Transcriptomic analysis reveals abnormal muscle repair and remodeling in survivors of critical illness with sustained weakness

Abstract

ICU acquired weakness (ICUAW) is a common complication of critical illness characterized by structural and functional impairment of skeletal muscle. The resulting physical impairment may persist for years after ICU discharge, with few patients regaining functional independence. Elucidating molecular mechanisms underscoring sustained ICUAW is crucial to understanding outcomes linked to different morbidity trajectories as well as for the development of novel therapies. Quadriceps muscle biopsies and functional measures of muscle strength and mass were obtained at 7 days and 6 months post-ICU discharge from a cohort of ICUAW patients. Unsupervised co-expression network analysis of transcriptomic profiles identified discrete modules of co-expressed genes associated with the degree of muscle weakness and atrophy in early and sustained ICUAW. Modules were enriched for genes involved in skeletal muscle regeneration and extracellular matrix deposition. Collagen deposition in persistent ICUAW was confirmed by histochemical stain. Modules were further validated in an independent cohort of critically ill patients with sepsis-induced multi-organ failure and a porcine model of ICUAW, demonstrating disease-associated conservation across species and peripheral muscle type. Our findings provide a pathomolecular basis for sustained ICUAW, implicating aberrant expression of distinct skeletal muscle structural and regenerative genes in early and persistent ICUAW.

Figure 1

Differentially expressed genes in ICUAW (a). Heat map of 695 gene probes differentially expressed between ICUAW at day 7 and ICUAW at month 6 post-ICU versus healthy controls. Differential expression was assessed at a false positive discovery rate (FDR) <0.05 and fold change >1.0. Scaled expression values are color coded according to the legend below the heat map. The top bars indicate patient variables: group (purple, ICUAW day 7; pink, ICUAW month 6; grey, control), age and sex (values are color coded according to respective legend to the right of the heat map). (b) Venn diagram of differentially expressed probes in ICUAW day 7 post-ICU (left) and ICUAW 6 months ICU (right). Number of overlapping genes shared between day 7 and month 6 are shown within the four squares within the yellow diamond; number of genes exclusively differentially expressed in ICUAW day 7 (left) or ICUAW month 6 post-ICU (right) are shown in the 4 squares outside the yellow diamond.

Figure 2. Weighted gene correlation network identifies eleven ICUAW-associated modules.

Association with ICUAW for each module, represented by the module eigenegene (first principal component). Direction of differential expression: A positive sign (+) indicates upregulation, and negative sign (−) downregulation of ICUAW samples compared to controls. Ten modules are significantly associated with ICUAW day 7 post-ICU at FDR <0.05 (Differential expression [DE] P-value: red boxes in the left-hand column indicate significant DE in ICUAW day 7 post-ICU,), and three modules are associated with ICUAW month 6 post-ICU (red boxes in right column). Grey boxes in left and right columns indicate no significant DE in ICUAW at day 7 and month 6 post-ICU discharge, respectively. Module phenotype correlations (p-value in brackets) for muscle strength (based on highest absolute value of either MRCSS or peak torque), muscle mass, and physical function (FIM score). Significantly enriched gene ontology or human phenotype ontology terms, based on FDR <0.05 (representative examples listed). Significantly over-represented transcription factor binding sites (TFBS) based on P-value < 9.1 × 10⁻⁴ (three most significant TFBS above cutoff shown per module).

Figure 3. Module 1 and 3 are associated with ICUAW at day 7 and month 6 post-ICU discharge, respectively.

(a,e) Module eigengene values (y-axis) across samples (x-axis), Purple, ICUAW day 7 post ICU; pink, ICUAW month 6 post ICU; grey, controls. P-values of linear mixed effects regression with age and sex as fixed effects. (b,f) Module eigengene values (y-axis) vs. clinical measurements (x-axis). Correlation R-values calculated using Turkey’s Biweight correlation, p-values adjusted for multiple comparisons. (c,g) Relevant gene ontology categories enriched in the M1 and M3 modules. (d,h) Top 50 most highly connected genes in the M1 and M3 modules. i Low magnification (10x) representative photomicrographs of vastus lateralis muscle cross sections from a healthy control (left panel) and a sustained ICUAW patient at month 6 (right panel) stained with Picro-Sirius Red Stain. Muscle stains yellow while collagen stains red.

Figure 4. Modules M1 and M3 from human ICUAW patients are preserved in two independent data sets.

Summary preservation statistic based on permutation testing Z summary score, using the human ICUAW patients as a reference. The y axis displays the Z score for each module in (a) human sepsis-induced multiple organ dysfunction and (b). Pig model of ICUAW; numbers beside each module (colored circle) indicate the corresponding module in the reference dataset. The x-axis indicates the number of genes in the module. Z scores of less than 2 (bottom red line) implies no evidence for module preservation, while scores exceeding 5 (green line) and exceeding 10 implies moderate and strong evidence for module preservation, respectively.