Fig. 2: High-resolution respirometry measurement of complex I (CI)-linked respiratory capacity at physiological conditions and at maximum capacity in frozen mouse cardiac tissue using the TSIT method.

A Representative trace of frozen left ventricular (LV) tissue. Basal respiratory capacity was determined at 2 mM malate (M), 100 µM NAD⁺, and 10 µM cytochrome c (Cytc). CI-linked respiratory capacity (purple) was determined at titration of 15 µl of 20 mM acetyl-CoA to mimic the physiological condition. Maximum CI-linked respiratory capacity (CI_max, olive-green) was determined with stepwise titration of 20 μl of 10 mM NADH. The initial NADH titration caused increase in O₂ flux that was not maintained, likely due to consumption by enzymes such as malate dehydrogenase and nicotinamide nucleotide transhydrogenase. Continued titration led to a steady-state O₂ flux, which was used for quantification. Complex II-linked respiratory capacity was determined at 1 µM rotenone (rot) and titration of 20 µl of 1 M succinate (S). Mitochondrial respiratory capacity was calculated as O₂ flux subtracted from residual oxygen consumption, which was determined at an additional 5 mM malonate (mna) and 5 µM antimycin A (ama). B Quantified O₂ flux showed that the CI_max (olive-green) was roughly 4 times that of the CI-linked respiratory capacity (purple) mediated by acetyl-CoA in frozen LV tissue (n = 3). Data are expressed as mean ± standard error of the mean. A two tailed unpaired Student t-test was used. **P < 0.01. **P < 0.01.