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

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

Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH.

Background: Mutations are found in 10-20% of idiopathic PAH (IPAH) patients, but none are consistently identified in connective tissue disease-associated PAH (APAH), which accounts for ∼45% of PAH cases. TET2 mutations, a cause of clonal hematopoiesis of indeterminant potential (CHIP), predispose to an inflammatory type of PAH. We now examine mutations in another CHIP gene, DNMT3A , in PAH. Methods: We assessed DNMT3A mutation prevalence in PAH Biobank subjects as compared with controls, first using whole exome sequencing (WES)-derived CHIP calls in 1832 PAH Biobank patients versus 7509 age-and sex-matched gnomAD controls. We then performed deep, targeted panel sequencing of CHIP genes on a subset of 710 PAH Biobank patients and compared the prevalence of DNMT3A mutations therein to an independent pooled control cohort (N = 3645). In another cohort of 80 PAH patients and 41 controls, DNMT3A mRNA expression was studied in peripheral blood mononuclear cells (PBMCs). Finally, we evaluated the development of PAH in a conditional, hematopoietic, Dnmt3a knockout mouse model. Results: DNMT3A mutations were more frequent in PAH cases versus control subjects in the WES dataset (OR 2.60, 95% CI: 1.71-4.27). Among PAH patients, 33 had DNMT3A variants, most of whom had APAH (21/33). While 21/33 had somatic mutations (female:male 17:4), germline variants occurred in 12/33 (female:male 11:1). Hemodynamics were comparable with and without DNMT3A mutations (mPAP=58±21 vs. 52±18 mmHg); however, patients with DNMT3A mutations were unresponsive to acute vasodilator testing. Targeted panel sequencing identified that 14.6% of PAH patients had CHIP mutations (104/710), with DNMT3A accounting for 49/104. There was a significant association between all CHIP mutations and PAH in analyses adjusted for age and sex (OR 1.40, 95% CI: 1.09-1.80), though DNMT3A CHIP alone was not significantly enriched (OR:1.15, 0.82-1.61). DNMT3A expression was reduced in patient-derived versus control PAH-PBMCs. Spontaneous PAH developed in Dnmt3a -/- mice, and it was exacerbated by 3 weeks of hypoxia. Dnmt3a -/- mice had increased lung macrophages and elevated plasma IL-13. The IL-1ß antibody canakinumab attenuated PAH in Dnmt3a -/- mice. Conclusions: Germline and acquired DNMT3A variants predispose to PAH in humans. DNMT3A mRNA expression is reduced in human PAH PBMCs. Hematopoietic depletion of Dnmt3a causes inflammatory PAH in mice. DNMT3A is a novel APAH gene and may be a biomarker and therapeutic target.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia.

RATIONALE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. OBJECTIVES: We examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. METHODS: We used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. RESULTS: SARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. CONCLUSIONS: Coronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

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.

Mitochondria in human neutrophils mediate killing of Staphylococcus aureus.

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.

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice.

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) generated by recording both pressure and volume during cardiac catheterization are vital when assessing both systolic and diastolic cardiac function. Left and right heart function are closely related, reflected in ventricular interdependence. Thus, recording biventricular function in the same animal is important to get a complete assessment of cardiac function. In this protocol, a closed chest approach to cardiac catheterization consistent with the way catheterization is performed in patients is adopted in mice. While challenging, the closed chest strategy is a more physiological approach, because opening the chest results in major changes in preload and afterload that create artifacts, most notably a fall in systemic blood pressure. While high-resolution echocardiography is used to assess rodents, cardiac catheterization is invaluable, particularly when assessing diastolic pressures in both ventricles. Described here is a procedure to perform invasive, closed chest, sequential left and right ventricular pressure-volume (PV) loops in the same animal. PV loops are acquired using admittance technology with a mouse pressure-volume catheter and pressure-volume system acquisition. The procedure is described, beginning with the neck dissection, which is required to access the right jugular vein and the right carotid artery, to the insertion and positioning of the catheter, and finally the data acquisition. Then, the criteria required to ensure the acquisition of high-quality PV loops are discussed. Finally, the analysis of the left and right ventricular PV loops and the different hemodynamic parameters available to quantify systolic and diastolic ventricular function are briefly described.

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.