Biology subjects

North, K. N.

Publications and source records attributed to North, K. N..

8 recordsLinked to original sources

bioRxiv2024

alpha-Actinin-3 deficiency protects from the effects of acute cold exposure through altered skeletal muscle Ca2+ and OXPHOS signaling.

Summary / AbstractA nonsense polymorphism in the ACTN3 gene (R577X, rs1815739) results in the loss of the fast skeletal muscle fiber protein -actinin-3 in an estimated 1.5 billion humans worldwide. Homozygosity for this common polymorphism (ACTN3 577XX) does not cause disease but is detrimental to sprint performance in elite athletes. Recently, we reported that ACTN3 577XX humans and Actn3 knockout mice show improved cold tolerance. This was not due to an increase in brown adipose tissue activity or a greater muscle shivering response, but was associated with an increased abundance of slow myosin heavy chain muscle fibers in XX individuals, which in turn increased muscle tone to improve cold tolerance. Using the Actn3 knockout mouse we have now analyzed the molecular and physiological impacts of acute cold exposure (4{degrees}C, for 5 hours) on skeletal muscle. In addition, we used statistical inference of publicly available archaic (Neanderthal and Denisovan) and modern human DNA data to estimate the age of the ACTN3 X-allele and to identify a novel haplotype associated with this variant that provides support for genetic selection in some populations of modern humans. Our results indicate that skeletal muscle of Actn3 knockout mice exhibit higher mitochondrial oxidative phosphorylation abundance and activity than wild-type controls. Following acute cold exposure, we identified reductions in gene ontology pathways associated with metabolism and protein breakdown, which impact muscle mass loss and improved muscle fatigue resistance. Together this provides further evidence for improved cold tolerance in Actn3 knockout mice and ACTN3 577XX individuals. Furthermore, we confirm that the X-allele first appeared in modern humans [~]135,000 years ago, with this variant increasing in frequency in European and Asian populations from as early as 42,000 years ago, which aligns with modern human migration out of Africa. Taken together these data provide additional molecular and functional evidence for a protective advantage of -actinin-3 deficiency following cold exposure, highlighting the importance of skeletal muscle in mammalian thermogenesis and its potential role in the evolution of modern-day humans.

cell biology↗

bioRxiv2024

ACTN3 genotype influences androgen response in skeletal muscle

AbstractAndrogens are vital for the maintenance of muscle mass and their anabolic effects are primarily exerted through the androgen receptor (AR). Accumulating evidence in humans and mice suggests that circulating androgens, AR and androgen response are influenced by ACTN3 (- actinin-3), also known as "the gene for speed". One in 5 people worldwide are -actinin-3 deficient due to homozygous inheritance of a common null polymorphism (577X) in ACTN3. In this study, we show that -actinin-3 deficiency decreases baseline AR in skeletal muscles of mice and humans, in both males and females, and that AR expression directly correlates with ACTN3 in a dosage dependent manner. We further demonstrate in Actn3 knockout mice that - actinin-3 deficiency increases muscle wasting induced by androgen deprivation and reduces the muscle hypertrophic response to dihydrotestosterone and this is mediated by differential activation of pathways regulating amino acid metabolism, intracellular transport, MAPK signalling, autophagy, mitochondrial activity and calcineurin signalling. Gene set enrichment and protein analyses indicate that the absence of -actinin-3 results in a failure to coactivate many of these pathways in response to changes in androgens, and relies on leveraging mitochondrial remodelling and calcineurin signalling to restore muscle homeostasis. We further identified 7 genes that are androgen sensitive and -actinin-3-dependent in expression, and whose functions correspond to these processes. Our results highlight the pivotal role of - actinin-3 in various processes associated with the regulation of protein turnover and muscle mass, and suggest that ACTN3 genotype is a genetic modifier of androgen action in skeletal muscle.

genetics↗

bioRxiv2024

Modern Insights into Muscle Glycogen Phosphorylase Activity

Recent identification of new human muscle glycogen phosphorylation sites has renewed interest in understanding human variations in the regulation of glycogen metabolism and glucose homeostasis. This paper presents a detailed method for the measurement of glycogen phosphorylase (GPh) activity in skeletal muscle. Our approach incorporates modifications to existing radiolabelling assays, optimizing specificity and sensitivity while enabling the assessment of both active and total enzyme activity levels. The utilization of radioisotope tracers and scintillation counting ensures accurate quantification of GPh activity, which we use to validate a previously published reduction in GPh activity in an Actn3 deficient mouse model. Moreover, we introduce a step-by-step guide for data acquisition, highlight the use of appropriate homogenization, discuss the need for allosteric activators/inhibitors and the importance of assay optimization to record a GPh activity assay for skeletal muscle. In conclusion, our refined method not only contributes to a deeper understanding of glycogen metabolism in muscle tissue but also provides a framework for future investigations, underscoring its role in advancing research on glycogen utilization and glucose homeostasis. NEW & NOTEWORTHYThe study optimizes the glycogen phosphorylase radiolabelled activity assay, unveiling nuances in muscle homogenization, sample dilution, and caffeine inclusion. The research introduces standardized conditions, enhancing assay reliability and reproducibility across mouse strains to reveal sex specific variations in GPh activity and underscore novel distinctions in an Actn3 deficient mouse model. These findings advance our understanding of muscle glycogen metabolism, offering a crucial tool for researchers and facilitating meaningful inter-laboratory comparisons.

biochemistry↗

bioRxiv2021

Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics

BackgroundA common polymorphism (R577X) in the ACTN3 gene results in complete absence of the Z-disc protein -actinin-3 from fast-twitch muscle fibres in ~16% of the worlds population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0+ 20% stretch did not cause eccentric damage. In contrast, L0+ 30% stretch produced a significant ~40% deficit in maximum force; here we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this. MethodsSingle fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means {+/-} SD and tested for significance using ANOVA. ResultsWe show that the absence of -actinin-3 does not affect the unloaded maximum speed of contraction, visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically contracted. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar lattice shifts. ConclusionsAbsence of -actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that, in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0+ 30%, but not, L0+ 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why -actinin-3 deficient humans have been reported to have a faster decline in muscle function with increasing age, that is; as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional -actinin-3 deficient fibres will be increased, resulting in fibres breakages.

physiology↗

bioRxiv2021

Loss of α-actinin-3 confers protection from eccentric contraction damage in fast-twitch EDL muscles from aged mdx dystrophic mice by reducing pathological fibre branching

The common null polymorphism (R577X) in the ACTN3 gene is present in over 1.5 billion people worldwide and results in the absence of the protein -actinin-3 from the Z-discs of fast-twitch skeletal muscle fibres. We have previously reported that this polymorphism is a modifier of dystrophin deficient Duchenne Muscular Dystrophy. To investigate the mechanism underlying this we use a double knockout (dk)Actn3KO/mdx (dKO) mouse model which lacks both dystrophin and sarcomere -actinin-3. We used dKO mice and mdx dystrophic mice at 12 months (aged) to investigate the correlation between morphological changes to the fast-twitch dKO EDL and the reduction in force deficit produced by an in vitro eccentric contraction protocol. In the aged dKO mouse we found a marked reduction in fibre branching complexity that correlated with protection from eccentric contraction induced force deficit. Complex branches in the aged dKO EDL fibres (28%) were substantially reduced compared to aged mdx EDL fibres (68%) and this correlates with a graded force loss over three eccentric contractions for dKO muscles ([~]35% after first contraction, [~]66% overall) compared to an abrupt drop in mdx upon the first eccentric contraction ([~]73% after first contraction, [~]89% after three contractions). In dKO protection from eccentric contraction damage was linked with a doubling of SERCA1 pump density the EDL. We propose that the increased oxidative metabolism of fast-twitch glycolytic fibres characteristic of the null polymorphism (R577X) and increase in SR Ca2+ pump proteins reduces muscle fibre branching and decreases susceptibility to eccentric injury in the dystrophinopathies.

genetics↗

bioRxiv2021

Lifespan analysis of dystrophic mdx fast-twitch muscle morphology and its impact on contractile function

Duchenne muscular dystrophy is caused by the absence of the protein dystrophin from skeletal muscle and is characterized by progressive cycles of necrosis/regeneration. Using the dystrophin deficient mdx mouse model we studied the morphological and contractile chronology of dystrophic skeletal muscle pathology in fast twitch EDL muscles from animals 4-22 months of age containing 100% regenerated muscle fibers. Catastrophically, the older age groups lost [~]80% of their maximum force after one eccentric contraction of 20% strain, with the greatest loss [~]93% recorded in senescent 22 month old mdx mice. In old age groups there was minimal force recovery [~]24% after 120 minutes, correlated with a dramatic increase in the number and complexity of branched fibers. This data supports our two-stage model where a "tipping point" is reached when branched fibers rupture irrevocably on eccentric contraction. These findings have important implications for pre-clinical drug studies and genetic rescue strategies.

physiology↗

bioRxiv2020

ACTN3 genotype influences skeletal muscle mass regulation and response to dexamethasone

Homozygosity for the common ACTN3 null polymorphism (ACTN3 577X) results in -actinin-3 deficiency in ~20% of humans worldwide and is linked to reduced sprint and power performance in both elite athletes and the general population. -Actinin-3 deficiency is also associated with reduced muscle mass and strength, increased risk of sarcopenia in the elderly, and altered response to muscle wasting induced by denervation and immobilisation. ACTN3 genotype is also a disease modifier for Duchenne muscular dystrophy (DMD), with -actinin-3 deficiency associated with slower disease progression. Here we show that -actinin-3 plays a key role in the regulation of protein synthesis and breakdown signalling in skeletal muscle, and its influence on muscle mass begins during early postnatal muscle development. Actn3 genotype also influences the skeletal muscle response to the glucocorticoid dexamethasone. Following acute dexamethasone exposure, transcriptomic analyses by RT-qPCR and RNA-sequencing show reduced atrophy signalling (Mstn, Tmem100, mRas, Fbxo32, Trim63) and anti-inflammatory response in -actinin-3 deficient mice compared to wild-type. -Actinin-3 deficiency also protects against muscle wasting following prolonged daily treatment with dexamethasone in female, but not male mice. In combination, these data suggest that ACTN3 R577X is a pharmacogenetic variant influencing the anti-inflammatory and muscle wasting response to glucocorticoid therapy.

genetics↗

bioRxiv2020

Loss of α-actinin-3 during human evolution provides superior cold resilience and muscle heat generation

The fast skeletal muscle protein -actinin-3 is absent in 1.5 billion people worldwide due to homozygosity for a nonsense polymorphism in the ACTN3 gene (R577X) 1. The prevalence of the 577X allele increased as modern humans moved to colder climates, suggesting a link between -actinin-3 deficiency and improved cold tolerance 1,2. Here, we show that humans lacking -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 towards 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 3,4. 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 clear 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.

genetics↗