Charles L. Hoppel, M.D. Pharmacology and Medicine Case Western Reserve University

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

• What is a Jedi Knight?

• A perspective on the Dark Side of the Force.



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

• 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)

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)

• 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









Oxidative Phosphorylationmitochondria

ADPsubstrates70 µM ADP 70 µM

ADP2 mM ADP

200 µM DNP

oxygen consumption

time

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

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









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

Patient 1: Oxidative PhosphorylationnA oxygen/min/mg mito prot

I

II

III IVQQ CC1/2O1/2O22

HH22OO

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

I

II

III IVQQ CC

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

I

II

III IVQQ CC

Patient 1 Summary

• ETC Complex I defect NADH-linked oxidationN Succinate oxidationN Quinol oxidationN Cyto c oxidation

Complex I/III Complex I

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 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


I

II


HH22OO


I

II

III IVQQ CC


I

II

III IVQQ CC

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

Patient 2 Summary

• ETC Complex III defectN NADH-linked oxidation Succinate oxidation Quinol oxidationN Cyto c oxidation

Complex I/III Complex III

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


I

II


HH22OO


I

II

III IVQQ CC


I

II

III IVQQ CC

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

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









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



CoomassieG-250

Restain

ComplexI

NDUFB8

ComplexIII

Rieske

ComplexIV

COX4

Complex IV

(Complex III)2

Complex I






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






Supercomplexes


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






Supercomplexes


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




ComplexIV

Activity

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









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

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

**

**

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

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

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

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.

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

Documents

Charles L. Hoppel, M.D. Pharmacology and Medicine Case Western Reserve University