Aging impairs skeletal muscle mitochondrial bioenergetic function

Abstract

This study investigated the influence of age on the functional status of mitochondria isolated from skeletal muscle of C57BL/6 mice aged 3 and 18 months. We hypothesized that skeletal muscle mitochondria isolated from aged animals will exhibit a decreased respiratory function. Mitochondrial respiratory functional measures (ie, State 3 and 4 respiration, respiratory control ratio and number of nanomoles of ADP phosphorylated by nanomoles of O(2) consumed per mitochondrion) and biochemical markers of oxidative damage (aconitase activity, protein carbonyl derivatives, sulfhydryl groups, and malondialdehyde) were measured in isolated mitochondrial suspensions. Along with traditional tests of mitochondrial function, an in vitro repetitive ADP-stimulation test was used to evaluate the mitochondrial capacity to reestablish the homeostatic balance between successive ADP stimulations. The number of mitochondria per mitochondrial suspension, calculated by transmission electron microscopy, was used to normalize functional and biochemical data. Our results confirm the existence of an age-associated decline in mitochondrial function of mixed skeletal muscle, which is significantly correlated with higher levels of mitochondrial oxidative damage.

Figure 1.

Methodological procedures to quantify the mitochondrial concentration in each suspension. (A) The number of mitochondria per micrometer was assessed by counting the mitochondria that were situated under the four lines crossing the center of the micrograph; the final number of mitochondria per micrometer was established as the mean value of the four counts. The number of mitochondria per square meter was evaluated by counting the total mitochondria present in each micrograph. (B) Assessment of the correction factor for centrifugation-induced compaction, in order to adjust the data drawn from the microscopic evaluation of the pellet to the real volume of the mitochondrial suspension.

Figure 2.

Repetitive ADP-stimulation test. See text for detailed description.

Figure 3.

Functional data obtained from skeletal muscle mitochondria isolated from young and mature animals with Complex I–linked substrates, pyruvate (5 mM) and malate (2 mM), in the repetitive ADP-stimulation test. Data are mean ± standard deviation. State 3 and State 4 respiratory rates are expressed as nanomoles of O₂ consumed per mitochondrion. Filled bars: young animals; open bars: mature animals. *Age differences (p < .05); ⁺significantly different from first cycle; ^#significantly different from first and second cycle; †significantly different from all ADP cycles (p < .05).

Figure 4.

Schematic representation of the Assay IV (6 ADP cycles) from the repetitive ADP-stimulation test in one young animal (A) and one mature animal (B).

Figure 5.

Acute biochemical alterations induced by the repetitive ADP-stimulation test in skeletal muscle mitochondria isolated from young and mature animals. Data are mean ± standard deviation. All data are expressed per mitochondrion. Data from protein carbonyl derivatives and cytochrome c are expressed as optical density arbitrary units per mitochondrion. Filled bars: young animals; open bars: mature animals. *Age differences (p < .05); ⁺significantly different from prestimulation (p < .05).