Mitochondrial oxidative stress mediates induction of autophagy and hypertrophy in angiotensin-II treated mouse hearts

Abstract

Autophagy is characterized by recycling of cellular organelles and can be induced by several stimuli, including nutrient deprivation and oxidative stress. As a major site of free radical production during oxidative phosphorylation, mitochondria are believed to be primary targets of oxidative damage during stress. Our recent study demonstrated that angiotensin II increases cardiac mitochondrial reactive oxygen species (ROS) production, causes a decline of mitochondrial membrane potential in cardiomyocytes and increases cardiac mitochondrial protein oxidative damage and mitochondrial DNA deletions. The deleterious effects of angiotensin II on mitochondria are associated with an increase in autophagosomes and increased signaling of mitochondrial biogenesis, interpreted as an attempt to replenish the damaged mitochondria and restore energy production. Direct evidence for the central role of mitochondrial ROS was investigated by comparing the effect on mice overexpressing catalase targeted to mitochondria (mCAT) and mice overexpressing peroxisomal targeted catalase (pCAT, the natural site of catalase) challenged by angiotensin II or Gαq overexpression. The mCAT, but not pCAT, mice are resistant to cardiac hypertrophy, fibrosis and mitochondrial damage, biogenesis and autophagy induced by angiotensin II, as well as heart failure induced by overexpression of Gαq.

Angiotensin II Induces Mitochondrial Autophagy and Biogenesis in Mouse Hearts through Mitochondrial ROS

Wild-type (WT), mCAT and pCAT mice were treated with continuous angiotensin II (1.1 mg/kg/d) delivered by subcutaneous osmotic minipump, which increased systolic and diastolic blood pressure by ∼25 mmHg. After 4 weeks of angiotensin II exposure, the protein carbonyl content of the cardiac mitochondria significantly increased by greater than four-fold in WT mice, indicative of increased oxidative damage. As the mtDNA deletion frequency has been shown to have an inverse correlation with mitochondrial function in heart and skeletal muscle, we found that the frequency of mitochondrial DNA deletions measured by the random mutation capture assay also significantly increased by greater than four-fold in WT mice after angiotensin II treatment. Both of these increases were significantly ameliorated in mCAT mice.

Transmission electron microscopy analyses demonstrated that angiotensin II significantly increases mitochondrial damage (shown as loss of cristae and electron density). This increased damage is associated with increased numbers of autophagosomes with the characteristic double-membrane structure seen by electron microscopy, as well as increased numbers of autolysosomes. Western blot analysis of LC3 also revealed a significant increase in the LC3-II/-I ratio, an indicator of autophagy. All of the above changes are attenuated by mCAT. ROS-induced mitochondrial damage and turnover is expected to result in increased signaling for mitochondrial biogenesis, a principal regulator of which is peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α). PGC-1α was previously shown to be transcriptionally upregulated by ROS. Four-week treatment with angiotensin II significantly increases expression of PGC-1α by 84%, with a concomitant increase in expression of PGC1α target genes, including TFAM, ERRα, NRF1 and NRF2. In contrast, angiotensin II-treated mCAT mice show little activation of PGC1α or its target genes, suggesting that the preservation of mitochondrial function by mCAT prevents activation of mitochondrial biogenesis signaling.

Overexpression of mCAT, but not pCAT, Blocks Angiotensin-Induced Cardiac Hypertrophy and Gαq Overexpression-Induced Heart Failure

To demonstrate that mitochondria are the primary target of oxidative damage, we compared the protective effects of overexpression of pCAT versus mCAT. Angiotensin II significantly increases heart weight and echocardiographic left ventricular mass index (LVMI) in WT mice. Consistent with this, both diastolic function and myocardial performance are significantly impaired. The pCAT mice show no differences from their WT littermates in all of the above parameters. In contrast, mCAT mice show appreciable protection from angiotensin-induced hypertrophy (size, heart weight and LVMI) and the decline of diastolic function and myocardial performance, compared to WT littermates.

Direct evidence of the protection of mitochondrial function conferred by mCAT to angiotensin-treated or Gαq-overexpressed hearts was shown by analysis of in situ state 3 (maximal stimulated) mitochondrial respiration, determined by measuring the O₂ consumption of saponin-permeabilized cardiac muscle in the presence of excess substrate (pyruvate and malate) and ADP. Four-week exposure to angiotensin II induces a 28% decline in state 3 respiration in WT and pCAT hearts, while mCAT hearts had substantially better preservation of state 3 mitochondrial respiration. Hearts from mice with Gαq overexpression show a 30% reduction in state 3 respiration and this is also preserved in mCAT/Gαq, but not pCAT/Gαq, mouse hearts. The fact that mCAT, but not pCAT, substantially ameliorates all of the above changes suggests that these events are mediated by mitochondrial ROS.

Proposed Mechanisms of Mitochondrial ROS Amplification

At the molecular level, angiotensin II binds to angiotensin II receptor I, a Gαq coupled-receptor, which subsequently activates NADPH oxidase at the cell membrane (NOX2-p47-p67 complexes) as well as NOX4 in the mitochondria. The ROS from either activated NOX2 or NOX4 might increase mitochondrial ROS production, however, protection by mCAT, but not pCAT, suggests that ROS produced directly in mitochondria by NOX4 might be the principal mediator of angiotensin II and Gαq-induced cardiac hypertrophy and failure. Mitochondrial processes can act to amplify and modulate this signal. These mechanisms might include ROS-induced ROS release and a ROS-mitochondrial DNA damage vicious cycle. For example, ROS production can increase electron leakage from the electron transport chain, further stimulating ROS production. This mechanism also agrees with our observations that primary damage to mitochondrial DNA in mitochondrial mutator mice is sufficient to elevate ROS, causes cardiac hypertrophy and potentiates the angiotensin II effect that leads to heart failure. Breaking the ROS vicious cycle within mitochondria by mCAT is effective to attenuate mitochondrial damage, autophagy, cardiac hypertrophy and failure, whether caused by angiotensin II, Gαq overexpression or mitochondrial mutator mice.

While clinical trials applying cellular antioxidants to attenuate the progression of cardiovascular diseases have shown equivocal benefits, this study provides a strong rationale to investigate the potential application of mitochondrial-targeted antioxidant drugs in the treatment or prevention of hypertensive cardiomyopathy and heart failure.

Punctum to: Dai DF, Johnson SC, Villarin JJ, Chin MT, Nieves-Cintrón M, Chen T, et al. Mitochondrial oxidative stress mediates angiotensin II-induced cardiac hypertrophy and G{alpha}q overexpression-induced heart failure. Circ Res. 2011;108:837–846. doi: 10.1161/CIRCRESAHA.110.232306.