Transcript The Mitochondrial Bioenergetics Core Resource

The Mitochondrial Bioenergetics
Core Resource
Anthony Molina
Gerontology and Geriatrics
The Mitochondrial Bioenergetics Core Facility
-Established in July, 2014.
-Managed by Dr. Manish Bharadwaj
-Institutionally supported- Mitochondrial Bioenergetics is central to key
aspects of the WFSM Strategic Plan (Cancer, Aging/AD,
Diabetes/Obesity/Metabolism)
-Continued growth of the Facility has been supported by the Aging
Center, AD Center, Cardiology, CDOM, and industry partnerships
-The Facility is utilized by faculty across WFSM & beyond: Cardiology,
Gerontology/Geriatrics, Molecular Medicine, Nephrology, Pulmonary,
CDOM, WFIRM, Cancer Center, Primate Center, Kulynych Center,
Public Health Sciences, Psychiatry, Biochemistry
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Goal: To provide affordable access to state of the art
bioenergetic profiling technologies and expertise
-Highly comprehensive mitochondrial profiling to support basic, translational,
and clinical research.
-In-depth scientific and technical support to ensure that researchers
undertake properly designed experiments and get the most out of their data.
-Custom assay development based on tissue/cell type of interest and research
question.
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Examining Mitochondrial Bioenergetics
What to measure?
• ATP
• Mito Membrane Potential
• Mito/Cellular Respiration
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Mitochondrial Respirometry
Chance and Williams 1955
Mitochondria Make A Comeback
Mar 1999
Primary Equipment
-Accredited Facility
-Plate based: 24 and 96 well versions
-Simultaneously measure respiration and
glycolysis
-Strategic Partner
-4 High Resolution Respirometry units
-Fluorometry module permits
simultaneous measurement of ATP,
calcium, H2O2, MMP.
Bioenergetic Profiling
Seahorse XF Data
(Courtesy of Seahorse Bioscience)
Oroboros HRR Data
(Pesta and Gnaiger 2012)
Services Available
Service
Equipment
Used
Whole cell respirometry: analysis of mitochondrial function in
Seahorse XF
cell lines and primary cells
Whole cell glycolytic function
Seahorse XF
Fuel utilization assays (fatty acids, glucose, amino acids, etc.)
Functional profiling of isolated mitochondria
(ETC coupling, sequential analysis of ETC complexes)
Mitochondrial isolation
Mitochondrial function in permeabilized cells/tissues
Unit of
Measure
24 samples
24 samples
Seahorse XF
24 samples
Seahorse XF
24 samples
In-House
Protocol
XF 24-3
3 samples
24 samples
Permeabilized tissue respirometry (e.g. muscle, fat)
High Resolution Respirometry
-Mitochondrial efficiency
-Analysis of uncoupled respiration
-Mitochondrial activation
-Integrated Fluoromentry
ETC profiling (relative expression of ETC complexes)
Oroboros O2K
2 samples
Oroboros O2K
2 samples
Western Blot
24 samples
mtDNA copy number
PCR
10 samples
Example Studies
Basic
-Mims J, Bansal N, Bharadwaj M, Zhao W, Molina AJ, Tsang AW, Furdui CM (2015) Energy Metabolism in a Matched Model
of Radiation Resistance for Head and Neck Squamous Cell Cancer. Radiat Res. 183(3):291-304. doi: 10.1667/RR13828.1.
-Bharadwaj MS, Zhou Y, Jiao Y, Molina AJ, Criswell T, Lu B (2014) Mitochondrial respiration and glucose metabolism are essentially
normal in Immp2l mutant mice. Redox Biol. 2014 Aug 28;2C:1008-1015. doi: 10.1016/j.redox.2014.08.006
-Liu TF, Vachharajani V, Millet P, Bharadwaj MS, Molina AJ, McCall CE. (2014) Sequential Actions of SIRT1-RelB-SIRT3 Coordinate
Nuclear-Mitochondrial Communication during Immunometabolic Adaptation to Acute Inflammation and Sepsis. J Biol Chem. pii:
jbc.M114.566349.
Translational/Clinical
-Molina AJ, Bharadwaj MS, Van Horn CG, Nicklas BJ, Lyles MF, Eggebeen J, Haykowsky MJ, Brubaker PH, Kitzman DW. (2016)
Skeletal Muscle Mitochondrial Content, Oxidative Capacity, and Mfn2 Expression are Reduced in Older Patients with Heart Failure
and Preserved Ejection Fraction and are Related to Exercise Intolerance. JACC HF (in press)
-Tyrrell DJ, Bharadwaj MS, Van Horn CG, Marsh AP, Nicklas BJ, Molina AJ. (2015) Blood-cell bioenergetics are associated with
physical function and inflammation in overweight/obese older adults. Exp Gerontol. 2015 Jul 29;70:84-91. PubMed PMID:
26226578.
-Bharadwaj MS, Tyrrell DJ, Leng I, Demons JL, Lyles MF, Carr JJ, Nicklas BJ, Molina AJ. (2015) Relationships between Mitochondrial
Content and Bioenergetics with Obesity, Body Composition and Fat Distribution in Healthy Older Adults. BMC Obesity, 2015;2:40.
-Tyrrell D, Bharadwaj MS, Van Horn CG, Krtichevsky SK, Nicklas BJ, Molina AJ. (2014) Respirometric Profiles of Muscle Mitochondria
and Blood Cells are Associated with Differences in Gait Speed Among Community-Dwelling Older Adults.. J Gerontol Med Sci. Jul
16. pii: glu096
Assay Development
-Bharadwaj MS, Lyles M, Demons J, Rogers G, Molina AJ (2014) Preparation and respirometric assessment of mitochondria isolated
from skeletal muscle tissue obtained by percutaneous needle biopsy. J Vis Exp. 2015 Feb 7;(96). doi: 10.3791/52350.
Assays developed through our research program
are made available through collaboration
-Blood Based Bioenergetic Profiling
-Minimally invasive approach for analyzing mitochondrial function
-Suitable for a wide array of patients (older, frail)
-Repeatable and suitable for serial assessments
-Diagnostic and prognostic applications
-Potential applications in personalized medicine
-Certain cell types stored for later analysis, paving the way for large
scale multi-center studies
Measures of mitochondrial function, performed in circulating cells,
are able to integrate cumulative effects (environment, genetics,
nutrition, lifestyle, etc.) on bioenergetic capacity
Premise: circulating cells are continuously exposed to a myriad of
circulating factors that mediate bioenergetic capacity across
multiple tissues
Can blood cells recapitulate the bioenergetic capacity of other tissues?
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Blood cell respirometry recapitulates skeletal muscle
bioenergetic capacity
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PBMC respiration is associated with
gait speed
SRC
Max
Basal
Correlation
r
p-value
r
p-value
r
p-value
Pearson
0.61
0.01*
0.59
0.01*
0.43
0.09
Partial w/ Age
0.60
0.02*
0.57
0.02*
0.42
0.11
Partial w/ Gender
0.54
0.03*
0.51
0.05*
0.33
0.22
Partial w/ BMI
0.61
0.01*
0.59
0.02*
0.43
0.10
(Tyrrell 2014)
PBMC respiration is associated
multiple measures of physical
ability
PBMC SRC
Correlation
exSPPB
r
p-value
Knee Extensor
Strength
r
p-value
Grip Strength
r
p-value
Muscle Quality
r
p-value
Pearson
0.59
0.02
0.60
0.03
0.54
0.04
0.56
0.04
Partial w/ Age
0.59
0.02
0.59
0.04
0.59
0.04
0.54
0.06
Partial w/ Gender
0.55
0.04
0.70
0.01
0.53
0.05
0.49
0.09
Partial w/ BMI
0.59
0.02
0.59
0.04
0.57
0.03
0.55
0.05
Tyrrell 2015
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Is this specific to muscle
-Hypothesis: Highly metabolically active tissues are susceptible to differences in
systemic bioenergetic capacity - measured in blood cells
-These are the same tissues most susceptible to age-related bioenergetic decline
The adult brain
-Accounts for ~2% of total body weight
-Utilizes ~20% of the total body O2 consumption at rest
-Requires ~120g of glucose daily (~420Kcal)
-~60% of total body glucose utilization.
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Blood cells mirror differences in brain (frontal cortex)
bioenergetic capacity
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Establishing Wake Forest as a Hub for Mitochondrial
Bioenergetics Research
The WFSM Bioenergetics Facility plays an active role in international efforts to advance
mitochondrial bioenergetics research
-Standardizing protocols
-Defining mitochondrial health
-Workshops and Seminars
-MitoFIT
-Diagnostic monitoring of a mitochondrial fitness score.
-Medical University of Innsbruck, University of Zurich, University of
Barcelona, University of Ulm, Sanford Burnham, University of Auckland, ECU,
University of Cape Town, and WFSM
-2015 to 2018; € 1.26 Mill; Fonds Tiroler Zukunftsstiftung
-MitoEAGLE
-An EU led effort to establish a well coordinated network of laboratories focused on
understanding the effects of Evolution, Age, Gender, Lifestyle and Environment
(EAGLE) on mitochondrial bioenergetics
-The WFSM Bioenergetics Facility was selected as 1 of 2 non EU member labs.
-2016-2020; € 4 Mill, European Cooperation in Science and Technology (COST) Action
CA15203
The Mito Team:
Daniel Tyrrell
Manish Bharadwaj PhD
John Stone
Stephen Dozier
Gargi Mahapatra PhD
Megan Henderson
Monique Otero
*In our first 1.5 years:
-6 NIH grants (6 currently in
review)
-2 Foundation Grants
-2 Center grants
-Lots of pilot grants…
Our early data indicate the respiratory profiles of blood
cells are related to cognitive impairment and AD risk
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unpublished
Acknowledgements
Sticht Center on Aging
The Mito Team:
Daniel Tyrrell
Manish Bharadwaj PhD
John Stone
Stephen Dozier
Gargi Mahapatra PhD
Megan Henderson
Monique Otero
Stephen Kritchevsky PhD
Barb Nicklas PhD
Suzanne Craft PhD
Dalane Kitzman MD
Jeff Williamson MD
Ramon Casanova PhD
Wake Forest Primate Center
Tom Register PhD
Carol Shively PhD
Matt Jorgensen PhD
The Biopsy Team
Mary Lyles MD
Jamehl Demons MD
Karin Murphy
Heather Gregory
Funding
NIH/NIA, AHA,
Pepper Center,
Kulynych Center,
Wake Forest Innovations
A Basic Approach for Profiling Cellular Bioenergetics by Respirometry
Fuel: (Glucose,
Pyruvate, Succinate)
O2
Oligomycin
C3
C1
FCCP
C4
Rotenone/AA
C5
C2
0.006
0.005
0.004
0.003
0.002
0.001
0
0
10
20
30
40
50
60
70
80
90
100
Improved Blood Cell respiration in response to a promising
intervention strategy
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Blood cell respiration in related to AD pathology
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Comparable to invasive skeletal muscle respirometry
(Tyrrell 2014)
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The relationship is maintained with
isolated mitochondria
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What about cardiac muscle
mitochondria?
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