Reversible Thiol Oxidation Increases Mitochondrial Electron Transport Complex Enzyme Activity but Not Respiration in Cardiomyocytes from Patients with End-Stage Heart Failure

Figures

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  Figure 1. DTT treatment improves mitochondrial complex I and III enzyme activity. Enzyme activities of complexes of the electron transport system were assessed in isolated cardiomyocyte mitochondria from patients with HFrEF. The 5 mM DTT treatment (gray) improved activity of complexes I and III compared to controls (white) but had no effect on complex II or IV. Enzyme activities compared via paired t-test. * p < 0.05.

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  Figure 2. Creatine kinase clamp used to control mitochondrial energetic demand. Example tracing of the creatine kinase clamp in which PCr is titrated to control the mitochondrial energetic demand (ΔGATP) (see Table 3). ΔGATP is then plotted against oxygen consumption (JO2) in which the slope of the relationship represents the mitochondrial oxidative phosphorylation (OXPHOS) conductance.

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  Figure 3. OXPHOS conductance does not improve with DTT treatment Saponin-permeabilized cardiomyocyte bundles from patients with HFrEF did not experience improvements in mitochondrial respiration under various energetic states following treatment with 5 mM DTT (A). Energetic demand (ΔGATP) was manipulated through titrations of PCr (see Table 2) and plotted against the rate of oxygen consumption (JO2) (B) to determine OXPHOS conductance (C), which was also unaffected by DTT treatment. JO2 and OXPHOS conductance of control and DTT-treated samples compared via paired t-test.

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  Figure 4. Mitochondrial ADP kinetics are unchanged by DTT treatment. (A) ADP concentrations corresponding to each energetic state were determined using the bioenergetics calculator from Fisher-Wellman et al. (2018) [18] (see Table 3) to determine Michaelis–Menten kinetics and the effects of 5 mM DTT treatment from respiration experiments presented in Figure 3. DTT treatment had no effect on maximal respiration (JO2Max; compared via paired t-test) (B) or ADP sensitivity ([ADP]50; compared via paired Wilcoxon signed-rank test) (C).