Selective downregulation of mitochondrial electron transport chain activity and increased oxidative stress in human atrial fibrillation

Abstract

Mitochondria are critical for maintaining normal cardiac function, and a deficit in mitochondrial energetics can lead to the development of the substrate that promotes atrial fibrillation (AF) and its progression. However, the link between mitochondrial dysfunction and AF in humans is still not fully defined. The aim of this study was to elucidate differences in the functional activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes and oxidative stress in right atrial tissue from patients without (non-AF) and with AF (AF) who were undergoing open-heart surgery and were not significantly different for age, sex, major comorbidities, and medications. The overall functional activity of the electron transport chain (ETC), NADH:O2 oxidoreductase activity, was reduced by 30% in atrial tissue from AF compared with non-AF patients. This was predominantly due to a selective reduction in complex I (0.06 ± 0.007 vs. 0.09 ± 0.006 nmol·min(-1)·citrate synthase activity(-1), P = 0.02) and II (0.11 ± 0.012 vs. 0.16 ± 0.012 nmol·min(-1)·citrate synthase activity(-1), P = 0.003) functional activity in AF patients. Conversely, complex V activity was significantly increased in AF patients (0.21 ± 0.027 vs. 0.12 ± 0.01 nmol·min(-1)·citrate synthase activity(-1), P = 0.005). In addition, AF patients exhibited a higher oxidative stress with increased production of mitochondrial superoxide (73 ± 17 vs. 11 ± 2 arbitrary units, P = 0.03) and 4-hydroxynonenal level (77.64 ± 30.2 vs. 9.83 ± 2.83 ng·mg(-1) protein, P = 0.048). Our findings suggest that AF is associated with selective downregulation of ETC activity and increased oxidative stress that can contribute to the progression of the substrate for AF.

Keywords: 4-hydroxynonenal protein adducts; atrial fibrillation; electron transport chain complexes; humans; mitochondria; oxidative phosphorylation; oxidative stress; superoxide.

Fig. 1.

Measurement of mitochondrial oxidative phosphorylation complex activity. Complex I activity was measured using exogenous β-nicotinamide adenine dinucleotide, reduced form (NADH) as donor and 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone (Q₁) as acceptor of electrons; rotenone (Rot) was added to quantify rotenone-sensitive NADH-decylubiquinone oxidoreductase activity. Complex II activity was determined using succinate as donor and 2,6-dichlorophenolindophenol (DCPIP) as acceptor; succinate-decylubiquinone 2,6-dichlorphenolindophenol reductase activity was determined in the presence of 2-thenoyl-trifluoroacetone (TTFA). Complex III activity was measured using decylubiquinol (DBH₂) as donor and the oxidized form of cytochrome c (Cyt c) as acceptor; antimycin A (AA) was used to distinguish the reduction of Cyt c catalyzed by the ubiquinol-cytochrome c reductase from the nonenzymatic reduction of Cyt c. Complex IV activity was estimated by using the reduced form of Cyt c (red Cyt c) as the electron donor for oxygen. Complex V activity was measured by coupled reactions using lactate dehydrogenase (LDH) and pyruvate kinase (PK) as the coupling enzymes; oligomycin A (Oligom) was added to determine the oligomycin-sensitive ATPase activity. The wavelength of absorbance is summarized for each complex. NAD⁺, oxidized form of β-nicotinamide adenine dinucleotide; PEP, phosphoenolpyruvate; ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate; P_i, inorganic phosphate; Q, endogenous ubiquinone; CI–CV, mitochondrial OXPHOS complexes.

Fig. 2.

Activity of NADH:O₂-oxidoreductase in the right atria of patients with and without atrial fibrillation. The activity was defined with a Clark electrode and normalized to citrate synthase (CS) activity. Data are means ± SE; n = 5 for non-atrial fibrillation (nAF) group, n = 7 for atrial fibrillation (AF) group. *P < 0.05.

Fig. 3.

Functional activities of mitochondrial oxidative phosphorylation complexes in the right atria of patients without (nAF) and with (AF) atrial fibrillation. Data are expressed in nanomoles per minute per citrate synthase activity and presented as means ± SE; n = 23 for nAF and n = 31 for AF. *P < 0.05.

Fig. 4.

Correlation between functional activity of oxidative phosphorylation complexes and body mass index (BMI). ○, non-atrial fibrillation (nAF); ●, atrial fibrillation (AF). Functional activity is expressed in nanomoles per minute per citrate synthase activity. *P < 0.05.

Fig. 5.

Separation and identification of mitochondrial complexes from patients without and with atrial fibrillation. A: representative gel of separated mitochondrial oxidative phosphorylation (OXPHOS) complexes I–V in native form by 1-dimensional BN-PAGE electrophoresis. B: identification of OXPHOS complexes after transferring proteins from BN-PAGE gel to PVDF membrane with MitoProfile Total OXPHOS human antibody cocktail. C and D: Western blot of individual subunits of OXPHOS complexes I–V using MitoProfile Total OXPHOS human antibody cocktail (C) and densitometry of bands from immunoblot corresponding to mitochondrial complexes normalized to density of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band (D). MW, molecular weight of the marker; nAF, without atrial fibrillation (n = 11); AF, with atrial fibrillation (n = 11); C I–C V, complexes I–V. Data are means ± SE. *P < 0.05.

Fig. 6.

Differences in protein expression level of mitochondrial OXPHOS complexes I–V from patients without and with atrial fibrillation. A: immunoblots of representative subunits of complexes I–V using MitoProfile Total OXPHOS human antibody cocktail. B: densitometry of bands from immunoblot corresponding to mitochondrial complexes normalized to density of cytochrome c band. nAF, without atrial fibrillation (n = 5); AF, with atrial fibrillation (n = 5); C I–C V, complexes I–V. Data are means ± SE. *P < 0.05.

Fig. 7.

Difference in 4-HNE protein adducts and mitochondrial superoxide level in the right atria between patients without and with atrial fibrillation. A: the level of 4-HNE was normalized to protein concentration in tissue supernatant; n = 7 for non-atrial fibrillation (nAF), n = 8 for atrial fibrillation (AF). B: representative images of changes in mitochondrial superoxide production in permeabilized myofibers from nAF and AF patients. Fibers were stained with 2 μg/ml Hoechst (λ_ex/λ_em = 350/461 nm) and 2.5 μM MitoSOX Red (λ_ex/λ_em = 510/580) for 30 min and imaged by time-lapse laser scanning confocal fluorescence microscopy (Olympus FV1200) with a ×10 objective. Antimycin A (AA) at 10 μM was applied to stimulate superoxide production in mitochondria. C: difference in fluorescence intensity of MitoSOX Red before and after antimycin A (15 min) exposure. Data are means ± SE; n = 8 for nAF and n = 5 for AF. *P < 0.05.