Figure 1 - Extracellular Tuning of Mitochondrial Respiration Leads to Aortic Aneurysm

Mitochondrial function decline in a mouse model of Marfan syndrome. A through D, RNA-sequencing analysis of aortas from 4 Fbn1 C1039G/+ mice with 3 Fbn1 +/+ littermates (24-week-old male mice). B, Top 10 significantly changed canonical pathways predicted by ingenuity pathway analysis based on differentially regulated genes. Metabolism-related pathways are highlighted in blue lettering; P<0.01. C, Activation or inhibition of upstream regulators predicted by ingenuity pathway analysis (-2>bias-corrected z-score>4; P<0.05). The predicted inhibition of Tfam is highlighted. D, Expression of genes encoding mitochondrial complexes (Co) and fatty acid oxidation enzymes, and genes related to mitochondrial function and glycolysis; P < 0.05. E, Quantitative reverse transcription polymerase chain reaction analysis of Tfam mRNA expression and quantitative polymerase chain reaction analysis of relative mtDNA content in aortic extracts from 20-week-old Fbn1 C1039G/+ and Fbn1 +/+ male mice. F through I, Primary murine vascular smooth muscle cells transduced with shFbn1 or shControl for 5 days. F, Quantitative reverse transcription polymerase chain reaction of Hif1a, Pdk1, and representative immunoblots analysis, and relative quantification of Hif1a and Pdk1 protein levels. G, Quantitative reverse transcription polymerase chain reaction of Tfam and Ppargc1a and representative immunoblot analysis and quantification of Pgc1α, Tfam, Mt-Nd1, and Mt-Co1. H, Quantitative polymerase chain reaction analysis of relative mtDNA content in shFbn1-and shControl-transduced vascular smooth muscle cells. I, OCR in shFbn1 and shControl vascular smooth muscle cells at basal respiration and after addition of the complex V inhibitor oligomycin (I) and fluoro carbonyl cyanide phenylhydrazone (II) to measure maximal respiration, followed by a combination of rotenone and antimycin A (III). J, Levels of extracellular lactate in the supernatant from shFbn1 and shControl vascular smooth muscle cells. Actin was used as total protein loading control. Data are mean±SEM Statistical significance was assessed by Student t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Fbn1 +/+ mice (E), shControl (F through J). Acaa1b indicates 3-ketoacyl-CoA thiolase B, peroxisomal; Acsl5, Acyl-CoA Synthetase Long Chain Family Member 5; Akt, serine/threonine kinase 1; Atpaf2, ATP synthase mitochondrial F1 complex assembly factor 2; Ccn2, cellular communication network factor 2; CoI-V, mitochondrial complexes I-V; Cox8b, cytochrome c oxidase subunit 8B; Cycs, cytochrome c, somatic; Echs1, enoyl-CoA hydratase, short chain 1; Erk, extracellular signal regulated kinases; Fbn1, fibrillin-1; Hadh, hydroxyacyl-CoA dehydrogenase; Hif1a, hypoxia-inducible factor 1 α; Idh3g, isocitrate dehydrogenase (NAD(+)) 3 non-catalytic subunit gamma; Ivd, isovaleryl-CoA dehydrogenase; Mrtfa, b, myocardin related transcription factor A, B; Mt-Atp6, mitochondrially-encoded ATP synthase membrane subunit 6; Mt-Co1, 2, mitochondrially encoded cytochrome c oxidase I /II; Mt-Nd1-4I, mitochondrially encoded NADH dehydrogenase 1-4; Myc, Myc protoncogen, myelocytomatosis oncogene; Ndufa9, 10, NADH:ubiquinone oxidoreductase subunit A9-10; Ndufb8, NADH:ubiquinone oxidoreductase subunit B8; Ndufs4, 8, NADH:ubiquinone oxidoreductase core subunit S4,8; Ndufv1-3, NADH:ubiquinone oxidoreductase core subunit V1-3; Nos2, nitric oxide synthase 2; (Continued)