Figure 2. BCKA oxidation in heart mitochondria requires both bicarbonate and physiological ATP-free energy of hydrolysis.

Isolated heart mitochondria were used for all experiments. (A) Representative trace of JO₂ during a phosphocreatine (PCR) titration in the absence and presence of 2 mM bicarbonate. (B) Quantification of data from (A) depicting the relationship between JO₂ and ΔG_ATP in mitochondria energized with KMV in the presence and absence of bicarbonate. Rates to the left of the dotted line correspond to mitochondria alone (‘mt’). (C) Representative trace of JO₂ during a bicarbonate titration in the presence of the CK or hexokinase clamp. (D) JO₂ quantification of the bicarbonate titration from the data depicted in (C). (E) Mitochondrial JO₂ without exogenous carbon substrates or saturating KMV in the absence and presence of either FCCP (2 μM) or oligomycin (Oligo; 2 μM). (F) Calculated changed in ATP, ADP, PCR, and Cr at different ΔG_ATP (−12.94 kcal/mol vs. −14.84 kcal/mol). (G) Heart mitochondria incubated at ΔG_ATP of −12.94 kcal/mol versus. −14.84 kcal/mol and energized with P/M/G/S/O (5/2/5/5/0.2 mM; ‘Multi’), P/M/G/S/O plus FCCP (2 μM; ‘FCCP’) or KMV alone (‘KMV’). (H) Comparison of KMV-supported respiration versus maximal respiratory flux (G compare ‘FCCP’ to ‘KMV’ at ΔG_ATP of −14.84 kcal/mol). (I) Dehydrogenase profiling in permeabilized cardiac mitochondria. BCKDH is highlighted in the red filled circle. Data are expressed as Log2 JNADH/JNADPH. Data are mean ± SEM, n = 3–7/group, *P < 0.05, **P < 0.001, ***P < 0.0001.