Cardiolipin Remodeling by ALCAT1 Regulates Dilated Cardiomyopathy Through Oxidative Stress and Mitophagy

Yuguang (Roger) Shi [email protected] Cardiolipin Biosynthetic and Remodeling Pathways

Phosphatidic Acid CTP CPC PPi CDP-Diacylglycerol De novo G-3-P PGS Synthesis CMP Phosphatidylglyerophosphate

PP Pi LPGAT CLS LPG Phosphatidylglycerol LPG (ER) CDP-DAG (Mitochondria) CLS CMP

Remodeling Cardiolipin cPLA2 ALCAT1 Lysocardiolipin Mitochondrial Theory of Aging

Dr. Denham Harman (born February 14, 1916)

Mfn

Harman, D (1956) J. Gerontol.11 (3): 298–300

Harman, D (1972). "A biologic clock: the mitochondria?" J. Am. Geriatrics Society 20 (4): 145–147 Pathological Remodeling of Cardiolipin Is a Common Defect in Aging-related Diseases

ROS Mito.Dysfunction & Pathological Cardiolipin Remodeling

Oxidative Stress and Cardiolipin Peroxidation Diabetes and Obesity Increase DHA Content in Cardiolipin

STZ-Diabetic Mouse Type 2 Diabetic Mouse

Diabetic Rosiglitazone

Han et al, Biochemistry. 2007 May 29;46(21):6417-6428. Pan et al, Vascul Pharmacol. 2006 Jul;45(1):65-71 InverseInverse Correlation Relationship of Lifespan between with Polyunsaturated DHA Content Phospholipids in Cardiolipin Content In and Mitochondria Lifespan DHA Content in Cardiolipin Is Associated with ROS Production Rate

DHA (C22:6)

Physiol Rev (2007) 87: 1175–1213 Carcinogenesis (2002), 23 :11 1919-1926

InverseInverse Correlation Relationship of Lifespan with between Polyunsaturated ROS Phospholipids Production Content and InLifespan Mitochondria

Nat Genet. 2004 Nov;36(11):1153-8. Oxidative Stress, Metabolic Diseases, and Cardiolipin Remodeling

Diabetes, Obesity, and CV Diseases

Oxidative Stress

ROS Physiological Pathological

Cardiolipin Cardiolipin Cardiolipin (Good,C18:2) (oxidized) (Bad, C22:6)

TAZ cPLA2 cPLA2 ALCAT1 MLCLAT

Lyso-CL C18:2-CoA C22:6-CoA

Mitochondrial Recovery Mitochondrial Dysfunction ALCAT1 Is Predominantly Expressed in Tissues with High Basal Metabolic Rate ALCAT1 Catalyzes Acylation of Lysocardiolipin

O OH O O O P O O P O O OH P O O O O O P O O O OH O O P P O O O O O O O O O O O O O O O O O O O O O O O O O O O FA-Ox Acyl-CoA CoA O O

O cPLA2 ALCAT1

CL-Ox MLCL CL The Recombinant ALCAT1 Protein Is Localized in the Mitochondria-Associated Membrane (MAM)

ALCAT Enzyme Activity 7 6 5 4 3

CL 2 nmol/min/mg protein 1 0

ALCAT Up-Regulation of ALCAT1 Enzyme Activity and mRNA Expression by the Onset of Obesity, Diabetes, and Heart Disease

Obesity B B

B Diabetes Cardiomyopathy Oxidative Stress B ALCAT1 Catalyzes Pathological Remodeling of Cardiolipin Commonly Associated with Diabetes and Obesity

A B C

8 Vector 0.12 Vector 5 Vector

ALCAT ALCAT ALCAT

4 6 0.09 3 4 0.06 2 2 0.03 Total CL Level CL Total

(nmol/mg protein) 1 Fatty Acyl Content Acyl in CL Fatty (nmol CL /mg protein) (nmolCL (nmol/mg protein) 0 Content Acyl in CL Fatty 0 0 18:1 18:2 20:3 20:4 22:6 Vector ALCAT Generation of Mice With Targeted Deletion of ALCAT1 Gene

Exon 2 BamHI EcoRV BamHI EcoRV WT Allele 9.3 kb 17.1 kb WT #6 #7 #8 #17

23.1 kb EcoRV EcoRV BamHI WT Targeting KO NEO Construct 5’ arm –4.2kb 3’ arm – 10kb 9.4 kb

6.5 kb EcoRV BamHI EcoRV BamHI EcoRV P2 P1 Targeted NEO Allele 7kb 13.6kb 5’ probe 3’ probe

WT -/- -/- -/- +/- +/- +/- ALCAT1

β-actin Increased Linoleic Acid Content in Cardiolipin in the Heart of ALCAT1 Knockout Mice

MOLECULAR SPECIES Column # from primary MS 1 18:2-18:2-18:2-16:1 CL molecular species (nmol/mg protein) 18:2-18:2-18:1-16:1, 18:2-18:2-18:2- 2 16:0 (1:1) 18:2-18:1-18:1-16:1, 18:2-18:2-18:1- 3 16:0 (1:1) 7.00 4 18:3-18:2-18:2-18:2 5 18:2-18:2-18:2-18:2 ** 6 18:2-18:2-18:2-18:1 Control 7 18:2-18:2-18:1-18:1 6.00 8 18:2-18:1-18:1-18:1 9 18:2-18:2-16:1-22:6 ALCAT1 KO 18:2-18:2-18:2-20:4, 18:2-18:1-16:1- 10 22:6, 18:2-18:2-16:0-22:6 (2:2:1) 5.00 11 18:2-18:2-18:2-20:3 18:2-18:2-18:1-20:3, 18:2-18:2-18:2- 12 20:2 13 18:1-18:2-18:2-20:2 4.00 18:1-18:2-18:2-20:1, 18:1-18:1-18:2- 14 20:2 (1:1) 18:1-18:1-18:2-20:1, 18:1-18:1-18:0- 15 20:3 (2:1) 16 18:2-18:2-18:3-22:6 3.00 17 18:2-18:2-18:2-22:6 18:2-18:2-18:2-22:5, 18:1-18:2-18:2- 18 22:6 (1:1)

nmol/mg protein nmol/mg 18:2-18:2-18:1-22:5, 18:2-18:1-18:1- 2.00 19 22:6 (2:1) 18:2-18:2-20:4-22:6, 18:2-20:4-20:4- 20 20:4 (1:1) 21 18:2-18:2-20:3-22:6 1.00 18:2-18:1-20:3-22:6, 18:2-18:2-20:2- 22 22:6 18:1-18:1-20:3-22:6, 18:1-18:2-20:2- 23 22:6 0.00 18:1-18:1-20:2-22:6, 18:1-18:2-20:1- 24 22:6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 25 18:2-18:3-22:6-22:6 26 18:2-18:2-22:6-22:6 27 18:2-18:1-22:6-22:6 CL molecular species 18:2-18:0-22:6-22:6, 18:1-18:1-22:6- 28 22:6 ALCAT Knockout Mice Are Resistant to the Onset of Diet-Induced Obesity

A B 80 +/+ ** 40 -/-

** +/+ 60 -/- * * ）

（ 40 30

Weight(g) 20 ** Percentage 20 0 0 1 2 3 4 5 6 7 8 Fat Lean Time(week) Improved Glucose Tolerance and Insulin Sensitivity in ALCAT1 Knockout Mice

A B Glucose Tolerance Test Insulin Tolerance Test

300 * 120 +/+ * * -/- * 200 * 80 * 100 +/+ 40 -/- Blood glucose Blood glucose (%) Blood glucose (%) glucose Blood 0 0 0 30 60 90 120 0 30 60 90 120 Time (min) Time (min) ALCAT1 Deficiency Improves Insulin Signaling in ALCAT1 Knockout Mice

A Cultured Adipocytes

WT KO

Insulin (nM) 0 1 10 0 1 10 p-Akt2

Akt2 (total)

Sk. Muscle B WT KO Insulin - - - + + + - - - + + + p-Akt Thr308

p-Akt Ser473

t-Akt Oxidative Stress and Mitochondrial Aging ALCAT1 Deficiency Prevents Lipid Peroxidation In Liver

40 WT 30 * KO

MDA(umol/g) 10

0 Liver muscle Fat Overexpression of ALCAT1 in C2C12 Cells Increases Oxygen Consumption Rate and Mitochondrial Proton Leakage

A B

oligomycin rotenone 12 Vector 120 Vector )

10 ALCAT ALCAT 100 8 80 * * ** * * * * 6 * * * * 60 * * 4 40 * 2 inChanges OCR(%) 20 (natom/min/million cells (natom/min/million Oxygen Consumption Oxygen Rate 0 0 Control Oligomycin FCCP -14 -7 0 7 14 21 28 35 42 Time (min) ALCAT1 Overexpression Depletes Mitochondrial Number and Increases Mitochondrial Mutation Rate in C2C12 Cells

D E 1.5

1.0 ** N N 0.5

Vector ALCAT1 0.0 Relative copy mtDNA number Vector ALCAT1 F G M 20 ) -4 * M 10 15 ×

M 10

M 5 Mutations/bp( Vector ALCAT1 0 Vector ALCAT1 ALCAT1 Deficiency Increases Mitochondrial Number and Decreases Mutation Rate in Skeletal Muscle

5 ** 4

3 WT 2

0 Relative copy mtDNA number WT KO

1.25 )

-4 1.00 10 × 0.75 KO 0.50 **

0.25 Mutations/bp(

0.00 WT KO Mitochondrial Fusion and Fission in Quality Control

EMBO J 2008; (27): 433-446 Overexpression of ALCAT1 in C2C12 Cells Causes Mitochondrial Fragmentation

Vector ALCAT1 100 Tubular Mix 80 Frag

20 Percentage (%) cell of 0 Vector ALCAT1

D E Overexpression of ALCAT1 in C2C12 Cells Causes Mitochondrial Swelling

VECTOR ALCAT1

0.6 µM

ALCAT1 Deficiency Increases Expression of MFN2 in MEF

MEFs C2C12 WT ALCAT1 KO WT 1.5 MFN1 ALCAT1 KO

MFN1 ** MFN2 1.0 1 0.62 ** ** MFN2 OPA1 1 0.26 0.5 OPA1 Calnexin

1 0.88 Relative protein level Prohibitin β-actin 0.0

1 1.05 β-actin MFN1 MFN2 OPA1 Actin CalnexinProhibitin ALCAT1 Overexpression Imparis Mitochondrial Fusion

mtEGFP mtRFP Overlay Boxed A B C D

Vector

E F G H

ALCAT1

I J K L

ALCAT1/ MFN2 MFN1 and MFN2, but not OPA1 Rescue Mitochondrial Fusion Defects Caused by ALCAT1 Overexpression in C2C12 Cells ALCAT1 Deficiency Prevents Mitochondrial Fragmentation in MEFs In Response to Oxidative Stress

WT KO

WT+ROS KO+ROS Mitochondrial Fusion and Fission in Quality Control The Working Model of ALCAT1 and Mitochondrial Dysfunction