Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia.

Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.

Hyperoxia but not AOX expression mitigates pathological cardiac remodeling in a mouse model of inflammatory cardiomyopathy.

Constitutive expression of the chemokine Mcp1 in mouse cardiomyocytes creates a model of inflammatory cardiomyopathy, with death from heart failure at age 7-8 months. A critical pathogenic role has previously been proposed for induced oxidative stress, involving NADPH oxidase activation. To test this idea, we exposed the mice to elevated oxygen levels. Against expectation, this prevented, rather than accelerated, the ultrastructural and functional signs of heart failure. This result suggests that the immune signaling initiated by Mcp1 leads instead to the inhibition of cellular oxygen usage, for which mitochondrial respiration is an obvious target. To address this hypothesis, we combined the Mcp1 model with xenotopic expression of the alternative oxidase (AOX), which provides a sink for electrons blocked from passage to oxygen via respiratory complexes III and IV. Ubiquitous AOX expression provided only a minor delay to cardiac functional deterioration and did not prevent the induction of markers of cardiac and metabolic remodeling considered a hallmark of the model. Moreover, cardiomyocyte-specific AOX expression resulted in exacerbation of Mcp1-induced heart failure, and failed to rescue a second cardiomyopathy model directly involving loss of cIV. Our findings imply that mitochondrial involvement in the pathology of inflammatory cardiomyopathy is multifaceted and complex.

Phenotypic effects of dietary stress in combination with a respiratory chain bypass in mice.

The alternative oxidase (AOX) from Ciona intestinalis was previously shown to be expressible in mice and to cause no physiological disturbance under unstressed conditions. Because AOX is known to become activated under some metabolic stress conditions, resulting in altered energy balance, we studied its effects in mice subjected to dietary stress. Wild-type mice (Mus musculus, strain C57BL/6JOlaHsd) fed a high-fat or ketogenic (high-fat, low-carbohydrate) diet show weight gain with increased fat mass, as well as loss of performance, compared with chow-fed animals. Unexpectedly, AOX-expressing mice fed on these metabolically stressful, fat-rich diets showed almost indistinguishable patterns of weight gain and altered body composition as control animals. Cardiac performance was impaired to a similar extent by ketogenic diet in AOX mice as in nontransgenic littermates. AOX and control animals fed on ketogenic diet both showed wide variance in weight gain. Analysis of the gut microbiome in stool revealed a strong correlation with diet, rather than with genotype. The microbiome of the most and least obese outliers reared on the ketogenic diet showed no consistent trends compared with animals of normal body weight. We conclude that AOX expression in mice does not modify physiological responses to extreme diets.

Alternative Oxidase Attenuates Cigarette Smoke-induced Lung Dysfunction and Tissue Damage.

Cigarette smoke (CS) exposure is the predominant risk factor for the development of chronic obstructive pulmonary disease (COPD) and the third leading cause of death worldwide. We aimed to elucidate whether mitochondrial respiratory inhibition and oxidative stress are triggers in its etiology. In different models of CS exposure, we investigated the effect on lung remodeling and cell signaling of restoring mitochondrial respiratory electron flow using alternative oxidase (AOX), which bypasses the cytochrome segment of the respiratory chain. AOX attenuated CS-induced lung tissue destruction and loss of function in mice exposed chronically to CS for 9 months. It preserved the cell viability of isolated mouse embryonic fibroblasts treated with CS condensate, limited the induction of apoptosis, and decreased the production of reactive oxygen species (ROS). In contrast, the early-phase inflammatory response induced by acute CS exposure of mouse lung, i.e., infiltration by macrophages and neutrophils and adverse signaling, was unaffected. The use of AOX allowed us to obtain novel pathomechanistic insights into CS-induced cell damage, mitochondrial ROS production, and lung remodeling. Our findings implicate mitochondrial respiratory inhibition as a key pathogenic mechanism of CS toxicity in the lung. We propose AOX as a novel tool to study CS-related lung remodeling and potentially to counteract CS-induced ROS production and cell damage.

Expression of the Alternative Oxidase Influences Jun N-Terminal Kinase Signaling and Cell Migration.

Downregulation of Jun N-terminal kinase (JNK) signaling inhibits cell migration in diverse model systems. In Drosophila pupal development, attenuated JNK signaling in the thoracic dorsal epithelium leads to defective midline closure, resulting in cleft thorax. Here we report that concomitant expression of the Ciona intestinalis alternative oxidase (AOX) was able to compensate for JNK pathway downregulation, substantially correcting the cleft thorax phenotype. AOX expression also promoted wound-healing behavior and single-cell migration in immortalized mouse embryonic fibroblasts (iMEFs), counteracting the effect of JNK pathway inhibition. However, AOX was not able to rescue developmental phenotypes resulting from knockdown of the AP-1 transcription factor, the canonical target of JNK, nor its targets and had no effect on AP-1-dependent transcription. The migration of AOX-expressing iMEFs in the wound-healing assay was differentially stimulated by antimycin A, which redirects respiratory electron flow through AOX, altering the balance between mitochondrial ATP and heat production. Since other treatments affecting mitochondrial ATP did not stimulate wound healing, we propose increased mitochondrial heat production as the most likely primary mechanism of action of AOX in promoting cell migration in these various contexts.

Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology.

Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.

Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies.

The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.