Anne-Marie Koop

4 159 Mitochondrial measurements Mitochondria were isolated from fresh RV tissue subjected to 12 weeks of pressure load by PAB, as dscribed in the supplemental methods. Mitochondrial respiration was measured by the oxygen consumption rate with either pyruvate and malate, or palmitoyl CoA and malate as substrate, in the presence of ADP in a stirred, 2-channel high-resolution respirometry (Oroboros, Innsbruck, Austra). The different states, including the ADP-driven state 3 (State 3) as well uncoupled state 3 (State 3u), representing mitochondrial conditions (ADP or respectively ATP-rich environment, and intact respectively absence of membrane gradient), were analysed as described in the supplemental methods. Oxygen consumption rate was corrected for protein content. Citrate synthase activity kit (Sigma Aldrich, USA) was used as a marker of mitochondrial density. Assessment triglyceride level plasma Triglyceride (TG) levels in plasma were measured by enzymatic methods using commercial kits according to the manufacturer’s instruction (Roche Diagnostics, Mannheim, Germany). Assessment of cardiac lipid content Lipidomics as performed on snap frozen RV tissue subjected to 12 weeks of pressure load by PAB. Sample work up and semi-quantitive analysis of the lipodome was perfomed as previously described. 32,33