Cardiac mitochondria in heart failure: decrease in respirasomes and oxidative phosphorylation

Abstract

Aims: Mitochondrial dysfunction is a major factor in heart failure (HF). A pronounced variability of mitochondrial electron transport chain (ETC) defects is reported to occur in severe acquired cardiomyopathies without a consistent trend for depressed activity or expression. The aim of this study was to define the defect in the integrative function of cardiac mitochondria in coronary microembolization-induced HF.

Methods and results: Studies were performed in the canine coronary microembolization-induced HF model of moderate severity. Oxidative phosphorylation was assessed as the integrative function of mitochondria, using a comprehensive variety of substrates in order to investigate mitochondrial membrane transport, dehydrogenase activity and electron-transport coupled to ATP synthesis. The supramolecular organization of the mitochondrial ETC also was investigated by native gel electrophoresis. We found a dramatic decrease in ADP-stimulated respiration that was not relieved by an uncoupler. Moreover, the ADP/O ratio was normal, indicating no defect in the phosphorylation apparatus. The data point to a defect in oxidative phosphorylation within the ETC. However, the individual activities of ETC complexes were normal. The amount of the supercomplex consisting of complex I/complex III dimer/complex IV, the major form of respirasome considered essential for oxidative phosphorylation, was decreased.

Conclusions: We propose that the mitochondrial defect lies in the supermolecular assembly rather than in the individual components of the ETC.

Figure 1

State 3 respiratory rates of heart subsarcolemmal mitochondria (A) and interfibrillar mitochondria (B). State 3 was induced by 200 µM ADP when mitochondria oxidize complex I substrates (Glutamate+Malate and Pyruvate+Malate), and 100 µM ADP when mitochondria oxidize complexes II (Succinate+rotenone), III (Duroquinol, DHQ+rotenone), and IV via cytochrome c (TMPD ascorbate+rotenone). M, malate. *P < 0.05 control (n = 5) vs. heart failure (n = 5). Mean ± SEM.

Figure 2

Uncoupled (200 µM dinitrophenol) respiratory rates of heart subsarcolemmal mitochondria (A) and interfibrillar mitochondria (B). Substrates that donate reducing equivalents to complexes I (Glutamate), II (Succinate+rotenone), III (Duroquinol, DHQ+rotenone), and IV via cytochrome c (TMPD ascorbate+rotenone). *P < 0.05 control (n = 5) vs. heart failure (n = 5). Mean ± SEM.

Figure 3

The activity of electron transport chain enzymes in isolated heart subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The enzyme activity was measured in detergent-solubilized, freshly isolated SSM and IFM from control (n = 5) and heart failure (n = 5) dogs, and expressed as nmol/min/mg mitochondrial protein or as the first-order rate constant (k = 1/min/mg mitochondrial protein) for complex IV. NCR, rotenone sensitive NADH-Cytochrome c Reductase; C I, complex I; NFR, NADH-Ferricyanide Reductase; SCR, antimycin A-sensitive Succinate-Cytochrome c Reductase. C II, TTFA-sensitive complex II; C II+Q, TTFA-sensitive Complex II with exogenous coenzyme Q analogue; C III, complex III; C IV, complex IV. Mean ± SEM.

Figure 4

The activity of cytochrome c oxidase in heart subsarcolemmal mitochondria (A) and interfibrillar mitochondria (B) determined polarographically. C, cytochrome c. A, asolectin. Mean ± SEM.

Figure 5

Cytochrome content in heart subsarcolemmal mitochondria (A) and interfibrillar mitochondria (B). *P < 0.05 control vs. HF. Mean ± SEM.

Figure 6

Separation of supramolecular assemblies of mitochondrial oxidative phosphorylation complexes by 1D BN-PAGE in subsarcolemmal mitochondria (SSM) of control and heart failure hearts. (A) Dog heart SSM solubilized with digitonin using a 6:1 digitonin/protein ratio (g/g) and separated by one-dimensional BN-PAGE. C I, C II, C III, C IV, and C V (right) indicate mitochondrial complexes I–V and correspond to bands 1–5 (left). Bands 6–9 (left) indicate supermolecular complexes. Band 8 indicates supercomplex CI-CIII₂-CIV. (B) Complex V (C V). (C) The density of the band corresponding to supercomplex CI-CIII₂-CIV (arrows) and of the bands corresponding to complexes I, III, and IV were normalized to the density of complex V band. *P < 0.05 control vs. HF. Mean ± SEM. One-dimensional BN-PAGE, one-dimensional blue native PAGE.

Figure 7

Separation of supramolecular assemblies of mitochondrial oxidative phosphorylation complexes by one-dimensional BN-PAGE in interfibrillar mitochondria (IFM) of control and heart failure hearts. (A) Dog heart IFM solubilized with digitonin using a 6:1 digitonin/protein ratio (g/g) and separated by 1D BN-PAGE. C I, C II, C III, C IV, and C V (right) indicate mitochondrial complexes I–V and correspond to bands 1–5 (left). Bands 6–9 (left) indicate supermolecular complexes. Band 8 (arrows) indicates supercomplex CI-CIII₂-CIV. (B) Complex V (C V). (C) The density of the band corresponding to supercomplex CI-CIII₂-CIV (arrows) and of the bands corresponding to complexes I, III, and IV were normalized to the density of complex V band. *P < 0.05 control vs. HF. #P = 0.07 control vs. HF. Mean ± SEM. One-dimensional BN-PAGE, one-dimensional blue native PAGE.