Proposal

Narrative

High-resolution tissue respirometry (HRR) is an analytical technique used to investigate mitochondrial physiology and pathophysiology. Understanding mitochondria is important to biomedicine because they contribute to healthy lifespan, age-related disease, and disease progression. By using tailored protocols containing a specified sequence of substrates, uncouplers, and inhibitors, HRR is capable of assessing a variety of respiratory states including maximal oxidative phosphorylation (OXPHOS) capacity (i.e. convergent electron flux through complex I+II), OXPHOS capacity of the individual respiratory chain subunits, maximal electron transfer system capacity, and leak respiration (i.e. a surrogate of proton leak) in tissue homogenates, isolated mitochondria, or permeabilized cells. Assessments with HRR require only a small amount of tissue to obtain accurate results. Less than 5 mg of fresh muscle tissue or <500,000 cells are needed (each sample is assayed in duplicate), equating to 10-fold less compared to conventional instruments. Specific applications of HRR include: • Diagnosis of acquired and genetic mitochondrial diseases • Diagnosis of myopathies and neuromuscular pathologies • Study of pathologies with reduced cellular respiration • Studies on cell death • Aging and senescence • Oxidative stress • Cancer research • Pharmacological testing • Responses to environmental stress To benefit students and faculty members, this project proposes two aims: 1) Support the healthy aging pillar by making high-resolution tissue respirometry available to the Florida Atlantic University community In the immediate term, HRR will contribute directly to research initiatives in the Department of Exercise Science and Health Promotion. The department houses 5 principal investigators engaged in aging and biochemistry related research projects that will be enhanced by HRR. These investigators have obtained or are in the process of attaining full member status at the Institute for Healthy Aging and Lifespan Studies (I-HeAL). To promote research inquiry, HRR will be used by undergraduate and graduate trainees conducting research under the supervision of investigators in the department, which typically number 5-6 per investigator. The potential reach of HRR within the department is further amplified given the student body approaches 1,500 undergraduates. In the near-to-medium term, this project aims to advance the mission of the healthy aging pillar at FAU. Mitochondrial dysfunction is implicated in the onset and/or progression of several-age related diseases, and we predict that HRR will be a welcome resource for FAU investigators engaged in studies of mitochondrial physiology. An estimated 10 or more faculty members in the Colleges of Science and Medicine conduct research pertinent to HRR, and interdisciplinary collaborations that yield impactful research which raises the visibility of I-HeAL and other pillars at FAU are strong possibilities. 2) Enrichment of curriculum by incorporating high-resolution respirometry to study mitochondrial physiology and pathophysiology The Department of Exercise Science and Health Promotion instructs nearly 1,500 undergraduates and approximately 60 graduate students. The project manager is responsible for teaching required undergraduate and graduate courses which address mitochondrial physiology to a significant extent. To supplement course material traditionally delivered in lecture format, HRR will be used to provide a hands-on experience that encourages student engagement and reinforces the critical role of this cellular organelle. Analysis and interpretation of HRR data for the purpose of disease detection will be particularly relevant for students that plan to pursue a career in medicine or graduate study in the biomedical and exercise sciences. Furthermore, by incorporating HRR into the curriculum, students will have acquired a sophisticated and specialized bench technique that we anticipate will enhance post-graduate opportunities. Overall, we anticipate that high-resolution respirometry in the Department of Exercise Science and Health Promotion will increase student research output, facilitate collaborations across campus, advance the mission of our new Institute for Healthy Aging and Lifespan Studies, and enrich the curriculum of our undergraduate and graduate student body.

Facilities

Hardware Requirements

High-resolution respirometry will require the specialized devices listed below from Oroboros Instruments (Innsbruck, Austria). 1. O2k-Core (Quantity 2) 2. Power-O2k Core Unit (Quantity 2) 3. Fluorometer attachments The O2k-Core and Power-O2k core unit (items 1 and 2) make up a two machine system that can simultaneously process multiple tissue samples. Two each of items 1 and 2 are requested to create a high-throughput system that will allows us to process an even greater number of samples in a single experiment such that data generation is increased, and technician operating time is reduced. The overall impact of having this high-throughput system is faster completion of research projects and dissemination of findings via publication. The fluorometer attachment (item 3) will allow reactive oxygen species emission to be measured at the same time as respiration, thereby providing us with more robust information on how mitochondria are functioning.

Software Requirements

Not applicable

Personnel Costs

Not applicable

Other Costs

Not applicable

Timeline

Upon project approval in March 2017, the devices will be purchased from Oroboros Instruments. Projected delivery time is approximately 6 weeks, with an additional few weeks required for set-up and calibration. Around May-June 2017, the devices will be used immediately by investigators and student trainees in the Department of Exercise Science and Health Promotion on approved projects. In the near-to-medium term (through early/mid 2018), the project manager and department leadership will liaise with the Director of I-HeAL and various faculty in the Colleges of Science and Medicine to establish collaborative opportunities. Although the elapsed time from equipment purchase to discussion of collaboration spans one year (ending ~August 2018), we anticipate fruitful, interdisciplinary research output for many years.

Sustainability

The devices for this project are expected to be permanent pieces of laboratory equipment with a long shelf-life extending well beyond the initial project period. Regular maintenance will be performed to ensure that the devices function properly. The project manager has start-up funding available that can be used to supply the necessary consumable products such as reagents and laboratory plastics for the foreseeable future. It is also anticipated that the preliminary data generated from high-resolution respirometry will be used for grant applications that may provide additional funding to supply the needed consumable products in the longer-term.

Resource Matching

Not applicable

Implementing Organization

The high-resolution respirometry system will be housed in the Department of Exercise Science and Health Promotion. The project manager and research staff in the department will be responsible for setting-up, operating, and maintaining the devices.