Essential amino acid supplementation alters the p53 transcriptional response and cytokine gene expression following total knee arthroplasty.

Reducing muscle atrophy following orthopedic surgery is critical during the postoperative period. Our previous work in patients who underwent total knee arthroplasty (TKA) showed that the vast majority of atrophy occurs within 2 wk following surgery and that essential amino acid (EAA) supplementation attenuates this atrophy. We used RNA-sequencing (RNA-seq) to identify genes associated with atrophy after TKA with and without EAAs. Analysis of overrepresented gene-ontology terms revealed that p53 signaling and the cytokine-cytokine receptor pathways were highly upregulated after TKA. Relative to the placebo group, the EAA group had altered expression of p53 regulators such as MDM2. This altered expression may account for differences between groups in timing of upregulation of some p53 targets such as apoptosis genes, and may account for the reduction in muscle loss in the subjects receiving EAAs. Furthermore, we observed altered expression of a large number of cytokine-signaling genes including TNFRSF12A, which plays a critical role in muscle atrophy, myogenesis, fibrosis, and the noncanonical NF-κB pathway.NEW & NOTEWORTHY Total knee arthroplasty is the most frequently performed inpatient surgical procedure for those over 45 yr in the United States. Following surgery, patients lose a large amount of muscle, which impacts functional mobility. Previously, our laboratory found that supplementing patients' diets with essential amino acids (EAAs) reduces postsurgical muscle loss. Here, our goal was to characterize the transcriptional changes associated with surgery with and without EAA supplementation to uncover the underlying mechanisms by which EAAs attenuate this muscle loss.

Cellular and morphological changes with EAA supplementation before and after total knee arthroplasty.

Investigate the underlying cellular basis of muscle atrophy (Placebo) and atrophy reduction (essential amino acid supplementation, EAAs) in total knee arthroplasty (TKA) patients by examining satellite cells and other key histological markers of inflammation, recovery, and fibrosis. Forty-one subjects (53-76 yr) scheduled for TKA were randomized into two groups, ingesting 20 g of EAAs or placebo, twice-daily, for 7 days before TKA and for 6 wk after surgery. A first set of muscle biopsies was obtained from both legs before surgery in the operating room, and patients were randomly assigned and equally allocated to have two additional biopsies at either 1 or 2 wk after surgery. Biopsies were processed for gene expression and immunohistochemistry. Satellite cells were significantly higher in patients ingesting 20 g of essential amino acids twice daily for the 7 days leading up to surgery compared with Placebo (operative leg P = 0.03 for satellite cells/fiber and P = 0.05 for satellite cell proportions for Type I-associated cells and P = 0.05 for satellite cells/fiber for Type II-associated cells.) Myogenic regulatory factor gene expression was different between groups, with the Placebo Group having elevated MyoD expression at 1 wk and EAAs having elevated myogenin expression at 1 wk. M1 macrophages were more prevalent in Placebo than the EAAs Group. IL-6 and TNF-α transcripts were elevated postsurgery in both groups; however, TNF-α declined by 2 wk in the EAAs Group. EAAs starting 7 days before surgery increased satellite cells on the day of surgery and promoted a more favorable inflammatory environment postsurgery.NEW & NOTEWORTHY Clinical studies by our group indicate that the majority of muscle atrophy after total knee arthroplasty (TKA) in older adults occurs rapidly, within the first 2 wks. We have also shown that essential amino acid supplementation (EAAs) before and after TKA mitigates muscle atrophy; however, the mechanisms are unknown. These results suggest that satellite cell numbers are elevated with EAA ingestion before surgery, and after surgery, EAA ingestion positively influences markers of inflammation. Combined, these data may help inform further studies designed to address the accelerated sarcopenia that occurs in older adults after major surgery.

Essential Amino Acid Supplementation Mitigates Muscle Atrophy After Total Knee Arthroplasty: A Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Substantial muscle atrophy occurs after total knee arthroplasty (TKA), resulting in decreased strength and impaired mobility. We sought to determine whether perioperative supplementation with essential amino acids (EAA) would attenuate muscle atrophy following TKA and whether the supplements were safe for ingestion in an older surgical population. METHODS: We performed a double-blind, placebo-controlled, randomized trial of 39 adults (age range, 53 to 76 years) undergoing primary unilateral TKA who ingested 20 g of EAA (n = 19) or placebo (n = 20) twice daily for 7 days preoperatively and for 6 weeks postoperatively. At baseline and 6 weeks postoperatively, magnetic resonance imaging (MRI) scans were obtained to measure quadriceps and hamstrings muscle volume. Secondary outcomes included functional mobility and strength. Data on physical activity, diet, and patient-reported outcomes (Veterans RAND 12-Item Health Survey and Knee injury and Osteoarthritis Outcome Score) were collected. Safety was determined through blood tests evaluating blood urea nitrogen, creatinine, creatinine clearance, homocysteine, and renal and liver function. Laboratory values at baseline, on the day of surgery, and at 2 days, 2 weeks, and 6 weeks postoperatively were compared between treatment groups. Analysis of covariance models, with baseline values as covariates, were used to evaluate outcomes between treatment groups. P values were adjusted for multiple tests. RESULTS: Compared with baseline, the EAA group had significantly less decrease in mean quadriceps muscle volume compared with the placebo group in the involved leg (-8.5% ± 2.5% compared with -13.4% ± 1.9%; p = 0.033) and the contralateral leg (-1.5% ± 1.6% compared with -7.2% ± 1.4%; p = 0.014). The hamstrings also demonstrated a greater muscle-volume-sparing effect for the EAA group than for the placebo group in the involved leg (-7.4% ± 2.0% compared with -12.2% ± 1.4%; p = 0.036) and contralateral leg (-2.1% ± 1.3% compared with -7.5% ± 1.5%; p = 0.005). There were no differences between the groups in terms of functional measures or strength. Blood chemistry values varied significantly between assessments periods but did not statistically differ between groups. CONCLUSIONS: The results of the present study suggest that EAA supplementation is safe and reduces the loss of muscle volume in older adults recovering from TKA. LEVEL OF EVIDENCE: Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Adaptive remodeling of skeletal muscle energy metabolism in high-altitude hypoxia: Lessons from AltitudeOmics.

Metabolic responses to hypoxia play important roles in cell survival strategies and disease pathogenesis in humans. However, the homeostatic adjustments that balance changes in energy supply and demand to maintain organismal function under chronic low oxygen conditions remain incompletely understood, making it difficult to distinguish adaptive from maladaptive responses in hypoxia-related pathologies. We integrated metabolomic and proteomic profiling with mitochondrial respirometry and blood gas analyses to comprehensively define the physiological responses of skeletal muscle energy metabolism to 16 days of high-altitude hypoxia (5260 m) in healthy volunteers from the AltitudeOmics project. In contrast to the view that hypoxia down-regulates aerobic metabolism, results show that mitochondria play a central role in muscle hypoxia adaptation by supporting higher resting phosphorylation potential and enhancing the efficiency of long-chain acylcarnitine oxidation. This directs increases in muscle glucose toward pentose phosphate and one-carbon metabolism pathways that support cytosolic redox balance and help mitigate the effects of increased protein and purine nucleotide catabolism in hypoxia. Muscle accumulation of free amino acids favor these adjustments by coordinating cytosolic and mitochondrial pathways to rid the cell of excess nitrogen, but might ultimately limit muscle oxidative capacity in vivo Collectively, these studies illustrate how an integration of aerobic and anaerobic metabolism is required for physiological hypoxia adaptation in skeletal muscle, and highlight protein catabolism and allosteric regulation as unexpected orchestrators of metabolic remodeling in this context. These findings have important implications for the management of hypoxia-related diseases and other conditions associated with chronic catabolic stress.

Transcriptional profiling and muscle cross-section analysis reveal signs of ischemia reperfusion injury following total knee arthroplasty with tourniquet.

Total knee arthroplasty (TKA) is the most common and cost-effective treatment for older adults with long-standing osteoarthritis. Projections indicate that nearly 3.5 million older adults will undergo this procedure annually by the year 2030. Thus, understanding the factors that lead to optimal outcomes is of great clinical interest. In the majority of cases, tourniquet is applied during surgery to maintain a clear surgical field, however, there is debate as to whether this intervention is completely benign. In particular, muscle atrophy is a significant factor in preventing full functional recovery following surgery, and some evidence suggests that tourniquet application and the associated ischemia-reperfusion injury that results contributes to muscle atrophy. For this reason, we examined tissue level changes in muscle in TKA patients following surgery and found that there was a significant increase in cross-sectional area of muscle fibers of all types. Furthermore, to detect changes not evident at the tissue level, we performed NextSeq analysis to assess the transcriptional landscape of quadriceps muscle cells following TKA with tourniquet and found 72 genes that were significantly upregulated. A large proportion of those genes regulate cell stress pathways, suggesting that muscle cells in our cohort of older adults were capable of mounting a significant response to cell stress. Furthermore, factors related to complement were upregulated, suggesting tourniquet may play a role in priming cells to ischemia reperfusion injury. Therefore, our analysis reveals potential harms of tourniquet during TKA, thus suggesting that surgeons should consider limiting its use.

Identification of genetic modifiers of CagA-induced epithelial disruption in Drosophila.

Helicobacter pylori strains containing the CagA protein are associated with high risk of gastric diseases including atrophic gastritis, peptic ulcers, and gastric cancer. CagA is injected into host cells via a Type IV secretion system where it activates growth factor-like signaling, disrupts cell-cell junctions, and perturbs host cell polarity. Using a transgenic Drosophila model, we have shown that CagA expression disrupts the morphogenesis of epithelial tissues such as the adult eye. Here we describe a genetic screen to identify modifiers of CagA-induced eye defects. We determined that reducing the copy number of genes encoding components of signaling pathways known to be targeted by CagA, such as the epidermal growth factor receptor (EGFR), modified the CagA-induced eye phenotypes. In our screen of just over half the Drosophila genome, we discovered 12 genes that either suppressed or enhanced CagA's disruption of the eye epithelium. Included in this list are genes involved in epithelial integrity, intracellular trafficking, and signal transduction. We investigated the mechanism of one suppressor, encoding the epithelial polarity determinant and junction protein Coracle, which is homologous to the mammalian Protein 4.1. We found that loss of a single copy of coracle improved the organization and integrity of larval retinal epithelia expressing CagA, but did not alter CagA's localization to cell junctions. Loss of a single copy of the coracle antagonist crumbs enhanced CagA-associated disruption of the larval retinal epithelium, whereas overexpression of crumbs suppressed this phenotype. Collectively, these results point to new cellular pathways whose disruption by CagA are likely to contribute to H. pylori-associated disease pathology.

Helicobacter pylori CagA disrupts epithelial patterning by activating myosin light chain.

Helicobacter pylori infection is a leading cause of ulcers and gastric cancer. We show that expression of the H. pylori virulence factor CagA in a model Drosophila melanogaster epithelium induces morphological disruptions including ectopic furrowing. We find that CagA alters the distribution and increases the levels of activated myosin regulatory light chain (MLC), a key regulator of epithelial integrity. Reducing MLC activity suppresses CagA-induced disruptions. A CagA mutant lacking EPIYA motifs (CagA(EPISA)) induces less epithelial disruption and is not targeted to apical foci like wild-type CagA. In a cell culture model in which CagA(EPISA) and CagA have equivalent subcellular localization, CagA(EPISA) is equally potent in activating MLC. Therefore, in our transgenic system, CagA is targeted by EPIYA motifs to a specific apical region of the epithelium where it efficiently activates MLC to disrupt epithelial integrity.

Bugs inside Bugs: what the fruit fly can teach us about immune and microbial balance in the gut.

In their coexistence with microbes, animals must fortify themselves against the onslaught of pathogens while maintaining a healthful consortium of resident commensals. In a recent paper in Science, Ryu and colleagues report that in fruit flies the Caudal transcription factor regulates appropriate immune responses to commensal bacteria, and that when these immune responses are perturbed, the gut microbial consortium shifts to one that is harmful to its host.

Tbx16 cooperates with Wnt11 in assembling the zebrafish organizer.

The organizer, the signaling center that specifies vertebrate axial polarity and the nervous system, is a dorsal midline mesodermal domain in the gastrula that will form prechordal plate and anterior notochord. We show that in zebrafish the organizer is not a single domain when it first arises in the nascent mesoderm at the onset of gastrulation. Rather, in the presumptive prechordal plate region, the organizer is subdivided into two side-by-side cellular fields. Within minutes, concurrent medial and anterior cellular movements merge, or 'coalesce', the two fields to form the well-known singular midline field. Coalescence forms a symmetrical domain because the cell movements on the left and right sides initiate simultaneously and occur synchronously. However, in embryos with reduced function of the T-box transcription factor Tbx16 (Spadetail) or its genetic target paraxial protocadherin (Papc), synchrony is lost, coalesence is disrupted, and the midline domain is misshaped. Furthermore, with combined loss of Tbx16 and Wnt11 (Silberblick), coalesence is essentially absent. Possibly as a consequence, both the anterior movement of presumptive prechordal plate and organizer function, as assayed by eye-field separation, are disrupted. Our findings thus reveal that Tbx16, in combination with Wnt11, are critical components not only in morphogenesis but also in initial assembly of the organizer.