Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.     

Short-term CR decreases cardiac mitochondrial oxidant production but increases carbonyl content

Lifelong caloric restriction (CR) reduces the rate of mitochondrial oxidant production and the accumulation of oxidized proteins and prevents some of the age-associated decline in 20S proteasome activity. However, few studies have investigated how rapidly the beneficial effects of CR take place. We investigated whether 2 mo of CR in 6-mo-old rats would be of sufficient duration to elicit these beneficial changes. Mitochondrial oxidant production was significantly diminished in the CR rats compared with the ad libitum-fed animals. Short-term CR also caused a significant decrease in mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities, but there were no differences in cytosolic SOD and GPX activities, whereas mitochondrial and cytosolic catalase (CAT) activity was increased with CR. However, protein carbonyl content was significantly elevated in both the mitochondrial and cytosolic fractions from CR rats. Of the three major 20S proteasome activities (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolase), the peptidylglutamyl-peptide hydrolase activity was significantly elevated in the CR animals, possibly because of the fact that there were more oxidized proteins to be degraded. Although fewer oxidants were produced in the CR animals, it is possible that the ability to scavenge oxidants was transiently suppressed because of the reduction in mitochondrial antioxidant enzyme activities, which may explain the observed increases in carbonyl content.     

Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction

The production of ATP is vital for muscle contraction, chemiosmotic homeostasis, and normal cellular function. Many studies have measured ATP content or qualitative changes in ATP production, but few have quantified ATP production in vivo in isolated mitochondria. Because of the importance of understanding the energy capacity of mitochondria in biology, physiology, cellular dysfunction, and ultimately, disease pathologies and normal aging, we modified a commercially available bioluminescent ATP determination assay for quantitatively measuring ATP content and rate of ATP production in isolated mitochondria. The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. The stabilities of the reaction mixture as well as relevant ATP standards were quantified. The luminescent signals of the reaction mixture and a 0.5 microM ATP standard decreased linearly at rates of 2.16 and 1.39% decay/min, respectively. For a 25 microM ATP standard, the luminescent signal underwent a logarithmic decay, due to intrinsic deviations from the Beer-Lambert law. Moreover, to test the functionality of isolated mitochondria, they were incubated with 1 and 5 mM oligomycin, an inhibitor of oxidative phosphorylation. The rate of ATP production in the mitochondria declined by 34 and 83%, respectively. Due to the sensitivity and stability of the assay and methodology, we were able to quantitatively measure in vivo the effects of age and caloric restriction on the ATP content and production in isolated mitochondria from the brain and liver of young and old Fischer-344 rats. In both tissues, neither age nor caloric restriction had any significant effect on the ATP content or the rate of ATP production. This study introduces a highly sensitive, reproducible, and quick methodology for measuring ATP in isolated mitochondria.     

Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis

Generation of reactive oxygen species and mitochondrial dysfunction has been implicated in doxorubicin-induced cardiotoxicity. This study examined pro-apoptotic mitochondrial cell death signals in an H9C2 myocyte rat cell line and in isolated rat heart mitochondria exposed to doxorubicin. Mitochondrial and cellular viability were assessed using an MTT viability assay (formazan product formed by functional mitochondrial dehydrogenases) and calcein AM dye (fluoresces upon cleavage by cytosolic esterases). Mitochondrial dysfunction followed by cell death was observed using nM concentrations of doxorubicin. Significant doxorubicin-induced cell death was not apparent until after 6 h following doxorubicin exposure using the calcein AM assay. The involvement of apoptosis is evidenced by an increase in TUNEL (terminal (TdT)-mediated dUTP-biotin nick end labeling)-positive nuclei following doxorubicin treatment. Furthermore, doxorubicin administered to isolated mitochondria induced a rapid increase in superoxide production, which persisted for at least 1 h and was followed by increased cytochrome c efflux. In addition, caspase-3 activity was increased with doxorubicin administration in the H9C2 myocyte cell line. An oxidant-mediated threshold of mitochondrial death may be required for doxorubicin-induced apoptosis.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Long-term treadmill exercise improves spatial memory of male APPswe/PS1dE9 mice by regulation of BDNF expression and microglia activation

Increasing evidence suggests that physical activity could delay or attenuate the symptoms of Alzheimer''s disease (AD). But the underlying mechanisms are still not fully understood. To investigate the effect of long-term treadmill exercise on the spatial memory of AD mice and the possible role of β-amyloid, brain-derived neurotrophic factor (BDNF) and microglia in the effect, male APPswe/PS1dE9 AD mice aged 4 months were subjected to treadmill exercise for 5 months with 6 sessions per week and gradually increased load. A Morris water maze was used to evaluate the spatial memory. Expression levels of β-amyloid, BDNF and Iba-1 (a microglia marker) in brain tissue were detected by immunohistochemistry. Sedentary AD mice and wildtype C57BL/6J mice served as controls. The results showed that 5-month treadmill exercise significantly decreased the escape latencies (P < 0.01 on the 4th day) and improved the spatial memory of the AD mice in the water maze test. Meanwhile, treadmill exercise significantly increased the number of BDNF-positive cells and decreased the ratios of activated microglia in both the cerebral cortex and the hippocampus. However, treadmill exercise did not significantly alleviate the accumulation of β-amyloid in either the cerebral cortex or the hippocampus of the AD mice (P > 0.05). The study suggested that long-term treadmill exercise could improve the spatial memory of the male APPswe/PS1dE9 AD mice. The increase in BDNF-positive cells and decrease in activated microglia might underpin the beneficial effect.     

Age-related differences in apoptosis with disuse atrophy in soleus muscle

Muscle atrophy is associated with a loss of muscle fiber nuclei, most likely through apoptosis. We investigated age-related differences in the extent of apoptosis in soleus muscle of young (6 mo) and old (32 mo) male Fischer 344 x Brown Norway rats subjected to acute disuse atrophy induced by 14 days of hindlimb suspension (HS). HS-induced atrophy (reduction in muscle weight and cross-sectional area) was associated with loss of myofiber nuclei in soleus muscle of young, but not old, rats. This resulted in a significant decrease in the myonuclear domain (cross-sectional area per nucleus) in young and old rats, with changes being more pronounced in old animals. Levels of apoptosis (TdT-mediated dUTP nick end labeling and DNA fragmentation) were higher in soleus muscles of old control rats than young animals. Levels were significantly increased with HS in young and old rats, with the greatest changes in old animals. Caspase-3 activity in soleus muscle tended to be increased with age, but changes were not statistically significant (P=0.052). However, with HS, caspase-3 activity significantly increased in young, but not old, rats. Immunohistochemistry showed that the proapoptotic endonuclease G (EndoG, a mitochondrion-specific nuclease) was localized in the subsarcolemmal mitochondria in control muscles, and translocation to the nucleus occurred in old, but not young, control animals. There was no difference between EndoG total protein content in young and old control rats, but EndoG increased almost fivefold in soleus muscle of old, but not young, rats after HS. These results show that deregulation of myonuclear number occurs in old skeletal muscle and that the pathways involved in apoptosis are distinct in young and old muscles. Apoptosis in skeletal muscle is partly mediated by the subsarcolemmal mitochondria through EndoG translocation to the nucleus in response to HS.