Integration of cellular bioenergetics with mitochondrial quality control and autophagy

Abstract

Bioenergetic dysfunction is emerging as a cornerstone for establishing a framework for understanding the pathophysiology of cardiovascular disease, diabetes,cancer and neurodegeneration. Recent advances in cellular bioenergetics have shown that many cells maintain a substantial bioenergetic reserve capacity, which is a prospective index of ‘ healthy ’ mitochondrial populations.The bioenergetics of the cell are likely regulated by energy requirements and substrate availability. Additionally,the overall quality of the mitochondrial population and the relative abundance of mitochondria in cells and tissues also impinge on overall bioenergetic capacity and resistance to stress. Because mitochondria are susceptible to damage mediated by reactive oxygen/nitrogen and lipid species, maintaining a ‘ healthy ’ population of mitochondria through quality control mechanisms appears to be essential for cell survival under conditions of pathological stress. Accumulating evidence suggest that mitophagy is particularly important for preventing amplification of initial oxidative insults, which otherwise would further impair the respiratory chain or promote mutations in mitochondrial DNA (mtDNA). The processes underlying the regulation of mitophagy depend on several factors, including the integrity of mtDNA, electron transport chain activity, and the interaction and regulation of the autophagic machinery. The integration and interpretation of cellular bioenergetics in the context of mitochondrial quality control and genetics is the theme of this review.

Figures

Figure 1. Regulation of mitochondrial quality control and the response to oxidative stress

An existing mitochondrial population is shown subjected to a pathological stress with the formation of reactive oxygen, nitrogen and lipid species (ROS/RNS/RLS). This oxidative stress damages mtDNA impairing the ability of the organelle to replace damaged electron transport proteins and decreasing bioenergetic reserve capacity; the resulting increased mitochondrial ROS then oxidatively damages previously unmodified mitochondria. The damaged mitochondria are turned over by a mitophagic mechanism that then suppresses this vicious cycle. The mitochondrial population is now renewed through mitochondrial biogenesis. The bioenergetic reserve capacity is essential for resistance to oxidative stress and supplying ATP demand. Once the bioenergetic reserve is depleted, bioenergetic failure occurs and the cell is programmed for cell death.

Figure 2. The cellular bioenergetic profile

This mitochondrial stress test can be used to measure several indices of mitochondrial function in intact cells in real time and allows for the identification of critical respiratory defects. A typical experiment is shown in which basal oxygen consumption rate (OCR) is allowed to stabilize before the sequential addition of oligomycin, FCCP, and antimycin A and/or rotenone with a measurement of changes in OCR as indicated. This time course is annotated to show the relative contribution of non-respiratory chain oxygen consumption, ATP-linked oxygen consumption, the maximal OCR after the addition of FCCP, and the reserve capacity of the cells.

Figure 3. Cellular bioenergetics in different cell types

Extracellular flux analysis using the mitochondrial stress test assay: Panel A: Adult rat cardiomyocytes, primary hepatocytes or bovine aortic endothelial cells were subject to a metabolic stress test as shown, and the OCR rates were normalized to protein. Panel B: in this analysis basal OCR was established as 100% OCR and the proportion of ATP-linked OCR, proton leak and non-mitochondrial oxygen consumption is shown. Panel C: in this analysis maximal OCR is established as 100% and the oxygen consumption is apportioned between ATP-linked OCR, proton leak, reserve capacity and non-mitochondrial O2 consumption. The RCR for basal and maximal OCR is reported together with the stateapparent for each cell type.

Figure 4. Possible interpretation tree for the mitochondrial bioenergetic profile

In cells, differences in basal oxygen consumption rate (OCR) could be due to several factors including ATP turnover reactions, proton leak, non-mitochondrial (N.M.) oxygen consumption, or damage to the electron transport chain. This can be tested by adding oligomycin, and some potential outcomes are shown. Next, the changes in the maximal capacity (and the reserve capacity) can be identified using the FCCP-stimulated rate. An increase or a decrease in this rate compared with controls could be due to changes in substrate availability, mitochondrial mass, or electron transport chain (ETC) integrity. Lastly, the effects of non-mitochondrial sources on oxygen consumption may be examined.

Figure 5. Glycolysis stress test

This extracellular flux assay demonstrates how basal glycolytic rate, maximal glycolytic rate, and the glycolytic reserve capacity can be determined. After measurement of the basal extracellular acidification rate (ECAR), oligomycin can be added, which generally increases glycolytic rate in response to loss of mitochondrial ATP production. The addition antimycin A/rotenone, depending on experimental conditions and cell type, may further increase the ECAR, giving an index of lactate production occurring when all mitochondrial electron transport is inhibited. Non-glycolytic extracellular acidification (i.e., acidification not due to lactate production) can then be measured by introducing koningic acid (KA; an inhibitor of glyceraldehyde-3-dehydrogenase) to inhibit glycolysis; alternatively, 2-deoxyglucose (2-DG) may be used. This assay may be prefaced by addition of oxidants or other stressors.

Figure 6. Hypothetical presentation of electronic energy utilization between mitochondria having mtDNA haplotypes representing Northern or Sub-Saharan origins in terms of latitude

Under conditions of high energy (ATP) demand, differences in energy utilization exist between mitochondria having different mtDNAs that manifest in differences in electrons used to generate ATP, oxidants and heat as illustrated by the arrows and pie charts. Under conditions of low energy (ATP) demand and excessive caloric intake (*), these profiles shift to decreased use of electronic energy for ATP generation and greater production of oxidants, with those mitochondria having sub-Saharan latitude mtDNAs generating more oxidants compared with those with Northern latitude mtDNAs as indicated by the arrows and pie charts.

Figure 7. General pathway for activation of autophagy

Autophagy is mediated by the interaction or activation of several components including Atg1/Atg13/FIP200 and a ubiquitin-like conjugation pathway involving Atg 3, 5, 7 and 22. This leads to the lipidation of LC3I, converting it to LC3II. LC3II is inserted into autophagosomal membranes and is essential for autophagosomal formation. Autophagosomal expansion is also stimulated by Beclin/VPS34 complex activities. Autophagosomes then fuse with lysosomes and the content of autophagosomes are degraded in the acidic environment of the lysosomes. Nix and BNIP can target to mitochondria and bind to LC3II via a LIR consensus sequence and are thus important for mitophagy. ERK and AMPA activities have been shown to stimulate mitophagy. Mitochondrial fission and fusion proteins such as Drp1, Fis1, Opa1, and Mfn1/2 are also critical in regulating mitophagy.

Figure 8. Regulation of mitophagy in response to mitochondrial membrane depolarization

Mitochondria with normal membrane potential and active presenilin-associated rhomboid-like protein (PARL) prevent mitochondrial PINK1 accumulation. When mitochondria become damaged, mitochondrial membrane potential decreases, PARL is inactivated, and mitochondrial PINK1 is stabilized and accumulates in the affected mitochondria. This recruits Parkin, which promotes the ubiquitination of Drp1 and Mfn, as well as a large number of other mitochondrial proteins. Ubiquitinated proteins can be recognized by p62 which binds LC3 and may help traffic damaged mitochondria to autophagosomes for degradation.

Figure 9. Mitochondrial quality control and xanthine oxidase in the heart

The increase in VO leads to increased usage of ATP, resulting in increased levels of ADP and AMP. ADP and AMP are then degraded to hypoxanthine (HX) via purine catabolism. XO reacts with HX forming superoxide and hydrogen peroxide (H2O2) which damage mitochondria, leading to bioenergetic dysfunction and increased ATP catabolism. Collectively, this causes increased electron leak to form more ROS and decreased ATP production. The increase in mitochondrial ROS leads to increased conversion of xanthine dehydrogenase (XDH) to xanthine oxidase. The enhanced ROS generation from mitochondria and XO cause further damage to the mitochondria and left ventricular dysfunction. Mitochondrially targeted ubiquinone (Mito Q) inhibits the conversion of XDH to XO which may depend on mitochondrial H2O2.