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Charles L. Hoppel, M.D.
Pharmacology and MedicineCase Western Reserve UniversityCenter for Mitochondrial Diseases
Center for Inherited Disorders of Energy Metabolism
2008 McKim Conference, Duluth, MN September 17,2008
Clinical Phenotype of Clinical Phenotype of Mitochondrial DiseaseMitochondrial Disease
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• What is a Jedi Knight?
• A perspective on the Dark Side of the Force.
Clinical Phenotype of Clinical Phenotype of Mitochondrial DiseaseMitochondrial Disease
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Clinical Phenotype of Clinical Phenotype of Mitochondrial DiseaseMitochondrial Disease
• Why study Mitochondria
• Mitochondrial Oxidative Phosphorylation
• Mitochondrial function in Patients
• Respirasomes ?? Supercomplexes!!
• Respirasomes in Heart Failure
• Summary and Conclusions
2008 McKim Conference, Duluth, MN September 17,2008
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Star WarsEpisode I
What makes a JEDI Knight?How do you become a JEDI Knight?
Adapted from G. Lucas,Adapted from G. Lucas, STAR WARS, Episode I (1999) STAR WARS, Episode I (1999)
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• Chip with a blood sample–Anakin Skywalker
• Midi-chlorian test
• Count is over twenty thousand
• No one has a count that high NOT even Master Yoda!
Adapted from G. Lucas,Adapted from G. Lucas, STAR WARS, Episode I (1999)STAR WARS, Episode I (1999)
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Midi-chlorians
• Microscopic life-forms – reside with cells
– Communicate with the FORCE
• We are symbiots with the midi-chlorians(living together with material advantage)
Adapted from G. Lucas,Adapted from G. Lucas, STAR WARS, Episode I (1999)STAR WARS, Episode I (1999)
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• Without midi-chlorians– Life cannot exist and
– Have no knowledge of the FORCE.
• Midi-chlorians– Speak to us
– Telling us the will of the FORCE
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Clinical Phenotype of Clinical Phenotype of Mitochondrial DiseaseMitochondrial Disease
• Why study Mitochondria
• Mitochondrial Oxidative Phosphorylation
• Mitochondrial function in Patients
• Respirasomes ?? Supercomplexes!!
• Respirasomes in Heart Failure
• Summary and Conclusions
2008 McKim Conference, Duluth, MN September 17,2008
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Oxidative Phosphorylationmitochondria
ADPsubstrates70 µM ADP 70 µM
ADP2 mM ADP
200 µM DNP
oxygen consumption
time
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Localize Sites of Damage to Electron Localize Sites of Damage to Electron Transport Chain by Use of Transport Chain by Use of Selective Substrates Selective Substrates
Cyt. Cyt. cc
HH++
IIIIIIIVIVIIII
Cyt. Cyt. c1c1
Cyt. Cyt. aa11aa3 3 (Cu)(Cu)
HH++
HH++
HH++
HH++
HH++
NADNAD++NADHNADH
SuccinateSuccinate
FumarateFumarate
ee--
ee--
ee--ee-- ee--
ee--
½O½O22 + 2H + 2H++ HH22OO
ee--
Coenzyme Coenzyme QQ
Cyt. Cyt. bb
ee--
II
AT PaseAT Pase
HH++
HH++
ADPADPATPATP
POPO442-2-
ADPADP
ANTANT
GlutamateGlutamate
DHQDHQ
TMPD-ascTMPD-asc
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Mitochondrial Oxidation Studies
Substrate Translocase Enzyme(s) Reducing EQ.
Pyruvate Pyruvate PDC NADH
Glutamate Glutamate Glutamate DH NADH
Palmitoyl- l-carnitine
Translocase β-oxidation NADH/FADH
Succinate Dicarboxylate Succinate DH FADH
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Clinical Phenotype of Clinical Phenotype of Mitochondrial DiseaseMitochondrial Disease
• Why study Mitochondria
• Mitochondrial Oxidative Phosphorylation
• Mitochondrial function in Patients
• Respirasomes ?? Supercomplexes!!
• Respirasomes in Heart Failure
• Summary and Conclusions
2008 McKim Conference, Duluth, MN September 17,2008
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Patient 1 56 yo man CC: myalgia and fatigue 2002 1 month after starting Lipitor (statin) Generalized fatigue, myalgia and minimal
weakness stopped Lipitor in early 2003 Intermittently elevated CK 506 U/L (N=0-
232) Continued symptoms off lipitor 5 years!! Exercises regularly
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Patient 1 RX:
Niaspan Tricor Metformin Glucotrol Lisinopril
Total Cholesterol 188 LDL 119 HDL 34.4
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Patient 1: Oxidative PhosphorylationnA oxygen/min/mg mito prot
I
II
III IVQQ CC1/2O1/2O22
HH22OO
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Patient 1: Skeletal Muscle Mitochondria ETC nmoles/min/mg mito prot
I
II
III IVQQ CC
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Patient 1: Skeletal Muscle ETC mol/min/g wet wt
I
II
III IVQQ CC
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Patient 1 Summary
• ETC Complex I defect NADH-linked oxidationN Succinate oxidationN Quinol oxidationN Cyto c oxidation
Complex I/III Complex I
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Patient 1 Summary• ETC Complex I defect
Statin – Induced?
What type of mechanism?
Statin – uncovered a primary defect?
In a 52 yo man!!!!
Statin – Induced coenzyme Q deficiency?
Complex I
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Patient 2 (Zinn)• 20 yo
age 3 yr: bilateral hearing loss age 12 yr: exercise intoleranceage 17 yr: cardiomyopathyplasma lactate 4.9 mML/P ratio 211995 biopsy - focal mito1996 biopsy - normal
negative for mDNA analysis
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Patient 2: Oxidative PhosphorylationnA oxygen/min/mg mito prot
I
II
III IVQQ CC1/2O1/2O22
HH22OO
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Patient 2: Skeletal Muscle Mitochondria ETC nmoles/min/mg mito prot
I
II
III IVQQ CC
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Patient 2: Skeletal Muscle ETC mol/min/g wet wt
I
II
III IVQQ CC
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Patient 2
0
100
200
300
400
500
600
700
800
900
1000
Glutamate
Pyruvate
Palm-ca
rn2-K
G
Palm-CoA
Succinate
TMPD/Asc
Duroquinol
Pt 2
Controls
nA
O/m
in/m
g p
rote
in
High ADP Respiration
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Patient 2 Summary
• ETC Complex III defectN NADH-linked oxidation Succinate oxidation Quinol oxidationN Cyto c oxidation
Complex I/III Complex III
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Patient 3• 3 1/2 yo girl
Björnstad syndrome Sensorineural deafnessPili tortiReferred for muscle biopsy and isolation of
skeletal muscle mitochondriaAlso studied in Seidman Lab at Harvard
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Patient 3: Oxidative PhosphorylationnA oxygen/min/mg mito prot
I
II
III IVQQ CC1/2O1/2O22
HH22OO
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Patient 3: Skeletal Muscle Mitochondria ETC nmoles/min/mg mito prot
I
II
III IVQQ CC
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Patient 3: Skeletal Muscle ETC mol/min/g wet wt
I
II
III IVQQ CC
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Patient 3 Summary
• ETC Complex III defectN NADH-linked oxidationN Succinate oxidation Quinol oxidationN Cyto c oxidation
Complex I/III and II/III Complex III
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Original Article Missense Mutations in the BCS1L Gene as a Cause
of the Björnstad Syndrome
J. Travis Hinson, B.A., Valeria R. Fantin, Ph.D., Jost Schönberger, M.D., Noralv Breivik, M.D., Geir Siem, M.D., Barbara McDonough, R.N., Pankaj Sharma, M.D.,
Ph.D., Ivan Keogh, M.D., Ricardo Godinho, M.D., Ph.D., Felipe Santos, M.D., Alfonso Esparza, M.D., Yamileth Nicolau, M.D., Edgar Selvaag, M.D., Ph.D., M.H.A., Bruce H.
Cohen, M.D., Charles L. Hoppel, M.D., Lisbeth Tranebjærg, M.D., Ph.D., Roland D. Eavey, M.D., S.M., J.G. Seidman, Ph.D., and Christine E. Seidman, M.D.
N Engl J MedVolume 356(8):809-819
February 22, 2007
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Conclusion BCS1L mutations cause disease phenotypes ranging
from highly restricted pili torti and sensorineural hearing loss (the Björnstad syndrome) to profound multisystem organ failure (complex III deficiency and the GRACILE syndrome)
All BCS1L mutations disrupted the assembly of mitochondrial respirasomes (the basic unit for respiration in human mitochondria), but the clinical expression of the mutations was correlated with the production of reactive oxygen species
Mutations that cause the Björnstad syndrome illustrate the exquisite sensitivity of ear and hair tissues to mitochondrial function, particularly to the production of reactive oxygen species
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Clinical Phenotype of Clinical Phenotype of Mitochondrial DiseaseMitochondrial Disease
• Why study Mitochondria
• Mitochondrial Oxidative Phosphorylation
• Mitochondrial function in Patients
• Respirasomes ?? Supercomplexes!!
• Respirasomes in Heart Failure
• Summary and Conclusions
2008 McKim Conference, Duluth, MN September 17,2008
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Individual Complexes
Triton X-100, 3 mg / mg protein
CoomassieG-250
Restain
What is Blue Native Electrophoresis?
Nonionic detergents - stabilize hydrophobic membrane protein complexesNative PAGE (no SDS) - acrylamide gradient from 3-4% down to 12-20%
Complexes “stick” at gel pore size ~ complex diameterAdd Coomassie G-250 - makes all such complexes negatively charged
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Individual Complexes
Triton X-100, 3 mg / mg protein
CoomassieG-250
Restain
ComplexI
NDUFB8
ComplexIII
Rieske
ComplexIV
COX4
Complex IV
(Complex III)2
Complex I
What is Blue Native Electrophoresis?
Nonionic detergents - stabilize hydrophobic membrane protein complexesNative PAGE (no SDS) - acrylamide gradient from 3-4% down to 12-20%
Complexes “stick” at gel pore size ~ complex diameterAdd Coomassie G-250 - makes all such complexes negatively charged
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Individual Complexes
Triton X-100, 3 mg / mg protein
Supercomplexes
Digitonin, 6 mg / mg protein
CoomassieG-250
Restain
ComplexI
NDUFB8
ComplexIII
Rieske
ComplexIV
COX4
CoomassieG-250
Restain
Complex IV
(Complex III)2
Complex I
Supercomplexes
What is Blue Native Electrophoresis?
Nonionic detergents - stabilize hydrophobic membrane protein complexesNative PAGE (no SDS) - acrylamide gradient from 3-4% down to 12-20%
Complexes “stick” at gel pore size ~ complex diameterAdd Coomassie G-250 - makes all such complexes negatively charged
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Individual Complexes
Triton X-100, 3 mg / mg protein
Supercomplexes
Digitonin, 6 mg / mg protein
CoomassieG-250
Restain
ComplexI
NDUFB8
ComplexIII
Rieske
ComplexIV
COX4
CoomassieG-250
Restain
Complex I
NDUFB8
Complex III
Rieske
ComplexIV
COX4
Complex IV
(Complex III)2
Complex I
Supercomplexes
What is Blue Native Electrophoresis?
Nonionic detergents - stabilize hydrophobic membrane protein complexesNative PAGE (no SDS) - acrylamide gradient from 3-4% down to 12-20%
Complexes “stick” at gel pore size ~ complex diameterAdd Coomassie G-250 - makes all such complexes negatively charged
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Individual Complexes
Triton X-100, 3 mg / mg protein
Supercomplexes
Digitonin, 6 mg / mg protein
CoomassieG-250
Restain
ComplexI
NDUFB8
ComplexIII
Rieske
ComplexIV
COX4
CoomassieG-250
Restain
Complex I
NDUFB8
Complex III
Rieske
ComplexIV
COX4
Complex IV
(Complex III)2
Complex I
Supercomplexes
What is Blue Native Electrophoresis?
Nonionic detergents - stabilize hydrophobic membrane protein complexesNative PAGE (no SDS) - acrylamide gradient from 3-4% down to 12-20%
Complexes “stick” at gel pore size ~ complex diameterAdd Coomassie G-250 - makes all such complexes negatively charged
ComplexIV
Activity
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Side view Top view
Side view (matrix -> bottom of page) Cartoon with associated factors
Q
CytCO2
NADH
IIII III IV
H+ H+ H+
What are supercomplexes?
S1 architecture at ~ 4 Angstrom resolution
Schäfer E, Seelert H et al. (2006) J. Biol. Chem. 281, 15370-5
Schäfer E, Dencher NA et al. (2007) Biochemistry 46, 12579-85.
S0 + 1 to 4 (Complex IV monomer) –> “respirasome” supercomplexes S1 through S4 (Complex I monomer) + (Complex III dimer) –> core S0 supercomplex
White = Complex I
Red = (Complex III)2
Green = Complex IV
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Clinical Phenotype of Clinical Phenotype of Mitochondrial DiseaseMitochondrial Disease
• Why study Mitochondria
• Mitochondrial Oxidative Phosphorylation
• Mitochondrial function in Patients
• Respirasomes ?? Supercomplexes!!
• Respirasomes in Heart Failure
• Summary and Conclusions
2008 McKim Conference, Duluth, MN September 17,2008
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0
500
1000
1500
2000
2500
nA
O/m
in/m
g p
rote
in
control
heart failure
SSM
*
**
**
Glutam ate+M Pyruvate+M DHQ Succinate TMPD+ascorbate
0
500
1000
1500
2000
2500
nA
O/m
in/m
g p
rote
in
IFM
**
*** Glutamate+M Pyruvate+M DHQ Succinate TMPD+ascorbate
State 3 respiratory rates
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Uncoupled (200 µM dinitrophenol) respiration
0
500
1000
1500
2000
2500
Glutamate Succinate+Rotenone DHQ+Rotenone TMPDasc+Rotenone
nA
O/m
in/m
g p
rote
in
control
heart failure
SSM
*
**
*
0
500
1000
1500
2000
2500
Glutamate Succinate+Rotenone DHQ+Rotenone TMPDasc+Rotenone
nA
O/m
in/m
g p
rote
in
control
heart failure
IFM
**
**
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ETC complexes in isolated heart mitochondria
0
1000
2000
3000
4000
5000
nm
ol/m
in/m
g
control
heartfailure
NCR
SSM IFM
0
200
400
600
800
1000
nm
o/m
in/m
g
C I
SSM IFM 0
1000
2000
3000
4000
5000
nm
o/m
in/m
g
NFR
SSM IFM
0
2000
4000
6000
8000
10000
12000
14000
16000
nm
ol/m
in/m
g
C III
SSM IFM 0
2000
4000
6000
8000
10000
k=1/
min
/mg
C IV
SSM IFM
0
100
200
300
400
500
600
nm
ol/m
in/m
g
SCR
SSM IFM
0
10
20
30
40
50
60
70
80
90
nm
ol/m
in/m
g
C II
SSM IFM 0
20
40
60
80
100
120
nm
ol/m
in/m
g
C II+Q
SSM IFM
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kDa1700
1000
750 500
200
130
control heart failure
98765
43
2
1
1D-B
N-P
AG
E
C I
C VC III
C IV
C II
0
200
400
600
800
1000
arb
itra
ry u
nit
s/m
g p
rote
in
Control
HF
C V
IFM
0
0.5
1
1.5
2
Control
HF
*
#
CI-CIII2-CIV/CV CI/CV CIII/CV CIV/CV
IFM
ETC supercomplexes in heart IFM
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1D-B
N-P
AG
E
C I
C VC III
C IV
C II
control heart failure control heart failure
SSM IFM
ETC individual complexes in heart mitochondria
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CONCLUSION
In HF the mitochondrial defect lies in the supermolecular assembly of the ETC in functional respirasomes.
We propose that phosphorylation of specific complex IV subunits is involved in disruption of supercomplex assembly of the mitochondrial ETC in HF.
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PPG - LaboratoryPPG - Laboratory• Mariana Rosca, M.D.Mariana Rosca, M.D.• Edwin Vazquez, B.S.Edwin Vazquez, B.S.• Janos Kerner, Ph.D.Janos Kerner, Ph.D.• Margaret Chandler, Ph.D.Margaret Chandler, Ph.D.• William Parland, Ph.D.William Parland, Ph.D.• David Kehres, Ph.D.David Kehres, Ph.D.• Hiral PatelHiral Patel• Colin NaplesColin Naples
AcknowledgementsAcknowledgementsCollaboratorsCollaborators• Hani Sabbah, Ph.D.Hani Sabbah, Ph.D.
Henry Ford HospitalHenry Ford Hospital
Detroit, MIDetroit, MI
• William Stanley, Ph.D.William Stanley, Ph.D.University of MarylandUniversity of Maryland
Baltimore, MDBaltimore, MD
Center for Inherited Disorders of Energy Metabolism Clinical
• Bruce Cohen, MD• Sumit Parikh, MD• Douglas Kerr, MD,PhD• Shawn McCandless
EPrint EPrint
Cardiovascular Research Cardiovascular Research
August 18, 2008August 18, 2008
Funding Support:Funding Support:PPG P01 HL074237PPG P01 HL074237 P01 AG015885P01 AG015885 R01 HL 64848R01 HL 64848