Genome-wide association analyses of physical activity and sedentary behavior provide insights into underlying mechanisms and roles in disease prevention

Although physical activity and sedentary behavior are moderately heritable, little is known about the mechanisms that influence these traits. Combining data for up to 703,901 individuals from 51 studies in a multi-ancestry meta-analysis of genome-wide association studies yields 99 loci that associate with self-reported moderate-to-vigorous intensity physical activity during leisure time (MVPA), leisure screen time (LST) and/or sedentary behavior at work. Loci associated with LST are enriched for genes whose expression in skeletal muscle is altered by resistance training. A missense variant in ACTN3 makes the alpha-actinin-3 filaments more flexible, resulting in lower maximal force in isolated type IIA muscle fibers, and possibly protection from exercise-induced muscle damage. Finally, Mendelian randomization analyses show that beneficial effects of lower LST and higher MVPA on several risk factors and diseases are mediated or confounded by body mass index (BMI). Our results provide insights into physical activity mechanisms and its role in disease prevention

Genome-wide association analyses of physical activity and sedentary behavior provide insights into underlying mechanisms and roles in disease prevention

Although physical activity and sedentary behavior are moderately heritable, little is known about the mechanisms that influence these traits. Combining data for up to 703,901 individuals from 51 studies in a multi-ancestry meta-analysis of genome-wide association studies yields 99 loci that associate with self-reported moderate-to-vigorous intensity physical activity during leisure time (MVPA), leisure screen time (LST) and/or sedentary behavior at work. Loci associated with LST are enriched for genes whose expression in skeletal muscle is altered by resistance training. A missense variant in ACTN3 makes the alpha-actinin-3 filaments more flexible, resulting in lower maximal force in isolated type IIA muscle fibers, and possibly protection from exercise-induced muscle damage. Finally, Mendelian randomization analyses show that beneficial effects of lower LST and higher MVPA on several risk factors and diseases are mediated or confounded by body mass index (BMI). Our results provide insights into physical activity mechanisms and its role in disease prevention. Multi-ancestry meta-analyses of genome-wide association studies for self-reported physical activity during leisure time, leisure screen time, sedentary commuting and sedentary behavior at work identify 99 loci associated with at least one of these traits

Genome-wide association analyses of physical activity and sedentary behavior provide insights into underlying mechanisms and roles in disease prevention

Although physical activity and sedentary behavior are moderately heritable, little is known about the mechanisms that influence these traits. Combining data for up to 703,901 individuals from 51 studies in a multi-ancestry meta-analysis of genome-wide association studies yields 99 loci that associate with self-reported moderate-to-vigorous intensity physical activity during leisure time (MVPA), leisure screen time (LST) and/or sedentary behavior at work. Loci associated with LST are enriched for genes whose expression in skeletal muscle is altered by resistance training. A missense variant in ACTN3 makes the alpha-actinin-3 filaments more flexible, resulting in lower maximal force in isolated type IIA muscle fibers, and possibly protection from exercise-induced muscle damage. Finally, Mendelian randomization analyses show that beneficial effects of lower LST and higher MVPA on several risk factors and diseases are mediated or confounded by body mass index (BMI). Our results provide insights into physical activity mechanisms and its role in disease prevention.publishedVersionPeer reviewe

Invloed van het alfa-actinin-3 R577X polymorfisme op skeletale spiereigenschappen

The association between the ACTN3 genotype and human performance has been intensively studied in a number of studies in populations of varied ethnicity. The R allele was found to be advantageous and the X allele wasdetrimental in sports which require muscle strength and power. A meta-analysis of these data has given a PThe generation of the Actn3-/- knockout mouse made it possible to study the effects of α-actinin-3 deficiency on skeletal muscle function. Compared to wild type mice, the KO mice show lower muscle mass, muscle strength, fiber diameter and an enhanced recovery from fatigue. In addition, KO fast muscle fibers have an increased activity of aerobic enzymes. These data suggest the development of slow-twitch characteristics in fast fibers in the absence of α-actinin-3. These observations have yet to be confirmed in humans.Our research group has already reported parallel study results in humans. A first doctoral project focused on the genotype-related differences in fiber-type distribution and differential mRNA responses in muscle damage markers, inflammatory response and repair markers to an acute eccentric exercise bout. Also a set of oxidative markers was tested to explore changes towards more oxidative characteristics in α-actinin-3 deficient fastfibers. New research questions following these initial studieshave lead to the design of the present research project. Here we will focus on the contractile and morphological differences of single fibers between ACTN3 577XX and RR subjects. 1. Contractile property differences between ACTN3 577XX and RR carriers using single fiber studies. The presence of α-actinin-3 and α-actinin-2 in type IIa and IIx fibresmight counteract mechanical-stress related damage to the Z-line of fastfibers in RR carriers compared to IIa/IIx fibers with only α-actinin-2. This might present a mechanical quality in RR carriers thatis compatible with improved responses to high power, concentric and eccentric muscle work that is typical for e.g. sprint trained athletes. To get more insight into the bio-mechanical differences, contractile properties and calcium-dependency of α-actinin-3 deficient and α-actinin-3 expressing type IIa/IIx fibers, single muscle fibers will be studied. We will collaborate with the research group of Prof. M.Francaux, Catholic University of Louvain, who has extensive experience with experiments in single human muscle fibers.Methods: Muscle biopsies are taken from the right m. quadriceps vastus lateralis and put in a special skinning solution on ice. After removal of fat and other non-muscle tissues, the muscle tissue is stored in skinning solution and single fibers are isolated stepwise. Within a time span of 5-6 weeks after the muscle biopsies, single muscle fibers are subjected to a series of mechanical tests in a pre-determined order. On the day of an experiment, a single fiber will be mounted between an isometric force transducer (model 400A, Aurora Scientific, Ontario, Canada) and the arm of a high-speed motor (model 312B, Aurora Scientific) by means of two connectors. The motor is operated either in length (slack tests and passive stretch tests) or in force mode (isotonic contractions) via a high speed digital controller (model 600A, Aurora Scientific). Once mounted, the fiber can be rapidly transferred between a relaxing or an activating solution. The setup is build over the stage of an inverted microscope (Axiovert25C, Zeiss, Germany) so that the fiber can be viewed with a magnification of 400x. All experiments are performed at 15°C. Collected data are analysed with custom made software (Labview, National instruments). Beforethe start of the mechanical tests, sarcomere length is adjusted to 2.5μm by use of a calibrated ocular micrometer (x400). Subsequently, a digital camera (Camedia C3020 Z, Olympus) is connected to the microscopeand interfaced to a PC, allowing picture capture of the fiber while briefly suspended in the air (±5s). Next, fiber width, CSA and fiber length(FL) are determined. Subsequently, single fiber P0 (mN) and V0 (FL/s) (slack test) are measured. After the slack test the single fiber force-velocity relationship is determined (FL/s). Afterwards, the fiber is subjected to a progressive stretch-release protocol (passive tension measurement). The last mechanical test measures the calcium sensitivity of the fiber applying different pCa and calculating the pCa50% (concentration atwhich half-maximal activation occurs), activation threshold and Hill plot coefficients n1 and n2. After these mechanical tests, fiber MHC isoform is determined by SDS-PAGE. Although the initial focus is on the IIx fibers, with a higher alpha-actinin-3 content, the number of type IIx fibers might be limited, and ACTN3 R577X genotype effects might only be testable for IIa fibers. We hypothesize that P0, V0, force/velocity relationship and power in IIx and IIa fibers of 577RR subjects is higher compared to 577XX subjects. Whether compensatory characteristics are present in type I fibers remains unclear. However, no differences between contractile properties of type I fibers between both groups are hypothesized. As running these single fiber experiments is highly labour intensive, 6-8 subjects within the XX and RR genotype group will be studied (aiming to study about 120 IIa+IIx fibers in both groups). As Malisoux etal. were able to find alterations in the mechanical properties of single fibers after a short training period, we expect this technique to be sensitive enough to detect the differences between genotype groups. 2. Structural/mechanical property differences between ACTN3 577XX and RR carriers using electron microscopy visualization of the Z-line To further investigate the structural/mechanical function of α-actinin-3 in humans, electron microscopic (EM) visualization of the Z-line structure could give additional insights in the physical properties. Presently, it remains unclear whether the presence/absence of α-actinin-3 is related to Z-line width differences in fast muscle fibers or differential structural damage occurring after eccentricexercise. This study will be performed in collaboration with the EM core facility at the Center for Human Genetics (UZ Leuven), lead by Prof. Peter Baatsen.Methods: A previously performed pilot study shows that EM visualization of the muscle Z-line is feasible but requires careful sampling and treatment of the biopsy. Therefore, part of this project will include the optimalization of the EM protocol. At the moment, we plan to use part of the muscle biopsies taken for the single fiberstudy for the optimalization+ of the EM study protocol. Our goal is to determine differences in sarcomere ultrastructure as a result of the ACTN3 polymorphism. Therefore the biopsy will be embedded in a relaxation solution. The samples will be stabilized by chemical fixation containing aldehydes and osmium tetroxide. Afterwards, they are contrastedwith solutions of heavy metal components and dehydrated through a graded series of ethanol to remove water. After coating of the samples in resin, ultrathin sections (60-90 nm) are cut with glass knives using an ultramicrotome (Leica UM EC7). These are transferred to specimen support grids and examined in the transmission electron microscope (Jeol Jem 2100). The EM can be operated at a range of acceleration voltages of 40 to 200 kV and features an Oxford INCA Energy Dispersive X-ray Spectroscopy (EDS) system and a GATAN Tridiem Electron Energy Loss Spectroscopy (EELS) filter/spectrometer for element mapping and image filtering. The morphology of the Z-lines will be examined and assessed with the identity of each sample concealed until the analysis had been completed.When the EM protocol is optimized, biopsies of rectus femoris will be taken from the test subjects after a strenuous eccentric exercise bout to induce Z-disc damage. Sequential analysis of one RR and one XX biopsy will beperformed and repeated in 4 sets of RR/XX paired subjects.status: publishe

History-dependent force, angular velocity and muscular endurance in ACTN3 genotypes

This study aimed at determining the influence of the ACTN3 R577X polymorphism on muscle strength and muscle endurance in non-athletic young men.status: publishe

History-dependent force, angular velocity and muscular endurance in ACTN3 genotypes

This study aimed at determining the influence of the ACTN3 R577X polymorphism on muscle strength and muscle endurance in non-athletic young men

Role of Alpha-actinin-3 in Contractile Properties of Human Single Muscle Fibers: A Case Series Study in Paraplegics

A common nonsense polymorphism in the ACTN3 gene results in the absence of α-actinin-3 in XX individuals. The wild type allele has been associated with power athlete status and an increased force output in numeral studies, though the mechanisms by which these effects occur are unclear. Recent findings in the Actn3(-/-) (KO) mouse suggest a shift towards 'slow' metabolic and contractile characteristics of fast muscle fibers lacking α-actinin-3. Skinned single fibers from the quadriceps muscle of three men with spinal cord injury (SCI) were tested regarding peak force, unloaded shortening velocity, force-velocity relationship, passive tension and calcium sensitivity. The SCI condition induces an 'equal environment condition' what makes these subjects ideal to study the role of α-actinin-3 on fiber type expression and single muscle fiber contractile properties. Genotyping for ACTN3 revealed that the three subjects were XX, RX and RR carriers, respectively. The XX carrier's biopsy was the only one that presented type I fibers with a complete lack of type II(x) fibers. Properties of hybrid type II(a)/II(x) fibers were compared between the three subjects. Absence of α-actinin-3 resulted in less stiff type II(a)/II(x) fibers. The heterozygote (RX) exhibited the highest fiber diameter (0.121±0.005 mm) and CSA (0.012±0.001 mm(2)) and, as a consequence, the highest peak force (2.11±0.14 mN). Normalized peak force was similar in all three subjects (P = 0.75). Unloaded shortening velocity was highest in R-allele carriers (P<0.001). No difference was found in calcium sensitivity. The preservation of type I fibers and the absence of type II(x) fibers in the XX individual indicate a restricted transformation of the muscle fiber composition to type II fibers in response to long-term muscle disuse. Lack of α-actinin-3 may decrease unloaded shortening velocity and increase fiber elasticity.status: publishe

Role of alpha-actinin-3 in contractile properties of human single muscle fibers: a case series study in paraplegics.

A common nonsense polymorphism in the ACTN3 gene results in the absence of α-actinin-3 in XX individuals. The wild type allele has been associated with power athlete status and an increased force output in numeral studies, though the mechanisms by which these effects occur are unclear. Recent findings in the Actn3(-/-) (KO) mouse suggest a shift towards 'slow' metabolic and contractile characteristics of fast muscle fibers lacking α-actinin-3. Skinned single fibers from the quadriceps muscle of three men with spinal cord injury (SCI) were tested regarding peak force, unloaded shortening velocity, force-velocity relationship, passive tension and calcium sensitivity. The SCI condition induces an 'equal environment condition' what makes these subjects ideal to study the role of α-actinin-3 on fiber type expression and single muscle fiber contractile properties. Genotyping for ACTN3 revealed that the three subjects were XX, RX and RR carriers, respectively. The XX carrier's biopsy was the only one that presented type I fibers with a complete lack of type II(x) fibers. Properties of hybrid type II(a)/II(x) fibers were compared between the three subjects. Absence of α-actinin-3 resulted in less stiff type II(a)/II(x) fibers. The heterozygote (RX) exhibited the highest fiber diameter (0.121±0.005 mm) and CSA (0.012±0.001 mm(2)) and, as a consequence, the highest peak force (2.11±0.14 mN). Normalized peak force was similar in all three subjects (P = 0.75). Unloaded shortening velocity was highest in R-allele carriers (P<0.001). No difference was found in calcium sensitivity. The preservation of type I fibers and the absence of type II(x) fibers in the XX individual indicate a restricted transformation of the muscle fiber composition to type II fibers in response to long-term muscle disuse. Lack of α-actinin-3 may decrease unloaded shortening velocity and increase fiber elasticity

Role of alpha-actinin-3 in contractile properties of human single muscle fibers: a case series study in paraplegics.

A common nonsense polymorphism in the ACTN3 gene results in the absence of α-actinin-3 in XX individuals. The wild type allele has been associated with power athlete status and an increased force output in numeral studies, though the mechanisms by which these effects occur are unclear. Recent findings in the Actn3(-/-) (KO) mouse suggest a shift towards 'slow' metabolic and contractile characteristics of fast muscle fibers lacking α-actinin-3. Skinned single fibers from the quadriceps muscle of three men with spinal cord injury (SCI) were tested regarding peak force, unloaded shortening velocity, force-velocity relationship, passive tension and calcium sensitivity. The SCI condition induces an 'equal environment condition' what makes these subjects ideal to study the role of α-actinin-3 on fiber type expression and single muscle fiber contractile properties. Genotyping for ACTN3 revealed that the three subjects were XX, RX and RR carriers, respectively. The XX carrier's biopsy was the only one that presented type I fibers with a complete lack of type II(x) fibers. Properties of hybrid type II(a)/II(x) fibers were compared between the three subjects. Absence of α-actinin-3 resulted in less stiff type II(a)/II(x) fibers. The heterozygote (RX) exhibited the highest fiber diameter (0.121±0.005 mm) and CSA (0.012±0.001 mm(2)) and, as a consequence, the highest peak force (2.11±0.14 mN). Normalized peak force was similar in all three subjects (P = 0.75). Unloaded shortening velocity was highest in R-allele carriers (P<0.001). No difference was found in calcium sensitivity. The preservation of type I fibers and the absence of type II(x) fibers in the XX individual indicate a restricted transformation of the muscle fiber composition to type II fibers in response to long-term muscle disuse. Lack of α-actinin-3 may decrease unloaded shortening velocity and increase fiber elasticity

The stiffness response of type IIa fibres after eccentric exercise-induced muscle damage is dependent on ACTN3 r577X polymorphism.

The aim of the study was to determine the effect of α-actinin-3 (ACTN3) deficiency (XX) on muscle damage induced by an eccentric exercise bout. In this purpose, 4 RR and 4 XX individuals performed an intensive eccentric knee flexion exercise on an isokinetic dynamometer. Muscle biopsies, blood and pain scores were taken before and after the exercise to determine the extent of the exercise-induced damage and the effect of the ACTN3 R577X polymorphism. Maximal isometric strength of the quadriceps and single fibre properties were compared before and after the exercise. The drop in maximal isometric strength of the quadriceps at 45° knee flexion following the eccentric exercise bout was on average 37% 24 h post-exercise. The decrease in force was also apparent in isolated type II fibres (8%; P = 0.02), but not in type I fibres (P = 0.88). Creatine kinase and myoglobin plasma levels increased in all participants at least by 55% and 87%, respectively (P < 0.05). In addition, mRNA levels of markers for muscle regeneration and muscle remodelling increased after the eccentric exercise (P < 0.05), however, independently from ACTN3 R577X genotype. The mRNA level of nuclear factor of activated T-cells 1 (NFATc1) decreased after the eccentric exercise only in XX genotypes (P < 0.05). The stiffness of type II, but not type I muscle fibres increased only in RR individuals after the eccentric exercise (P < 0.05). While no major effect of α-actinin-3 deficiency on susceptibility to muscle damage was found acutely, the increased stiffness response in fast RR fibres might be a protection mechanism from muscle damage during a subsequent eccentric exercise bout