RESUMEN
The protein α-actinin-3 expressed in fast-twitch skeletal muscle fiber is absent in 1.5 billion people worldwide due to homozygosity for a nonsense polymorphism in ACTN3 (R577X). The prevalence of the 577X allele increased as modern humans moved to colder climates, suggesting a link between α-actinin-3 deficiency and improved cold tolerance. Here, we show that humans lacking α-actinin-3 (XX) are superior in maintaining core body temperature during cold-water immersion due to changes in skeletal muscle thermogenesis. Muscles of XX individuals displayed a shift toward more slow-twitch isoforms of myosin heavy chain (MyHC) and sarcoplasmic reticulum (SR) proteins, accompanied by altered neuronal muscle activation resulting in increased tone rather than overt shivering. Experiments on Actn3 knockout mice showed no alterations in brown adipose tissue (BAT) properties that could explain the improved cold tolerance in XX individuals. Thus, this study provides a mechanism for the positive selection of the ACTN3 X-allele in cold climates and supports a key thermogenic role of skeletal muscle during cold exposure in humans.
Asunto(s)
Actinina/genética , Termogénesis/genética , Tejido Adiposo Pardo/metabolismo , Animales , Temperatura Corporal/genética , Codón sin Sentido/genética , Evolución Molecular , Humanos , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/metabolismo , Selección Genética/genéticaRESUMEN
Loss of expression of ACTN3, due to homozygosity of the common null polymorphism (p.Arg577X), is underrepresented in elite sprint/power athletes and has been associated with reduced muscle mass and strength in humans and mice. To investigate ACTN3 gene dosage in performance and whether expression could enhance muscle force, we performed meta-analysis and expression studies. Our general meta-analysis using a Bayesian random effects model in elite sprint/power athlete cohorts demonstrated a consistent homozygous-group effect across studies (per allele OR = 1.4, 95% CI 1.3-1.6) but substantial heterogeneity in heterozygotes. In mouse muscle, rAAV-mediated gene transfer overexpressed and rescued α-actinin-3 expression. Contrary to expectation, in vivo "doping" of ACTN3 at low to moderate doses demonstrated an absence of any change in function. At high doses, ACTN3 is toxic and detrimental to force generation, to demonstrate gene doping with supposedly performance-enhancing isoforms of sarcomeric proteins can be detrimental for muscle function. Restoration of α-actinin-3 did not enhance muscle mass but highlighted the primary role of α-actinin-3 in modulating muscle metabolism with altered fatiguability. This is the first study to express a Z-disk protein in healthy skeletal muscle and measure the in vivo effect. The sensitive balance of the sarcomeric proteins and muscle function has relevant implications in areas of gene doping in performance and therapy for neuromuscular disease.
Asunto(s)
Actinina/genética , Músculo Esquelético/fisiología , Anaerobiosis , Animales , Animales Recién Nacidos , Atletas , Calcineurina/metabolismo , Dependovirus/metabolismo , Regulación hacia Abajo/genética , Estudio de Asociación del Genoma Completo , Heterocigoto , Homocigoto , Humanos , Ratones Endogámicos C57BL , Fatiga Muscular , Fibras Musculares Esqueléticas/metabolismo , Tamaño de los Órganos , Oxidación-ReducciónRESUMEN
We used gene editing to introduce DNA sequences encoding the tdTomato fluorescent protein into the α -skeletal actin 1 (ACTA1) locus to develop an ACTA1-tdTomato induced pluripotent stem cell reporter line for monitoring differentiation of skeletal muscle. This cell line will be used to better understand skeletal muscle maturation and development in vitro as well as provide a useful tool for drug screening and the evaluation of novel therapeutics for the treatment of skeletal muscle disease.
Asunto(s)
Sistemas CRISPR-Cas , Células Madre Pluripotentes Inducidas , Proteína Fluorescente Roja , Humanos , Sistemas CRISPR-Cas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Actinas/genética , Actinas/metabolismo , Músculo Esquelético/metabolismoRESUMEN
To produce an in vitro model of nemaline myopathy, we reprogrammed the peripheral blood mononuclear cells (PBMCs) of a patient with a heterozygous p.Gly148Asp mutation in exon 3 of the ACTA1 gene to iPSCs. Using CRISPR/Cas9 gene editing we corrected the mutation to generate an isogenic control line. Both the mutant and control show a normal karyotype, express pluripotency markers and could differentiae into the three cell states that represent embryonic germ layers (endoderm, mesoderm and neuroectoderm) and the dermomyotome (precursor of skeletal muscle). When differentiated these cell lines will be used to explore disease mechanisms and evaluate novel therapeutics.