Cardiolipin Remodeling by ALCAT1 Regulates Dilated Cardiomyopathy Through Oxidative Stress and Mitophagy. Yuguang (Roger) Shi
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1 Cardiolipin Remodeling by ALCAT1 Regulates Dilated Cardiomyopathy Through xidative Stress and Mitophagy Yuguang (Roger) Shi
2 Cardiolipin Biosynthetic and Remodeling Pathways Phosphatidic Acid De novo Synthesis CTP CPC PPi CDP-Diacylglycerol G-3-P PGS CMP Phosphatidylglyerophosphate LPG (ER) LPGAT PP Pi Phosphatidylglycerol CDP-DAG CLS CMP CLS LPG (Mitochondria) Remodeling Cardiolipin cpla2 ALCAT1 Lysocardiolipin
3 Mitochondrial Theory of Aging Dr. Denham Harman (born February 14, 1916) Mfn Harman, D (1956) J. Gerontol.11 (3): Harman, D (1972). "A biologic clock: the mitochondria?" J. Am. Geriatrics Society 20 (4):
4 Pathological Remodeling of Cardiolipin Is a Common Defect in Aging-related Diseases RS Mito.Dysfunction & Pathological Cardiolipin Remodeling
5 xidative Stress and Cardiolipin Peroxidation
6 Diabetes and besity Increase DHA Content in Cardiolipin STZ-Diabetic Mouse Type 2 Diabetic Mouse Diabetic Rosiglitazone Han et al, Biochemistry May 29;46(21): Pan et al, Vascul Pharmacol Jul;45(1):65-71
7 Inverse Correlation Relationship of Lifespan between with Polyunsaturated DHA Content Phospholipids in Cardiolipin Content In and Mitochondria Lifespan
8 DHA Content in Cardiolipin Is Associated with RS Production Rate DHA (C22:6) Physiol Rev (2007) 87: Carcinogenesis (2002), 23 :
9 Inverse Inverse Correlation Relationship of Lifespan with between Polyunsaturated RS Phospholipids Production Content and In Lifespan Mitochondria Nat Genet Nov;36(11):
10 xidative Stress, Metabolic Diseases, and Cardiolipin Remodeling Diabetes, besity, and CV Diseases xidative Stress Physiological Cardiolipin (Good,C18:2) TAZ MLCLAT C18:2-CoA Mitochondrial Recovery cpla2 RS Cardiolipin (oxidized) Lyso-CL cpla2 ALCAT1 Pathological Cardiolipin (Bad, C22:6) C22:6-CoA Mitochondrial Dysfunction
11 ALCAT1 Is Predominantly Expressed in Tissues with High Basal Metabolic Rate
12 ALCAT1 Catalyzes Acylation of Lysocardiolipin P H P FA-x H P P Acyl-CoA CoA P H P cpla2 ALCAT1 CL-x MLCL CL
13 The Recombinant ALCAT1 Protein Is Localized in the Mitochondria-Associated Membrane (MAM) 7 ALCAT Enzyme Activity CL ALCAT nmol/min/mg protein
14 Up-Regulation of ALCAT1 Enzyme Activity and mrna Expression by the nset of besity, Diabetes, and Heart Disease besity B B B Diabetes Cardiomyopathy B xidative Stress
15 ALCAT1 Catalyzes Pathological Remodeling of Cardiolipin Commonly Associated with Diabetes and besity A B C Fatty Acyl Content in CL (nmol/mg protein) Vector ALCAT 18:1 18:2 Fatty Acyl Content in CL (nmol/mg protein) Vector ALCAT 20:3 20:4 22:6 Total CL Level (nmol CL /mg protein) Vector ALCAT Vector ALCAT
16 Generation of Mice With Targeted Deletion of ALCAT1 Gene BamHI EcoRV Exon 2 BamHI EcoRV WT Allele 9.3 kb 17.1 kb WT #6 #7 #8 #17 Targeting Construct EcoRV BamHI EcoRV NE 5 arm 4.2kb 3 arm 10kb 23.1 kb 9.4 kb WT K Targeted Allele BamHI 5 probe P2 7kb EcoRV BamHI P1 NE EcoRV 13.6kb EcoRV 3 probe 6.5 kb WT -/- -/- -/- +/- +/- +/- ALCAT1 β-actin
17 Increased Linoleic Acid Content in Cardiolipin in the Heart of ALCAT1 Knockout Mice nmol/mg protein ** CL molecular species (nmol/mg protein) Control ALCAT1 K CL molecular species MLECULAR SPECIES Column # from primary MS 1 18:2-18:2-18:2-16:1 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) 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 7 18:2-18:2-18:1-18:1 8 18:2-18:1-18:1-18:1 9 18:2-18:2-16:1-22:6 18:2-18:2-18:2-20:4, 18:2-18:1-16: :6, 18:2-18:2-16:0-22:6 (2:2:1) 11 18:2-18:2-18:2-20:3 18:2-18:2-18:1-20:3, 18:2-18:2-18: : :1-18:2-18:2-20:2 18:1-18:2-18:2-20:1, 18:1-18:1-18: :2 (1:1) 18:1-18:1-18:2-20:1, 18:1-18:1-18: :3 (2:1) 16 18:2-18:2-18:3-22: :2-18:2-18:2-22:6 18:2-18:2-18:2-22:5, 18:1-18:2-18: :6 (1:1) 18:2-18:2-18:1-22:5, 18:2-18:1-18: :6 (2:1) 18:2-18:2-20:4-22:6, 18:2-20:4-20: :4 (1:1) 21 18:2-18:2-20:3-22:6 18:2-18:1-20:3-22:6, 18:2-18:2-20: :6 18:1-18:1-20:3-22:6, 18:1-18:2-20: :6 18:1-18:1-20:2-22:6, 18:1-18:2-20: : :2-18:3-22:6-22: :2-18:2-22:6-22: :2-18:1-22:6-22:6 18:2-18:0-22:6-22:6, 18:1-18:1-22: :6
18 ALCAT Knockout Mice Are Resistant to the nset of Diet-Induced besity A B Weight(g) /+ -/- * * ** Percentage ( % ) /+ -/- ** Fat ** Lean Time(week)
19 Improved Glucose Tolerance and Insulin Sensitivity in ALCAT1 Knockout Mice A Glucose Tolerance Test B Insulin Tolerance Test Blood glucose (%) /+ -/- * * * Blood glucose (%) /+ -/- * * * Time (min) Time (min)
20 ALCAT1 Deficiency Improves Insulin Signaling in ALCAT1 Knockout Mice A Cultured Adipocytes WT K Insulin (nm) p-akt2 Akt2 (total) B Insulin p-akt Thr308 p-akt Ser473 t-akt WT Sk. Muscle K
21 xidative Stress and Mitochondrial Aging
22 ALCAT1 Deficiency Prevents Lipid Peroxidation In Liver 40 MDA(umol/g) * WT K 0 Liver muscle Fat
23 verexpression of ALCAT1 in C2C12 Cells Increases xygen Consumption Rate and Mitochondrial Proton Leakage A B xygen Consumption Rate (natom/min/million cells) Vector ALCAT ** * Control ligomycin FCCP Changes in CR(%) oligomycin rotenone * Time (min) Vector ALCAT * * * * *
24 ALCAT1 verexpression Depletes Mitochondrial Number and Increases Mitochondrial Mutation Rate in C2C12 Cells D F M N Vector M Vector E G M M N ALCAT1 ALCAT1 Relative mtdna copy number Mutations/bp( 10-4 ) Vector Vector ** ALCAT1 * ALCAT1
25 ALCAT1 Deficiency Increases Mitochondrial Number and Decreases Mutation Rate in Skeletal Muscle WT Relative mtdna copy number WT ** K 1.25 K Mutations/bp( 10-4 ) ** 0.00 WT K
26 Mitochondrial Fusion and Fission in Quality Control EMB J 2008; (27):
27 verexpression of ALCAT1 in C2C12 Cells Causes Mitochondrial Fragmentation Vector ALCAT1 Percentage of cell (%) 100 Tubular Mix 80 Frag Vector ALCAT1 D E
28 verexpression of ALCAT1 in C2C12 Cells Causes Mitochondrial Swelling VECTR ALCAT1 0.6 µm
29 ALCAT1 Deficiency Increases Expression of MFN2 in MEF MFN1 MFN2 PA1 β-actin C2C MFN1 MFN2 PA1 Calnexin Prohibitin β-actin WT MEFs ALCAT1 K Relative protein level MFN1 MFN2 ** PA1 Calnexin WT ALCAT1 K ** ** Prohibitin Actin
30 ALCAT1 verexpression Imparis Mitochondrial Fusion A mtegfp mtrfp verlay Boxed B C D Vector E F G H ALCAT1 I J K L ALCAT1/ MFN2
31 MFN1 and MFN2, but not PA1 Rescue Mitochondrial Fusion Defects Caused by ALCAT1 verexpression in C2C12 Cells
32 ALCAT1 Deficiency Prevents Mitochondrial Fragmentation in MEFs In Response to xidative Stress WT K WT+RS K+RS
33 Mitochondrial Fusion and Fission in Quality Control
34 The Working Model of ALCAT1 and Mitochondrial Dysfunction
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