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Right ventricular failure (RVF) is a leading cause of morbidity and mortality in multiple cardiovascular diseases, but there are no approved treatments for RVF as therapeutic targets are not clearly defined. Contemporary transcriptomic/proteomic evaluations of RVF are predominately conducted in small animal studies, and data from large animal models are sparse. Moreover, a comparison of the molecular mediators of RVF across species is lacking. Here, we used transcriptomics and proteomics analyses to define the molecular pathways associated with cardiac MRI-derived values of RV hypertrophy, dilation, and dysfunction in pulmonary artery banded (PAB) piglets. Publicly available data from rat monocrotaline-induced RVF and pulmonary arterial hypertension patients with preserved or impaired RV function were used to compare the three species.Transcriptomic and proteomic analyses identified multiple pathways that were associated with RV dysfunction and remodeling in PAB pigs. Surprisingly, disruptions in fatty acid oxidation (FAO) and electron transport chain (ETC) proteins were different across the three species. FAO and ETC proteins and transcripts were mostly downregulated in rats, but were predominately upregulated in PAB pigs, which more closely matched the human data. Thus, the pig PAB metabolic molecular signature was more similar to human RVF than rodents. These data suggest there may be divergent molecular responses of RVF across species, and that pigs more accurately recapitulate the metabolic aspects of human RVF.

Background:Right ventricular dysfunction (RVD) is the leading cause of death in pulmonary arterial hypertension (PAH), but no RV-specific therapy exists. We showed microtubule-mediated junctophilin-2 dysregulation (MT-JPH2 pathway) causes t-tubule disruption and RVD in rodent PAH, but the druggable regulators of this critical pathway are unknown. GP130 (glycoprotein 130) activation induces cardiomyocyte microtubule remodeling in vitro; however, the effects of GP130 signaling on the MT-JPH2 pathway and RVD resulting from PAH are undefined.Methods:Immunoblots quantified protein abundance, quantitative proteomics defined RV microtubule-interacting proteins (MT-interactome), metabolomics evaluated the RV metabolic signature, and transmission electron microscopy assessed RV cardiomyocyte mitochondrial morphology in control, monocrotaline, and monocrotaline-SC-144 (GP130 antagonist) rats. Echocardiography and pressure-volume loops defined the effects of SC-144 on RV-pulmonary artery coupling in monocrotaline rats (8–16 rats per group). In 73 patients with PAH, the relationship between interleukin-6, a GP130 ligand, and RVD was evaluated.Results:SC-144 decreased GP130 activation, which normalized MT-JPH2 protein expression and t-tubule structure in the monocrotaline RV. Proteomics analysis revealed SC-144 restored RV MT-interactome regulation. Ingenuity pathway analysis of dysregulated MT-interacting proteins identified a link between microtubules and mitochondrial function. Specifically, SC-144 prevented dysregulation of electron transport chain, Krebs cycle, and the fatty acid oxidation pathway proteins. Metabolomics profiling suggested SC-144 reduced glycolytic dependence, glutaminolysis induction, and enhanced fatty acid metabolism. Transmission electron microscopy and immunoblots indicated increased mitochondrial fission in the monocrotaline RV, which SC-144 mitigated. GP130 antagonism reduced RV hypertrophy and fibrosis and augmented RV-pulmonary artery coupling without altering PAH severity. In patients with PAH, higher interleukin-6 levels were associated with more severe RVD (RV fractional area change 23±12% versus 30±10%,P=0.002).Conclusions:GP130 antagonism reduces MT-JPH2 dysregulation, corrects metabolic derangements in the RV, and improves RVD in monocrotaline rats.

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 pO(2)-responsive production of reactive oxygen species (ROS). These ROS are converted to a diffusible signaling molecule, hydrogen peroxide (H(2)O(2)), by superoxide dismutase (SOD2). H(2)O(2) exits the mitochondria and regulates ion channels and enzymes, altering plasma membrane potential, intracellular Ca(2+) and Ca(2+)-sensitization and controlling acute, adaptive, responses to hypoxia that involve changes in ventilation, vascular tone and neurotransmitter release. Subversion of this O(2)-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 pO(2). Epigenetic silencing of SOD2 by DNA methylation alters H(2)O(2) 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 Ca(2+), resulting from impaired mitochondrial calcium uniporter complex (MCUC) function, links abnormal mitochondrial metabolism and dynamics. MiR-mediated decreases in MCUC function reduce intramitochondrial Ca(2+), promoting Warburg metabolism, whilst increasing cytosolic Ca(2+), promoting fission. Epigenetically disordered mitochondrial O(2)-sensing, metabolism, dynamics, and Ca(2+) 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.

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.

Background: The impact of pulmonary hypertension (PH) on outcomes after surgical tricuspid valve replacement (TVR) and repair (TVr) is unclear. We sought to characterize PH in patients undergoing TVR/TVr, based on invasive hemodynamics and evaluate the effect of PH on mortality. Methods: We identified 86 consecutive patients who underwent TVR/TVr with invasive hemodynamic measurements within 3 months before surgery. We used Kaplan-Meier survival and restricted mean survival time (RMST) analyses to quantify the effects of PH on survival. Results: The mean age was 63 ± 13 years, 59% were female, 45% had TVR, 55% had TVr, 39.5% had isolated TVR/TVr, and 60.5% had TVR/TVr concomitant with other cardiac surgeries). Eighty-six percent of these patients had PH with a mean pulmonary artery pressure of 30 ± 10 mm Hg, pulmonary vascular resistance (PVR) of 2.5 (interquartile range: 1.5-3.9) Wood units (WU), pulmonary arterial compliance of 2.3 (1.6-3.6) mL/mm Hg, and pulmonary arterial elastance of 0.8 (0.6-1.2) mm Hg/mL. Cardiac output was mildly reduced at 4.0 ± 1.4 L/min, with elevated right-atrial pressure (14 ± 12 mm Hg) and pulmonary capillary wedge pressure (19 ± 7 mm Hg). Over a median follow-up of 6.3 years, 22% of patients died. Patients with PVR ≥ 2.5 WU had lower RMST over 5 years compared with patients with PVR < 2.5 WU. Conclusion: PH is common in patients undergoing TVR/TVr, with combined pre- and postcapillary being the most common type. PVR ≥ 2.5 WU is associated with lower survival at 5-year follow-up. Résumé: Contexte: On connaît mal les répercussions de l’hypertension pulmonaire (HP) chez les patients qui ont subi une intervention chirurgicale de remplacement de la valve tricuspide (RVT) ou de réparation de la valve tricuspide (rVT). Nous avons tenté de caractériser l’HP chez les patients ayant subi un RVT ou une rVT en fonction des paramètres de surveillance hémodynamique effractive et d’évaluer l’effet de l’HP sur la mortalité. Méthodologie: Nous avons relevé 86 patients consécutifs ayant subi un RVT ou une rVT qui avaient fait l’objet de mesures hémodynamiques effractives dans les trois mois précédant l’intervention chirurgicale. Pour quantifier les effets de l’HP sur la survie, nous avons analysé la survie au moyen de la méthode de Kaplan-Meier et de la survie moyenne restreinte. Résultats: Les patients avaient en moyenne 63 ± 13 ans; 59 % d’entre eux étaient des femmes; 45 % avaient subi un RVT et 55 %, une rVT; 39,5 % avaient subi seulement un RVT ou une rVT lors de l’intervention chirurgicale; 60,5 % avaient subi un RVT ou une rVT en même temps qu’une autre intervention cardiaque. Quatre-vingt-six pour cent de ces patients présentaient une HP avec une pression artérielle pulmonaire moyenne de 30 ± 10 mmHg, une résistance vasculaire pulmonaire (RVP) de 2,5 (intervalle interquartile : 1,5 à 3,9) unités de Wood (UW), une compliance artérielle pulmonaire de 2,3 (1,6 à 3,6) ml/mmHg et une élastance artérielle pulmonaire de 0,8 (0,6 à 1,2) mmHg/ml. On a observé une légère baisse du débit cardiaque à 4,0 ± 1,4 L/min, ainsi qu’une augmentation de la pression auriculaire droite (14 ± 12 mmHg) et de la pression artérielle pulmonaire d’occlusion (19 ± 7 mmHg). Sur une période médiane de suivi de 6,3 ans, 22 % des patients sont décédés. Le taux de survie moyenne restreinte à 5 ans était plus faible chez les patients présentant une RVP ≥ 2,5 UW que chez les patients présentant une RVP < 2,5 UW. Conclusion: L’HP est fréquente chez les patients subissant un RVT ou une rVT, le type le plus courant étant l’HP mixte (pré-capillaire et post-capillaire). Une RVP ≥ 2,5 UW est associée à un taux de survie à 5 ans plus faible.

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.

Supplemental Digital Content is available in the text., 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 TGFβ (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. Graphic Abstract: A graphic abstract is available for this article.

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%.

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-β1 (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.

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.

Supplemental Digital Content is available in the text., 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.

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.

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

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.

Background Neutrophils play a role in innate immunity and are critical for clearance of Staphylococcus aureus. Current understanding of neutrophil bactericidal effects is that NADPH oxidase produces reactive oxygen species (ROS), mediating bacterial killing. Neutrophils also contain numerous mitochondria; since these organelles lack oxidative metabolism, their function is unclear. We hypothesize that mitochondria in human neutrophils contribute to the bactericidal capacity of S. aureus. Methods and Findings: Using human neutrophils isolated from healthy volunteers (n = 13; 7 females, 6 males), we show that mitochondria are critical in the immune response to S. aureus. Using live-cell and fixed confocal, and transmission electron microscopy, we show mitochondrial tagging of bacteria prior to ingestion and surrounding of phagocytosed bacteria immediately upon engulfment. Further, we demonstrate that mitochondria are ejected from intact neutrophils and engage bacteria during vital NETosis. Inhibition of the mitochondrial electron transport chain at Complex III, but not Complex I, attenuates S. aureus killing by 50 ± 7%, comparable to the NADPH oxidase inhibitor apocynin. Similarly, mitochondrial ROS scavenging using MitoTEMPO attenuates bacterial killing 112 ± 60% versus vehicle control. Antimycin A treatment also reduces mitochondrial ROS production by 50 ± 12% and NETosis by 53 ± 5%. Conclusions We identify a previously unrecognized role for mitochondria in human neutrophils in the killing of S. aureus. Inhibition of electron transport chain Complex III significantly impairs antimicrobial activity. This is the first demonstration that vital NETosis, an early event in the antimicrobial response, occurring within 5 min of bacterial exposure, depends on the function of mitochondrial Complex III. Mitochondria join NADPH oxidase as bactericidal ROS generators that mediate the bactericidal activities of human neutrophils., Graphical abstract Image 1, Highlights • This study evaluates the role of neutrophil mitochondria in the immune response to Staphylococcus aureus. • Mitochondrial electron transport chain inhibition at Complex III significantly attenuates neutrophil bactericidal activity.

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 & 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, p3 mitochondria, n=9, p 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.

Introduction: Increase in mitotic fission in pulmonary arterial smooth muscle cells (PASMC) contributes to the obstructive vasculopathy in pulmonary arterial hypertension (PAH). PAH’s fissogenic phenotype is due, in part, to excessive mitochondrial fission caused by activation of dynamin related protein-1 (Drp1). Recent observation suggests that to complete mitochondrial fission, Drp1 requires assistance from the GTPase dynamin 2 (DNM2). In this study we explore the role of DNM2 in the pathogenesis of PAH. Hypothesis: In PAH, increased expression of DNM2 increases mitochondrial fission and proliferation of PASMC. Methods: Immunoblot was used to quantify the expression of DNM2 in control (n=4) and PAH PASMC (n=5). Immunofluorescence was used to quantify the expression of DNM2 in the pulmonary arteries of monocrotaline (MCT) vs control rats. Flow cytometry was used to assess the effects of manipulating the expression of DNM2 on cell proliferation, cell cycle progression and rates of unstimulated apoptosis. Mitochondrial network structure was assessed using confocal imaging of cells infected with Adv-TOM20 mNeon green. Results: The expression of DNM2 was increased in human PAH PASMC vs control PASMC. Increase in the expression of DNM2 expression was also observed in the pulmonary arteries of MCT-PAH rats. Silencing DNM2 inhibited mitochondrial fission in PAH PASMC. Furthermore, silencing DNM2 inhibited proliferation in PAH PASMC by blocking the cell cycle at G1/G0. Conversely, augmenting DNM2 in normal PASMC induced mitochondrial fission and accelerated cell proliferation, recapitulating the PAH phenotype. In silico analysis identified miR-124-3p as a putative negative regulator of DNM2. Augmenting miR-124-3p decreased DNM2 expression, inhibited proliferation and induced apoptosis in PAH PASMC. Conclusion: DNM2 promotes mitochondrial fission and regulates cell proliferation in human PAH PASMC. Decreased miR-124-3p expression may contribute to upregulation of DNM2 in PAH. This work highlights the importance of DNM2 as partner with Drp1 in regulation of mitochondrial fission and shows that like Drp1, DNM2 is increased in human PAH. DNM2 may constitute new therapeutic target for PAH.

Introduction: The ductus arteriosus (DA) allows placentally oxygenated blood to bypass the non-ventilated fetal lungs and enter the systemic circulation. The fetus’ systemic pO 2 is ~40 mmHg, maintaining the DA in a state of hypoxic vasodilation. As pO 2 rises, with the first breath, the DA constricts and achieves functional closure within minutes. We have identified a mitochondrial O 2 -sensor controlling functional closure within DA smooth muscle cells (DASMC). Increased pO 2 leads to increased reactive oxygen species (ROS) production by complexes I and III of the electron transport chain (ETC). These ROS are diffusible signaling molecules and inhibit redox sensitive potassium channels causing DASMC depolarization and vasoconstriction. We propose this reflects electron leak from ETC complexes. Using the term rabbit model, we investigated the differential function of ETC I vs III complex-specific electron leak suppressors (S1QEL vs S3QEL) on vascular tone in DA rings. Methods: DA’s from term rabbits (n=11 rabbits, n=29 individual DA rings) were mounted on a force transducer at ~800 mg in Krebs at a PO 2 of ~40mmHg. DA’s were subject to varying pO 2 levels (from 35-155 mmHg) and at peak O 2 constriction were treated with ETC leak suppressors, ETC complex inhibitors or vehicle controls (DMSO). Results: Exposure to normoxia caused an average 1100±62 mg increase in DA tension. S1QEL (50 μM) caused an average 20.9±6.2% reduction in DA tension (n=8) whereas S3QEL (100 μM) only reduced DA tension by 4.4±2.2% (n=4). Rotenone (complex I inhibitor) and antimycin A (complex III inhibitor) also relaxed the DA while vehicle had minimal effect (-2.6±1.5%). Conclusions: Electron leak from complex 1, but not complex 3, is implicated in DA O 2 -sensing.

Introduction: 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 increased oxygen (O 2 ) immediately post-partum, resulting in functional DA closure, with anatomic closure ensuing within the first days of life. Failure of DA closure results in patent ductus arteriosus (PDA), a common complication of extreme preterm birth. The DA response to O 2 , though modulated by the endothelium, is intrinsic to DA smooth muscle cells (DASMC). DA constriction is mediated by mitochondria-derived reactive oxygen species, which increase in proportion to PaO 2 . The molecular signature of the DASMC response to O 2 is not well understood. Methods & Results: DASMC were isolated from DA obtained from 10 infants at the time of congenital heart surgery. Cells were purified by flow cytometry, negatively sorted to eliminate fibroblasts (CD90+) or endothelial cells (CD31+). DASMC purity was confirmed by α-smooth muscle actin staining. Cells were grown for 96 hours in hypoxia (2.5% O 2 ) or normoxia (19% O 2 ) and confocal calcium imaging confirmed O 2 -responsiveness of all cell lines: O 2 exposure increased intracellular calcium 18.1%±4.4% and decreased cell length 27%±1.5% relative to hypoxic baseline. RNA sequencing (Seq) of the cells grown in hypoxia and normoxia revealed significant regulation of 1344 genes (corrected p2 , revealing enrichment of functional groups including mitochondria, cellular respiration and transcription. Multiple subunits of electron transport chain complex I, NDUF (NADH:ubiquinone oxidoreductase) family genes, are upregulated in hDASMC following normoxia. Upregulated NDUF genes were validated with qPCR (t-test p Conclusion: This first examination of the effects of O 2 on human DA transcriptomics supports a putative role for mitochondria as O 2 sensors, with mitochondrial subunits as potential future therapeutic targets for PDA.

The Ductus Arteriosus (DA) is a fetal vessel that connects the aorta to the pulmonary artery ensuring that placental oxygenated blood is diverted from the lungs to the systemic circulation. Following exposure to oxygen (O2), in the first few days of life, the DA responds with a functional closure that is followed by anatomical closure. Here, we study human DA smooth muscle cells (DASMC) taken from 10 term infants during congenital heart surgery. Purification of these cells using flow cytometry ensured a pure population of DASMCs, which we confirmed as responsive to O2. 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. These cells were maintained in either hypoxia (2.5% O2), mimicking in utero conditions or in normoxia (19% O2) mimicking neonate conditions. We then used 3’ RNAsequencing to identify the transcriptome of DASMCs in each condition [1]. In this paper, we present the full differentially regulated gene list from this experiment.

A 51-year-old woman with pulmonary arterial hypertension presented with progressive chest pain and dyspnea. Computed tomography imaging showed significant enlargement of her main pulmonary artery (PA) and was suggestive of left main coronary artery (LMCA) compression by the PA. The patient underwent percutaneous coronary intervention, which confirmed the diagnosis and a stent was deployed to the LMCA. Three months after the procedure the patient has near resolution of her symptoms. LMCA compression by an enlarged PA is an important cause of chest pain in patients with pulmonary arterial hypertension and can be managed safely and effectively with percutaneous coronary intervention and stenting.

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.

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.

Introduction: Female sex is associated with better right ventricular (RV) function in pulmonary hypertension (PH). The female sex hormone 17-beta-estradiol is postulated to mediate these differences, but the molecular mechanisms underlying these observations are incompletely defined. Interestingly, 17-beta-estradiol induces microtubule depolymerization in cell culture, which may be relevant to RV dysfunction because microtubule remodeling promotes RV dysfunction via dysregulation of junctophilin-2 (MT-JPH2 pathway). We speculate that 17-beta-estradiol modulates the MT-JPH2 pathway and preserves RV function in PH. Methods: Pressure-volume assessments quantified RV function in monocrotaline (MCT) and pulmonary artery-banded (PAB) rats. Immunoblots quantified the tubulin and junctophilin-2 protein content in RV extracts. Echocardiography quantified RV function by RV fractional area change for 379 human PH patients. Sedimentation experiments, fluorescence-based polymerization assessments, and total internal reflection fluorescence (TIRF) microscopy examined the effects of 17-beta-estradiol on microtubules. Results: Female sex results in better RV function and less dysregulation of the MT-JPH2 pathway in both MCT and PAB rats. Moreover, in human PH, female sex was associated with better RV function, which persisted after adjusting for afterload. 17-beta-estradiol inhibited microtubule polymerization in vitro and TIRF microscopy showed 17-beta-estradiol localized to microtubule tips and prevented further microtubule polymerization. Conclusions: Preclinical and human studies show that females are better able to tolerate RV pressure overload. There are blunted microtubule-mediated t-tubule remodeling and preserved RV function in female MCT and PAB rats. In human PH, females have better RV function. These findings may be due to 17-beta-estradiol directly regulating microtubule dynamics as shown by sedimentation and polymerization assays and TIRF microscopy. These results provide additional insights into sex-differences in RV function in PH.