RESUMEN
KEY POINTS: Histamine is a primordial signalling molecule, capable of activating cells in an autocrine or paracrine fashion via specific cell surface receptors, in a variety of pathways that probably predate its more recent role in innate and adaptive immunity. Although histamine is normally associated with pathological conditions or allergic and anaphylactic reactions, it may contribute beneficially to the normal changes that occur within skeletal muscle during the recovery from exercise. We show that the human response to exercise includes an altered expression of thousands of protein-coding genes, and much of this response appears to be driven by histamine. Histamine may be an important molecular transducer contributing to many of the adaptations that accompany chronic exercise training. ABSTRACT: Histamine is a primordial signalling molecule, capable of activating cells in an autocrine or paracrine fashion via specific cell surface receptors. In humans, aerobic exercise is followed by a post-exercise activation of histamine H1 and H2 receptors localized to the previously exercised muscle. This could trigger a broad range of cellular adaptations in response to exercise. Thus, we exploited RNA sequencing to explore the effects of H1 and H2 receptor blockade on the exercise transcriptome in human skeletal muscle tissue harvested from the vastus lateralis. We found that exercise exerts a profound influence on the human transcriptome, causing the differential expression of more than 3000 protein-coding genes. The influence of histamine blockade post-exercise was notable for 795 genes that were differentially expressed between the control and blockade condition, which represents >25% of the number responding to exercise. The broad histamine footprint on the human exercise transcriptome crosses many cellular functions, including inflammation, vascular function, metabolism, and cellular maintenance.
Asunto(s)
Ejercicio Físico/fisiología , Histamina/fisiología , Transcriptoma , Adulto , Femenino , Hemodinámica , Antagonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H1 no Sedantes/farmacología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Humanos , Rodilla/fisiología , Masculino , Músculo Esquelético/fisiología , Ranitidina/farmacología , Receptores Histamínicos H1/fisiología , Receptores Histamínicos H2/fisiología , Terfenadina/análogos & derivados , Terfenadina/farmacología , Adulto JovenRESUMEN
Early-life exposure to maternal obesity or a maternal calorically dense Western-style diet (WSD) is strongly associated with a greater risk of metabolic diseases in offspring, most notably insulin resistance and metabolic dysfunction-associated steatotic liver disease (MASLD). Prior studies in our well-characterized Japanese macaque model demonstrated that offspring of dams fed a WSD, even when weaned onto a control (CTR) diet, had reductions in skeletal muscle mitochondrial metabolism and increased skeletal muscle insulin resistance compared to offspring of dams on CTR diet. In the current study, we employed a nested design to test for differences in gene expression in skeletal muscle from lean 3-year-old adolescent offspring from dams fed a maternal WSD in both the presence and absence of maternal obesity or lean dams fed a CTR diet. We included offspring weaned to both a WSD or CTR diet to further account for differences in response to post-weaning diet and interaction effects between diets. Overall, we found that a maternal WSD fed to dams during pregnancy and lactation was the principal driver of differential gene expression (DEG) in offspring muscle at this time point. We identified key gene pathways important in insulin signaling including PI3K-Akt and MAP-kinase, regulation of muscle regeneration, and transcription-translation feedback loops, in both male and female offspring. Muscle DEG showed no measurable difference between offspring of obese dams on WSD compared to those of lean dams fed WSD. A post-weaning WSD effected offspring transcription only in individuals from the maternal CTR diet group but not in maternal WSD group. Collectively, we identify that maternal diet composition has a significant and lasting impact on offspring muscle transcriptome and influences later transcriptional response to WSD in muscle, which may underlie the increased metabolic disease risk in offspring.
Asunto(s)
Histamina , Acondicionamiento Físico Humano , Volumen Espiratorio Forzado , Humanos , TranscriptomaRESUMEN
Elevated CO(2) levels (hypercapnia) frequently occur in patients with obstructive pulmonary diseases and are associated with increased mortality. However, the effects of hypercapnia on non-neuronal tissues and the mechanisms that mediate these effects are largely unknown. Here, we develop Drosophila as a genetically tractable model for defining non-neuronal CO(2) responses and response pathways. We show that hypercapnia significantly impairs embryonic morphogenesis, egg laying, and egg hatching even in mutants lacking the Gr63a neuronal CO(2) sensor. Consistent with previous reports that hypercapnic acidosis can suppress mammalian NF-kappaB-regulated innate immune genes, we find that in adult flies and the phagocytic immune-responsive S2* cell line, hypercapnia suppresses induction of specific antimicrobial peptides that are regulated by Relish, a conserved Rel/NF-kappaB family member. Correspondingly, modest hypercapnia (7-13%) increases mortality of flies inoculated with E. faecalis, A. tumefaciens, or S. aureus. During E. faecalis and A. tumefaciens infection, increased bacterial loads were observed, indicating that hypercapnia can decrease host resistance. Hypercapnic immune suppression is not mediated by acidosis, the olfactory CO(2) receptor Gr63a, or by nitric oxide signaling. Further, hypercapnia does not induce responses characteristic of hypoxia, oxidative stress, or heat shock. Finally, proteolysis of the Relish IkappaB-like domain is unaffected by hypercapnia, indicating that immunosuppression acts downstream of, or in parallel to, Relish proteolytic activation. Our results suggest that hypercapnic immune suppression is mediated by a conserved response pathway, and illustrate a mechanism by which hypercapnia could contribute to worse outcomes of patients with advanced lung disease, who frequently suffer from both hypercapnia and respiratory infections.
Asunto(s)
Infecciones Bacterianas/inmunología , Dióxido de Carbono/farmacología , Drosophila melanogaster/inmunología , Drosophila melanogaster/microbiología , Inmunidad Innata/efectos de los fármacos , Acidosis/complicaciones , Acidosis/inmunología , Animales , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Infecciones Bacterianas/complicaciones , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/crecimiento & desarrollo , Regulación de la Expresión Génica/efectos de los fármacos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Hipercapnia/complicaciones , Hipercapnia/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/inmunología , Óxido Nítrico/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Análisis de Supervivencia , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Artroplastia de Reemplazo de Rodilla , Aminoácidos Esenciales , Artroplastia de Reemplazo de Rodilla/efectos adversos , Citocinas/genética , Suplementos Dietéticos , Expresión Génica , Humanos , Músculo Esquelético , Proteína p53 Supresora de Tumor/genéticaRESUMEN
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.
Asunto(s)
Artroplastia de Reemplazo de Rodilla/efectos adversos , Perfilación de la Expresión Génica/métodos , Músculo Cuádriceps/patología , Daño por Reperfusión/diagnóstico , Daño por Reperfusión/genética , Torniquetes/efectos adversos , Anciano , Artroplastia de Reemplazo de Rodilla/tendencias , Femenino , Redes Reguladoras de Genes/genética , Humanos , Masculino , Persona de Mediana Edad , Daño por Reperfusión/etiología , Torniquetes/tendencias , Transcripción Genética/genética , Resultado del TratamientoRESUMEN
Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the causal mutation by traditional mapping and candidate gene sequencing is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in Caenorhabditis elegans mutants. First, we report our use of restriction site-associated DNA (RAD) polymorphism markers for rapidly mapping mutations after chemical mutagenesis and mutant isolation. Second, we describe our use of genomic interval pull-down sequencing (GIPS) to selectively capture and sequence megabase-sized portions of a mutant genome. Together, these two methods provide a rapid and cost-effective approach for positional cloning of C. elegans mutant loci, and are also applicable to other genetic model systems.
Asunto(s)
Caenorhabditis elegans/genética , Análisis Mutacional de ADN/métodos , ADN/genética , Genoma/genética , Mapeo Restrictivo/métodos , Animales , ADN/metabolismo , Análisis Mutacional de ADN/economía , Sitios Genéticos/genética , Polimorfismo Genético/genética , Mapeo Restrictivo/economíaRESUMEN
To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.
Asunto(s)
Alelos , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Genes de Helminto/genética , Genes Letales/genética , Mutación/genética , Temperatura , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Larva/genética , Datos de Secuencia Molecular , Fenotipo , Análisis de Secuencia de ADNRESUMEN
Activation of heat shock proteins (Hsps) is critical to adaptation to low oxygen levels (hypoxia) and for enduring the oxidative stress of reoxygenation. Hsps are known to be regulated by heat shock factor (Hsf), but our results demonstrate an unexpected regulatory link between the oxygen-sensing and heat shock pathways. Hsf transcription is up-regulated during hypoxia due to direct binding by hypoxia-inducible factor-1 (HIF-1) to HIF-1 response elements in an Hsf intron. This increase in Hsf transcripts is necessary for full Hsp induction during hypoxia and reoxygenation. The HIF-1-dependent increase in Hsps has a functional impact, as reduced production of Hsps decreases viability of adult flies exposed to hypoxia and reoxygenation. Thus, HIF-1 control of Hsf transcriptional levels is a regulatory mechanism for sensitizing heat shock pathway activity in order to maximize production of protective Hsps. This cross-regulation represents a mechanism by which the low oxygen response pathway has assimilated complex new functions by regulating the key transcriptional activator of the heat shock pathway.