Ex Vivo Cardiac Metabolic Profiling

Myocardial substrate utilization was measured ex vivo through isolated working mouse heart perfusions, as described previously (Bray et al. 2008 (link); Durgan et al. 2011a (link); Tsai et al. 2010 (link); Tsai, et al. 2013 (link)). All hearts were perfused in the working mode (non-recirculating manner) for 40 minutes with a preload of 12.5mmHg and an afterload of 50mmHg. Standard Krebs–Henseleit buffer was supplemented with 8mM glucose, 0.4mM oleate conjugated to 3% BSA (fraction V, fatty acid-free; dialyzed), 10µU/ml insulin (basal/fasting concentration), 2mM β-hydroxybutyrate, 0.2mM acetoacetate, 0.05mM L-carnitine, and 0.13mM glycerol. Metabolic flux were assessed through the use of distinct radiolabeled tracers: 1) [U-¹⁴C]-β-hydroxybutyrate (0.04mCi/L; β-hydroxybutyrate oxidation); 2) [U-¹⁴C]-glucose (0.12mCi/L; glycolysis, glucose oxidation); and 3) [9,10-³H]-oleate (0.067mCi/L;β-oxidation). Measures of cardiac metabolism (i.e., β-hydroxybutyrate oxidation and oxygen consumption) and function (i.e., cardiac power and rate pressure product) were determined as described previously (Bray et al. 2008 (link); Durgan et al. 2011a (link); Tsai et al. 2010 (link); Tsai et al. 2013 (link)). At the end of the perfusion period, hearts were snap-frozen in liquid nitrogen and stored at −80°C prior to analysis. Data are presented as steady state values (i.e., values during the last 10 minutes of the perfusion protocol).