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.

BACKGROUND: Drp1 (dynamin-related protein 1), a large GTPase, mediates the increased mitochondrial fission, which contributes to hyperproliferation of pulmonary artery smooth muscle cells in pulmonary arterial hypertension (PAH). We developed a potent Drp1 GTPase inhibitor, Drpitor1a, but its specificity, pharmacokinetics, and efficacy in PAH are unknown. METHODS: Drpitor1a’s ability to inhibit recombinant and endogenous Drp1 GTPase was assessed. Drpitor1a’s effects on fission were studied in control and PAH human pulmonary artery smooth muscle cells (hPASMC) and blood outgrowth endothelial cells (BOEC). Cell proliferation and apoptosis were studied in hPASMC. Pharmacokinetics and tissue concentrations were measured following intravenous and oral drug administration. Drpitor1a’s efficacy in regressing monocrotaline-PAH was assessed in rats. In a pilot study, Drpitor1a reduced PA remodeling only in females. Subsequently, we compared Drpitor1a to vehicles in control and monocrotaline-PAH females. RESULTS: Drp1 GTPase activity was increased in PAH hPASMC. Drpitor1a inhibited the GTPase activity of recombinant and endogenous Drp1 and reversed the increased fission, seen in PAH hPASMC and PAH BOEC. Drpitor1a inhibited proliferation and induced apoptosis in PAH hPASMC without affecting electron transport chain activity, respiration, fission/fusion mediator expression, or mitochondrial Drp1 translocation. Drpitor1a did not inhibit proliferation or alter mitochondrial dynamics in normal hPASMC. Drpitor1a regressed monocrotaline-PAH without systemic vascular effects or toxicity. CONCLUSIONS: Drpitor1a is a specific Drp1 GTPase inhibitor that reduces mitochondrial fission in PAH hPASMC and PAH BOEC. Drpitor1a reduces proliferation and induces apoptosis in PAH hPASMC and regresses monocrotaline-PAH. Drp1 is a therapeutic target in PAH, and Drpitor1a is a potential therapy with an interesting therapeutic sexual dimorphism.

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.

Background and Aims: Multiple germline gene variants promote familial and idiopathic pulmonary arterial hypertension (PAH); however, none are consistently identified in associated PAH with connective tissue disease (APAH-CTD). Moreover, the role of somatic variants in genes mediating clonal haematopoiesis of indeterminate potential (CHIP) in PAH is unknown. Here, somatic and germline DNMT3A variants and CHIP gene variants in PAH were evaluated. Methods: Exome sequencing (ES) was compared between PAH Biobank participants (n = 1832 European ancestry/2572 total), vs. gnomAD controls (7509 European ancestry/141 456 total). Subsequently, targeted panel sequencing (TPS) of 22 CHIP genes, including DNMT3A, was performed in PAH (n = 1659) vs. controls (n = 3644). Somatic CHIP variants in the UK Biobank using ES (controls = 448 239; PAH = 2559) were also assessed. DNMT3A mRNA expression was measured in peripheral blood mononuclear cells (PBMCs) of patients with scleroderma APAH-CTD (n = 50), idiopathic PAH (n = 30), scleroderma without PAH (n = 19), and healthy controls (n = 41). Hemodynamic were evaluated in haematopoietic Dnmt3a-knockout mice. Results: Predicted deleterious germline DNMT3A variants were increased in subjects of European ancestry (6/1832) vs. controls (6/7509) (relative risk [RR] = 4.1, P = .018). In the entire PAH Biobank cohort (n = 2572), DNMT3A germline and somatic variants were further enriched (PAH: 1.28% vs. controls: .43%, P = 1.65 × 10−10). Eight DNMT3A mutations (.39%) were likely germline (female/male: 7/1) and 25 (.82%) likely somatic (female/male: 21/4), including 13/33 APAH-CTD participants. TPS identified CHIP in 242 PAH subjects (48% DNMT3A). DNMT3A- and all-CHIP variants were associated with PAH after correcting for age, sex, and age–CHIP interactions (odds ratio [OR]: 25.44, P = 4.50 × 10−5; OR: 23.35, P = 2.87 × 10−8, respectively). In the UK Biobank, CHIP mutations were increased in PAH (PAH = 5.35% vs. Control = 3.45%, P ≤ .0001). DNMT3A was reduced in PAH-PBMCs (area under curve [AUC] = .82) (P < .0001). Haematopoietic Dnmt3a-knockout in mice caused inflammatory PAH, which was attenuated by IL-1β antibody therapy. Conclusions: Germline DNMT3A variants and somatic variants of DNMT3A and CHIP genes increase the risk of PAH, including APAH-CTD, promote inflammation, and constitute potential biomarkers and therapeutic targets.

Editorial: The effects of mitochondrial dysfunction on the cell cycle

BACKGROUND: DRP1 (dynamin-related protein 1) mediates mitochondrial fission and permits rapid cell cycle progression in hyperproliferative cells by coordinating nuclear and mitochondrial division, a process called mitotic fission. However, DRP1 alone appears insufficient to complete fission, and the link between fission and cell cycle progression is unknown. We hypothesize that DNM2 (dynamin 2) interacts with DRP1 to complete mitochondrial fission and regulate cell cycle progression. We show that DNM2 is upregulated in pulmonary artery smooth muscle cells (PASMCs) in human and rodent pulmonary arterial hypertension (PAH), contributing to disease pathophysiology. METHODS: Mitochondrial morphology, protein colocalization, and fission were assessed using stimulated emission depletion microscopy, protein interactions by immunoprecipitation, and transcriptomics by RNA sequencing. DNM2 was quantified in PASMC and lungs from patients with PAH and rats with pulmonary hypertension (PH), induced by monocrotaline or sugen5416/hypoxia. siDNM2’s effects on cell proliferation, cell cycle progression, and apoptosis were assessed by flow cytometry. Single-cell RNA sequencing was performed on publicly available data sets. siDNM2 was nebulized to monocrotaline- and sugen5416/hypoxia-PH rats, and disease regression was quantified by cardiac catheterization and histology. RESULTS: DNM2 is increased in PAH PASMC. DNM2 interacts with DRP1 via its GTPase domain, permitting mitochondrial translocation and promoting fission. siDNM2 inhibits fission and cell proliferation and increases apoptosis. siDNM2 causes G1/G0 blockade by downregulating RGCC (regulator of cell cycle) with downstream effects on CDK (cyclin-dependent kinase) 4, cyclin D1, and p27 ^kip1 . Conversely, augmenting DNM2 in normal PASMC induces fission and accelerates proliferation. Upregulation of DNM2 in PAH is due to decreased miR-124-3p (microRNA-124-3p) and activation of STAT3 (signal transducer and activator of transcription 3). An miR-124-3p-STAT3-DNM2-DRP1-RGCC pathway accelerates mitotic fission and is upregulated in PASMC, airway epithelium, endothelial cells, fibroblasts, and macrophages in PAH. Nebulized siDNM2 regresses established PH in vivo in rats of both sexes. CONCLUSIONS: DNM2 is a mediator in the terminal steps of DRP1-dependent fission and constitutes a novel therapeutic target in PAH.