Oxygen sensing, mitochondrial biology and experimental therapeutics for pulmonary hypertension and cancer

The homeostatic oxygen sensing system (HOSS) optimizes systemic oxygen delivery. Specialized tissues utilize a conserved mitochondrial sensor, often involving NDUFS2 in complex I of the mitochondrial electron transport chain, as a site of pO2-responsive production of reactive oxygen species (ROS). These ROS are converted to a diffusible signaling molecule, hydrogen peroxide (H2O2), by superoxide dismutase (SOD2). H2O2 exits the mitochondria and regulates ion channels and enzymes, altering plasma membrane potential, intracellular Ca2+ and Ca2+-sensitization and controlling acute, adaptive, responses to hypoxia that involve changes in ventilation, vascular tone and neurotransmitter release. Subversion of this O2-sensing pathway creates a pseudohypoxic state that promotes disease progression in pulmonary arterial hypertension (PAH) and cancer. Pseudohypoxia is a state in which biochemical changes, normally associated with hypoxia, occur despite normal pO2. Epigenetic silencing of SOD2 by DNA methylation alters H2O2 production, activating hypoxia-inducible factor 1α, thereby disrupting mitochondrial metabolism and dynamics, accelerating cell proliferation and inhibiting apoptosis. Other epigenetic mechanisms, including dysregulation of microRNAs (miR), increase pyruvate dehydrogenase kinase and pyruvate kinase muscle isoform 2 expression in both diseases, favoring uncoupled aerobic glycolysis. This Warburg metabolic shift also accelerates cell proliferation and impairs apoptosis. Disordered mitochondrial dynamics, usually increased mitotic fission and impaired fusion, promotes disease progression in PAH and cancer. Epigenetic upregulation of dynamin-related protein 1 (Drp1) and its binding partners, MiD49 and MiD51, contributes to the pathogenesis of PAH and cancer. Finally, dysregulation of intramitochondrial Ca2+, resulting from impaired mitochondrial calcium uniporter complex (MCUC) function, links abnormal mitochondrial metabolism and dynamics. MiR-mediated decreases in MCUC function reduce intramitochondrial Ca2+, promoting Warburg metabolism, whilst increasing cytosolic Ca2+, promoting fission. Epigenetically disordered mitochondrial O2-sensing, metabolism, dynamics, and Ca2+ homeostasis offer new therapeutic targets for PAH and cancer. Promoting glucose oxidation, restoring the fission/fusion balance, and restoring mitochondrial calcium regulation are promising experimental therapeutic strategies

Biventricular Increases in Mitochondrial Fission Mediator (MiD51) and Proglycolytic Pyruvate Kinase (PKM2) Isoform in Experimental Group 2 Pulmonary Hypertension-Novel Mitochondrial Abnormalities

Introduction: Group 2 pulmonary hypertension (PH), defined as a mean pulmonary arterial pressure ≥25 mmHg with elevated pulmonary capillary wedge pressure >15 mmHg, has no approved therapy and patients often die from right ventricular failure (RVF). Alterations in mitochondrial metabolism, notably impaired glucose oxidation, and increased mitochondrial fission, contribute to right ventricle (RV) dysfunction in PH. We hypothesized that the impairment of RV and left ventricular (LV) function in group 2 PH results in part from a proglycolytic isoform switch from pyruvate kinase muscle (PKM) isoform 1 to 2 and from increased mitochondrial fission, due either to upregulation of expression of dynamin-related protein 1 (Drp1) or its binding partners, mitochondrial dynamics protein of 49 or 51 kDa (MiD49 or 51).Methods and Results: Group 2 PH was induced by supra-coronary aortic banding (SAB) in 5-week old male Sprague Dawley rats. Four weeks post SAB, echocardiography showed marked reduction of tricuspid annular plane systolic excursion (2.9 ± 0.1 vs. 4.0 ± 0.1 mm) and pulmonary artery acceleration time (24.3 ± 0.9 vs. 35.4 ± 1.8 ms) in SAB vs. sham rats. Nine weeks post SAB, left and right heart catheterization showed significant biventricular increases in end systolic and diastolic pressure in SAB vs. sham rats (LV: 226 ± 15 vs. 103 ± 5 mmHg, 34 ± 5 vs. 7 ± 1 mmHg; RV: 40 ± 4 vs. 22 ± 1 mmHg, and 4.7 ± 1.5 vs. 0.9 ± 0.5 mmHg, respectively). Picrosirius red staining showed marked biventricular fibrosis in SAB rats. There was increased muscularization of small pulmonary arteries in SAB rats. Confocal microscopy showed biventricular mitochondrial depolarization and fragmentation in SAB vs. sham cardiomyocytes. Transmission electron microscopy confirmed a marked biventricular reduction in mitochondria size in SAB hearts. Immunoblot showed marked biventricular increase in PKM2/PKM1 and MiD51 expression. Mitofusin 2 and mitochondrial pyruvate carrier 1 were increased in SAB LVs.Conclusions: SAB caused group 2 PH. Impaired RV function and RV fibrosis were associated with increases in mitochondrial fission and expression of MiD51 and PKM2. While these changes would be expected to promote increased production of reactive oxygen species and a glycolytic shift in metabolism, further study is required to determine the functional consequences of these newly described mitochondrial abnormalities

Mitochondrial Genetic Background Modulates Bioenergetics and Susceptibility to Acute Cardiac Volume Overload

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial–nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the ‘mitochondrial paradigm’ for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress

A method for assessing mitochondrial bioenergetics in whole white adipose tissues

Obesity is a primary risk factor for numerous metabolic diseases including metabolic syndrome, type II diabetes (T2DM), cardiovascular disease and cancer. Although classically viewed as a storage organ, the field of white adipose tissue biology is expanding to include the consideration of the tissue as an endocrine organ and major contributor to overall metabolism. Given its role in energy production, the mitochondrion has long been a focus of study in metabolic dysfunction and a link between the organelle and white adipose tissue function is likely. Herein, we present a novel method for assessing mitochondrial bioenergetics from whole white adipose tissue. This method requires minimal manipulation of tissue, and eliminates the need for cell isolation and culture. Additionally, this method overcomes some of the limitations to working with transformed and/or isolated primary cells and allows for results to be obtained more expediently. In addition to the novel method, we present a comprehensive statistical analysis of bioenergetic data as well as guidelines for outlier analysis

Transcriptomic Signature of Right Ventricular Failure in Experimental Pulmonary Arterial Hypertension: Deep Sequencing Demonstrates Mitochondrial, Fibrotic, Inflammatory and Angiogenic Abnormalities

Right ventricular failure (RVF) remains the leading cause of death in pulmonary arterial hypertension (PAH). We investigated the transcriptomic signature of RVF in hemodynamically well-phenotyped monocrotaline (MCT)-treated, male, Sprague-Dawley rats with severe PAH and decompensated RVF (increased right ventricular (RV) end diastolic volume (EDV), decreased cardiac output (CO), tricuspid annular plane systolic excursion (TAPSE) and ventricular-arterial decoupling). RNA sequencing revealed 2547 differentially regulated transcripts in MCT-RVF RVs. Multiple enriched gene ontology (GO) terms converged on mitochondria/metabolism, fibrosis, inflammation, and angiogenesis. The mitochondrial transcriptomic pathway is the most affected in RVF, with 413 dysregulated genes. Downregulated genes included TFAM (−0.45-fold), suggesting impaired mitochondrial biogenesis, CYP2E1 (−3.8-fold), a monooxygenase which when downregulated increases oxidative stress, dehydrogenase/reductase 7C (DHRS7C) (−2.8-fold), consistent with excessive autonomic activation, and polypeptide N-acetyl-galactose-aminyl-transferase 13 (GALNT13), a known pulmonary hypertension (PH) biomarker (−2.7-fold). The most up-regulated gene encodes Periostin (POSTN; 4.5-fold), a matricellular protein relevant to fibrosis. Other dysregulated genes relevant to fibrosis include latent-transforming growth factor beta-binding protein 2 (LTBP2), thrombospondin4 (THBS4). We also identified one dysregulated gene relevant to all disordered transcriptomic pathways, ANNEXIN A1. This anti-inflammatory, phospholipid-binding mediator, is a putative target for therapy in RVF-PAH. Comparison of expression profiles in the MCT-RV with published microarray data from the RV of pulmonary artery-banded mice and humans with bone morphogenetic protein receptor type 2 (BMPR2)-mutations PAH reveals substantial conservation of gene dysregulation, which may facilitate clinical translation of preclinical therapeutic and biomarkers studies. Transcriptomics reveals the molecular fingerprint of RVF to be heavily characterized by mitochondrial dysfunction, fibrosis and inflammation

The methyl donor S-adenosylmethionine prevents liver hypoxia and dysregulation of mitochondrial bioenergetic function in a rat model of alcohol-induced fatty liver disease

Background: Mitochondrial dysfunction and bioenergetic stress play an important role in the etiology of alcoholic liver disease. Previous studies from our laboratory show that the primary methyl donor S-Adenosylmethionine (SAM) minimizes alcohol-induced disruptions in several mitochondrial functions in the liver. Herein, we expand on these earlier observations to determine whether the beneficial actions of SAM against alcohol toxicity extend to changes in the responsiveness of mitochondrial respiration to inhibition by nitric oxide (NO), induction of the mitochondrial permeability transition (MPT) pore, and the hypoxic state of the liver. Methods: For this, male Sprague-Dawley rats were pair-fed control and alcohol-containing liquid diets with and without SAM for 5 weeks and liver hypoxia, mitochondrial respiration, MPT pore induction, and NO-dependent control of respiration were examined. Results: Chronic alcohol feeding significantly enhanced liver hypoxia, whereas SAM supplementation attenuated hypoxia in livers of alcohol-fed rats. SAM supplementation prevented alcohol-mediated decreases in mitochondrial state 3 respiration and cytochrome c oxidase activity. Mitochondria isolated from livers of alcohol-fed rats were more sensitive to calcium-mediated MPT pore induction (i.e., mitochondrial swelling) than mitochondria from pair-fed controls, whereas SAM treatment normalized sensitivity for calcium-induced swelling in mitochondria from alcohol-fed rats. Liver mitochondria from alcohol-fed rats showed increased sensitivity to NO-dependent inhibition of respiration compared with pair-fed controls. In contrast, mitochondria isolated from the livers of SAM treated alcohol-fed rats showed no change in the sensitivity to NO-mediated inhibition of respiration. Conclusion: Collectively, these findings indicate that the hepato-protective effects of SAM against alcohol toxicity are mediated, in part, through a mitochondrial mechanism involving preservation of key mitochondrial bioenergetic parameters and the attenuation of hypoxic stress

Mitochondrial – nuclear genetic interaction modulates whole body metabolism, adiposity and gene expression in vivoResearch in context

We hypothesized that changes in the mitochondrial DNA (mtDNA) would significantly influence whole body metabolism, adiposity and gene expression in response to diet. Because it is not feasible to directly test these predictions in humans we used Mitochondrial-Nuclear eXchange mice, which have reciprocally exchanged nuclear and mitochondrial genomes between different Mus musculus strains. Results demonstrate that nuclear-mitochondrial genetic background combination significantly alters metabolic efficiency and body composition. Comparative RNA sequencing analysis in adipose tissues also showed a clear influence of the mtDNA on regulating nuclear gene expression on the same nuclear background (up to a 10-fold change in the number of differentially expressed genes), revealing that neither Mendelian nor mitochondrial genetics unilaterally control gene expression. Additional analyses indicate that nuclear-mitochondrial genome combination modulates gene expression in a manner heretofore not described. These findings provide a new framework for understanding complex genetic disease susceptibility. Keywords: Mitochondrial DNA, Obesity, Mitochondrial – nuclear exchange, Metabolism, Gene expression, Nuclear–mitochondrial interaction, Mitochondrial gene therapy, Adipos

Mitochondria in the pulmonary vasculature in health and disease : oxygen-sensing, metabolism, and dynamics

In lung vascular cells, mitochondria serve a canonical metabolic role, governing energy homeostasis. In addition, mitochondria exist in dynamic networks, which serve noncanonical functions, including regulation of redox signaling, cell cycle, apoptosis, and mitochondrial quality control. Mitochondria in pulmonary artery smooth muscle cells (PASMC) are oxygen sensors and initiate hypoxic pulmonary vasoconstriction. Acquired dysfunction of mitochondrial metabolism and dynamics contribute to a cancer‐like phenotype in pulmonary arterial hypertension (PAH). Acquired mitochondrial abnormalities, such as increased pyruvate dehydrogenase kinase (PDK) and pyruvate kinase muscle isoform 2 (PKM2) expression, which increase uncoupled glycolysis (the Warburg phenomenon), are implicated in PAH. Warburg metabolism sustains energy homeostasis by the inhibition of oxidative metabolism that reduces mitochondrial apoptosis, allowing unchecked cell accumulation. Warburg metabolism is initiated by the induction of a pseudohypoxic state, in which DNA methyltransferase (DNMT)‐mediated changes in redox signaling cause normoxic activation of HIF‐1α and increase PDK expression. Furthermore, mitochondrial division is coordinated with nuclear division through a process called mitotic fission. Increased mitotic fission in PAH, driven by increased fission and reduced fusion favors rapid cell cycle progression and apoptosis resistance. Downregulation of the mitochondrial calcium uniporter complex (MCUC) occurs in PAH and is one potential unifying mechanism linking Warburg metabolism and mitochondrial fission. Mitochondrial metabolic and dynamic disorders combine to promote the hyperproliferative, apoptosis‐resistant, phenotype in PAH PASMC, endothelial cells, and fibroblasts. Understanding the molecular mechanism regulating mitochondrial metabolism and dynamics has permitted identification of new biomarkers, nuclear and CT imaging modalities, and new therapeutic targets for PAH

Electron Leak From the Mitochondrial Electron Transport Chain Complex I at Site IQ Is Crucial for Oxygen Sensing in Rabbit and Human Ductus Arteriosus

Background As partial pressure of oxygen (pO2) rises with the first breath, the ductus arteriosus (DA) constricts, diverting blood flow to the pulmonary circulation. The DA's O2 sensor resides within smooth muscle cells. The DA smooth muscle cells’ mitochondrial electron transport chain (ETC) produces reactive oxygen species (ROS) in proportion to oxygen tension, causing vasoconstriction by regulating redox‐sensitive ion channels and enzymes. To identify which ETC complex contributes most to DA O2 sensing and determine whether ROS mediate O2 sensing independent of metabolism, we used electron leak suppressors, S1QEL (suppressor of site IQ electron leak) and S3QEL (suppressor of site IIIQo electron leak), which decrease ROS production by inhibiting electron leak from quinone sites IQ and IIIQo, respectively. Methods and Results The effects of S1QEL, S3QEL, and ETC inhibitors (rotenone and antimycin A) on DA tone, mitochondrial metabolism, O2‐induced changes in intracellular calcium, and ROS were studied in rabbit DA rings, and human and rabbit DA smooth muscle cells. S1QEL's effects on DA patency were assessed in rabbit kits, using micro computed tomography. In DA rings, S1QEL, but not S3QEL, reversed O2‐induced constriction (P=0.0034) without reducing phenylephrine‐induced constriction. S1QEL did not inhibit mitochondrial metabolism or ETC‐I activity. In human DA smooth muscle cells, S1QEL and rotenone inhibited O2‐induced increases in intracellular calcium (P=0.02 and 0.001, respectively), a surrogate for DA constriction. S1QEL inhibited O2‐induced ROS generation (P=0.02). In vivo, S1QEL prevented O2‐induced DA closure (P<0.0001). Conclusions S1QEL, but not S3QEL, inhibited O2‐induced rises in ROS and DA constriction ex vivo and in vivo. DA O2 sensing relies on pO2‐dependent changes in electron leak at site IQ in ETC‐I, independent of metabolism. S1QEL offers a therapeutic means to maintain DA patency