistic analyses. MEASUREMENTS AND MAIN RESULTS: Early SAA1 expression was significantly higher in skeletal muscle of CIM compared to non-CIM patients. Immunohistochemistry showed SAA1 accumulations in muscle of CIM patients at the early time point, which resolved later. SAA1 expression was induced by IL-6 and tumor necrosis factor-alpha in human and mouse myocytes in vitro. Inflammation-induced muscular SAA1 accumulation was reproduced in a sepsis mouse model. CONCLUSIONS: Skeletal muscle contributes to general inflammation and acute-phase response in CIM patients. Muscular SAA1 could be important for CIM pathogenesis. TRIAL REGISTRATION: ISRCTN77569430

Early <i>SAA1</i> expression correlated with muscle membrane excitability of critically ill patients.

<p>A multivariate analysis was performed to test, if <i>SAA1</i> and <i>SAA4</i> expressions were correlated with clinical CIM parameters. <i>SAA1</i> (left) and <i>SAA4</i> (right) expressions measured in early biopsy specimens were inversely correlated with compound muscle action potential on direct muscle stimulation (dmCMAP).</p

Sepsis and proinflammatory cytokines increase muscular SAA1 expression and protein content <i>in vivo</i> and <i>in vitro</i>.

<p>(A) Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in mice for 24 h (n = 5). Sham operated mice were used as controls (n = 5). RT-PCR was used to measure <i>SAA1</i> expression in <i>gastrocnemius plantaris</i> and <i>tibialis anterior</i> muscles, which was normalized to <i>GAPDH</i> expression. **<i>P</i><0.01, *<i>P</i><0.05. (B) Immunohistochemistry of SAA1 (green) on <i>gastrocnemius plantaris</i> muscle of sham and CLP treated mice (24 h treatment). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue); scale bar 50 μm. (C) Mass-spectrometry was used to quantitate SAA1, SAA2 and SAA4 in dialysates of <i>vastus medialis</i> of sham (n = 8) and CLP (n = 8) 24 h after surgery. ***<i>P</i><0.001, *<i>P</i><0.05. (D) Differentiated mouse skeletal myotubes were treated with murine recombinant IL-6 (100 ng/ml) or murine recombinant TNF-α (10 ng/ml) for 16 h. RT-PCR was used to measure <i>SAA1</i> expression, which was normalized to <i>GAPDH</i>; *<i>P</i><0.05. (E) Immunocytochemistry of SAA1 (green) on differentiated murine myotubes following treatment with murine recombinant IL-6 (100 ng/ml), murine recombinant TNF-α (10 ng/ml) or a combination of both (IL-6, 100 ng/ml; TNF-α, 10 ng/ml) for 16 h is shown. Nuclei were stained with DAPI (blue); scale bar 50 μm.</p

A-SAA serum levels and <i>IL-6</i> and <i>TNF-α</i> expression in skeletal muscle of critically ill patients.

<p>(A) Serum levels of acute phase SAA (A-SAA) measured by ELISA in healthy controls (n = 6), critically ill patients (ICUs, n = 30), non-CIM (n = 19) and CIM (n = 11) patients. Serum samples were obtained at days 2 to 3 after ICU admission. **<i>P</i><0.01, *<i>P</i><0.05. (B) RT-PCR analyses of <i>IL-6</i> and <i>TNF-α</i> expressions in skeletal muscle from critically ill patients at early (day 5) and late (day 15) time points. <i>Glyceraldehyde-3 phosphate dehydrogenase</i> (<i>GAPDH</i>) expression was used as reference. (C) RT-PCR analyses of <i>IL-6</i> and <i>TNF-α</i> expression at early and late time points in CIM and non-CIM patients. Data are presented as box plots showing median, 25^th and 75^th percentiles. Wilcoxon tests were performed between early and late biopsy specimens and Mann-Whitney tests for the respective time points and controls; ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, or n.s. (not statistically significant).</p

Muscular <i>SAA1</i> and <i>SAA4</i> expression and accumulation in CIM patients.

<p>Control values (no ICU subjects) were set to one and means were indicated as dashed lines. (A) RT-PCR analyses of <i>SAA1</i> and <i>SAA4</i> expression at early (day 5) and late (day 15) time points in <i>vastus lateralis</i> muscle of critically ill patients, and (B) CIM and non-CIM patients. <i>Glyceraldehyde-3 phosphate dehydrogenase</i> (<i>GAPDH</i>) expression was used as reference. Data are presented as box plots showing median, 25^th and 75^th percentiles. Wilcoxon tests were performed between early and late biopsy specimens and Mann-Whitney tests for the respective time points and controls. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, or n.s. (not statistically significant). (C) Immunohistochemistry of SAA1 (green) and the membrane marker laminin (red) on skeletal muscle biopsy specimens from control subjects, non-CIM and CIM patients. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue); scale bar 50 μm. (D) Higher magnification of the merged picture from CIM patient in (C) to illustrate colocalization of SAA1 (green) and laminin (red) at the cell membrane, accumulation of SAA1 in the interstitium and around myofibers; scale bar 50 μm.</p

Study protocol.

<p>Study protocol.</p

Characterization of critically ill patients.

<p>ICU indicates intensive care unit; BMI, body mass index; ARDS, acute respiratory distress syndrome; SOFA, Sequential Organ Failure Assessment; SAPS-II, Simplified Acute Physiology Score II; MRC, Medical Research Council; RASS, Richmond Agitation Sedation Scale. Results are expressed as medians with inter-quartile range or as absolute numbers with percentages. Differences are calculated between patients with excitable (non-CIM) and non-excitable (CIM) muscle membrane. Mann-Whitney test.</p><p>*<i>P</i><0.05,</p>†<p><i>P</i><0.01.</p

IL-6, TNF-α and LPS increased <i>SAA1</i> expression and protein content in human skeletal muscle cells <i>in vitro</i>.

<p>(A) Differentiated human skeletal myotubes were treated with human recombinant IL-6 (100 ng/ml), TNF-α (10 ng/ml), or a combination of both (IL-6, 100 ng/ml; TNF-α, 10 ng/ml) for 16 h. RT-PCR was used to measure <i>SAA1</i> expression, which was normalized to <i>beta-2-microglobulin</i> expression. Relative gene expression by fold-induction of <i>SAA1</i> expression (above column) is shown. **<i>P</i><0.01, *<i>P</i><0.05. (B) Immunocytochemistry of SAA1 (green) on differentiated human myotubes following treatment with human recombinant IL-6 (100 ng/ml), human recombinant TNF-α (10 ng/ml), and both cytokines (IL-6, 100 ng/ml; TNF-α, 10 ng/ml) together for 16 h is shown. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue); scale bar 50 μm. (C) Human skeletal myotubes were treated with lipopolysaccharide (LPS, 1 μg/ml) for 16 h. RT-PCR was used to measure <i>SAA1</i> expression, which was normalized to <i>Glyceraldehyde-3 phosphate dehydrogenase</i> (<i>GAPDH</i>); *<i>P</i><0.05. (D) Immunocytochemistry of SAA1 (green) on human myotubes following LPS treatment (1 μg/ml) for 16 h. Nuclei were stained with DAPI (blue); scale bar 50 μm.</p