Impact of the alpha-actinin-3 R577X polymorphism on skeletal muscle properties

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.

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