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.

Dynamin-related protein 1 is a critical regulator of mitochondrial calcium homeostasis during myocardial ischemia/reperfusion injury.

Dynamin-related protein 1 (Drp1) is a cytosolic GTPase protein that when activated translocates to the mitochondria, meditating mitochondrial fission and increasing reactive oxygen species (ROS) in cardiomyocytes. Drp1 has shown promise as a therapeutic target for reducing cardiac ischemia/reperfusion (IR) injury; however, the lack of specificity of some small molecule Drp1 inhibitors and the reliance on the use of Drp1 haploinsufficient hearts from older mice have left the role of Drp1 in IR in question. Here, we address these concerns using two approaches, using: (a) short-term (3 weeks), conditional, cardiomyocyte-specific, Drp1 knockout (KO) and (b) a novel, highly specific Drp1 GTPase inhibitor, Drpitor1a. Short-term Drp1 KO mice exhibited preserved exercise capacity and cardiac contractility, and their isolated cardiac mitochondria demonstrated increased mitochondrial complex 1 activity, respiratory coupling, and calcium retention capacity compared to controls. When exposed to IR injury in a Langendorff perfusion system, Drp1 KO hearts had preserved contractility, decreased reactive oxygen species (ROS), enhanced mitochondrial calcium capacity, and increased resistance to mitochondrial permeability transition pore (MPTP) opening. Pharmacological inhibition of Drp1 with Drpitor1a following ischemia, but before reperfusion, was as protective as Drp1 KO for cardiac function and mitochondrial calcium homeostasis. In contrast to the benefits of short-term Drp1 inhibition, prolonged Drp1 ablation (6 weeks) resulted in cardiomyopathy. Drp1 KO hearts were also associated with decreased ryanodine receptor 2 (RyR2) protein expression and pharmacological inhibition of the RyR2 receptor decreased ROS in post-IR hearts suggesting that changes in RyR2 may have a role in Drp1 KO mediated cardioprotection. We conclude that Drp1-mediated increases in myocardial ROS production and impairment of mitochondrial calcium handling are key mechanisms of IR injury. Short-term inhibition of Drp1 is a promising strategy to limit early myocardial IR injury which is relevant for the therapy of acute myocardial infarction, cardiac arrest, and heart transplantation.

Macrophage-NLRP3 Activation Promotes Right Ventricle Failure in Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) often results in death from right ventricular failure (RVF). NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3)-macrophage activation may promote RVF in PAH. Objectives: Evaluating the contribution of the NLRP3 inflammasome in RV macrophages to PAH RVF. Methods: Rats with decompensated RV hypertrophy (monocrotaline [MCT] and Sugen-5416 hypoxia [SuHx]) were compared with compensated RV hypertrophy rats (pulmonary artery banding). Echocardiography and right heart catheterization were performed. Macrophages, atrial natriuretic peptides, and fibrosis were evaluated by microscopy or flow cytometry. NLRP3 inflammasome activation and cardiotoxicity were confirmed by immunoblot and in vitro strategies. MCT rats were treated with SC-144 (a GP130 antagonist) or MCC950 (an NLRP3 inhibitor). Macrophage-NLRP3 activity was evaluated in patients with PAH RVF. Measurements and Main Results: Macrophages, fibrosis, and atrial natriuretic peptides were increased in MCT and SuHx RVs but not in left ventricles or pulmonary artery banding rats. Although MCT RV macrophages were inflammatory, lung macrophages were antiinflammatory. CCR2+ macrophages (monocyte-derived) were increased in MCT and SuHx RVs and highly expressed NLRP3. The macrophage-NLRP3 pathway was upregulated in patients with PAH with decompensated RVs. Cultured MCT monocytes showed NLRP3 activation, and in coculture experiments resulted in cardiomyocyte mitochondrial damage, which MCC950 prevented. In vivo, MCC950 reduced NLRP3 activation and regressed pulmonary vascular disease and RVF. SC-144 reduced RV macrophages and NLRP3 content, prevented STAT3 (signal transducer and activator of transcription 3) activation, and improved RV function without regressing pulmonary vascular disease. Conclusions: NLRP3-macrophage activation occurs in the decompensated RV in preclinical PAH models and patients with PAH. Inhibiting GP130 or NLRP3 signaling improves RV function. The concept that PAH RVF results from RV inflammation rather than solely from elevated RV afterload suggests a new therapeutic paradigm.

PINK1-induced phosphorylation of mitofusin 2 at serine 442 causes its proteasomal degradation and promotes cell proliferation in lung cancer and pulmonary arterial hypertension.

Impaired mitochondrial fusion, due in part to decreased mitofusin 2 (Mfn2) expression, contributes to unrestricted cell proliferation and apoptosis-resistance in hyperproliferative diseases like pulmonary arterial hypertension (PAH) and non-small cell lung cancer (NSCLC). We hypothesized that Mfn2 levels are reduced due to increased proteasomal degradation of Mfn2 triggered by its phosphorylation at serine 442 (S442) and investigated the potential kinase mediators. Mfn2 expression was decreased and Mfn2 S442 phosphorylation was increased in pulmonary artery smooth muscle cells from PAH patients and in NSCLC cells. Mfn2 phosphorylation was mediated by PINK1 and protein kinase A (PKA), although only PINK1 expression was increased in these diseases. We designed a S442 phosphorylation deficient Mfn2 construct (PD-Mfn2) and a S442 constitutively phosphorylated Mfn2 construct (CP-Mfn2). The effects of these modified Mfn2 constructs on Mfn2 expression and biological function were compared with those of the wildtype Mfn2 construct (WT-Mfn2). WT-Mfn2 increased Mfn2 expression and mitochondrial fusion in both PAH and NSCLC cells resulting in increased apoptosis and decreased cell proliferation. Compared to WT-Mfn2, PD-Mfn2 caused greater Mfn2 expression, suppression of proliferation, apoptosis induction, and cell cycle arrest. Conversely, CP-Mfn2 caused only a small increase in Mfn2 expression and did not restore mitochondrial fusion, inhibit cell proliferation, or induce apoptosis. Silencing PINK1 or PKA, or proteasome blockade using MG132, increased Mfn2 expression, enhanced mitochondrial fusion and induced apoptosis. In a xenotransplantation NSCLC model, PD-Mfn2 gene therapy caused greater tumor regression than did therapy with WT-Mfn2. Mfn2 deficiency in PAH and NSCLC reflects proteasomal degradation triggered by Mfn2-S442 phosphorylation by PINK1 and/or PKA. Inhibiting Mfn2 phosphorylation has potential therapeutic benefit in PAH and lung cancer.

Epigenetic Metabolic Reprogramming of Right Ventricular Fibroblasts in Pulmonary Arterial Hypertension: A Pyruvate Dehydrogenase Kinase-Dependent Shift in Mitochondrial Metabolism Promotes Right Ventricular Fibrosis.

RATIONALE: Right ventricular (RV) fibrosis in pulmonary arterial hypertension contributes to RV failure. While RV fibrosis reflects changes in the function of resident RV fibroblasts (RVfib), these cells are understudied. OBJECTIVE: Examine the role of mitochondrial metabolism of RVfib in RV fibrosis in human and experimental pulmonary arterial hypertension. METHODS AND RESULTS: Male Sprague-Dawley rats received monocrotaline (MCT; 60 mg/kg) or saline. Drinking water containing no supplement or the PDK (pyruvate dehydrogenase kinase) inhibitor dichloroacetate was started 7 days post-MCT. At week 4, treadmill testing, echocardiography, and right heart catheterization were performed. The effects of PDK activation on mitochondrial dynamics and metabolism, RVfib proliferation, and collagen production were studied in RVfib in cell culture. Epigenetic mechanisms for persistence of the profibrotic RVfib phenotype in culture were evaluated. PDK expression was also studied in the RVfib of patients with decompensated RV failure (n=11) versus control (n=7). MCT rats developed pulmonary arterial hypertension, RV fibrosis, and RV failure. MCT-RVfib (but not left ventricular fibroblasts) displayed excess mitochondrial fission and had increased expression of PDK isoforms 1 and 3 that persisted for >5 passages in culture. PDK-mediated decreases in pyruvate dehydrogenase activity and oxygen consumption rate were reversed by dichloroacetate (in RVfib and in vivo) or siRNA targeting PDK 1 and 3 (in RVfib). These interventions restored mitochondrial superoxide and hydrogen peroxide production and inactivated HIF (hypoxia-inducible factor)-1α, which was pathologically activated in normoxic MCT-RVfib. Redox-mediated HIF-1α inactivation also decreased the expression of TGF-ß1 (transforming growth factor-beta-1) and CTGF (connective tissue growth factor), reduced fibroblast proliferation, and decreased collagen production. HIF-1α activation in MCT-RVfib reflected increased DNMT (DNA methyltransferase) 1 expression, which was associated with a decrease in its regulatory microRNA, miR-148b-3p. In MCT rats, dichloroacetate, at therapeutic levels in the RV, reduced phospho-pyruvate dehydrogenase expression, RV fibrosis, and hypertrophy and improved RV function. In patients with pulmonary arterial hypertension and RV failure, RVfib had increased PDK1 expression. CONCLUSIONS: MCT-RVfib manifest a DNMT1-HIF-1α-PDK-mediated, chamber-specific, metabolic memory that promotes collagen production and RV fibrosis. This epigenetic mitochondrial-metabolic pathway is a potential antifibrotic therapeutic target.

The molecular mechanisms of oxygen-sensing in human ductus arteriosus smooth muscle cells: A comprehensive transcriptome profile reveals a central role for mitochondria.

The ductus arteriosus (DA) connects the fetal pulmonary artery and aorta, diverting placentally oxygenated blood from the developing lungs to the systemic circulation. The DA constricts in response to increases in oxygen (O2) with the first breaths, resulting in functional DA closure, with anatomic closure occurring within the first days of life. Failure of DA closure results in persistent patent ductus arteriosus (PDA), a common complication of extreme preterm birth. The DA's response to O2, though modulated by the endothelium, is intrinsic to the DA smooth muscle cells (DASMC). DA constriction is mediated by mitochondrial-derived reactive oxygen species, which increase in proportion to arterial partial pressure of oxygen (PaO2). The resulting redox changes inhibit voltage-gated potassium channels (Kv) leading to cell depolarization, calcium influx and DASMC constriction. To date, there has not been an unbiased assessment of the human DA O2-sensors using transcriptomics, nor are there known molecular mechanisms which characterize DA closure. DASMCs were isolated from DAs obtained from 10 term infants at the time of congenital heart surgery. Cells were purified by flow cytometry, negatively sorting using CD90 and CD31 to eliminate fibroblasts or endothelial cells, respectively. The purity of the DASMC population was confirmed by positive staining for α-smooth muscle actin, smoothelin B and caldesmon. Cells were grown for 96 h in hypoxia (2.5% O2) or normoxia (19% O2) and confocal imaging with Cal-520 was used to determine oxygen responsiveness. An oxygen-induced increase in intracellular calcium of 18.1% ± 4.4% and SMC constriction (-27% ± 1.5% shortening) occurred in all cell lines within five minutes. RNA sequencing of the cells grown in hypoxia and normoxia revealed significant regulation of 1344 genes (corrected p < 0.05). We examined these genes using Gene Ontology (GO). This unbiased assessment of altered gene expression indicated significant enrichment of the following GOterms: mitochondria, cellular respiration and transcription. The top regulated biologic process was generation of precursor metabolites and energy. The top regulated cellular component was mitochondrial matrix. The top regulated molecular function was transcription coactivator activity. Multiple members of the NADH-ubiquinone oxidoreductase (NDUF) family are upregulated in human DASMC (hDASMC) following normoxia. Several of our differentially regulated transcripts are encoded by genes that have been associated with genetic syndromes that have an increased incidence of PDA (Crebb binding protein and Histone Acetyltransferase P300). This first examination of the effects of O2 on human DA transcriptomics supports a putative role for mitochondria as oxygen sensors.

Identification of Long Noncoding RNA H19 as a New Biomarker and Therapeutic Target in Right Ventricular Failure in Pulmonary Arterial Hypertension.

BACKGROUND: Right ventricular (RV) function is the major determinant for both functional capacity and survival in patients with pulmonary arterial hypertension (PAH). Despite the recognized clinical importance of preserving RV function, the subcellular mechanisms that govern the transition from a compensated to a decompensated state remain poorly understood and as a consequence there are no clinically established treatments for RV failure and a paucity of clinically useful biomarkers. Accumulating evidence indicates that long noncoding RNAs are powerful regulators of cardiac development and disease. Nonetheless, their implication in adverse RV remodeling in PAH is unknown. METHODS: Expression of the long noncoding RNA H19 was assessed by quantitative PCR in plasma and RV from patients categorized as control RV, compensated RV or decompensated RV based on clinical history and cardiac index. The impact of H19 suppression using GapmeR was explored in 2 rat models mimicking RV failure, namely the monocrotaline and pulmonary artery banding. Echocardiographic, hemodynamic, histological, and biochemical analyses were conducted. In vitro gain- and loss-of-function experiments were performed in rat cardiomyocytes. RESULTS: We demonstrated that H19 is upregulated in decompensated RV from PAH patients and correlates with RV hypertrophy and fibrosis. Similar findings were observed in monocrotaline and pulmonary artery banding rats. We found that silencing H19 limits pathological RV hypertrophy, fibrosis and capillary rarefaction, thus preserving RV function in monocrotaline and pulmonary artery banding rats without affecting pulmonary vascular remodeling. This cardioprotective effect was accompanied by E2F transcription factor 1-mediated upregulation of enhancer of zeste homolog 2. In vitro, knockdown of H19 suppressed cardiomyocyte hypertrophy induced by phenylephrine, while its overexpression has the opposite effect. Finally, we demonstrated that circulating H19 levels in plasma discriminate PAH patients from controls, correlate with RV function and predict long-term survival in 2 independent idiopathic PAH cohorts. Moreover, H19 levels delineate subgroups of patients with differentiated prognosis when combined with the NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels or the risk score proposed by both REVEAL (Registry to Evaluate Early and Long-Term PAH Disease Management) and the 2015 European Pulmonary Hypertension Guidelines. CONCLUSIONS: Our findings identify H19 as a new therapeutic target to impede the development of maladaptive RV remodeling and a promising biomarker of PAH severity and prognosis.

Novel Mutations and Decreased Expression of the Epigenetic Regulator TET2 in Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy. Hereditary cases are associated with germline mutations in BMPR2 and 16 other genes; however, these mutations occur in <25% of patients with idiopathic PAH and are rare in PAH associated with connective tissue diseases. Preclinical studies suggest epigenetic dysregulation, including altered DNA methylation, promotes PAH. Somatic mutations of Tet-methylcytosine-dioxygenase-2 (TET2), a key enzyme in DNA demethylation, occur in cardiovascular disease and are associated with clonal hematopoiesis, inflammation, and adverse vascular remodeling. The role of TET2 in PAH is unknown. METHODS: To test for a role of TET2, we used a cohort of 2572 cases from the PAH Biobank. Within this cohort, gene-specific rare variant association tests were performed using 1832 unrelated European patients with PAH and 7509 non-Finnish European subjects from the Genome Aggregation Database (gnomAD) as control subjects. In an independent cohort of 140 patients, we quantified TET2 expression in peripheral blood mononuclear cells. To assess causality, we investigated hemodynamic and histological evidence of PAH in hematopoietic Tet2-knockout mice. RESULTS: We observed an increased burden of rare, predicted deleterious germline variants in TET2 in PAH patients of European ancestry (9/1832) compared with control subjects (6/7509; relative risk=6; P=0.00067). Assessing the whole cohort, 0.39% of patients (10/2572) had 12 TET2 mutations (75% predicted germline and 25% somatic). These patients had no mutations in other PAH-related genes. Patients with TET2 mutations were older (71±7 years versus 48±19 years; P<0.0001), were more unresponsive to vasodilator challenge (0/7 versus 140/1055 [13.2%]), had lower pulmonary vascular resistance (5.2±3.1 versus 10.5±7.0 Wood units; P=0.02), and had increased inflammation (including elevation of interleukin-1ß). Circulating TET2 expression did not correlate with age and was decreased in >86% of PAH patients. Tet2-knockout mice spontaneously developed PAH, adverse pulmonary vascular remodeling, and inflammation, with elevated levels of cytokines, including interleukin-1ß. Long-term therapy with an antibody targeting interleukin-1ß blockade resulted in regression of PAH. CONCLUSIONS: PAH is the first human disease related to potential TET2 germline mutations. Inherited and acquired abnormalities of TET2 occur in 0.39% of PAH cases. Decreased TET2 expression is ubiquitous and has potential as a PAH biomarker.

An epigenetic increase in mitochondrial fission by MiD49 and MiD51 regulates the cell cycle in cancer: Diagnostic and therapeutic implications.

Excessive proliferation and apoptosis-resistance are hallmarks of cancer. Increased dynamin-related protein 1 (Drp1)-mediated mitochondrial fission is one of the mediators of this phenotype. Mitochondrial fission that accompanies the nuclear division is called mitotic fission and occurs when activated Drp1 binds partner proteins on the outer mitochondrial membrane. We examine the role of Drp1-binding partners, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), as drivers of cell proliferation and apoptosis-resistance in non-small cell lung cancer (NSCLC) and invasive breast carcinoma (IBC). We also evaluate whether inhibiting MiDs can be therapeutically exploited to regress cancer. We show that MiD levels are pathologically elevated in NSCLC and IBC by an epigenetic mechanism (decreased microRNA-34a-3p expression). MiDs silencing causes cell cycle arrest through (a) increased expression of cell cycle inhibitors, p27Kip1 and p21Waf1 , (b) inhibition of Drp1, and (c) inhibition of the Akt-mTOR-p70S6K pathway. Silencing MiDs leads to mitochondrial fusion, cell cycle arrest, increased apoptosis, and tumor regression in a xenotransplant NSCLC model. There are positive correlations between MiD expression and tumor size and grade in breast cancer patients and inverse correlations with survival in NSCLC patients. The microRNA-34a-3p-MiDs axis is important to cancer pathogenesis and constitutes a new therapeutic target.

Identification of novel dynamin-related protein 1 (Drp1) GTPase inhibitors: Therapeutic potential of Drpitor1 and Drpitor1a in cancer and cardiac ischemia-reperfusion injury.

Mitochondrial fission is important in physiological processes, including coordination of mitochondrial and nuclear division during mitosis, and pathologic processes, such as the production of reactive oxygen species (ROS) during cardiac ischemia-reperfusion injury (IR). Mitochondrial fission is mainly mediated by dynamin-related protein 1 (Drp1), a large GTPase. The GTPase activity of Drp1 is essential for its fissogenic activity. Therefore, we aimed to identify Drp1 inhibitors and evaluate their anti-neoplastic and cardioprotective properties in five cancer cell lines (A549, SK-MES-1, SK-LU-1, SW 900, and MCF7) and an experimental cardiac IR injury model. Virtual screening of a chemical library revealed 17 compounds with high predicted affinity to the GTPase domain of Drp1. In silico screening identified an ellipticine compound, Drpitor1, as a putative, potent Drp1 inhibitor. We also synthesized a congener of Drpitor1 to remove the methoxymethyl group and reduce hydrolytic lability (Drpitor1a). Drpitor1 and Drpitor1a inhibited the GTPase activity of Drp1 without inhibiting the GTPase of dynamin 1. Drpitor1 and Drpitor1a have greater potency than the current standard Drp1 GTPase inhibitor, mdivi-1, (IC50 for mitochondrial fragmentation are 0.09, 0.06, and 10 µM, respectively). Both Drpitors reduced proliferation and induced apoptosis in cancer cells. Drpitor1a suppressed lung cancer tumor growth in a mouse xenograft model. Drpitor1a also inhibited mitochondrial ROS production, prevented mitochondrial fission, and improved right ventricular diastolic dysfunction during IR injury. In conclusion, Drpitors are useful tools for understanding mitochondrial dynamics and have therapeutic potential in treating cancer and cardiac IR injury.

Ndufs2, a Core Subunit of Mitochondrial Complex I, Is Essential for Acute Oxygen-Sensing and Hypoxic Pulmonary Vasoconstriction.

RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) optimizes systemic oxygen delivery by matching ventilation to perfusion. HPV is intrinsic to pulmonary artery smooth muscle cells (PASMCs). Hypoxia dilates systemic arteries, including renal arteries. Hypoxia is sensed by changes in mitochondrial-derived reactive oxygen species, notably hydrogen peroxide (H2O2) ([H2O2]mito). Decreases in [H2O2]mito elevate pulmonary vascular tone by increasing intracellular calcium ([Ca2+]i) through reduction-oxidation regulation of ion channels. Although HPV is mimicked by the Complex I inhibitor, rotenone, the molecular identity of the O2 sensor is unknown. OBJECTIVE: To determine the role of Ndufs2 (NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 2), Complex I's rotenone binding site, in pulmonary vascular oxygen-sensing. METHODS AND RESULTS: Mitochondria-conditioned media from pulmonary and renal mitochondria isolated from normoxic and chronically hypoxic rats were infused into an isolated lung bioassay. Mitochondria-conditioned media from normoxic lungs contained more H2O2 than mitochondria-conditioned media from chronic hypoxic lungs or kidneys and uniquely attenuated HPV via a catalase-dependent mechanism. In PASMC, acute hypoxia decreased H2O2 within 112±7 seconds, followed, within 205±34 seconds, by increased intracellular calcium concentration, [Ca2+]i. Hypoxia had no effects on [Ca2+]i in renal artery SMC. Hypoxia decreases both cytosolic and mitochondrial H2O2 in PASMC while increasing cytosolic H2O2 in renal artery SMC. Ndufs2 expression was greater in PASMC versus renal artery SMC. Lung Ndufs2 cysteine residues became reduced during acute hypoxia and both hypoxia and reducing agents caused functional inhibition of Complex I. In PASMC, siNdufs2 (cells/tissue treated with Ndufs2 siRNA) decreased normoxic H2O2, prevented hypoxic increases in [Ca2+]i, and mimicked aspects of chronic hypoxia, including decreasing Complex I activity, elevating the nicotinamide adenine dinucleotide (NADH/NAD+) ratio and decreasing expression of the O2-sensitive ion channel, Kv1.5. Knocking down another Fe-S center within Complex I (Ndufs1, NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 1) or other mitochondrial subunits proposed as putative oxygen sensors (Complex III's Rieske Fe-S center and COX4i2 [cytochrome c oxidase subunit 4 isoform 2] in Complex IV) had no effect on hypoxic increases in [Ca2+]i. In vivo, siNdufs2 significantly decreased hypoxia- and rotenone-induced constriction while enhancing phenylephrine-induced constriction. CONCLUSIONS: Ndufs2 is essential for oxygen-sensing and HPV.

Metabolic Syndrome Exacerbates Pulmonary Hypertension due to Left Heart Disease.

RATIONALE: Pulmonary hypertension (PH) due to left heart disease (LHD), or group 2 PH, is the most prevalent form of PH worldwide. PH due to LHD is often associated with metabolic syndrome (MetS). In 12% to 13% of cases, patients with PH due to LHD display vascular remodeling of pulmonary arteries (PAs) associated with poor prognosis. Unfortunately, the underlying mechanisms remain unknown; PH-targeted therapies for this group are nonexistent, and the development of a new preclinical model is crucial. Among the numerous pathways dysregulated in MetS, inflammation plays also a critical role in both PH and vascular remodeling. OBJECTIVE: We hypothesized that MetS and inflammation may trigger the development of vascular remodeling in group 2 PH. METHODS AND RESULTS: Using supracoronary aortic banding, we induced diastolic dysfunction in rats. Then we induced MetS by a combination of high-fat diet and olanzapine treatment. We used metformin treatment and anti-IL-6 (interleukin-6) antibodies to inhibit the IL-6 pathway. Compared with sham conditions, only supracoronary aortic banding+MetS rats developed precapillary PH, as measured by both echocardiography and right/left heart catheterization. PH in supracoronary aortic banding+MetS was associated with macrophage accumulation and increased IL-6 production in lung. PH was also associated with STAT3 (signal transducer and activator of transcription 3) activation and increased proliferation of PA smooth muscle cells, which contributes to remodeling of distal PA. We reported macrophage accumulation, increased IL-6 levels, and STAT3 activation in the lung of group 2 PH patients. In vitro, IL-6 activates STAT3 and induces human PA smooth muscle cell proliferation. Metformin treatment decreased inflammation, IL-6 levels, STAT3 activation, and human PA smooth muscle cell proliferation. In vivo, in the supracoronary aortic banding+MetS animals, reducing IL-6, either by anti-IL-6 antibody or metformin treatment, reversed pulmonary vascular remodeling and improve PH due to LHD. CONCLUSIONS: We developed a new preclinical model of group 2 PH by combining MetS with LHD. We showed that MetS exacerbates group 2 PH. We provided evidence for the importance of the IL-6-STAT3 pathway in our experimental model of group 2 PH and human patients.

Endothelial BMPR2 Loss Drives a Proliferative Response to BMP (Bone Morphogenetic Protein) 9 via Prolonged Canonical Signaling.

OBJECTIVE: Pulmonary arterial hypertension is a disease of proliferative vascular occlusion that is strongly linked to mutations in BMPR2-the gene encoding the BMPR-II (BMP [bone morphogenetic protein] type II receptor). The endothelial-selective BMPR-II ligand, BMP9, reverses disease in animal models of pulmonary arterial hypertension and suppresses the proliferation of healthy endothelial cells. However, the impact of BMPR2 loss on the antiproliferative actions of BMP9 has yet to be assessed. Approach and Results: BMP9 suppressed proliferation in blood outgrowth endothelial cells from healthy control subjects but increased proliferation in blood outgrowth endothelial cells from pulmonary arterial hypertension patients with BMPR2 mutations. This shift from growth suppression to enhanced proliferation was recapitulated in control human pulmonary artery endothelial cells following siRNA-mediated BMPR2 silencing, as well as in mouse pulmonary endothelial cells isolated from endothelial-conditional Bmpr2 knockout mice (Bmpr2EC-/-). BMP9-induced proliferation was not attributable to altered metabolic activity or elevated TGFß (transforming growth factor beta) signaling but was linked to the prolonged induction of the canonical BMP target ID1 in the context of BMPR2 loss. In vivo, daily BMP9 administration to neonatal mice impaired both retinal and lung vascular patterning in control mice (Bmpr2EC+/+) but had no measurable effect on mice bearing a heterozygous endothelial Bmpr2 deletion (Bmpr2EC+/-) and caused excessive angiogenesis in both vascular beds for Bmpr2EC-/- mice. CONCLUSIONS: BMPR2 loss reverses the endothelial response to BMP9, causing enhanced proliferation. This finding has potential implications for the proposed translation of BMP9 as a treatment for pulmonary arterial hypertension and suggests the need for focused patient selection in clinical trials.

Clinical value of non-coding RNAs in cardiovascular, pulmonary, and muscle diseases.

Although a majority of the mammalian genome is transcribed to RNA, mounting evidence indicates that only a minor proportion of these transcriptional products are actually translated into proteins. Since the discovery of the first non-coding RNA (ncRNA) in the 1980s, the field has gone on to recognize ncRNAs as important molecular regulators of RNA activity and protein function, knowledge of which has stimulated the expansion of a scientific field that quests to understand the role of ncRNAs in cellular physiology, tissue homeostasis, and human disease. Although our knowledge of these molecules has significantly improved over the years, we have limited understanding of their precise functions, protein interacting partners, and tissue-specific activities. Adding to this complexity, it remains unknown exactly how many ncRNAs there are in existence. The increased use of high-throughput transcriptomics techniques has rapidly expanded the list of ncRNAs, which now includes classical ncRNAs (e.g., ribosomal RNAs and transfer RNAs), microRNAs, and long ncRNAs. In addition, splicing by-products of protein-coding genes and ncRNAs, so-called circular RNAs, are now being investigated. Because there is substantial heterogeneity in the functions of ncRNAs, we have summarized the present state of knowledge regarding the functions of ncRNAs in heart, lungs, and skeletal muscle. This review highlights the pathophysiologic relevance of these ncRNAs in the context of human cardiovascular, pulmonary, and muscle diseases.

Suppression of Superoxide-Hydrogen Peroxide Production at Site IQ of Mitochondrial Complex I Attenuates Myocardial Stunning and Improves Postcardiac Arrest Outcomes.

OBJECTIVES: Cardiogenic shock following cardiopulmonary resuscitation for sudden cardiac arrest is common, occurring even in the absence of acute coronary artery occlusion, and contributes to high rates of postcardiopulmonary resuscitation mortality. The pathophysiology of this shock is unclear, and effective therapies for improving clinical outcomes are lacking. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: C57BL/6 adult female mice. INTERVENTIONS: Anesthetized and ventilated adult female C57BL/6 wild-type mice underwent a 4, 8, 12, or 16-minute potassium chloride-induced cardiac arrest followed by 90 seconds of cardiopulmonary resuscitation. Mice were then blindly randomized to a single IV injection of vehicle (phosphate-buffered saline) or suppressor of site IQ electron leak, an inhibitor of superoxide production by complex I of the mitochondrial electron transport chain. Suppressor of site IQ electron leak and vehicle were administered during cardiopulmonary resuscitation. MEASUREMENTS AND MAIN RESULTS: Using a murine model of asystolic cardiac arrest, we discovered that duration of cardiac arrest prior to cardiopulmonary resuscitation determined postresuscitation success rates, degree of neurologic injury, and severity of myocardial dysfunction. Post-cardiopulmonary resuscitation cardiac dysfunction was not associated with myocardial necrosis, apoptosis, inflammation, or mitochondrial permeability transition pore opening. Furthermore, left ventricular function recovered within 72 hours of cardiopulmonary resuscitation, indicative of myocardial stunning. Postcardiopulmonary resuscitation, the myocardium exhibited increased reactive oxygen species and evidence of mitochondrial injury, specifically reperfusion-induced reactive oxygen species generation at electron transport chain complex I. Suppressor of site IQ electron leak, which inhibits complex I-dependent reactive oxygen species generation by suppression of site IQ electron leak, decreased myocardial reactive oxygen species generation and improved postcardiopulmonary resuscitation myocardial function, neurologic outcomes, and survival. CONCLUSIONS: The severity of cardiogenic shock following asystolic cardiac arrest is dependent on the length of cardiac arrest prior to cardiopulmonary resuscitation and is mediated by myocardial stunning resulting from mitochondrial electron transport chain complex I dysfunction. A novel pharmacologic agent targeting this mechanism, suppressor of site IQ electron leak, represents a potential, practical therapy for improving sudden cardiac arrest resuscitation outcomes.

Standards and Methodological Rigor in Pulmonary Arterial Hypertension Preclinical and Translational Research.

Despite advances in our understanding of the pathophysiology and the management of pulmonary arterial hypertension (PAH), significant therapeutic gaps remain for this devastating disease. Yet, few innovative therapies beyond the traditional pathways of endothelial dysfunction have reached clinical trial phases in PAH. Although there are inherent limitations of the currently available models of PAH, the leaky pipeline of innovative therapies relates, in part, to flawed preclinical research methodology, including lack of rigour in trial design, incomplete invasive hemodynamic assessment, and lack of careful translational studies that replicate randomized controlled trials in humans with attention to adverse effects and benefits. Rigorous methodology should include the use of prespecified eligibility criteria, sample sizes that permit valid statistical analysis, randomization, blinded assessment of standardized outcomes, and transparent reporting of results. Better design and implementation of preclinical studies can minimize inherent flaws in the models of PAH, reduce the risk of bias, and enhance external validity and our ability to distinguish truly promising therapies form many false-positive or overstated leads. Ideally, preclinical studies should use advanced imaging, study several preclinical pulmonary hypertension models, or correlate rodent and human findings and consider the fate of the right ventricle, which is the major determinant of prognosis in human PAH. Although these principles are widely endorsed, empirical evidence suggests that such rigor is often lacking in pulmonary hypertension preclinical research. The present article discusses the pitfalls in the design of preclinical pulmonary hypertension trials and discusses opportunities to create preclinical trials with improved predictive value in guiding early-phase drug development in patients with PAH, which will need support not only from researchers, peer reviewers, and editors but also from academic institutions, funding agencies, and animal ethics authorities.

Excess Protein O-GlcNAcylation Links Metabolic Derangements to Right Ventricular Dysfunction in Pulmonary Arterial Hypertension.

The hexosamine biosynthetic pathway (HBP) converts glucose to uridine-diphosphate-N-acetylglucosamine, which, when added to serines or threonines, modulates protein function through protein O-GlcNAcylation. Glutamine-fructose-6-phosphate amidotransferase (GFAT) regulates HBP flux, and AMP-kinase phosphorylation of GFAT blunts GFAT activity and O-GlcNAcylation. While numerous studies demonstrate increased right ventricle (RV) glucose uptake in pulmonary arterial hypertension (PAH), the relationship between O-GlcNAcylation and RV function in PAH is unexplored. Therefore, we examined how colchicine-mediated AMP-kinase activation altered HBP intermediates, O-GlcNAcylation, mitochondrial function, and RV function in pulmonary artery-banded (PAB) and monocrotaline (MCT) rats. AMPK activation induced GFAT phosphorylation and reduced HBP intermediates and O-GlcNAcylation in MCT but not PAB rats. Reduced O-GlcNAcylation partially restored the RV metabolic signature and improved RV function in MCT rats. Proteomics revealed elevated expression of O-GlcNAcylated mitochondrial proteins in MCT RVs, which fractionation studies corroborated. Seahorse micropolarimetry analysis of H9c2 cardiomyocytes demonstrated colchicine improved mitochondrial function and reduced O-GlcNAcylation. Presence of diabetes in PAH, a condition of excess O-GlcNAcylation, reduced RV contractility when compared to nondiabetics. Furthermore, there was an inverse relationship between RV contractility and HgbA1C. Finally, RV biopsy specimens from PAH patients displayed increased O-GlcNAcylation. Thus, excess O-GlcNAcylation may contribute to metabolic derangements and RV dysfunction in PAH.

Providing care for the 99.9% during the COVID-19 pandemic: How ethics, equity, epidemiology, and cost per QALY inform healthcare policy.

Managing healthcare in the Coronavirus Disease 2019 (COVID-19) era should be guided by ethics, epidemiology, equity, and economics, not emotion. Ethical healthcare policies ensure equitable access to care for patients regardless of whether they have COVID-19 or another disease. Because healthcare resources are limited, a cost per Quality Life Year (QALY) approach to COVID-19 policy should also be considered. Policies that focus solely on mitigating COVID-19 are likely to be ethically or financially unsustainable. A cost/QALY approach could target resources to optimally improve QALYs. For example, most COVID-19 deaths occur in long-term care facilities, and this problem is likely better addressed by a focused long-term care reform than by a society-wide non-pharmacological intervention. Likewise, ramping up elective, non-COVID-19 care in low prevalence regions while expanding testing and case tracking in hot spots could reduce excess mortality from non-COVID-19 diseases and decrease adverse financial impacts while controlling the epidemic. Globally, only ∼0.1% of people have had a COVID-19 infection. Thus, ethical healthcare policy must address the needs of the 99.9%.

Epigenetic Dysregulation of the Dynamin-Related Protein 1 Binding Partners MiD49 and MiD51 Increases Mitotic Mitochondrial Fission and Promotes Pulmonary Arterial Hypertension: Mechanistic and Therapeutic Implications.

BACKGROUND: Mitotic fission is increased in pulmonary arterial hypertension (PAH), a hyperproliferative, apoptosis-resistant disease. The fission mediator dynamin-related protein 1 (Drp1) must complex with adaptor proteins to cause fission. Drp1-induced fission has been therapeutically targeted in experimental PAH. Here, we examine the role of 2 recently discovered, poorly understood Drp1 adapter proteins, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), in normal vascular cells and explore their dysregulation in PAH. METHODS: Immunoblots of pulmonary artery smooth muscle cells (control, n=6; PAH, n=8) and immunohistochemistry of lung sections (control, n=6; PAH, n=6) were used to assess the expression of MiD49 and MiD51. The effects of manipulating MiDs on cell proliferation, cell cycle, and apoptosis were assessed in human and rodent PAH pulmonary artery smooth muscle cells with flow cytometry. Mitochondrial fission was studied by confocal imaging. A microRNA (miR) involved in the regulation of MiD expression was identified using microarray techniques and in silico analyses. The expression of circulatory miR was assessed with quantitative reverse transcription-polymerase chain reaction in healthy volunteers (HVs) versus patients with PAH from Sheffield, UK (plasma: HV, n=29, PAH, n=27; whole blood: HV, n=11, PAH, n=14) and then confirmed in a cohort from Beijing, China (plasma: HV, n=19, PAH, n=36; whole blood: HV, n=20, PAH, n=39). This work was replicated in monocrotaline and Sugen 5416-hypoxia, preclinical PAH models. Small interfering RNAs targeting MiDs or an miR mimic were nebulized to rats with monocrotaline-induced PAH (n=4-10). RESULTS: MiD expression is increased in PAH pulmonary artery smooth muscle cells, which accelerates Drp1-mediated mitotic fission, increases cell proliferation, and decreases apoptosis. Silencing MiDs (but not other Drp1 binding partners, fission 1 or mitochondrial fission factor) promotes mitochondrial fusion and causes G1-phase cell cycle arrest through extracellular signal-regulated kinases 1/2- and cyclin-dependent kinase 4-dependent mechanisms. Augmenting MiDs in normal cells causes fission and recapitulates the PAH phenotype. MiD upregulation results from decreased miR-34a-3p expression. Circulatory miR-34a-3p expression is decreased in both patients with PAH and preclinical models of PAH. Silencing MiDs or augmenting miR-34a-3p regresses experimental PAH. CONCLUSIONS: In health, MiDs regulate Drp1-mediated fission, whereas in disease, epigenetic upregulation of MiDs increases mitotic fission, which drives pathological proliferation and apoptosis resistance. The miR-34a-3p-MiD pathway offers new therapeutic targets for PAH.

Assessment of Right Ventricular Function in the Research Setting: Knowledge Gaps and Pathways Forward. An Official American Thoracic Society Research Statement.

BACKGROUND: Right ventricular (RV) adaptation to acute and chronic pulmonary hypertensive syndromes is a significant determinant of short- and long-term outcomes. Although remarkable progress has been made in the understanding of RV function and failure since the meeting of the NIH Working Group on Cellular and Molecular Mechanisms of Right Heart Failure in 2005, significant gaps remain at many levels in the understanding of cellular and molecular mechanisms of RV responses to pressure and volume overload, in the validation of diagnostic modalities, and in the development of evidence-based therapies. METHODS: A multidisciplinary working group of 20 international experts from the American Thoracic Society Assemblies on Pulmonary Circulation and Critical Care, as well as external content experts, reviewed the literature, identified important knowledge gaps, and provided recommendations. RESULTS: This document reviews the knowledge in the field of RV failure, identifies and prioritizes the most pertinent research gaps, and provides a prioritized pathway for addressing these preclinical and clinical questions. The group identified knowledge gaps and research opportunities in three major topic areas: 1) optimizing the methodology to assess RV function in acute and chronic conditions in preclinical models, human studies, and clinical trials; 2) analyzing advanced RV hemodynamic parameters at rest and in response to exercise; and 3) deciphering the underlying molecular and pathogenic mechanisms of RV function and failure in diverse pulmonary hypertension syndromes. CONCLUSIONS: This statement provides a roadmap to further advance the state of knowledge, with the ultimate goal of developing RV-targeted therapies for patients with RV failure of any etiology.