Figure 1.
Study protocol.
Table 1.
Characterization of critically ill patients.
Figure 2.
Muscular SAA1 and SAA4 expression and accumulation in CIM patients.
Control values (no ICU subjects) were set to one and means were indicated as dashed lines. (A) RT-PCR analyses of SAA1 and SAA4 expression at early (day 5) and late (day 15) time points in vastus lateralis muscle of critically ill patients, and (B) CIM and non-CIM patients. Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) expression was used as reference. Data are presented as box plots showing median, 25th and 75th percentiles. Wilcoxon tests were performed between early and late biopsy specimens and Mann-Whitney tests for the respective time points and controls. ***P<0.001, **P<0.01, *P<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.
Figure 3.
A-SAA serum levels and IL-6 and TNF-α expression in skeletal muscle of critically ill patients.
(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. **P<0.01, *P<0.05. (B) RT-PCR analyses of IL-6 and TNF-α expressions in skeletal muscle from critically ill patients at early (day 5) and late (day 15) time points. Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) expression was used as reference. (C) RT-PCR analyses of IL-6 and TNF-α expression at early and late time points in CIM and non-CIM patients. Data are presented as box plots showing median, 25th and 75th percentiles. Wilcoxon tests were performed between early and late biopsy specimens and Mann-Whitney tests for the respective time points and controls; ***P<0.001, **P<0.01, *P<0.05, or n.s. (not statistically significant).
Figure 4.
IL-6, TNF-α and LPS increased SAA1 expression and protein content in human skeletal muscle cells in vitro.
(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 SAA1 expression, which was normalized to beta-2-microglobulin expression. Relative gene expression by fold-induction of SAA1 expression (above column) is shown. **P<0.01, *P<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 SAA1 expression, which was normalized to Glyceraldehyde-3 phosphate dehydrogenase (GAPDH); *P<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.
Figure 5.
Early SAA1 expression correlated with muscle membrane excitability of critically ill patients.
A multivariate analysis was performed to test, if SAA1 and SAA4 expressions were correlated with clinical CIM parameters. SAA1 (left) and SAA4 (right) expressions measured in early biopsy specimens were inversely correlated with compound muscle action potential on direct muscle stimulation (dmCMAP).
Figure 6.
Sepsis and proinflammatory cytokines increase muscular SAA1 expression and protein content in vivo and in vitro.
(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 SAA1 expression in gastrocnemius plantaris and tibialis anterior muscles, which was normalized to GAPDH expression. **P<0.01, *P<0.05. (B) Immunohistochemistry of SAA1 (green) on gastrocnemius plantaris 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 vastus medialis of sham (n = 8) and CLP (n = 8) 24 h after surgery. ***P<0.001, *P<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 SAA1 expression, which was normalized to GAPDH; *P<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.