Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Veterinary Medicine
Non Technical Summary
Exercise-induced muscle damage is a common condition in equine athletes. The condition is typically recognized soon after a bout of training exercise as varying degrees of muscle soreness and damage. In severe cases, the amount of muscle damage can create secondary conditions such as shock and kidney failure that can be life-threatening. Progress has been made in identifying genetic defects in specific horse breeds that predispose horses to developing severe forms of the condition (Recurrent Exertional Rhabdomyolysis or RER), but non-genetic factors still pose a risk for ALL athletic horses to develop exercise-induced muscle damage. One important factor may be heat. Working muscle can get very hot - over 110oF - and studies have shown that when muscle gets that hot, the processes used by the muscle to convert nutrients and oxygen into the energy that powers the muscle become inefficient, creating a vicious cycle of producing more and more heat just to maintain the same exercise performance. Our hypothesis is that excessive heat build-up in working muscle is a cause of exercise-induced muscle damage. We will use new technology called high resolution respirometry to determine how increased temperatures cause progressive failure of equine muscle, and identify the specific changes that occur in equine muscle during training to make that muscle more resistant to heat-related injury, fatigue, and failure. We expect that our studies will identify changes in specific proteins withint the muscle cells that replace less heat-tolerant proteins, as well as the development of a "pressure-relief valve" within the energy centers of the muscle that prevents damage during high workloads. Using these studies, we will not only provide answers to why horses can "tie-up" even when they do not possess a genetic defect, but also provide the tools to understand why horses with genetic defects are more prone to tying-up.Successful conditioning of a horse for athletic activities requires the careful application of stress - too little stress and there is little or no improvement in exercise capacity, but too much stress results in damage to the horse. Like other tissues that must be altered to improve athletic performance, muscle requires this careful balance and tying-up is believed to be the result of too much stress for what the muscle can accept. When a horse ties-up, the trainer must provide for proper recovery, which leads to additional costs and time in training. Conversely, it is likely that optimal training intensity for muscle conditioning may not be consistently delivered in an effort to avoid triggering an episode of tying-up. This necessarily cautious approach causes training to take longer and cost more than it would otherwise require if it was known precisely what causes tying up and thus being able to have greater confidence in the amount of stress that can be placed safely on a horse in training. The proposed studies will provide the sort of information that will lead to greater understanding of the causes for tying-up, and in doing so provide a greater level of confidence in the process of conditioning horses for athletic performance. And in doing so, reduce the episodes of exercise- or training-induced damage while increasing the efficiency with which horses are conditioned.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Specific Aim I: Quantify the relationship between physiological hyperthermia and mitochondrial leak using high resolution respirometry of equine skeletal muscle biopsies.Specific Aim 2: Use high resolution respirometry to determine the temperature threshold of different enzymatic elements of the equine skeletal muscle mitochondrial oxidative phosphorylation system.Specific Aim 3: Quantify the relationship between oxidative phosphorylation and pH using high resolution respirometry of equine skeletal muscle biopsies.Specific Aim 4: Determine whether aerobic conditioning will result in improved tolerance of skeletal muscle mitochondria to hyperthermiaSpecific Aim 5: Determine whether aerobic conditioning will result in improved tolerance of skeletal muscle mitochondria to acidosis.
Project Methods
Skeletal muscle samples will be obtained through sterile percutaneous biopsy of the semintendinosus muscle. The biopsy samples will be placed in cold biopsy preservation solution and immediately transported to the laboratory for further processing. Pieces of the biopsy will be dissected under low magnification to remove blood and connective tissue, and individual muscle fibers will be partially separated in order to improve the diffusion of substances in and out of the muscle fibers. The partially dissected muscle fibers will be chemically permeabilized and washed twice with mitochondrial incubation media. After calibration and the measurement of instrument-derived oxygen flux, the appropriate amount of permeabilized tissue will be weighed and added to the reaction chamber to begin the analysis using 4 distinct Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocols.SUIT1 is structured for the specific assessment of oxidative phosphorylation supported by Complex I. Pyruvate (5 mM), glutamate (10 mM), and malate (0.5 mM) will be added to the reaction chamber and the resulting plateau of sample mass-specific oxygen flux (JO2) will be State IV or leak respiration. Adenosine diphosphate (15 mM) will be added to produce oxygen flux supported by the enzymes of the Kreb's cycle and oxidized through Complex I. Cytochrome c (10µM) will be added to determine whether loss of cytochrome c has occurred in vivo, during processing, or during incubation due to increased permeability of the outer mitochondrial membrane. The pH of the incubation media will be lowered with repeated small titrations of lactic acid (1 mM) every 5 minutes until pH has decreased to 6.2. After achieving a pH of 6.2, the pH of the incubation chamber will be raised back to 7.1 with repeated small titrations of dilute NaOH.SUIT2 is structured for the specific assessment of oxidative phosphorylation supported by Complex II. A small amount of rotenone will be added to block Complex I, then succinate (10 mM) will be added to the reaction chamber to produce leak respiration supported by Complex II. From this point, the SUIT will proceed similar to SUIT1: adenosine diphosphate (15 mM) will be added to produce oxygen flux supported by Complex II, followed by cytochrome c (10 µM). The pH of the incubation in the proposed studies will be lowered with repeated small titrations of lactic acid (1 mM) every 5 minutes until pH has decreased to 6.2. After achieving a pH of 6.2, the pH of the incubation chamber will be raised back to 7.1 with repeated small titrations of dilute NaOH.SUIT3 is structured to optimize the measurement of relatively high rates of oxygen flux that are likely to be produced by maximal activity of equine skeletal muscle mitochondria. The combined substrates of SUITs 1 & 2 (pyruvate, glutamate, malate, and succinate) will be added to the reaction chamber and the resulting plateau of sample mass-specific oxygen flux will be leak respiration for the combination of Complex I and Complex II. The SUIT will then proceed with the sequential additions of adenosine diphosphate and cytochrome c, resulting in maximal oxidative phosphorylation. The pH of the incubation media will then be lowered and raised in the same stepwise fashion as described for in earlier methods.SUIT4 is similar to SUIT3, but with the addition of uncoupling titrations to assess the limiting effect of adenosine triphosphate synthase on maximum oxygen flux. The combined substrates of SUITs 1 & 2 (pyruvate, glutamate, malate, and succinate) will be added to the reaction chamber and the resulting plateau of sample mass-specific oxygen flux will be leak respiration for the combination of Complex I and Complex II. The SUIT will then proceed with the sequential additions of ADP and cytochrome c, resulting in maximal oxidative phosphorylation. Uncoupler (in 0.5 μM steps) will be added to uncouple the phosphorylation system from the Electron Transfer System (ETS), with peak oxygen flux representing the maximal capacity of ETS. Electron flow will be blocked with the individual titrations of rotenone, malonic acid, and antimycin A. The low level of oxygen flux after the addition of all three inhibitors will be subtracted from the other JO2 values obtained from that sample incubation to produce corrected leak, maximal oxidative phosphorylation, and ETS oxygen flux values.