Decreased cytochrome c oxidase subunit VIIa in aged rat heart mitochondria: immunocytochemistry

Abstract

Aging decreases oxidative phosphorylation through cytochrome oxidase (COX) in cardiac interfibrillar mitochondria (IFM) in 24-month old (aged) rats compared to 6-month old adult Fischer 344 rats, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Immunoelectron microscopy (IEM) reveals in aged rats a 25% reduction in cardiac COX subunit VIIa in cardiac IFM, but not in SSM. In contrast, the content of subunit IV remains unchanged in both SSM and IFM, irrespective of age. These subunits are localized mainly on cristae membranes. In contrast, semi-quantitative immunoblotting, which detects denatured protein, indicates that the content of COX VIIa is similar in IFM and SSM from both aged and adult hearts. IEM provides a sensitive method for precise localizing and quantifying specific mitochondrial proteins. The lack of immunoreaction of COX VIIa subunit by IEM in aged IFM is not explained by a reduction in protein, but rather by a masking phenomenon or by an in situ change in protein structure affecting COX activity.

Fig. 1

Immunoblot analysis of COX VIIaHL monoclonal antibody. The immunoblot experiment was performed as described in Materials and Methods, except each well was loaded with 43 μg mitochondrial protein and 10% Tricine gel. As a quality control, the membrane also was incubated with COX IV. The two rows were from the same immunoblot, but are based on different exposure times.

Fig. 2

Immunoelectron microscopic analysis of COX IV (A, E, I, M) and COX VIIa (BD, FH, JL, and NP) in isolated cardiac SSM and IFM from 6 months. (AD, and IL) and 24 months. (EH, and MP) Fisher 344 rats. Gold particles (5 nm) indicate the COX IV or COX VIIa contents of the mitochondria. Vertical line separates these mitochondria labeled for COX IV and COX VIIa. Scale bar = 1μm; all micrographs on this plate are at the same magnification.

Fig. 3

Histogram showing the average number of gold particles per μm² mitochondrion (Y-axis; mean ± SD) in vitro. Electron micrographs of 50 mitochondria were obtained from each experiment and the total number of gold particles counted for each mitochondrion. Three independent experiments were performed using anti-COX IV and COX VIIaHL antibodies on isolated SSM and IFM from three adults and three aged rats (total = 150 organelles). Each bar indicates the mean number of gold particles from 150 mitochondria±SD * A decrease in the expression of subunit COX VIIa in IFM in the 24-month rat compared with the expression of subunit COX VIIa in the 6-month rat heart was determined using parametric one-way ANOVA test (P < 0.05).

Fig. 4

IEM of COX VIIa in serial but not consecutive sections of an isolated cardiac IFM (A–E) from 6 months. Fischer 344 rat. Gold particles (5nm) indicate COX VIIa in the mitochondrion. Scale bar = 1 μm; all micrographs are at the same magnification.

Fig. 5

Representative immunoelectron microscopic analysis of COX IV (A, C, E, and G) and COX VIIa [B, D, F, and H] in in situ mitochondria from 6 months. SSM (A and B), 6 months. IFM (E and F), 24 months. SSM (C and D), and 24 months. IFM (G and H). Gold particles (5nm) indicate the COX IV or COX VIIa contents. Myofibrils (MF). Plasma membrane (PM). Because of aldehyde fixation without osmium postfixation, limiting membranes of these mitochondria are variously discernible. Scale bar = 1μm; all micrographs are at the same magnification.

Fig. 6

Histogram showing the average number of gold particles per mitochondrion (Y-axis; mean ± SD) in vivo. Electron micrographs of 50 mitochondria were obtained from each experiment and the total number of gold particles counted for each mitochondrion. Three independent experiments using anti-COX IV and COX VIIaHL antibodies on isolated SSM and IFM from three adults and three aged rats were performed (total = 150 organelles). Each bar indicates the mean number of gold particles from 150 mitochondria±SD. * A decrease in the expression of subunit COX VIIa in IFM in the 24-month rat compared with the expression of subunit COX VIIa in the 6-month rat heart was determined using parametric one-way ANOVA test (P < 0.05).

Fig. 7

Immunoelectron microscopic analysis of COX VIIa in isolated cardiac IFM from 24 months. Sections were dephosphorylated with non-specific alkaline phosphatase. A: Alkaline phosphatase treatment; B: Buffer without alkaline phosphatase; C: No treatment. The results are identical in all three cases. ***Bar = 0.5 μm.

Fig. 8

Quantification of densitometry measurements of COX VIIa bands of western blots. A: Representative Western blots of COX IV and COX VIIa bands from SSM and IFM of three 24-month F344 rats are compared to those from SSM and IFM of one typical 6-month F344 rat. B: Protein bands of COX VIIa and IV were quantified by densitometry using NIH Image J 1.32 software (http://rsb.info.nih.gov/ij/). In the histogram, the plotted value is the ratio of the density of COX VIIa in the 24-month mitochondria to the density of COX VIIa in the 6 months; comparison is based on SSM and IFM in the two different age groups (6 months, N = 8; 24 months, N = 8). The horizontal line at 1 refers to the 6-month mitochondria, and aids in comparing the 24-month mitochondria with their younger counterparts. This figure contains the data from the rats used in the IEM study plus those used in the experiments originally performed for immunoblotting only.