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

Neutrophil-mediated innate immune resistance to bacterial pneumonia is dependent on Tet2 function

We thank Clare A. Edward for technical assistance and Catherine M. Andary for being a second scorer for IF and histopathological analysis. We also thank Elsa N. Bou Ghanem and Manmeet Bhalla for their assistance with the neutrophil killing assays. DMEB was funded through the Canadian Research Chairs program and CIHR. CQ was supported by a CIHR Postdoctoral Fellowship Award. JB was supported by a MIRA fellowship. This study was supported by a project grant from the CIHR (PJT-156291).Peer reviewe

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

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

Mitochondrial-nuclear DNA mismatch matters

Could different nuclear DNA–mitochondrial DNA combinations affect disease severity?</jats:p

Abstract 13283: Mitochondrial DNA Damage and Remodelling of Nucleoid Bodies in Pulmonary Artery Smooth Muscle Cells From Human Patients With Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is an obstructive pulmonary vasculopathy characterized by pulmonary artery smooth muscle cell (PASMC) hyperproliferation. PAH-PASMC have mitochondrial defects, including increased fragmentation, and increased production of reactive oxygen species (ROS). Mitochondrial-derived ROS can cause DNA damage. Mitochondrial DNA (mtDNA) is surrounded by replication proteins which form nucleoid bodies (NB). Hypothesis: We hypothesize that mtDNA damage is increased and NB structure altered in human PAH PASMC. Methods &amp; Results: We profiled mtDNA and NB composition and integrity in control and PAH PASMC (n = 5/group). mtDNA content was assessed via confocal immunofluorescence of discrete extranuclear double-stranded DNA colocalized with mitochondria. Lower mtDNA content was observed in PAH PASMC vs control (1.8±0.2 vs 3.5±0.2 mtDNAs/μm 3 mitochondria, p&lt;0.0001). Reduced mtDNA in PAH vs control was validated by qPCR (0.69±0.07 vs 1.00±0.07 ng mtDNA/μg cellular protein, n=4-5, p=0.0182). siRNA targeting of Mitofusin2 (Mfn2) in normal PASMC to mimic the mitochondrial fragmentation of PAH, reduced total mtDNA compared to control (1.05±0.06 vs 3.5±0.2 mtDNA/μm 3 mitochondria, n=9, p&lt;0.0001), suggesting increased fission reduces mtDNA expression. Amplification of an 8.8kb mtDNA fragment, a measure of mtDNA damage, revealed increased damage in PAH vs control (14.3±3.9 vs 28.7±2.4 pg mtDNA, n=4, p=0.02). PAH PASMC exhibited higher expression of NB proteins vs control (n=5/measurement): TFAM (+96.7±23.3%, p=0.0032), SSBP (+63.4±14.4%, p=0.0023), and POLG (+59.0±24.0%, p=0.0397). These results were confirmed via immunoblotting for TFAM (+161.9±39.3%, n=4-5, p=0.0045) . Immunofluorescence verified increased TFAM in PAH PASMC vs control. Conclusions: We report a paradoxical upregulation of NB proteins despite reduced mtDNA content and elevated mtDNA damage in PAH. We speculate there is an unsuccessful compensatory attempt to restore mtDNA levels by increasing the expression of mtDNA’s replication apparatus in PAH PASMC. </jats:p

A method for assessing mitochondrial bioenergetics in whole white adipose tissues

AbstractObesity 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