Ilse Vanhorebeek

As compared with withholding parenteral nutrition (PN) for one week (late-PN), early supplementation of insufficient enteral nutrition with PN (early-PN) i

Critically ill patients requiring treatment in the intensive care unit (ICU) suffer from muscle weakness that persists for years. As compared with healthy subjects, skeletal muscle of patients biopsied five years post‐ICU revealed an abnormal transcriptome partially associated with poor muscle strength. We now hypothesized that skeletal muscle of long‐term ICU survivors is “epigenetically aged”, as determined by a muscle‐specific epigenetic clock, and that such accelerated epigenetic aging contributes to their long‐term muscle weakness. Muscle DNA‐methylation data from former ICU patients at 5‐year follow‐up ( N = 118) and healthy controls ( N = 160), aged 18–89 years, were analyzed by the MEATv2 epigenetic clock. First, epigenetic age (DNAmAge), epigenetic minus chronological age (AADiff) and epigenetic age acceleration (AAResid) were compared between 97 former patients and 97 controls, propensity score‐matched for age and sex. Next, the impact of any muscle‐specific epigenetic aging of ICU survivors was investigated, via multivariable models, as a potential contributor to the altered transcriptome and reduced muscle strength. Former ICU patients showed a significantly higher muscle DNAmAge, AADiff, and AAResid than matched controls. In adjusted models, higher muscle DNAmAge, AADiff, or AAResid did not substantially contribute to differentially expressed muscle RNAs in former patients as compared with controls and was not associated with the poor long‐term muscle strength. In conclusion, five years after ICU discharge, former patients showed accelerated epigenetic aging in skeletal muscle. However, the muscle‐specific epigenetic clock did not capture molecular changes that are associated with long‐term muscle weakness, which highlights the need for other muscle‐specific biological predictors of age‐related physical impairment. Trail Registration: ClinicalTrials.gov: NCT00512122

Background: Many children admitted to a pediatric intensive-care-unit (PICU) show impaired physical function years later. Testing physical function clinically is time-consuming and expensive, hampering routine follow-up. We investigated whether subjective parent-reported physical health-related quality-of-life correlates with and may replace objectively measured physical function of former PICU-patients. Methodology: This secondary analysis of the PEPaNIC-RCT included 517 former PICU-patients with physical function tests and parent-reported health-related quality-of-life 4 years later. Parents scored physical function, bodily pain, general health and physical role functioning with questionnaires. Physical function tests included handgrip-strength, timed up-and-go-test, 6-minute-walk-test, and accelerometry. Questionnaire-items and test-scores were correlated with Pearson/Spearman correlation. Results: Parent-reported physical function correlated moderately with daily time in sedentary state (ρ=-0.300, p < 0.0001), daily sedentary bouts ( R =-0.330, p < 0.0001) and daily steps/hour monitored ( R = 0.303, p < 0.0001), and weakly with handgrip-strength, physical activity energy expenditure, time in certain activity-intensity and daily steps (ρ/ R = 0.185–0.289, p ≤ 0.019). Parent-reported general health (ρ/ R =-0.228 to 0.296, p ≤ 0.033) and physical role functioning (ρ/ R =-0.269 to 0.240, p ≤ 0.038) correlated weakly with roughly the same measured physical functions. Parent-reported bodily pain correlated weakly with daily sedentary bouts ( R =-0.143, p = 0.024) and daily steps/hour monitored ( R = 0.145, p = 0.022). Correlations among physical function test scores were mostly weak. Conclusions: Several aspects of subjective parent-reported physical health-related quality-of-life correlated with former PICU-patients’ performance on physical function tests. The mostly weak strength of the correlations suggest that interrogated physical function cannot simply replace clinical testing. Nevertheless, when resources for clinical testing are lacking, interrogating physical function could still be valuable to get an idea about long-term physical functioning.

The PEPaNIC RCT showed that early supplementation of insufficient enteral nutrition by parenteral nutrition (early-PN) worsened outcome of critically ill children as compared with withholding PN for 1 week (late-PN). The best timing to initiate nutritional support in the pediatric intensive care unit (PICU) remains unclear. In adults, declining phosphate levels may identify patients who are particularly harmed by early-PN. We therefore assessed whether early hypophosphatemia in critically ill children may indicate metabolic intolerance to nutrition. In this secondary analysis of the PEPaNIC RCT (n = 1440), we investigated whether development of hypophosphatemia statistically interacts with the randomized intervention for its impact on clinical outcome, adjusting for baseline risk factors. Outcomes of interest included the incidence of new infections and the duration of PICU dependency as primary endpoints, 90-day mortality as safety endpoint, and duration of mechanical ventilation and of hospital stay as secondary endpoints. Subsequently, the impact of early-PN vs. late-PN in patients with and without early hypophosphatemia was assessed. Analyses were performed for phosphate abnormalities on PICU day 1 and 2, with and without imputing 20.3% (day 1) or 22.0% (day 2) missing phosphate data. Of patients with available phosphate measurements, 19.9% (211/1060) and 27.5% (243/883) developed hypophosphatemia on day 1 and day 2, respectively. Day 1 hypophosphatemia did not interact with the randomized intervention for any studied outcome. On day 2, hypophosphatemia interacted with randomization for risk of new infection (P = 0.031), likelihood of earlier live PICU discharge (P = 0.030), and time to live weaning from mechanical ventilation (P = 0.025). Harm by early-PN was more pronounced in patients with hypophosphatemia than in those without. Results after imputing missing phosphate data were similar, with an additional interaction for 90-day mortality (P = 0.038) revealing higher mortality with early-PN in patients with hypophosphatemia. Development of hypophosphatemia may identify critically ill children who are particularly harmed by early-PN. This opens perspectives for its use as a biomarker of metabolic intolerance to enhanced nutrition, which requires further investigation. ClinicalTrials.gov NCT01536275, registered February 2012.

Background: Critically ill patients requiring intensive care unit (ICU) admission suffer from muscle weakness that persists for years. Recently, altered RNA expression was documented in muscle of former ICU patients 5 years after critical illness that suggested disrupted mitochondrial function, disturbed lipid metabolism and fibrosis, of which many associated with the former patients' long‐term loss of muscle strength. We hypothesized that abnormal DNA methylation detectable years after critical illness associates with these abnormal RNA expression patterns, as a potential biological basis for the persistent loss of muscle strength. Methods: Genome‐wide DNA methylation was assessed (Infiniumv2‐HumanMethylationEPIC‐BeadChips) in skeletal muscle biopsies from 118 former ICU patients harvested 5 years after critical illness (79.6% male, median 58 years, median BMI 27.3 kg/m ² ) and 30 controls who never required ICU admission (76.7% male, median 61 years, median BMI 26.4 kg/m ² ). Differentially methylated positions (DMPs) in former patients versus controls were identified, adjusting for age, sex, and BMI (minfi‐package in R, Benjamini–Hochberg false‐discovery‐rate < 0.05), followed by pathway over‐representation of affected genes. Spearman correlations between DMP methylation and RNA expression were compared among groups of RNA with Z‐test and Kolmogorov–Smirnov test. Risk factors for abnormal DNA methylation were identified with multivariable linear regression. Results: As compared with controls, former ICU patients showed 7379 DMPs (average difference 2.6% ranging up to 24.9%). They were associated with 1334 unique genes, enriched for muscle contraction, vascular development, cell differentiation and signal transduction. DMPs correlated more strongly with differentially expressed RNAs (DERNAs) than with non‐differentially expressed RNAs (18.1% vs. 1.7% correlations with |rho| > 0.3, p < 2.2 × 10 ⁻¹⁶ ). Such correlations were more abundant among DERNAs associated with reduced muscle strength vs. those not associated (24.4% vs. 12.5%), also within the previously identified disrupted pathways (mitochondrial function 23.3% vs. 10.9%, lipid metabolism 15.9% vs. 7.2%, fibrosis 44.3% vs. 5.8%, all p < 2.2 × 10 ⁻¹⁶ ). Older age, female sex, in‐ICU treatment with glucocorticoids, benzodiazepines, early parenteral nutrition and opioids and insulin and antipsychotic medication at follow‐up were most notably associated with more abnormal DNA methylation. Conclusions: Abnormal DNA methylation in muscle biopsied 5 years after critical illness associated with long‐term altered RNA expression that has been linked to lower muscle strength. These data suggest a possible epigenetic basis for this long‐term sequel after critical illness. Abnormal DNA methylation was also found to associate with (possibly) avoidable risk factors during and after ICU stay. These findings may open perspectives for prevention and possibly treatment of long‐term muscle weakness after critical illness.

Background: Suppression of the peroxisome proliferator‐activated receptor alpha (PPARα) has been related to poor outcomes in sepsis and may compromise ketogenesis during critical illness. Infusion of 3‐hydroxybutyrate (3HB) was shown to attenuate muscle weakness in septic mice. We hypothesise that endogenous ketogenesis induced by pharmacological PPARα activation, either alone or combined with ketogenic nutrition, is safe and can also mitigate muscle weakness in septic mice. Methods: In a fluid‐resuscitated, antibiotic‐treated mouse model of prolonged sepsis, we first (Study 1) assessed the safety and effectiveness (impact on ketosis and muscle weakness) of the PPARα agonist pemafibrate (1 mg/kg/d, n = 16), versus placebo ( n = 15) combined with standard balanced parenteral nutrition (PN), composed of glucose, amino acids and long‐chain triglycerides (LCT) (balanced‐TPN). We subsequently (Study 2) evaluated the impact of pemafibrate combined with four types of PN on ketosis and muscle weakness: balanced‐TPN ( n = 18), TPN with extra LCTs (TPN + LCT, n = 18), low‐dose pure LCT emulsion (Low‐LCT, n = 16) and high‐dose pure LCT emulsion (High‐LCT n = 18). Carbohydrates and amino acids were omitted in the pure LCT groups. Healthy control mice (HC, n = 19) served as controls. Ex vivo muscle force was measured as the primary outcome. Metabolic, inflammatory and microstructural parameters were assessed on plasma and in muscle and liver tissue by targeted metabolomics, gene expression analysis, biochemical and metabolite assays and histological assessment. Results: Pemafibrate treatment with balanced‐TPN upregulated hepatic gene expression of PPARα ( Ppara ) and its downstream genes ( Cd36 , Cpt1a , Atgl , Acadl, Hadha, Acox1, and Hmgcs2 ) ( p < 0.0001) and was well tolerated. However, pemafibrate treatment with the use of balanced‐TPN administration did not induce detectable ketosis or improve muscle weakness. In combination with pemafibrate, TPN + LCT also did not induce ketosis, nor did it affect muscle weakness. In contrast, 3‐hydroxybutyrate plasma concentrations increased with High‐LCT (95‐fold) and Low‐LCT (10‐fold) ( p < 0.0001) in combination with pemafibrate, yet muscle force declined further (High‐LCT 25.0%, Low‐LCT 10.7% of HC, p < 0.0001). Blood glucose was lowered with pure High‐LCT and Low‐LCT (High‐LCT 86.9%, Low‐LCT 55.1% of TPN, p < 0.05), while plasma lipids and LC‐carnitines were increased ( p < 0.0001). Markers of hepatic protein catabolism were upregulated with High‐LCT and Low‐LCT ( p < 0.007), while muscle glycolytic intermediates ( p < 0.0001) and ATP levels ( p < 0.0001) were depleted. Conclusions: In septic mice, pemafibrate combined with balanced‐TPN or lipid‐rich TPN induced PPARα activation but did not result in ketosis nor affect muscle weakness. Pemafibrate combined with pure LCTs induced ketosis in sepsis but worsened muscle weakness, possibly explained by muscular bioenergetic failure.

Intensive care unit acquired weakness (ICUAW) is common in critically ill patients, contributing to substantial morbidity. Major trials and novel mechanistic findings published over the past years have advanced knowledge for the prevention and treatment of ICUAW. To streamline future research priorities, a multinational, interdisciplinary group of ICU clinicians, researchers, and people with lived experience convened to develop this evidence-based research agenda. Using a stepwise process including a systematic review with meta-analysis, two expert panel meetings, and a two-round modified Delphi method, we identified the top ten research priorities for ICUAW. Our report highlights the lack of reliable prognostic markers and mechanistic understanding that limit early diagnosis and treatment. Current evidence to treat ICUAW supports enhanced physical rehabilitation versus no rehabilitation, while higher dose enterally-delivered protein alone does not improve patient outcomes. However, the direct effects on muscles or appropriate dosing for patients with comorbidities remain largely unknown. The proposed ten key priority research questions integrate pathophysiology, diagnostics, treatment, and follow-up and emphasise personalised medicine and patient-centred outcomes over the continuum of recovery. Future research should focus on early prognostic markers, mechanisms of ICUAW, and identification of treatment responders allowing individualised dosing strategies tailored across the recovery trajectory. Defining meaningful outcomes, improving follow-up care, and integrating patient, family and caregiver priorities are essential. Advancing this agenda will require interdisciplinary collaboration and the use of emerging methods, including artificial intelligence, to support personalised and effective ICUAW care.

Purpose Critically ill patients requiring intensive care unit (ICU) admission suffer from muscle weakness that persists for years, compromising quality-of-life. The pathophysiology of this long-term weakness remains unclear. We hypothesized that former ICU-patients show a long-term abnormal RNA-expression profile, which may contribute to lower long-term strength and for which modifiable risk factors can be identified. Methods This pre-planned secondary analysis of the EPaNIC-trial compared muscle transcriptomes of 115 former ICU-patients 5 years after critical illness and 30 matched controls with RNA-sequencing, followed by pathway over-representation and differential co-expression analyses of the differentially expressed RNAs. We used multivariable linear regression analyses to identify which of the abnormal RNA-expressions associated with the long-term muscle strength of the patients and to identify potential risk factors for the abnormal RNA-expressions. Results In former patients, 234 down-regulated and 116 up-regulated RNAs were identified after adjustment for age, sex, and BMI. Pathway over-representation and further molecular and histological analyses indicated impaired mitochondrial energy metabolism, disturbed lipid metabolism, and increased collagen formation/fibrosis in former patients. Abnormal muscle RNA-expression in former patients correlated with lower long-term muscle strength. Several treatments given in-ICU and at 5-year follow-up associated with abnormal RNA-expression, most notably in-ICU early parenteral nutrition (early PN) and glucocorticoid use. Conclusion Abnormal RNA-expression profiles 5 years after critical illness suggest disrupted mitochondrial function, disturbed lipid metabolism, and fibrosis, associated with lower long-term muscle strength and partly attributable to possibly avoidable risk factors. These findings open perspectives for prevention and possibly treatment of long-term muscle weakness after critical illness. Trial registration number and date ClinicalTrials.gov-NCT00512122, July 31, 2007.