Anne-Marie Koop

4 165 The ratio of both State 3 at 5 weeks ( suppl. figure 1c ), and State 3u at 5 and 12 weeks ( figure 2a, third graph ) shifted in favour of the use of carbohydrates over fatty acids. To test whether these changes correlated with changed mitochondrial content, citrate synthase was assessed, which was not different ( figure 2b ). The expression of carnitine palmitoyltransferase 1b (CPT1B), fatty acid transporter on the mitochondrial membrane, did not change in rats with PAB as compared with control, whereas expression of glucose transporter 4 (GLUT4) was increased at all time points ( figure 2c ). At this stage of disease, metabolic regulators peroxisome proliferator-activated receptor alpha (PPARα ) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α ) remained unchanged ( suppl. table 2 ). Medium chain acyl CoA dehydrogenase (MCAD) mRNA levels decreased at 5 weeks ( figure 2c ). RV pressure load induced changes in intra-cardiac lipid content The RV lipid content was determined at the 12 weeks time point by semi-quantitative measurements of lipids such as TG, DG, Cer, cardiolipin (CL), phosphaphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG) and phosphatidic acid (PA) ( suppl. table 3 and 4 ). These were also assessed per major class (e.g. all TG), per cluster (e.g. TG within cluster 62) and as individual lipid species (e.g. TG(42:4), representing the lipid (number of carbon atoms : number of double bonds) ). PAB induced several changes in RV lipid content ( figure 3 ). In the major class analyses, RV content of DG, one of the non-phospholipids, was decreased 12 weeks after PAB as compared with RVs from control rats ( figure 3a ). TG and Cer showed a negative trend ( figure 3a ). Less uniform were the changes in lipid content for the phospholipids ( figure 3b ). RV cardiolipin content was decreased at 12 weeks after PAB compared to controls, whereas PC and PE were not decreased. RV content of precursor phospholipids PI, PG and PA did not show any differences between the PAB and control groups ( figure 3c ). Zooming in at the individual level, the heat map of significantly changed non-phospholipids showed a uniform decrease with exception of Cer(d34:0) and Cer(d36:0) ( figure 3d ). The heat map of individual cardiolipins showed lower levels of CL(72:8) and CL(72:9)( figure 3e ). CL(72:8), tetralinoleoylcardiolipin, dominated the decrease of cardiolipins, whereas the sum of other, less abundant, cardiolipin species appeared to be increased ( figure 3f ). Within TG and DG species, PAB induced a shift from (poly-)unsaturated fatty acids (PUFA’s) to more saturated fatty acids (see data supplement for additional boxplots and complete heat maps), e.g. TG 62 cluster and DG 42 ( figure 3g ). Plasma total TG levels were unchanged Tetralinoleoylcardiolipin, an essential lipid in theinner mitochondrial membrane and essential for mitochondrial energy production, decreasesdin right ventricular pressure overload.