Aging augments mitochondrial susceptibility to heat stress

Abstract

The pathophysiology of aging is accompanied by a decline in tolerance to environmental stress. While mitochondria are primary suspects in the etiology of aging, little is known about their ability to tolerate perturbations to homeostasis in older organisms. To investigate the role of mitochondria in the increased susceptibility to heat stress that accompanies aging, young and old Fischer 344 rats underwent a heat stress protocol known to elicit exaggerated cellular damage with aging. At either 2 or 24 h after heat stress, livers were removed from animals, and hepatic mitochondria were isolated. Electron microscopy revealed extensive morphological damage to mitochondria from young and, to a greater extent, old rats after heat stress. There was also a significant loss of cytochrome c from old, but not young, mitochondria and a persistent increase in 4-hydroxynonenal-modified proteins in old vs. young mitochondria exposed to heat stress. Electron paramagnetic resonance measurements of superoxide indicate greater superoxide production from mitochondria of old compared with young animals and suggest that mitochondrial integrity was altered during heat stress. The mitochondrial stress response, which functions to correct stress-induced damage to mitochondrial proteins, was also blunted in old rats. Delayed and reduced levels of heat shock protein 60 (Hsp60), the main inducible mitochondrial stress protein, were observed in old compared with young mitochondria after heat stress. Additionally, the amount of Hsp10 protein increased in young, but not old, rat liver mitochondria after hyperthermic challenge. Taken together, these data suggest that mitochondria in old animals are more vulnerable to incurring and less able to repair oxidative damage that occurs in response to a physiologically relevant heat stress.

Fig. 1.

Heat stress causes structural damage to mitochondria in young and, to a larger extent, old rat livers. Mitochondria were isolated from young and old control and heat-stressed rats and immediately fixed. After dehydration, embedding, and cutting, transmission electron microscopy was used to capture representative micrographs. These images revealed morphological damage in old control mitochondria, while young control mitochondria had normal morphology (top). Extensive damage was observed in isolated mitochondria (e.g., severe swelling, loss of cristae, disruption of membranes) from old and young animals after heating at the 2-h time point (middle) and in old animals only at the 24-h time point (bottom). Scale bar = 10 μM.

Fig. 2.

Effects of age and heat stress on mitochondrial superoxide production. Top: height of the 5-dimethyl-1-pyrroline N-oxide (DMPO)-OH signal, a result of the reaction of superoxide with the spin-trap DMPO, was measured on an EPR spectrometer. Values represent means ± SE of 4–6 animals in each group. *P < 0.05 compared with young at same time point. †P < 0.05 compared with nonheated age-matched control. #P < 0.05 compared with 2-h time point. Bottom: examples of EPR experiments from young and old rat mitochondria incubated with the Complex I substrate NADH alone (spectrum A); NADH + rotenone (spectrum B); and NADH + antimycin A (spectrum C). Spectrum D for young and old represent a repeat of trial C (NADH + antimycin A) in the presence of the superoxide scavenger SOD. All EPR signals were virtually eliminated with addition of SOD. Y, young; O, old; C, nonheated control.

Fig. 3.

Lipid peroxidation is increased in mitochondria after heat stress. Mitochondria were isolated from liver samples obtained from young and old rats in control conditions and at 2 and 24 h after heat stress. Bottom: densitometry values for immunoblot results are presented. Mitochondrial 4-hydroxynonenal (4HNE) levels were unchanged from control levels for young and old rats at the 2-h time point. At 24-h after heat stress, 4HNE was significantly higher in both young and old mitochondria compared with control and 2-h groups. Four to six rats were analyzed per age group at each time point. The density of each lane was normalized to the density of Gel Code blue-stained gel. Values are means ± SE. †P < 0.05 compared with nonheated age-matched control. #P < 0.05 compared with 2-h time point. Top: representative 4HNE immunoblots for mitochondria from livers of young and old rats during a time course of recovery from a heat-stress protocol. Each lane represents a liver mitochondrial sample from a different rat with 30 μg of protein loaded per lane.

Fig. 4.

Aging increases the loss of cytochrome c from mitochondria after heat stress. Mitochondria were isolated from liver samples obtained from young and old rats in control conditions and at 2 and 24 h after heat stress. Upper left: densitometry values for immunoblot results for mitochondrial cytochrome c levels were decreased in old vs. young rats at the 2- and 24-h time points. Lower left: densitometry values for immunoblot results for cytosolic cytochrome c levels were increased in old vs. young rats at the control and 2-h time points. Four to six rats were analyzed per age group at each time point. The density of each lane was normalized to the density of Gel Code blue-stained gel. Values are expressed as means ± SE. *P < 0.05 compared with young at the same time point. †P < 0.05 compared with nonheated age-matched control. #P < 0.05 compared with 2-h time point. Representative cytochrome c immunoblots are presented for mitochondrial (upper right) and cytosolic (lower right) fractions from livers of young and old rats during a time course of recovery from a heat-stress protocol. Each lane represents a sample from a different rat with 30 μg of protein loaded per lane.

Fig. 5.

The stress protein Hsp60 is increased in mitochondria after heat stress. Mitochondria were isolated from liver samples obtained from young and old rats in control conditions and at 2 and 24 h after heat stress. Bottom: densitometry values for immunoblot results are presented. Mitochondrial Hsp60 levels were elevated compared with control at 2 and 24 h after heat stress in young rats. Hsp60 levels in old rat mitochondria were unchanged from control and significantly lower than young at 2 h. At 24 h after heat stress, Hsp60 was significantly higher in both young and old mitochondria compared with control. Four to six rats were analyzed per age group at each time point. The density of each lane was normalized to the density of Gel Code blue-stained gel. Values are expressed as means ± SE. *P < 0.05 compared with young at the same time point. †P < 0.05 compared with nonheated age-matched control. #P < 0.05 compared with 2-h time point. Top: representative Hsp60 immunoblots for mitochondria from livers of young and old rats during a time course of recovery from a heat-stress protocol. Each lane represents a liver mitochondrial sample from a different rat with 30 μg of protein loaded per lane.

Fig. 6.

Heat shock protein 10 (Hsp10) levels are elevated in young, but not old, mitochondria after heat stress. Mitochondria were isolated from liver samples obtained from young and old rats in control conditions and at 2 and 24 h after heat stress. Bottom: densitometry values for immunoblot results are presented. Mitochondrial Hsp10 levels in young rats were elevated compared with old at 2 h after heat stress and elevated compared with young control at 24 h. In old rat mitochondria, Hsp10 levels were unchanged from control at either time point after heat. The density of each lane was normalized to the density of Gel Code blue-stained gel. Values are expressed as means ± SE. *P < 0.05 compared with young at same time point. †P < 0.05 compared with nonheated age-matched control. Top: representative Hsp10 immunoblots for mitochondria from livers of young and old rats during a time course of recovery from a heat-stress protocol. Each lane represents a liver mitochondrial sample from a different rat with 30 μg of protein loaded per lane.

Fig. 7.

Age-related changes in mitochondrial Hsp70 (mtHsp70) levels in mitochondria after heat stress. Mitochondria were isolated from liver samples obtained from young and old rats in control conditions and at 2 and 24 h after heat stress. Bottom: densitometry values for immunoblot results are presented. Mitochondrial mtHsp70 levels in old rats were elevated compared with young in the control condition. At 2 h after heat stress, young rats showed elevated mtHsp70 levels compared with young control and old 2-h rats. No differences were observed at the 24-h time point. The density of each lane was normalized to the density of Gel Code blue-stained gel. Values are expressed as means ± SE. *P < 0.05 compared with young at the same time point. †P < 0.05 compared with nonheated age-matched control. Top: representative mtHsp70 immunoblots for mitochondria from livers of young and old rats during a time course of recovery from a heat-stress protocol. Each lane represents a liver mitochondrial sample from a different rat with 30 μg of protein loaded per lane.