Cytochrome c oxidase III as a mechanism for apoptosis in heart failure following myocardial infarction

Abstract

Cytochrome c oxidase (COX) is composed of 13 subunits, of which COX I, II, and III are encoded by a mitochondrial gene. COX I and II function as the main catalytic components, but the function of COX III is unclear. Because myocardial ischemia affects mitochondrial oxidative metabolism, we hypothesized that COX activity and expression would be affected during postischemic cardiomyopathy. This hypothesis was tested in a monkey model following myocardial infarction (MI) and subsequent pacing-induced heart failure (HF). In this model, COX I protein expression was decreased threefold after MI and fourfold after HF (P < 0.05 vs. sham), whereas COX II expression remained unchanged. COX III protein expression increased 5-fold after MI and further increased 10-fold after HF compared with sham (P < 0.05 vs. sham). The physiological impact of COX III regulation was examined in vitro. Overexpression of COX III in mitochondria of HL-1 cells resulted in an 80% decrease in COX I, 60% decrease in global COX activity, 60% decrease in cell viability, and threefold increase in apoptosis (P < 0.05). Oxidative stress induced by H2O2 significantly (P < 0.05) increased COX III expression. H2O2 decreased cell viability by 47 +/- 3% upon overexpression of COX III, but only by 12 +/- 5% in control conditions (P < 0.05). We conclude that ischemic stress in vivo and oxidative stress in vitro lead to upregulation of COX III, followed by downregulation of COX I expression, impaired COX oxidative activity, and increased apoptosis. Therefore, upregulation of COX III may contribute to the increased susceptibility to apoptosis following MI and subsequent HF.

Fig. 1.

Regulation of cytochrome c oxidase (COX) subunits during ischemic cardiomyopathy. COX I, II, and III proteins from the heart of sham-treated monkeys (n = 4), a monkey model of myocardial infarction (MI, n = 6), and a monkey model of myocardial infarction and subsequent heart failure (MI + HF, n = 9) were detected by immunoblotting, and protein abundance was calculated. A: COX I was significantly (P < 0.05) decreased in MI and HF monkeys. B: COX II remained unchanged in MI and HF monkeys. C: COX III was increased significantly, by 5-fold in MI vs. sham and further increased 2-fold in HF. Values are means ± SE. *P < 0.05 vs. sham.

Fig. 2.

Overexpression and localization of COX III in mitochondria of HL-1 cells. A: agarose gel showing that the pEF-BOS-COX VIII-COX III-FLAG plasmid (pEF-BOS-C8C3F, 4 μg) was successfully transfected and overexpressed in HL-1 cells as detected by RT-PCR. Total length of the DNA sequence is 880 bp, which consists of 150 bp of COX VIII (mitochondrial targeted peptide), 780 bp of site-directed mutated COX III genes, and 30 bp of FLAG genes. MW, molecular weight. B: dose-dependent overexpression of COX III as a function of plasmid amount was detected by quantitative real-time PCR (qPCR). Different amounts of PEF-BOS-C8C3F plasmid (2, 4 and 6 μg) were transfected into 6-cm plates and cultured for 48 h. β-Actin was used as an internal control. Each condition was measured in triplicate. Values are means ± SE for the triplicates. *P < 0.05 vs. vector. C: localization of overexpressed COX III (4 μg) was confirmed using a mitochondrial probe, MitoTracker Red (C1), and anti-FLAG-FITC antibody (C2) followed by merging of both images (C3), which demonstrates that overexpressed COX III was localized in mitochondria.

Fig. 3.

Effects of COX III overexpression on level of COX I and II and total activity of COX. Compared with cells transfected with empty vector, overexpression of COX III (4 μg) resulted in a decrease (P < 0.05) in COX I expression (A), a slight, but not significant (P = 0.09), increase in COX II expression (B), and a significant decrease in total COX activity (C). Values are means ± SE for experiments performed in triplicate. *P < 0.05 vs. vector.

Fig. 4.

Dose-dependent decrease in cell viability induced by COX III overexpression. A: cell viability, as measured by trypan blue, progressively decreased in response to increased concentrations (2, 4, and 8 μg) of pEF-BOS-C8C3F plasmid. Values are means ± SE for experiments performed in triplicate. *P < 0.05 vs. vector. B: changes in cellular morphology were induced by COX III overexpression as a consequence of decreased viability.

Fig. 5.

Overexpression of COX III induces apoptosis in HL-1 cardiac myocytes. A: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis of HL-1 cells exposed to increased concentrations (4 and 8 μg) of pEF-BOS-C8C3F plasmid. B: activity of caspase-3, as measured by ELISA, in HL-1 cells exposed to empty vector or 4 μg of pEF-BOS-C8C3F plasmid. OD A_450nm, optical density absorbance at 450 nm. Values are means ± SE for experiments performed in triplicate. *P < 0.05 vs. vector. C: caspase-3 cleavage, as measured by Western blotting, in HL-1 cells exposed to empty vector or 4 μg of pEF-BOS-C8C3F plasmid. Values are means ± SE for experiments performed in triplicate. *P < 0.05 vs. vector.

Fig. 6.

Reactive oxygen species (ROS) production in HL-1 cells exposed to COX III overexpression. ROS production was detected using the fluorescent marker 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA). ROS production was increased 2.4-fold by transfection with 4 μg of pEF-BOS-C8C3F plasmid compared with the empty vector. Values are means ± SE for experiments performed in triplicate. *P < 0.05 vs. vector.

Fig. 7.

Decreased cell viability upon COX III overexpression in the presence of H₂O₂ in HL-1 cells. Viability of cells transfected with the empty vector or with 4 μg of pEF-BOS-C8C3F plasmid was evaluated by trypan blue in response to oxidative stress induced by increasing concentrations of H₂O₂ (0, 50, 100 and 200 μM). Values are means ± SE for experiments performed in triplicate. *P < 0.05 vs. corresponding vector. #P < 0.05 vs. corresponding group at 0 μM H₂O₂. Inset: average H₂O₂-induced decrease in cell viability was greater with COX III overexpression than in the presence of the empty vector (*P < 0.05 vs. vector).

Fig. 8.

Expression of COX I and III in COX III transcripts in HL-1 cells treated with or without 200 μM H₂O₂ and in the presence or absence of COX III overexpression. A: transcription of COX I, as measured by quantitative real-time PCR, was not changed in the vector group treated with H₂O₂ but was significantly decreased upon COX III overexpression (4 μg of plasmid). B: COX III transcription was increased significantly after treatment with H₂O₂ in transfected (4 μg of plasmid) and nontransfected (vector) controls compared with the corresponding groups without H₂O₂ treatment. Values are means ± SE for experiments performed in triplicate. *P < 0.05 vs. corresponding vector. #P < 0.05 vs. corresponding group without H₂O₂.