Clinical Phenotype of Mitochondrial Diseasemckim.qsari.org/Presentations/Sept_16_McKim2008_09_Charles_Ho… · (statin) Generalized fatigue, myalgia and minimal weakness stopped Lipitor

11/14/2008

1

Charles L. Hoppel, M.D.

Pharmacology and Medicine

Case Western Reserve University

Center for Mitochondrial Diseases

Center for Inherited Disorders of Energy Metabolism

2008 McKim Conference, Duluth, MN September 17,2008

Clinical Phenotype of

Mitochondrial Disease

• What is a Jedi Knight?

• A perspective on the Dark Side of

the Force.


Mitochondrial Disease

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Mitochondrial Disease• Why study Mitochondria

• Mitochondrial Oxidative Phosphorylation

• Mitochondrial function in Patients

• Respirasomes ?? Supercomplexes!!

• Respirasomes in Heart Failure

• Summary and Conclusions


Star WarsEpisode I

What makes a JEDI Knight?

How do you become a JEDI Knight?

Adapted from G. Lucas, 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, STAR WARS, Episode I (1999)

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, 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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Oxidative Phosphorylation

mitochondriaADP

substrates

70 µM

ADP70 µM

ADP

2 mM ADP

200 µM D�P

oxygen

consumption

time

Localize Sites of Damage to Electron

Transport Chain by Use of Selective Substrates

Cyt. c

H+

IIIIVII

Cyt. c1

Cyt. a1a3 (Cu)

H+

H+

H+

H+

H+

�AD+

�ADH

Succinate

Fumarate

e-

e-

e-e- e-

e-

½O2 + 2H+H2O

e-

Coenzyme Q

Cyt. b

e-

I

AT Pase

H+

H+

ADPATP

PO42-

ADP

A�T

Glutamate

DHQ

TMPD-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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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

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 IVQ C

Substrate transporter DH ETC Rate Control

glutamate glutamate GDH I/III/IV 58 164±44

succinate dicarboxy SDH II/III/IV 268 295±40

duroquinol III/IV 450 588±74

TMPD/Asc IV 1461 952±169

Yield

(mg/g)

5.1 5.3±1.1

1/2O2

H2O

Patient 1: Skeletal Muscle Mitochondria ETC nmoles/min/mg mito prot

I

II

III IVQ C

Rot Sens NADH-Cytochr c red I-III 303 1377±554

NADH Ubiquinone Reductase I 74 228±73

Succ-cytochr c red II-III 127 309±154

Succinate Ubiquinone Reductase II 52 42±20

Decylubiquinol-cytochr c reductase III 2832 4512±1527

Aconitase 670 633±226

Citrate Synthase 1703 1949±704

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Patient 1: Skeletal Muscle ETC µmol/min/g wet wt

I

II

III IVQ C

Rot Sens NADH-Cytochr c red I-III 0.6 2.7±2.0

NADH ferricyanide red “I” 44.3 29.6±9.8

Succ-cytochr c red II-III 2.6 2.4±1.0

Succinate DH “II” 0.9 1.0±0.6

Decylubiquinol-cytochr c reductase III 14.9 19±10.1

Citrate Synthase 15.7 17.5±4.6

Patient 1 Summary

• ETC Complex I defect

� NADH-linked oxidation

N Succinate oxidation

N Quinol oxidation

N 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

Patient 2 (Zinn)

• 20 yoage 3 yr: bilateral hearing loss

age 12 yr: exercise intolerance

age 17 yr: cardiomyopathy

plasma lactate 4.9 mM

L/P ratio 21

1995 biopsy - focal � mito

1996 biopsy - normalnegative for mDNA

analysis

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I

II

III IVQ C





TMPD/Asc IV 797 440±178

Yield

(mg/g)

6.6 5.2±1.3

1/2O2

H2O


I

II

III IVQ C


NADH ferricyanide red “I” 1475 1620±364


Succinate DH “II” 116 75±30


Cyto. Oxidase IV 98271 73307±22667


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I

II

III IVQ C


NADH ferricyanide red “I” 16.1 41±14



Decylubiquinol-cytochr c reductase III 3.2 19±6

Cyto. Oxidase IV 81.1 137±47

Citrate Synthase 9.7 20±5

Patient 2

0

100

200

300

400

500

600

700

800

900

1000

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 defect

N NADH-linked oxidation

� Succinate oxidation

� Quinol oxidation

N Cyto c oxidation

� Complex I/III

� Complex III

Patient 3

• 3 1/2 yo girlBjörnstad syndrome

Sensorineural deafness

Pili torti

Referred for muscle biopsy and isolation of skeletal muscle mitochondria

Also studied in Seidman Lab at Harvard

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I

II

III IVQ C





TMPD/Asc IV 939 952±189

Yield

(mg/g)

2.1 5.3±1.1

1/2O2

H2O


I

II

III IVQ C


NADH Ubiquinone Reductase I 111 228±73


Succinate Ubiquinone Reductase II 72 42±20


Aconitase 543 633±226


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I

II

III IVQ C


NADH ferricyanide red “I” 36.4 29.6±9.8



Decylubiquinol-cytochr c reductase III 2.2 19±10.1

Citrate Synthase 16 17.5±4.6

Patient 3 Summary

• ETC Complex III defect

N NADH-linked oxidation

N Succinate oxidation

� Quinol oxidation

N 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

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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Individual Complexes

Triton X-100, 3 mg / mg protein

Coomassie

G-250

Restain

What is Blue Native Electrophoresis?

Nonionic detergents - stabilize hydrophobic membrane protein complexes

Native PAGE (no SDS) - acrylamide gradient from 3-4% down to 12-20%

Complexes “stick” at gel pore size ~ complex diameter

Add Coomassie G-250 - makes all such complexes negatively charged

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Coomassie

G-250

Restain

Complex

I

NDUFB8

Complex

III

Rieske

Complex

IV

COX4

Complex IV

(Complex III)2

Complex I








Supercomplexes

Digitonin, 6 mg / mg protein

Coomassie

G-250

Restain

Complex

I

NDUFB8

Complex

III

Rieske

Complex

IV

COX4

Coomassie

G-250

Restain

Complex IV

(Complex III)2

Complex I

Supercomplexes






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Supercomplexes


Coomassie

G-250

Restain

Complex

I

NDUFB8

Complex

III

Rieske

Complex

IV

COX4

Coomassie

G-250

Restain

Complex

I

NDUFB8

Complex

III

Rieske

Complex

IV

COX4

Complex IV

(Complex III)2

Complex I

Supercomplexes








Supercomplexes


Coomassie

G-250

Restain

Complex

I

NDUFB8

Complex

III

Rieske

Complex

IV

COX4

Coomassie

G-250

Restain

Complex

I

NDUFB8

Complex

III

Rieske

Complex

IV

COX4

Complex IV

(Complex III)2

Complex I

Supercomplexes






Complex

IV

Activity

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Side view Top view

Side view (matrix -> bottom of page) Cartoon with associated factors

Q

CytC

O2

NADH

I

III 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 �A 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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0

500

1000

1500

2000

2500

nA O/m

in/m

g protein

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 protein

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

nAO/min/mg protein

control

heart failure

SSM

*

**

*

0

500

1000

1500

2000

2500

Glutamate Succinate+Rotenone DHQ+Rotenone TMPDasc+Rotenone

nAO/min/mg protein

control

heart failure

IFM

**

**

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ETC complexes in isolated heart mitochondria

0

1000

2000

3000

4000

5000nmol/min/mg

control

heart

failure

NCR

SSM IFM

0

200

400

600

800

1000

nmo/min/mg

C I

SSM IFM 0

1000

2000

3000

4000

5000

nmo/min/mg

NFR

SSM IFM

0

2000

4000

6000

8000

10000

12000

14000

16000

nmol/min/mg

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

nmol/min/mg

SCR

SSM IFM

0

10

20

30

40

50

60

70

80

90

nmol/min/mg

C II

SSM IFM 0

20

40

60

80

100

120

nmol/min/mg

C II+Q

SSM IFM

kDa1700

1000

750

500

200

130

control heart failure

9

8

7

6

5

4

3

2

1

1D-BN-PAGE

C I

C V

C III

C IV

C II

0

200

400

600

800

1000

arbitrary units/m

g protein 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-BN-PAGE

C I

C V

C III

C IV

C II

control heart failure control heart failure

SSM IFM

ETC individual complexes in heart mitochondria

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 - Laboratory

• Mariana Rosca, M.D.

• Edwin Vazquez, B.S.

• Janos Kerner, Ph.D.

• Margaret Chandler, Ph.D.

• William Parland, Ph.D.

• David Kehres, Ph.D.

• Hiral Patel

• Colin Naples

AcknowledgementsCollaborators• Hani Sabbah, Ph.D.

Henry Ford Hospital

Detroit, MI

• William Stanley, Ph.D.

University of Maryland

Baltimore, MD

Center for Inherited Disorders of Energy Metabolism Clinical

• Bruce Cohen, MD• Sumit Parikh, MD

• Douglas Kerr, MD,PhD

• Shawn McCandless

EPrint

Cardiovascular Research

August 18, 2008

Funding Support:

PPG P01 HL074237

P01 AG015885

R01 HL 64848

Documents

Clinical Phenotype of Mitochondrial Diseasemckim.qsari.org/Presentations/Sept_16_McKim2008_09_Charles_Ho… · (statin) Generalized fatigue, myalgia and minimal weakness stopped Lipitor