Targeting Mitochondrial Free Radical Production
to Improve Aged Skeletal Muscle Function
Disclosure: Paid consultant for Stealth Biotherapeutics
David Marcinek
Department of Radiology
University of Washington
dmarc@uw.edu
Muscle Study Group
Buffalo, NY
September 23, 2014
Marriotto; Liao; NIDDK.
Multifactorial Origins of Sarcopenia
Reduced Physical
Activity
Oxidative
Damage
Neuromuscular
Junction Defects
Mitochondrial
Dysfunction
?
Decreased Anabolic
Hormones
Reduced Satellite
Cell Function
Inflammation
REDOX STRESS
OXIDATIVE DAMAGE
REDOX SIGNALING
STRUCTURAL CHANGES
Irreversible (carbonyls, HNE, …)
mtDNA mutations
Reversible redox modification
Morphology
MITOCHONDRIAL DEFECTS
Intrinsic to mitochondria
INHIBITION
Intact system
MUSCLE
DYSFUNCTION
Redox signaling
(adaptive)
Homeostasis
HEALTH
Redox Stress
(inhibition)
Oxidative Damage
OXIDATIVE STRESS
DISEASE
Marcinek and Siegel. Aging. 2013
Redox control of mitochondrial proteins
H+
Aon et al., Biochim Biophys Acta. 2010
Mailloux et al., JBC. 2011
Garcia et al., JBC. 2010
McLain et al., Free Radic Res. 2011
Taylor et al., JBC. 2003
Fratelli et al., PNAS. 2002
Han et al., Biochem. 2005
Odin et al., J Clin Invest. 2001
In Vivo Metabolic Spectroscopy
Optical Spectroscopy: O2 cons.
MR Spectroscopy: ATP prod.
Optics
Cuff
MR Coil
Hindlimb
Siegel et al., (2012) Mech Ageing Dev
Metabolic
Spectroscopy
Metabolic
Spectroscopy
PCr
CK
ATP
31P
NMR
MbO2
HbO2
Optical Spectra
Parallel effects of ox. stress and aging on in vivo energetics
#
2.5
P/O
2.0
*
*
1.5
1.0
0.5
0.0
PQ
5-7 mo
C
PQ
27-28 mo
ATPmax
(nmole/g/sec)
C
#
1000
800
600
*
400
200
0
C
Siegel et al. Mech Age Devel. 2012
PQ
5-7 mo
C
PQ
27-28 mo
Aging In Vivo Energetics Similar in Mice and Humans
FDI
Amara et al. PNAS. 2007
QUAD
Conley et al. J Physiol. 2000
Summary
1. Age-related mito deficits similar in mice and
elderly humans
2. Induced oxidative stress similar to agerelated deficits
Hypothesis
Acute manipulation of oxidative stress
will reverse mito deficits in aged muscle.
Mitochondrial-targeted peptide Bendavia (SS-31)
Szeto, et al. Antiox and Redox Sig., 2008
Zhao, et al., JBC. 2004
Anderson, et al., J Clin Invest. 2009
Bendavia interacts with cardiolipin (CL)
SS-31
POPC
•
•
•
•
TLCL
MLCL + SS-31
MLCL
CL interacts with each ETC complex
Necessary for cristae structure
Anchors supercomplexes
Preferentially oxidized by stress and disease
Birk et al. JASN 2013.
H2O2 Production
pmol H2O2/mg mito/min
Mitochondrial H2O2 Production
300
200
100
0
Mito
10000
A m p le x R e d ( A .U .)
Bendavia (SS-31)
Mito +
5 uM catalase
S ta n d a rd
0 .5 m M S S - 3 1
8000
2 m M S S -3 1
6000
4000
2000
0
0
1
2
3
4
H 2 O 2 c o n c e n t r a t io n ( m M )
5
Bendavia Reverses In Vivo Mitochondrial Deficits
Inject young and old mice with 3mg/kg SS-31 (~2μM) → in vivo
spectroscopy 1 hour later
ATPase
nmole ATP/g/s
40
**
30
##
P/O
3
20
**
10
2
##
0
C
SS-31
C
SS-31
Young
Old
1
O2 Consumption
10
nmole O2/g/s
8
0
6
C
4
2
SS-31
C
SS-31
Young
Old
(5 mo)
(27 mo)
0
C
SS-31
Young
C
SS-31
Old
Siegel et al., Aging Cell. 2013
Bendavia Reverses In Vivo Mitochondrial Deficits
1.0
1000
0.8
800
nmole ATP/g/s
PCr (normalized)
Inject young and old mice with 3mg/kg SS-31 (~2μM) → in vivo
spectroscopy 1 hour later
0.6
0.4
Old
0.2
0.0
50
100
150
Time (s)
200
**
600
##
400
200
Old, SS-31
0
ATPmax
250
0
C
SS-31
Young
C
SS-31
Old
Siegel et al., Aging Cell. 2013
Bendavia GSH redox state in aged muscle
20
###
15
GSH (mM)
*
10
5
0
C
SS-31
Young
C
Young
SS-31
Old
C
SS-31
Old
C
SS-31
GSH redox potential (mV)
-320
Siegel et al., Aging Cell. (2013)
-325
-330
-335
-340
*
Summary
1.
Reduces mito H2O2 – not a scavenger.
2.
Acute Bendavia reverses mito deficits.
3.
Does not effect healthy mito function
Function?
Does reversal of mitochondrial deficits translate
to improved skeletal muscle performance?
Improved muscle performance
Fraction of Resting Force
(force*time integral)
1 hr after single 3 mg/kg
1.0
Control
SS31
0.8
0.6
*
0.4
young
0.2
0.0
0
50
100
150
200
250
Time (s)
*
Siegel et al., Aging Cell. 2013 and unpublished
Improved Exercise Capacity in Old Mice
OLD (26 mo)
Control
SS-31
0.8
0.6
0.4
0.2
0.0
0
500
1000
1500
2000
Treadmill time (s)
1.0
Fraction Running
7 daily 3 mg/kg doses
Fraction Running
1.0
YOUNG (6 mo)
0.8
0.6
0.4
0.2
0.0
0
5000
10000
Time (s)
15000
Improved Exercise Capacity with 4 week Bendavia
Male
6 mo
Female
25 mo
6 mo
27 mo
Take Home Messages
1. Mitochondrial redox environment controls in vivo
energetics and function in aging skeletal muscle
2. Bendavia represents a new class of compound to
target mitochondrial oxidative stress.
3. Acute Bendavia improves age-related skeletal
muscle function and exercise tolerance.
Phase IIa test of acute Bendavia
PI – Baback Roshanravan, MD (UW)
Coord-Inv – Kevin Conley, PhD (UW)
(SPITM-201)
1. Randomized, double-blind, placebo-controlled
2. Single dose, pre, post measurements
3. Target n=20 per group
4. Muscle function, in vivo energetics
Acknowledgements
Marcinek Lab
Michael Siegel (OHSU)
Shane Kruse
Matt Campbell
Rudy Stuppard
Gary Knowels
Preston Kendrick
Lab Website:
www.blogs.uw.edu/marcinek
UW Nathan Shock Center
Peter Rabinovitch
• Ann Chiao
• Pabalu Karunadharma
• Nate Basisty
Mike MacCoss
Collaborators
Wei Jun Qian (PNNL)
Justin Percival (UW)
Terry Kavanagh (UW)
Collin White (UW)
Dave Brown (ECU)
Hazel Szeto (Cornel)
Stealth Biotherapeutics
Effect on Thiol Redox Status
Sohal and Orr. Free Rad Biol Med. 2012
Thiol oxidation: with age, with Bendavia (4wk)
C y s r e s id u e s w it h a lt e r e d S - g lu t .
1 .5
Increased S-glut with age
lo g 2 r a t i o
1 .0
Partial reversal with SS-31
0 .5
0 .0
50
100
150
-0 .5
In d iv id u a l C y s r e s id u e s
200
Thiol oxidation: with age, with Bendavia (SS-31, 4wk)
Glycogen Phosphorylase
GAPDH
PFK
Heat shock proteins
Cathepsins
Proteasome system
Ubiquitin system
Glutaredoxin 2 (mito)
Thioredoxin
Complex I
ANT
Citrate Synthase
SDH
UCP3
Tropomyosin α and β
Myosin LC
RyR1
Titin
Na+/K+ ATPase
Aged/Young
Total=118
Serum/extracellular
Protein homeostasis/redox system
Mitochondrial
Sarcomeric/calcium
Glycolytic
Other
Aged/Aged-SS-31
Total=188
Take Home Messages
1. Bendavia improves mitochondrial function and
exercise tolerance.
2. Bendavia restores redox homeostasis as well as
energy homeostasis.
3. Restoring redox homeostasis can effect cell
physiology through the thiol proteome in addition to
directly improving energetics.
Proton Motive Force
(predominantly DYm)
Pathological Mitochondrial
Physiological
Mitochondrial
Respiration
H+
H+
H+ +H+ +H+
H +H
H
Reactive Oxygen Species (ROS)
Cardiolipin
Peroxidized Cardiolipin
H+
H+
H+
Mitochondrial Matrix
H+ H+
H+
H+
H+
H+
Oxygen
H+
Parallel effects of PQ and aging on skeletal muscle energetics
2.5
1000
2.0
800
*
1.5
*
1.0
0.5
ATPmax
(nmole/g/sec)
In Vivo
P/O
#
0.0
PQ
5-7 mo
C
*
400
200
PQ
C
27-28 mo
PQ
5-7 mo
C
PQ
27-28 mo
Maximum
(state 3)
Proton leak
(state 4)
80
pmol O2/(s*mg)
pmol O2/(s*mg)
10
perm fibers
600
0
C
Ex Vivo
#
8
6
4
2
0
60
40
20
0
C
PQ2
Young
Siegel et al. Mech Age Devel. 2012
C
PQ2
Old
C
PQ2
Young
C
PQ2
Old