105 Regulation of ATP Binding Cassette Transporter A1 and Sterol Regulatory Element by Trans Fatty Acids Fei Shao1 1Saint Louis University Trans fatty acid consumption is associated with increased risk of cardiovascular disease, which is likely the result of adverse changes in plasma lipids and lipoprotein profile. These alterations in lipoprotein profile include increased low-density lipoprotein (LDL) levels and decreased high-density lipoprotein (HDL) levels. LDL and lipid oxidation is a key event in the initiation of pro-atherogenic foam cell production, which can be reversed by removal of cholesterol from foam cells. Since macrophage cholesterol efflux plays an important role in the atheroprotective role of HDL and cholesterol homeostasis, our studies have focused on the role of trans fatty acids in altering mechanisms responsible for macrophage cholesterol metabolism and efflux. We show that individual trans fatty acid species differentially regulate protein expression of the major cholesterol efflux transporter, ATP binding cassette transporter A1 (ABCA1) protein in mouse macrophage J774. the most abundant trans fatty acid elaidic acid (9t-18:1) upregulates ABCA1 protein expression as well as promoter activity of the transcriptional regulators of ABCA1 activity, Liver X receptor-α (LXRα) and Retinoic X Receptor-α (RXRα). in contrast, corresponding cis fatty acids decrease both ABCA1 protein expression and LXRα/RXRα promoter activity. Furthermore, cells transfected with sterol regulatory element (SRE) show increased SRE activity following exposure to elaidic acid in a dose dependent manner, which suggests that elaidic acid may increase activities of sterol regulatory element binding proteins (SREBPs) family. Similar responses were observed with other trans fatty acid species including trans-vaccenic acid, palmitelaidic acid. in contrast, cis fatty acids down-regulate SRE activity. Overall, these results provide new insights into disparate roles of cis and trans fatty acids on important mechanisms regulating cholesterol metabolism.

do i : 10.1016/j.freeradbiomed.2011.10.130 106 Heme Oxygenase 1 Promotes Murine Stem Cell Differentiation into Beating Cardiomyocytes Hagir B Suliman1, and Claude a Piantadosi1 1Duke University Medical Center Adaptive mitochondrial biogenesis involves the inducible heme oxygenase-1 (HO-1) enzyme and redox activation of transcription and replication of the mitochondrial genome (mtDNA). Mitochondrial DNA transcription and replication are also key regulatory events in myocardial cell differentiation and are highly dependent on nuclear-encoded transcription factors imported by the mitochondria. We tested the hypothesis that HO-1 induction would facilitate myocardial embryonic stem cell differentiation. Using a mouse embryonic stem cell, CGR8, we compared mitochondrial proliferation and the expression of mtDNA-specific transcription factors in undifferentiated CGR8 with spontaneously differentiated cells. CGR8 cells undergoing spontaneous differentiation in culture into beating cardiomyocytes initiate mitochondrial proliferation and mtDNA transcription. Undifferentiated, CGR8 cells have few mitochondria, but as they differentiate, they increase the expression of regulators of mitochondrial fusion and fission resulting in mitochondrial maturation and network expansion to provide an energetic supply to nascent sarcomeres. Overexpression of HO-1 in undifferentiated CGR8 cells accelerated the expression of NRF-1 and Tfam and cardiac-specific markers troponin I, β-myosin

heavy chain, atrial natriuretic peptide, and α-sarcomeric actinin. the expression of differentiation transcription factors NKX2.5 and GATA4 increased over time reaching the highest levels at 2 weeks. Moreover, nuclear localization of NKX2.5 and GATA4 correlated with expression of α-sarcomeric actinin. These changes were accompanied by an increase in myofibril organization and the number of beating clusters of cells. RNA silencing of HO-1 disrupted mitochondrial biogenesis and spontaneous differentiation. RNA silencing of NRF-1 or Tfam prevented mitochondrial organization and ATP production, causing deficient sarcomerogenesis and a decreased number of contractile clusters. These data establish that the mitochondrial biogenesis and oxidative metabolism are prerequisites for the differentiation of stem cells into a functional cardiac phenotype and are critical regulators of cardiogenesis. doi : 10.1016/j.freeradbiomed.2011.10.131

107 Thiocyanate: a Potent Modulator of Myeloperoxidase-Mediated Oxidation Jihan Talib1, David Pattison1, and Michael Davies1,2 1The Heart Research Institute, Sydney, Australia, 2University of Sydney, Sydney, Australia Oxidative damage to proteins catalysed by the heme enzyme myeloperoxidase (MPO) has been linked to multiple inflammatory pathologies, including atherosclerosis. Elevated levels of MPO and 3-chlorotyrosine, a specific product of MPO-catalysed oxidation, has been detected in human atherosclerotic lesions. MPO catalyses the oxidation, by H2O2, of chloride (Cl–), bromide (Br–) and thiocyanate ions (SCN–) to form the oxidants HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid) respectively. Enhanced generation of these oxidants has been linked to biological damage. SCN– which is present at elevated levels in smokers (≤ 250 μM compared to < 80 μM in controls), is the preferred substrate for MPO with up to 50 % of H2O2 consumed by MPO being used to oxidise SCN–. in addition, HOCl and HOBr oxidise SCN– to HOSCN, which is a reactive but thiol specific oxidant. in the current study, the oxidation of plasma protein residues by HOSCN and MPO/Cl–/H2O2/SCN– mixtures was investigated. It was found that the presence of elevated SCN– levels (100-200 μM), as detected in smokers, protected Met, Tyr, Trp, His and Lys from oxidation by HOCl, and in contrast enhanced the proportion of oxidised Cys residues from 40 % (when treated with HOCl alone) to 90 %. the presence of 100-200 μM SCN– levels also caused a ≥ 50 % decrease in the generation of the MPO biomarkers; 3-chlorotyrosine and di-tyrosine compared to oxidation by HOCl and MPO/Cl–/H2O2. These findings suggest that: (a) the utilisation of these compounds as biomarkers for MPO-damage may underestimate the true extent of MPO mediated oxidation and damage and (b) that enhanced Cys oxidation due to elevated concentrations of SCN– could be a contributing factor to smoking-enhanced atherosclerosis.

doi : 10.1016/j.freeradbiomed.2011.10.132

SFRBM 2011 S51