Authors Charlotte Cuffe (1), Elizabeth Attree (1), Victoria Lindsay-McGee (1,3), Emily Clark (4), Richard J Piercy (2), Androniki Psifidi (1,4)
Affiliations 1. Clinical Genetics Group, and 2. Comparative Neuromuscular Diseases Laboratory, Clinical Science and Services, Royal Veterinary College, UK 3. The Royal (Dick) School of Veterinary Studies, and 4. The Roslin Institute, University of Edinburgh, UK
Presentation Type Talk
Abstract
Mature equine muscle is made up of different ratios of three different types of fibres. These are characterised as Type 1 (Slow twitch-oxidative), 2A (Fast twitch-oxidative) and 2X (Fast twitch-glycolytic). The proportions of these fibre types depend on the function of the muscle. Muscle with a primarily stabilising function such as the sacrocaudalis dorsalis medialis (SCDM) has more than 80% Type 1 muscle fibres, while muscle with greater locomotor functions, such as the gluteus medius, has nearly 70% Type 2X muscle fibres (Hyytiäinen et al., 2014). Further, fibre type proportions change in response to training, and differ between breeds. In certain myopathies, glycolytic or oxidative muscle are preferentially affected. In exertional rhabdomyolysis (ER), glycolytic muscles, such as the semimembranosus (SM), are principally affected. Previous genetic studies in our group (Lindsay, 2022) identified genetic variation associated with ER in Warmbloods and Connemara ponies located in non-coding regions of the genome. Some of these variants overlapped with regulatory elements identified by ChIP-Sequencing of the equine longissimus dorsi muscle as part of the Functional Annotation of Animal Genomes (FAANG) project (Kingsley et al., 2019, ENA study accession PRJEB35307). We are further developing this line of research by comparing two different types of muscle, SCDM and SM from two horse breeds (Warmblood and Thoroughbred) using the assay for transposase-accessible chromatin with sequencing (ATAC-Seq). The SCDM is a primarily oxidative muscle, while the SM is a primarily glycolytic muscle. Therefore, using these muscles we expect to generate a baseline profile of differences between primarily oxidative and primarily glycolytic muscles in the horse. This profile will be informed by ATAC-seq and confirmed by RNA-seq data of the same muscles.
Samples from the SCDM and SM of four Warmblood and four Thoroughbred horses were collected and nuclei extracted using a Dounce homogeniser and cryopreserved in a sucrose buffer and 10% DMSO. ATAC-Seq libraries were prepared using an ATAC-Seq kit from Active Motif that we optimised for use in our two equine muscles. Initial quality control and low pass sequencing (3 million reads, 150bp paired-end, Illumina Novaseq 6000) results indicated efficient optimisation of the ATAC-seq protocol for our samples. We are currently analysing high-depth ATAC-seq data (200 million reads/sample, 150bp paired-end reads, Illumina NovaSeq 6000) to assess the differences in accessible chromatin regions between the two muscle types. We will use RNA-Seq data from the same samples to investigate how these differences in chromatin accessibility affect gene expression. The results of this work will inform future equine genetic studies related with muscle disease.