Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
College of Veterinary Medicine
Non Technical Summary
Muscle pain and soreness with exercise is the most common disorder of equine skeletal muscle, affecting between 3 and 10% of most equine breeds. Many cases of exertional muscle pain are due to inborn errors in muscle metabolism, muscle contraction or potentially structurally important muscle proteins. Our long-term goal is to identify the specific cause of equine muscle disorders, develop practical diagnostic tests, targeted treatment and management strategies. Through cooperation with referring veterinarians, we have traveled to farms and obtained frozen muscle samples from a family of Warmblood horses that possess a novel exertional muscle disorder and from healthy horses residing on the same farm. Affected horses have a history of exercise intolerance, gait abnormality and, in some cases, tying-up. The unique feature of muscle biopsies from affected horses include a striking disarray of myofilaments, which are normally kept in strategic alignment by a large number of proteins responsible for providing structure, shape and contractile movement to the cell (i.e. cytoskeletal proteins). We propose to use a broad-based proteomic approach called iTRAQ ((isobaric tags for relative and absolute quantification) to screen for those proteins that are present in an altered level in closely related Warmblood horses with and without this suspected MFM. We will also determine if there is a change in the expression of these or other muscle protein encoding genes by comparing differential gene expression using RNA-seq.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
We propose to use a combined proteomic and transcriptomic approach to narrow down the list of potentially causative genes for an exertional muscle disorder in a family of Warmblood horses.
Project Methods
1. Twenty mg samples of frozen muscle from each horse will be pulverized and submitted to the Center for Mass Spectrometry and Proteomics (CMSP) at the U of M. There they will be suspended in extraction buffer, normalized to equivalent protein concentrations, and digested with trypsin to generate the short peptides that will be labeled with the iTRAQ reagents. . Samples will be run as an 8 reagent package (4 cases and 4 controls) and a 4 reagent package ( 2 cases and 2 controls).2. Transcriptome sequencing of the same 12 horses will be performed. Total muscle RNA will be isolated, quantified and integrity of rRNA bands evaluated. Library construction and sequencing will be performed. Total RNA will be converted to a cDNA library. Reads will be mapped to the reference sequence, alignment will be assessed for mapping quality. Both the reference annotated transcriptome and a gene transfer format file generated will be used to determine differential expression. Differences in expression for each transcript will be assessed between affected and control samples.