Increased Drp1-mediated mitochondrial fission promotes proliferation and collagen production by right ventricular fibroblasts in experimental pulmonary arterial hypertension

Introduction: Right ventricular (RV) fibrosis contributes to RV failure in pulmonary arterial hypertension (PAH). The mechanisms underlying RV fibrosis in PAH and the role of RV fibroblasts (RVfib) are unknown. Activation of the mitochondrial fission mediator dynamin-related protein 1 (Drp1) contributes to dysfunction of RV myocytes in PAH through interaction with its binding partner, fission protein 1 (Fis1). However, the role of mitochondrial fission in RVfib and RV fibrosis in PAH is unknown. Objective: We hypothesize that mitochondrial fission is increased in RVfib of rats with monocrotaline (MCT)-induced PAH. We evaluated the contribution of Drp1 and Drp1–Fis1 interaction to RVfib proliferation and collagen production in culture and to RV fibrosis in vivo. Methods: Vimentin (+) RVfib were enzymatically isolated and cultured from the RVs of male Sprague–Dawley rats that received MCT (60 mg/kg) or saline. Mitochondrial morphology, proliferation, collagen production, and expression of Drp1, Drp1 binding partners and mitochondrial fusion mediators were measured. The Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1), P110, a competitive peptide inhibitor of Drp1–Fis1 interaction, and siRNA targeting Drp1 were assessed. Subsequently, prevention and regression studies tested the antifibrotic effects of P110 (0.5 mg/kg) in vivo. At week 4 post MCT, echocardiography and right heart catheterization were performed. The RV was stained for collagen. Results: Mitochondrial fragmentation, proliferation rates and collagen production were increased in MCT-RVfib versus control-RVfib. MCT-RVfib had increased expression of activated Drp1 protein and a trend to decreased mitofusin-2 expression. Mdivi-1 and P110 inhibited mitochondrial fission, proliferation and collagen III expression in MCT-RVfib. However, P110 was only effective at high doses (1 mM). siDrp1 also reduced fission in MCT-RVfib. Despite promising results in cell therapy, in vivo therapy with P110 failed to prevent or regress RV fibrosis in MCT rats, perhaps due to failure to achieve adequate P110 levels or to the greater importance of interaction of Drp1 with other binding partners. Conclusion: PAH RVfib have increased Drp1-mediated mitochondrial fission. Inhibiting Drp1 prevents mitochondrial fission and reduces RVfib proliferation and collagen production. This is the first description of disordered mitochondrial dynamics in RVfib and suggests that Drp1 is a potential new antifibrotic target

Ischemia-induced Drp1 and Fis1-mediated mitochondrial fission and right ventricular dysfunction in pulmonary hypertension

Right ventricular (RV) function determines prognosis in pulmonary arterial hypertension (PAH). We hypothesize that ischemia causes RV dysfunction in PAH by triggering dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. RV function was compared in control rats (n = 50) versus rats with monocrotaline-induced PAH (MCT-PAH; n = 60) both in vivo (echocardiography) and ex vivo (RV Langendorff). Mitochondrial membrane potential and morphology and RV function were assessed before or after 2 cycles of ischemia-reperfusion injury challenge (RV-IR). The effects of Mdivi-1 (25 μM), a Drp1 GTPase inhibitor, and P110 (1 μM), a peptide inhibitor of Drp1-Fis1 interaction, were studied. We found that MCT caused RV hypertrophy, RV vascular rarefaction, and RV dysfunction. Prior to IR, the mitochondria in MCT-PAH RV were depolarized and swollen with increased Drp1 content and reduced aconitase activity. RV-IR increased RV end diastolic pressure (RVEDP) and mitochondrial Drp1 expression in both control and MCT-PAH RVs. IR depolarized mitochondria in control RV but did not exacerbate the basally depolarized MCT-PAH RV mitochondria. During RV IR mdivi-1 and P110 reduced Drp1 translocation to mitochondria, improved mitochondrial structure and function, and reduced RVEDP. In conclusion, RV ischemia occurs in PAH and causes Drp1-Fis1-mediated fission leading to diastolic dysfunction. Inhibition of mitochondrial fission preserves RV function in RV-IR

MicroRNA-138 and microRNA-25 down-regulate mitochondrial calcium uniporter, causing the pulmonary arterial hypertension cancer phenotype

Rationale: Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by excessive pulmonary artery smooth muscle cell (PASMC) proliferation, migration, and apoptosis resistance. This cancer-like phenotype is promoted by increased cytosolic calcium ([Ca2+]cyto), aerobic glycolysis, and mitochondrial fission. Objectives: To determine how changes in mitochondrial calcium uniporter (MCU) complex (MCUC) function influence mitochondrial dynamics and contribute to PAH’s cancer-like phenotype. Methods: PASMCs were isolated from patients with PAH and healthy control subjects and assessed for expression of MCUC subunits. Manipulation of the pore-forming subunit, MCU, in PASMCs was achieved through small interfering RNA knockdown or MCU plasmid-mediated up-regulation, as well as through modulation of the upstream microRNAs (miRs) miR-138 and miR-25. In vivo, nebulized anti-miRs were administered to rats with monocrotaline-induced PAH. Measurements and Main Results: Impaired MCUC function, resulting from down-regulation of MCU and up-regulation of an inhibitory subunit, mitochondrial calcium uptake protein 1, is central to PAH’s pathogenesis. MCUC dysfunction decreases intramitochondrial calcium ([Ca2+]mito), inhibiting pyruvate dehydrogenase activity and glucose oxidation, while increasing [Ca2+]cyto, promoting proliferation, migration, and fission. In PAH PASMCs, increasing MCU decreases cell migration, proliferation, and apoptosis resistance by lowering [Ca2+]cyto, raising [Ca2+]mito, and inhibiting fission. In normal PASMCs, MCUC inhibition recapitulates the PAH phenotype. In PAH, elevated miRs (notably miR-138) down-regulate MCU directly and also by decreasing MCU’s transcriptional regulator cAMP response element–binding protein 1. Nebulized anti-miRs against miR-25 and miR-138 restore MCU expression, reduce cell proliferation, and regress established PAH in the monocrotaline model. Conclusions: These results highlight miR-mediated MCUC dysfunction as a unifying mechanism in PAH that can be therapeutically targeted

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

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

The Herpes Simplex Virus pUL16 and pUL21 Proteins Prevent Capsids from Docking at Nuclear Pore Complexes.

After entry into cells, herpes simplex virus (HSV) nucleocapsids dock at nuclear pore complexes (NPCs) through which viral genomes are released into the nucleoplasm where viral gene expression, genome replication, and early steps in virion assembly take place. After their assembly, nucleocapsids are translocated to the cytoplasm for final virion maturation. Nascent cytoplasmic nucleocapsids are prevented from binding to NPCs and delivering their genomes to the nucleus from which they emerged, but how this is accomplished is not understood. Here we report that HSV pUL16 and pUL21 deletion mutants accumulate empty capsids at the cytoplasmic face of NPCs late in infection. Additionally, prior expression of pUL16 and pUL21 prevented incoming nucleocapsids from docking at NPCs, delivering their genomes to the nucleus and initiating viral gene expression. Both pUL16 and pUL21 localized to the nuclear envelope, placing them in an appropriate location to interfere with nucleocapsid/NPC interactions

Supra-coronary aortic banding improves right ventricular function in experimental pulmonary arterial hypertension in rats by increasing systolic right coronary artery perfusion

Aim -- Pulmonary arterial hypertension (PAH) results in right ventricular (RV) dysfunction owing, in part, to RV ischemia. The relative contribution of RV microvascular rarefaction vs reduced right coronary artery perfusion pressure (RCA-PP) to RV ischemia remains unknown. We hypothesize that increasing RCA-PP improves RV function in PAH by increasing RV systolic perfusion. Methods -- Supra-coronary aortic banding (SAB) or sham surgery was performed on male Sprague-Dawley rats. Seven to ten days later, rats received either monocrotaline (MCT; 60 mg/kg) or saline. After 1 month, echocardiography, cardiac catheterization, 99mTc-sestamibi single-photon emission computed tomography (SPECT) and microsphere infusion studies were performed. The RV was harvested for measurement of hypertrophy (RVH), fibrosis and immunoblotting, and the lung was harvested for pulmonary artery (PA) histology. Results -- Supra-coronary aortic banding increased systolic pressures in proximal aorta and systolic RCA-PP in SAB + MCT vs MCT rats (114 ± 12 vs 5 ± 9 mm Hg), without altering diastolic RCA-PP. SAB + MCT rats had improved RV function vs MCT rats, evident from their significantly increased cardiac output (CO), RV free wall (RVFW) thickening, tricuspid annular plane systolic excursion (TAPSE) and RV-PA coupling indices. RV-PA coupling indices and CO correlated directly with systolic RCA-PP. RV perfusion was increased in SAB + MCT vs MCT rats and correlated well with CO; whereas microvascular rarefaction was unaltered. SAB + MCT rats had less RVH and fibrosis and lower PA pressures vs MCT rats. SAB + MCT rats had significantly lower RV pyruvate kinase muscle isoform 2/1 ratios than MCT rats, consistent with restoration of oxidative metabolism. Conclusion -- A SAB-induced increase in systolic RCA-PP improves RV perfusion and function in MCT rats. Maintaining systolic RCA perfusion can preserve RV function in PAH

Inhibiting pyruvate kinase muscle isoform 2 regresses group 2 pulmonary hypertension induced by supra-coronary aortic banding

Introduction: Group 2 pulmonary hypertension (PH) has no approved PH-targeted therapy. Metabolic remodelling, specifically a biventricular increase in pyruvate kinase muscle (PKM) isozyme 2 to 1 ratio, occurs in rats with group 2 PH induced by supra-coronary aortic banding (SAB). We hypothesize that increased PKM2/PKM1 is maladaptive and inhibiting PKM2 would improve right ventricular (RV) function. Methods: Male, Sprague-Dawley SAB rats were confirmed to have PH by echocardiography and then randomized to treatment with a PKM2 inhibitor (intraperitoneal shikonin, 2 mg/kg/day) versus 5% DMSO (n = 5/group) or small interfering RNA-targeting PKM2 (siPKM2) versus siRNA controls (n = 7/group) by airway nebulization. Results: Shikonin-treated SAB rats had milder PH (PAAT 32.1 ± 1.3 vs 22.1 ± 1.2 ms, P =.0009) and lower RV systolic pressure (RVSP) (31.5 ± 0.9 vs 55.7 ± 1.9 mm Hg, P <.0001) versus DMSO-SAB rats. siPKM2 nebulization reduced PKM2 expression in the RV, increased PAAT (31.7 ± 0.7 vs 28.0 ± 1.3 ms, P =.025), lowered RVSP (30.6 ± 2.6 vs 42.0 ± 4.0 mm Hg, P =.032) and reduced diastolic RVFW thickness (0.69 ± 0.04 vs 0.85 ± 0.06 mm, P =.046). Both shikonin and siPKM2 regressed PH-induced medial hypertrophy of small pulmonary arteries. Conclusion: Increases in PKM2/PKM1 in the RV contribute to RV dysfunction in group 2 PH. Chemical or molecular inhibition of PKM2 restores the normal PKM2/PKM1 ratio, reduces PH, RVSP and RVH and regresses adverse PA remodelling. PKM2 merits consideration as a therapeutic cardiac target for group 2 PH

Endogenously Generated Omega‐3 Fatty Acids Attenuate Vascular Inflammation and Neointimal Hyperplasia by Interaction With Free Fatty Acid Receptor 4 in Mice

Background: Omega‐3 polyunsaturated fatty acids (ω3 PUFAs) suppress inflammation through activation of free fatty acid receptor 4 (FFAR4), but this pathway has not been explored in the context of cardiovascular disease. We aimed to elucidate the involvement of FFAR4 activation by ω3 PUFAs in the process of vascular inflammation and neointimal hyperplasia in mice. Methods and Results: We used mice with disruption of FFAR4 (Ffar4−/−), along with a strain that synthesizes high levels of ω3 PUFAs (fat‐1) and a group of crossed mice (Ffar4−/−/fat‐1), to elucidate the role of FFAR4 in vascular dysfunction using acute and chronic thrombosis/vascular remodeling models. The presence of FFAR4 in vascular‐associated cells including perivascular adipocytes and macrophages, but not platelets, was demonstrated. ω3 PUFAs endogenously generated in fat‐1 mice (n=9), but not in compound Ffar4−/−/fat‐1 mice (n=9), attenuated femoral arterial thrombosis induced by FeCl3. Neointimal hyperplasia and vascular inflammation in the common carotid artery were significantly curtailed 4 weeks after FeCl3 injury in fat‐1 mice (n=6). This included greater luminal diameter and enhanced blood flow, reduced intima:media ratio, and diminished macrophage infiltration in the vasculature and perivascular adipose tissue compared with control mice. These effects were attenuated in the Ffar4−/−/fat‐1 mice. Conclusions: These results indicate that ω3 PUFAs mitigate vascular inflammation, arterial thrombus formation, and neointimal hyperplasia by interaction with FFAR4 in mice. Moreover, the ω3 PUFA–FFAR4 pathway decreases inflammatory responses with dampened macrophage transmigration and infiltration