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Projects

 

Translational Center for In Vivo Mitochondrial Medicine

 

Mitochondrial Dysfunction in Aged Muscle

 

MR & Optical Diagnosis of Mitochondrial Metabolism and Dysfunction

 

Skeletal Muscle Mitochondrial Adaptions to Two-Year Caloric Restriction

 

Mitochondrial Dysfunction In Vivo: Cellular Mechanisms and Reversibility

 

 
 

Translational Center for In Vivo Mitochondrial Medicine

Sponsor: NIH / NIA (ARRA "Grand Opportunity" grant program)

Principal Investigators: David J. Marcinek, Kevin E. Conley

The primary aim of this project is to establish a center for collaborative studies that exploits transformational in vivo diagnostic tools to reveal the roles of mitochondria and cell energetics in cell health and disease using mouse and human muscle model systems. Mitochondria are now widely recognized as a nexus between cell energetics, oxidative stress, and cell survival in age-related degenerative diseases, cancer, heart disease and diabetes. An explosion of genetic models of mitochondrial dysfunction and human disease models provides an extraordinary opportunity for innovative new tools to reveal mitochondria’s role in specific disease pathologies, especially in human tissues. Transformational tools that provide a new window on the cell to fully exploit these model systems are the core of the proposed center. The proposed initiative will fill the void in non-invasive approaches to study mitochondria in vivo to reveal how they ensure cell health but fail in symptomatic disease. Innovative magnetic resonance (MRS) and optical spectroscopic (OS) approaches permit measuring - for the first time in vivo - three key classes of energetic properties impacted by pathology: mitochondrial coupling (ATP/O2 or P/O) and capacity (O2max), cell glycolysis and ATP level, and cellular and vascular oxygenation (PO2). The proposed Translational Center will provide these tools and innovative approach for collaborative studies, provide new infrastructure for high throughput analyses, and new technical developments for improved measurements. Aim #1 establishes the Translational Center. Three steps are involved: 1) new personnel are hired and trained in the innovative tools and approaches, 2) new infrastructure is established to permit collaborative studies both locally and nationally and 3) new procedures and protocols are established to standardize the approach between mouse and human models for translational comparisons. Aim #2 integrates the spectroscopic tools for high throughput studies. OS and MRS probes are engineered to permit parallel spectroscopic measurements for increased throughput and expanded application for both mouse and human studies. New optical technical developments will greatly increase sensitivity and expand the subject populations available for our studies. Aim #3 disseminates these in vivo diagnostic methods to other sites involved in aging research. We propose to extend our success in dissemination to establish our in vivo diagnostic approach at 3 new research sites with high field clinical magnets and ongoing studies of aging human muscle. This will expand opportunities for critical breakthroughs as diverse research groups apply our innovative methods to a wide array of new experimental programs. The clinical relevance of the proposed center is the development of an approach that will transform our understanding of the role of mitochondrial dysfunction in disease by providing 1) new tools for disease diagnosis, 2) new insights into disease mechanisms, and 3) a new capability for monitoring interventions to halt or reverse disease symptoms. It will also promote economic stimulus through the hiring and retention of 5 new long-term positions and the purchase of 3 major pieces of equipment.

 

 

 
 

Mitochondrial Dysfunction in Aged Muscle

Sponsor: NIH / NIA

Principal Investigator: David J. Marcinek

Co-Investigators: Martin J. Kushmerick, Kevin E Conley

Mitochondria play a key role in linking cell respiration to cell survival and are critical elements in many age-related degenerative pathologies. The mitochondrial theory of aging proposes that oxidative damage leads to irreversible mitochondrial dysfunction and tissue degeneration with age.  New non-invasive methods developed in the last grant cycle have revealed significant mitochondrial uncoupling measured in vivo in aged mouse and human skeletal muscle that is at least partially reversible.  This proposal builds on these findings to evaluate:  1) the mechanisms underlying in vivo mitochondrial dysfunction with age, and 2) the reversibility of each component of mitochondrial dysfunction.  We employ state-of-the-art optical and magnetic resonance spectroscopic approaches to quantify in vivo deficits in mitochondrial ATP and O2 fluxes with age.  The biochemical bases of these deficits are determined from in vitro tissue analysis of the same mouse muscles.  We study wild-type mice over a range of ages to evaluate how accumulation of oxidative damage is related to mitochondrial defects and dysfunction in natural aging.  Transgenic models with altered antioxidant activities and uncoupling protein (UCP3) expression are used to identify the underlying mechanisms of this dysfunction.  Aim 1 tests the roles of oxidative damage and uncoupling protein activity in the loss of mitochondrial coupling (reduced P/O) with age in wild-type and transgenic mice.  Aim 2 determines how respiratory chain defects impair respiratory function in vivo.  We pair in vivo measurements of O2 flux with measures of oxidative damage to specific mitochondrial components at multiple ages in wild-type and transgenic mice.  Aim 3 tests the reversibility of the mitochondrial defects and dysfunction measured in Aims 1 and 2.  Endurance exercise is used to increase mitochondrial proliferation and turnover, thereby replacing damaged mitochondria and improving function.  The relevance of the proposed research to human health is two-fold. 1) The determination of the specific biochemical mechanisms leading to mitochondrial dysfunction will identify potential strategies to retard or reverse mitochondrial pathologies with age.  2) Our long-term goal is the development of non-invasive methods to diagnose mitochondrial dysfunction and follow the progress of interventions meant to reverse disability in the elderly.

 
   
 

MR & Optical Diagnosis of Mitochondrial Metabolism and Dysfunction

Sponser: NIH / NIAMS

Princiapl Investigator: Kevin E. Conley

Co-Investigators: Martin J. Kushmerick; Kenneth Schenkman

This application builds on our recent innovations involving a novel combination of magnetic resonance (MRS) and optical (OS) spectroscopic approaches for studying mitochondrial energetics in human muscle.  Muscle provides a unique window on the mitochondrial defects that are at the center of a growing number of metabolic and degenerative diseases (e.g., diabetes, neurodegeneration and aging).  In this competing renewal, we use the dual OS/MRS spectroscopy approach applied to skeletal muscle to develop diagnostic procedures for identifying defects in mitochondrial function.  The proposed studies build on progress made over the tenure of this grant that have led to two key achievements: 1) a combined OS/MRS strategy for measuring chemical content and dynamics that yield energy fluxes and 2) new optical analytical tools that open the visible spectrum for exploitation in vivo.  This progress has led to the first quantitative measurement of O2 uptake, which combined with ATP flux, measures mitochondrial coupling non-invasively in vivo.  Aim #1 expands on this mitochondrial functional measurement and capitalizes on our ability to use the visible spectrum to develop a measurement of mitochondrial capacity based on cytochrome aa3 content.  To achieve this, we will exploit new analytical methods that permit extending our optical spectroscopy into the visible wavelengths.  Aim #2 combines new optical and MRS methods to determine cellular and vascular oxygenation in vivo.  A novel optical wavelength shift analysis combined with quantification by MRS yields cellular oxygenation ([oxy-Mb]/total [Mb]) and blood oxygenation ([oxy-Hb]/total [Hb]).  These oxygenation measurements have the potential to reveal vascular dysfunction, as well as calibrate our O2 uptake determinations.  Aim #3 proposes to design, fabricate and implement a multimodal apparatus that will integrate optical spectroscopy with interleaved, multinuclear 1H and 31P MR spectroscopy.  This apparatus uses the technological advances in Aims #1 and #2 to permit new measurements and increased signal-to-noise that will allow studies of mitochondrial function in human muscle in a single session of <90 min.  Aim #4 translates this diagnostic tool to aging, which is accompanied by significant mitochondrial dysfunction and is a malady of considerable importance to the health of the nation.  The ability to conduct such a complete analysis in a clinically feasible session will provide a unique diagnostic probe of the nature and extent of dysfunction in metabolic and degenerative disorders.  Upon completion of this project we will be in position to translate our tools to standard wide bore 3T or other human magnets for use in a clinical setting.

 
   
 

Skeletal Muscle Mitochondrial Adaptions to Two-Year Caloric Restriction

Sponsor: Pennington Biomedical Research Center, LSU (NIH)

Principal Investigator: Kevin E. Conley

This project is one component of a 3 University constortium studying mitochnodrial functional and structural changes to calories restriction in non-obese subjects.  We will perform detailed studies of mitochondrial mass and energetics in 50 non-obese and 25 matched control subjects enrolled in the NIA U01 named “CALERIE” at baseline and after 1 and 2 years of a 25% calorie restriction.  Specific measurements made at the University of Washington include mitochondrial capacity and efficiency made using Innovative optical and magnetic resonance spectroscopic methods applied to non-invasively measure ATP synthesis and O2 uptake in human skeletal muscle.  Muscle biopsies taken from site of the MRS/OS determinations will be analyzed for mitochondrial content, myoglobin concentration and metabolite levels.  This collaborative project will be the first test of the CR-ROS-oxidative stress model of aging in humans over a two year period and the first to use a combined in vivo / in vitro approach to measure mitochondrial function during CR. These studies build upon a growing body of literature in rodents that supports the expected results and will also be the longest application of CR in man to test the durability of the effects of CR on mitochondrial structure and function.

 
   

Mitochondrial Dysfunction In Vivo: Cellular Mechanisms and Reversibility

Sponsor: The Ellison Medical Foundation

Principal Investigator: David J. Marcinek

The mitochondrial theory of aging proposes that the accumulation of oxidative damage with age results in mitochondrial dysfunction leading to tissue degeneration. This proposal tests the hypothesis that oxidative damage leads to reversible mitochondrial dysfunction in vivo in aging skeletal muscle.  The links among oxidative damage, mitochondrial dysfunction, and age remain controversial despite several decades of intense focus.  Studies on isolated mitochondria and cells have identified several potential mechanisms that could lead to mitochondrial dysfunction.  However, it is not clear the extent to which these molecular level changes with age result in mitochondrial dysfunction in vivo.  To measure mitochondrial function in intact skeletal muscle we have developed a unique approach, combining in vivo optical and magnetic resonance spectroscopy.  Specific aim 1 uses transgenic mouse models altering oxidative stress and uncoupling protein expression to determine the cellular mechanisms underlying in vivo dysfunction.  Oxidative stress in the skeletal muscle is manipulated using mouse models overexpressing catalase in mitochondria (mCAT) and null for cytoplasmic superoxide dismutase (SOD1).  Mice lacking the muscle specific isoform of UCP (UCP3) are used to determine if changes in UCP3 activity can account for reduced in vivo mitochondrial coupling with age.  Specific aim 2 tests whether exercise training can “turn back the clock” and reverse in vivo mitochondrial dysfunction in aged skeletal muscle.  The ability to combine non-invasive measures of mitochondrial function with modern genetic techniques is a significant breakthrough in the mission to understand how molecular level events lead to in vivo dysfunction in aging and disease.