DNA methylation alterations in muscle of critically ill patients

Abstract

Background: Intensive care unit (ICU)-acquired weakness can persist beyond ICU stay and has been associated with long-term functional impairment of ICU survivors. Recently, DNA methylation alterations were found in the blood of ICU patients, partially explaining long-term developmental impairment of critically ill children. As illness-induced aberrant DNA methylation theoretically could also be involved in long-term weakness, we investigated whether the DNA methylation signature in muscle of adult critically ill patients differs from that in muscle of healthy controls.

Methods: Genome-wide methylation was determined (Infinium® HumanMethylationEPIC BeadChips) in DNA extracted from skeletal muscle biopsies that had been collected on Day 8 ± 1 in ICU from 172 EPaNIC-trial patients [66% male sex, median age 62.7 years, median body mass index (BMI) 25.9 kg/m2 ] and 20 matched healthy controls (70% male sex, median age 58.0 years, median BMI 24.4 kg/m2 ). Methylation status of individual cytosine-phosphate-guanine (CpG) sites of patients and controls was compared with F-tests, using the Benjamini-Hochberg false discovery rate to correct for multiple comparisons. Differential methylation of DNA regions was assessed with bump hunting, with 1000 permutations assessing uncertainty, expressed as family-wise error rate. Gene expression was investigated for 10 representative affected genes.

Results: In DNA from ICU patients, 565 CpG sites, associated with 400 unique genes, were differentially methylated as compared with controls (average difference 3.2 ± 0.1% ranging up to 16.9%, P < 0.00005). Many of the associated genes appeared highly relevant for muscle structure and function/weakness, including genes involved in myogenesis, muscle regeneration, nerve/muscle membrane excitability, muscle denervation/re-innervation, axon guidance/myelination/degeneration/regeneration, synapse function, ion channelling with especially calcium signalling, metabolism (glucose, protein, and fat), insulin signalling, neuroendocrine hormone regulation, mitochondrial function, autophagy, apoptosis, oxidative stress, Wnt signalling, transcription regulation, muscle fat infiltration during regeneration, and fibrosis. In patients as compared with controls, we also identified two hypomethylated regions, spanning 18 and 3 CpG sites in the promoters of the HIC1 and NADK2 genes, respectively (average differences 5.8 ± 0.01% and 12.1 ± 0.04%, family-wise error rate <0.05). HIC1 and NADK2 play important roles in muscle regeneration and postsynaptic acetylcholine receptors and in mitochondrial processes, respectively. Nine of 10 investigated genes containing DNA methylation alterations were differentially expressed in patients as compared with controls (P ≤ 0.03).

Conclusions: Critically ill patients present with a different DNA methylation signature in skeletal muscle as compared with healthy controls, which in theory could provide a biological basis for long-term persistence of weakness in ICU survivors.

Trial registration: ClinicalTrials.gov: NCT00512122, registered on 31 July 2007.

Keywords: Critical illness; DNA methylation; Epigenetics; Intensive care unit-acquired muscle weakness; Muscle.

Conflict of interest statement

None declared.

All authors certify that they comply with the ethical guidelines for authorship and publishing in the Journal of Cachexia, Sarcopenia and Muscle. Institutional review board approval was obtained (ML4190). The study was performed in accordance with the 1964 Declaration of Helsinki and its later amendments. Written informed consent was acquired from all patients or their next of kin.

© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.

Figures

Figure 1

Consort diagram. Data file‐related problem refers to unrepairable damage to one of the IDAT data files, which became unreadable. DNA, deoxyribonucleic acid; EPaNIC, Early Parenteral Nutrition Completing Enteral Nutrition in Adult Critically Ill Patients; ICU, intensive care unit; RCT, randomized controlled trial.

Figure 2

Location of differentially methylated cytosine–phosphate–guanine (CpG) sites. (A) Distribution over the autosomes, (B) association with known gene transcripts, and (C) relative position to CpG islands are shown for the 565 CpG sites that were significantly differentially methylated in critically ill patients as compared with healthy controls. Gene transcript regions are divided into the promotor region, where proteins bind to initiate transcription of the gene; the 5′ untranslated region (5′‐UTR) region, which regulates translation; the gene body, which actually encodes the protein; the 3′ untranslated region (3′‐UTR) region, which is a regulatory unit; and the intergenic region, which is not related to a known gene transcript. In relation to CpG islands, the regions are divided into islands, which are regions with a high frequency of CpG sites; shores, which immediately flank the islands (within 2000 bp); shelves, which flank the shores (within 4000 bp from a CpG island); and open‐sea regions, which are not related to a CpG island. The height of the bars indicates the total number of CpG sites. Red and blue bars indicate the number of CpG sites that were respectively hypermethylated and hypomethylated in patients as compared with controls. In relation to gene transcripts, the 78 CpG sites that were associated with more than one gene section type are included for each of these associations.

Figure 3

Visualisation of the differentially methylated regions in patients vs. controls. Regions that are differentially methylated in muscle of critically ill patients as compared with healthy controls are visualised. The red line on the chromosome ideogram (top line of each panel) indicates the position of the differentially methylated region (DMR) relative to the chromosome. The light blue highlighted area shows the DMR. In blue and red, average cytosine–phosphate–guanine (CpG) methylation (beta‐value) and its confidence interval are shown for healthy controls and patients, respectively. At the bottom of each panel, the location of CpG sites evaluated by the microarray is indicated. Purple areas show locations of exons of known gene transcripts in this region. (A) The first DMR, which is located on chromosome 17 and spans 636 nucleotides containing 18 CpG sites. All 18 CpG sites were hypomethylated in patients as compared with controls. This region spans the hypermethylated in cancer 1 (HIC1) gene. (B) The second DMR, which is located on chromosome 5, spans 34 nucleotides, and contains three CpG sites that were hypomethylated in patients as compared with controls. This region spans the NAD kinase 2 (NADK2) gene.

Figure 4

Gene expression. mRNA expression of selected genes affected by DNA methylation alterations is shown for controls (n = 19) and patients (n = 162). Results are expressed relatively to the median of the healthy controls, with cancer susceptibility candidate gene 3 (CASC3) used as housekeeping gene. Boxes represent medians and inter‐quartile ranges, and whiskers are drawn to the furthest point within 1.5 × inter‐quartile range from the box. P‐values for comparison of expression between patients and controls are shown for each gene.