The Role of Mitochondrial Dynamics and Mitotic Fission in Regulating the Cell Cycle in Cancer and Pulmonary Arterial Hypertension: Implications for Dynamin-Related Protein 1 and Mitofusin2 in Hyperproliferative Diseases.

Mitochondria, which generate ATP through aerobic respiration, also have important noncanonical functions. Mitochondria are dynamic organelles, that engage in fission (division), fusion (joining) and translocation. They also regulate intracellular calcium homeostasis, serve as oxygen-sensors, regulate inflammation, participate in cellular and organellar quality control and regulate the cell cycle. Mitochondrial fission is mediated by the large GTPase, dynamin-related protein 1 (Drp1) which, when activated, translocates to the outer mitochondrial membrane (OMM) where it interacts with binding proteins (Fis1, MFF, MiD49 and MiD51). At a site demarcated by the endoplasmic reticulum, fission proteins create a macromolecular ring that divides the organelle. The functional consequence of fission is contextual. Physiological fission in healthy, nonproliferating cells mediates organellar quality control, eliminating dysfunctional portions of the mitochondria via mitophagy. Pathological fission in somatic cells generates reactive oxygen species and triggers cell death. In dividing cells, Drp1-mediated mitotic fission is critical to cell cycle progression, ensuring that daughter cells receive equitable distribution of mitochondria. Mitochondrial fusion is regulated by the large GTPases mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2), which fuse the OMM, and optic atrophy 1 (OPA-1), which fuses the inner mitochondrial membrane. Mitochondrial fusion mediates complementation, an important mitochondrial quality control mechanism. Fusion also favors oxidative metabolism, intracellular calcium homeostasis and inhibits cell proliferation. Mitochondrial lipids, cardiolipin and phosphatidic acid, also regulate fission and fusion, respectively. Here we review the role of mitochondrial dynamics in health and disease and discuss emerging concepts in the field, such as the role of central versus peripheral fission and the potential role of dynamin 2 (DNM2) as a fission mediator. In hyperproliferative diseases, such as pulmonary arterial hypertension and cancer, Drp1 and its binding partners are upregulated and activated, positing mitochondrial fission as an emerging therapeutic target.

Acquired disorders of mitochondrial metabolism and dynamics in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is an orphan disease of the cardiopulmonary unit that reflects an obstructive pulmonary vasculopathy and presents with hypertrophy, inflammation, fibrosis, and ultimately failure of the right ventricle (RVF). Despite treatment using pulmonary hypertension (PH)-targeted therapies, persistent functional impairment reduces the quality of life for people with PAH and death from RVF occurs in approximately 40% of patients within 5 years of diagnosis. PH-targeted therapeutics are primarily vasodilators and none, alone or in combination, are curative. This highlights a need to therapeutically explore molecular targets in other pathways that are involved in the pathogenesis of PAH. Several candidate pathways in PAH involve acquired mitochondrial dysfunction. These mitochondrial disorders include: 1) a shift in metabolism related to increased expression of pyruvate dehydrogenase kinase and pyruvate kinase, which together increase uncoupled glycolysis (Warburg metabolism); 2) disruption of oxygen-sensing related to increased expression of hypoxia-inducible factor 1α, resulting in a state of pseudohypoxia; 3) altered mitochondrial calcium homeostasis related to impaired function of the mitochondrial calcium uniporter complex, which elevates cytosolic calcium and reduces intramitochondrial calcium; and 4) abnormal mitochondrial dynamics related to increased expression of dynamin-related protein 1 and its binding partners, such as mitochondrial dynamics proteins of 49 kDa and 51 kDa, and depressed expression of mitofusin 2, resulting in increased mitotic fission. These acquired mitochondrial abnormalities increase proliferation and impair apoptosis in most pulmonary vascular cells (including endothelial cells, smooth muscle cells and fibroblasts). In the RV, Warburg metabolism and induction of glutaminolysis impairs bioenergetics and promotes hypokinesis, hypertrophy, and fibrosis. This review will explore our current knowledge of the causes and consequences of disordered mitochondrial function in PAH.

Efficacy of Drpitor1a, a Dynamin-Related Protein 1 inhibitor, in Pulmonary Arterial Hypertension.

Rationale: Dynamin-related protein 1 (Drp1), a large GTPase, mediates mitochondrial fission. Increased Drp1-mediated fission permits accelerated mitosis, contributing to hyperproliferation of pulmonary artery smooth muscle cells (PASMC), which characterizes pulmonary arterial hypertension (PAH). We developed a Drp1 inhibitor, Drpitor1a, and tested its ability to regress PAH. Objectives: Assess Drpitor1a's efficacy and toxicity in: a)normal and PAH human PASMC (hPASMC); b)normal rats versus rats with established monocrotaline (MCT)-induced PAH. Methods: Drpitor1a's effects on recombinant and endogenous Drp1-GTPase activity, mitochondrial fission, and cell proliferation were studied in hPASMCs (normal=3; PAH=5). Drpitor1a's pharmacokinetics and tissue concentrations were measured (n=3 rats/sex). In a pilot study (n=3-4/sex/dose), Drpitor1a (1mg/kg/48-hours, intravenous) reduced adverse PA remodeling only in females. Consequently, we compared Drpitor1a to vehicle in normal (n=6 versus 8) and MCT-PAH (n=9 and 11) females, respectively. Drpitor1a treatment began 17-days post-MCT with echocardiography and cardiac catheterization performed 28-29 days post-MCT. Results: Drpitor1a inhibited recombinant and endogenous Drp1 GTPase activity, which was increased in PAH hPASMC. Drpitor1a inhibited mitochondrial fission and proliferation and induced apoptosis, in PAH hPASMC but not normal hPASMC. Drpitor1a tissue levels were higher in female versus male RVs. In MCT-PAH females, Drpitor1a regressed PA obstruction, lowered pulmonary vascular resistance, and improved RV function, without hematologic, renal, or hepatic toxicity. Conclusions: Drpitor1a inhibits Drp1 GTPase, reduces mitochondrial fission, and inhibits cell proliferation in PAH hPASMC. Drpitor1a caused no toxicity in MCT-PAH and had no significant effect on normal rats or hPASMCs. Drpitor1a is a potential PAH therapeutic which displays an interesting therapeutic sexual dimorphism.

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.

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

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

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.

Oxygen sensing, mitochondrial biology and experimental therapeutics for pulmonary hypertension and cancer.

The homeostatic oxygen sensing system (HOSS) optimizes systemic oxygen delivery. Specialized tissues utilize a conserved mitochondrial sensor, often involving NDUFS2 in complex I of the mitochondrial electron transport chain, as a site of pO2-responsive production of reactive oxygen species (ROS). These ROS are converted to a diffusible signaling molecule, hydrogen peroxide (H2O2), by superoxide dismutase (SOD2). H2O2 exits the mitochondria and regulates ion channels and enzymes, altering plasma membrane potential, intracellular Ca2+ and Ca2+-sensitization and controlling acute, adaptive, responses to hypoxia that involve changes in ventilation, vascular tone and neurotransmitter release. Subversion of this O2-sensing pathway creates a pseudohypoxic state that promotes disease progression in pulmonary arterial hypertension (PAH) and cancer. Pseudohypoxia is a state in which biochemical changes, normally associated with hypoxia, occur despite normal pO2. Epigenetic silencing of SOD2 by DNA methylation alters H2O2 production, activating hypoxia-inducible factor 1α, thereby disrupting mitochondrial metabolism and dynamics, accelerating cell proliferation and inhibiting apoptosis. Other epigenetic mechanisms, including dysregulation of microRNAs (miR), increase pyruvate dehydrogenase kinase and pyruvate kinase muscle isoform 2 expression in both diseases, favoring uncoupled aerobic glycolysis. This Warburg metabolic shift also accelerates cell proliferation and impairs apoptosis. Disordered mitochondrial dynamics, usually increased mitotic fission and impaired fusion, promotes disease progression in PAH and cancer. Epigenetic upregulation of dynamin-related protein 1 (Drp1) and its binding partners, MiD49 and MiD51, contributes to the pathogenesis of PAH and cancer. Finally, dysregulation of intramitochondrial Ca2+, resulting from impaired mitochondrial calcium uniporter complex (MCUC) function, links abnormal mitochondrial metabolism and dynamics. MiR-mediated decreases in MCUC function reduce intramitochondrial Ca2+, promoting Warburg metabolism, whilst increasing cytosolic Ca2+, promoting fission. Epigenetically disordered mitochondrial O2-sensing, metabolism, dynamics, and Ca2+ homeostasis offer new therapeutic targets for PAH and cancer. Promoting glucose oxidation, restoring the fission/fusion balance, and restoring mitochondrial calcium regulation are promising experimental therapeutic strategies.

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.

Mitochondria in the Pulmonary Vasculature in Health and Disease: Oxygen-Sensing, Metabolism, and Dynamics.

In lung vascular cells, mitochondria serve a canonical metabolic role, governing energy homeostasis. In addition, mitochondria exist in dynamic networks, which serve noncanonical functions, including regulation of redox signaling, cell cycle, apoptosis, and mitochondrial quality control. Mitochondria in pulmonary artery smooth muscle cells (PASMC) are oxygen sensors and initiate hypoxic pulmonary vasoconstriction. Acquired dysfunction of mitochondrial metabolism and dynamics contribute to a cancer-like phenotype in pulmonary arterial hypertension (PAH). Acquired mitochondrial abnormalities, such as increased pyruvate dehydrogenase kinase (PDK) and pyruvate kinase muscle isoform 2 (PKM2) expression, which increase uncoupled glycolysis (the Warburg phenomenon), are implicated in PAH. Warburg metabolism sustains energy homeostasis by the inhibition of oxidative metabolism that reduces mitochondrial apoptosis, allowing unchecked cell accumulation. Warburg metabolism is initiated by the induction of a pseudohypoxic state, in which DNA methyltransferase (DNMT)-mediated changes in redox signaling cause normoxic activation of HIF-1α and increase PDK expression. Furthermore, mitochondrial division is coordinated with nuclear division through a process called mitotic fission. Increased mitotic fission in PAH, driven by increased fission and reduced fusion favors rapid cell cycle progression and apoptosis resistance. Downregulation of the mitochondrial calcium uniporter complex (MCUC) occurs in PAH and is one potential unifying mechanism linking Warburg metabolism and mitochondrial fission. Mitochondrial metabolic and dynamic disorders combine to promote the hyperproliferative, apoptosis-resistant, phenotype in PAH PASMC, endothelial cells, and fibroblasts. Understanding the molecular mechanism regulating mitochondrial metabolism and dynamics has permitted identification of new biomarkers, nuclear and CT imaging modalities, and new therapeutic targets for PAH. © 2020 American Physiological Society. Compr Physiol 10:713-765, 2020.

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.

Increased Drp1-Mediated Mitochondrial Fission Promotes Proliferation and Collagen Production by Right Ventricular Fibroblasts in Experimental Pulmonary Arterial Hypertension.

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

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.

Biventricular Increases in Mitochondrial Fission Mediator (MiD51) and Proglycolytic Pyruvate Kinase (PKM2) Isoform in Experimental Group 2 Pulmonary Hypertension-Novel Mitochondrial Abnormalities.

Introduction: Group 2 pulmonary hypertension (PH), defined as a mean pulmonary arterial pressure ≥25 mmHg with elevated pulmonary capillary wedge pressure >15 mmHg, has no approved therapy and patients often die from right ventricular failure (RVF). Alterations in mitochondrial metabolism, notably impaired glucose oxidation, and increased mitochondrial fission, contribute to right ventricle (RV) dysfunction in PH. We hypothesized that the impairment of RV and left ventricular (LV) function in group 2 PH results in part from a proglycolytic isoform switch from pyruvate kinase muscle (PKM) isoform 1 to 2 and from increased mitochondrial fission, due either to upregulation of expression of dynamin-related protein 1 (Drp1) or its binding partners, mitochondrial dynamics protein of 49 or 51 kDa (MiD49 or 51). Methods and Results: Group 2 PH was induced by supra-coronary aortic banding (SAB) in 5-week old male Sprague Dawley rats. Four weeks post SAB, echocardiography showed marked reduction of tricuspid annular plane systolic excursion (2.9 ± 0.1 vs. 4.0 ± 0.1 mm) and pulmonary artery acceleration time (24.3 ± 0.9 vs. 35.4 ± 1.8 ms) in SAB vs. sham rats. Nine weeks post SAB, left and right heart catheterization showed significant biventricular increases in end systolic and diastolic pressure in SAB vs. sham rats (LV: 226 ± 15 vs. 103 ± 5 mmHg, 34 ± 5 vs. 7 ± 1 mmHg; RV: 40 ± 4 vs. 22 ± 1 mmHg, and 4.7 ± 1.5 vs. 0.9 ± 0.5 mmHg, respectively). Picrosirius red staining showed marked biventricular fibrosis in SAB rats. There was increased muscularization of small pulmonary arteries in SAB rats. Confocal microscopy showed biventricular mitochondrial depolarization and fragmentation in SAB vs. sham cardiomyocytes. Transmission electron microscopy confirmed a marked biventricular reduction in mitochondria size in SAB hearts. Immunoblot showed marked biventricular increase in PKM2/PKM1 and MiD51 expression. Mitofusin 2 and mitochondrial pyruvate carrier 1 were increased in SAB LVs. Conclusions: SAB caused group 2 PH. Impaired RV function and RV fibrosis were associated with increases in mitochondrial fission and expression of MiD51 and PKM2. While these changes would be expected to promote increased production of reactive oxygen species and a glycolytic shift in metabolism, further study is required to determine the functional consequences of these newly described mitochondrial abnormalities.

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

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

MicroRNA-138 and MicroRNA-25 Down-regulate Mitochondrial Calcium Uniporter, Causing the Pulmonary Arterial Hypertension Cancer Phenotype.

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

The role of Drp1 adaptor proteins MiD49 and MiD51 in mitochondrial fission: implications for human disease.

Mitochondrial morphology is governed by the balance of mitochondrial fusion, mediated by mitofusins and optic atrophy 1 (OPA1), and fission, mediated by dynamin-related protein 1 (Drp1). Disordered mitochondrial dynamics alters metabolism, proliferation, apoptosis and mitophagy, contributing to human diseases, including neurodegenerative syndromes, pulmonary arterial hypertension (PAH), cancer and ischemia/reperfusion injury. Post-translational regulation of Drp1 (by phosphorylation and SUMOylation) is an established means of modulating Drp1 activation and translocation to the outer mitochondrial membrane (OMM). This review focuses on Drp1 adaptor proteins that also regulate fission. The proteins include fission 1 (Fis1), mitochondrial fission factor (Mff) and mitochondrial dynamics proteins of 49 kDa and 51 kDa (MiD49, MiD51). Heterologous MiD overexpression sequesters inactive Drp1 on the OMM, promoting fusion; conversely, increased endogenous MiD creates focused Drp1 multimers that optimize OMM scission. The triggers that activate MiD-bound Drp1 in disease states are unknown; however, MiD51 has a unique capacity for ADP binding at its nucleotidyltransferase domain. Without ADP, MiD51 inhibits Drp1, whereas ADP promotes MiD51-mediated fission, suggesting a link between metabolism and fission. Confusion over whether MiDs mediate fusion (by sequestering inactive Drp1) or fission (by guiding Drp1 assembly) relates to a failure to consider cell types used and to distinguish endogenous compared with heterologous changes in expression. We speculate that endogenous MiDs serve as Drp1-binding partners that are dysregulated in disease states and may be important targets for inhibiting cell proliferation and ischemia/reperfusion injury. Moreover, it appears that the composition of the fission apparatus varies between disease states and amongst individuals. MiDs may be important targets for inhibiting cell proliferation and attenuating ischemia/reperfusion injury.

Mechanism of Activation-Induced Downregulation of Mitofusin 2 in Human Peripheral Blood T Cells.

Mitofusin 2 (Mfn2), a mitochondrial protein, was shown to have antiproliferative properties when overexpressed. In this article, we show that activation of resting human peripheral blood T cells caused downregulation of Mfn2 levels. This downregulation of Mfn2 was blocked by different inhibitors (mTOR inhibitor rapamycin, PI3K inhibitor LY294002, and Akt inhibitor A443654), producing cells that were arrested in the G0/G1 stage of the cell cycle. Furthermore, the activation-induced downregulation of Mfn2 preceded the entry of the cells into the cell cycle, suggesting that Mfn2 downregulation is a prerequisite for activated T cell entry into the cell cycle. Accordingly, small interfering RNA-mediated knockdown of Mfn2 resulted in increased T cell proliferation. Overexpression of constitutively active AKT resulted in the downregulation of Mfn2, which can be blocked by a proteasome inhibitor. Akt-mediated downregulation of Mfn2 was via the mTORC1 pathway because this downregulation was blocked by rapamycin, and overexpression of wild-type, but not kinase-dead mTOR, caused Mfn2 downregulation. Our data suggested that activation-induced reactive oxygen species production plays an important role in the downregulation of Mfn2. Collectively, our data suggest that the PI3K-AKT-mTOR pathway plays an important role in activation-induced downregulation of Mfn2 and subsequent proliferation of resting human T cells.