The effects of mitochondrial dysfunction on the cell cycle

Hyperproliferative disorders, such as pulmonary arterial hypertension (PAH) and cancers, are characterized by excessive cell proliferation and resistance to apoptosis (Dasgupta et al., 2021). This “neoplastic phenotype” is due, at least in part, to acquired changes in mitochondrial metabolism. While perturbation of mitochondrial metabolism, notably a shift to aerobic glycolysis (Warburg phenomenon) contributes to the proliferation/apoptosis imbalance in cells from hyperproliferative disease origin, a newly recognized abnormality, namely, dysregulation of mitochondrial dynamics has been identified (Rehman et al., 2012). Mitochondria continuously join together (fusion) and divide (fission) thereby maintaining network quality control (Mao and Klionsky, 2013), mediating cell death (Tian et al., 2017) and regulating metabolism and the cell cycle (Chen et al., 2018). The major mediator of mitochondrial fission is dynamin-related protein 1 (Drp1); while fusion is meditated by mitofusin-1 and mitofusin-2 (Archer, 2013). Upon activation, Drp1 is recruited from the cytosol to the mitochondrial outer membrane (OMM) via interaction with its receptor proteins in a poorly understood multimerization reaction. In mammals, there are four proteins on the mitochondrial outer membrane that act as Drp1 receptors: mitochondrial fission 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) (Atkins et al., 2016). Mitotic fission coordinates mitochondrial and nuclear division, ensuring equitable distribution of mitochondria between daughter cells. Mitotic fission occurs via a Drp1-dependent process. Several studies have shown that inhibition of mitotic fission triggers a cell cycle checkpoint and results in cell cycle arrest and apoptosis (Chen et al., 2018) both in cancers and in non-malignant, hyperproliferative diseases such as PAH (Marsboom et al., 2012). Thus, mitotic fission is an appealing therapeutic target. It has also been shown that Drp1 expression is upregulated in hyperproliferative diseases and Drp1 is postranslationally activated (Marsboom et al., 2012; Rehman et al., 2012; Tian et al., 2018; Abu-Hanna et al., 2023). Inhibition of Drp1 regresses cancer and PAH in animal models (Marsboom et al., 2012; Rehman et al., 2012). In this special edition of Frontiers in Cell and Developmental Biology, four journal articles on The effects of mitochondrial dysfunction on the cell cycle were published: Two original research articles, one review, and one mini-review. Three articles are relevant to pulmonary hypertension (PH), and the other is related to diabetes

Colchicine depolymerizes microtubules, increases junctophilin-2, and improves right ventricular function in experimental pulmonary arterial hypertension

Background Pulmonary arterial hypertension (PAH) is a lethal disease characterized by obstructive pulmonary vascular remodeling and right ventricular (RV) dysfunction. Although RV function predicts outcomes in PAH, mechanisms of RV dysfunction are poorly understood, and RV‐targeted therapies are lacking. We hypothesized that in PAH, abnormal microtubular structure in RV cardiomyocytes impairs RV function by reducing junctophilin‐2 (JPH2) expression, resulting in t‐tubule derangements. Conversely, we assessed whether colchicine, a microtubule‐depolymerizing agent, could increase JPH2 expression and enhance RV function in monocrotaline‐induced PAH. Methods and Results Immunoblots, confocal microscopy, echocardiography, cardiac catheterization, and treadmill testing were used to examine colchicine's (0.5 mg/kg 3 times/week) effects on pulmonary hemodynamics, RV function, and functional capacity. Rats were treated with saline (n=28) or colchicine (n=24) for 3 weeks, beginning 1 week after monocrotaline (60 mg/kg, subcutaneous). In the monocrotaline RV, but not the left ventricle, microtubule density is increased, and JPH2 expression is reduced, with loss of t‐tubule localization and t‐tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves t‐tubule morphology in RV cardiomyocytes. Colchicine therapy diminishes RV hypertrophy, improves RV function, and enhances RV–pulmonary artery coupling. Colchicine reduces small pulmonary arteriolar thickness and improves pulmonary hemodynamics. Finally, colchicine increases exercise capacity. Conclusions Monocrotaline‐induced PAH causes RV‐specific derangement of microtubules marked by reduction in JPH2 and t‐tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves both t‐tubule architecture and RV function. Colchicine also reduces adverse pulmonary vascular remodeling. These results provide biological plausibility for a clinical trial to repurpose colchicine as a RV‐directed therapy for PAH

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

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

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

A Wireless Sensor Network Based Personnel Positioning Scheme in Coal Mines with Blind Areas

This paper proposes a novel personnel positioning scheme for a tunnel network with blind areas, which compared with most existing schemes offers both low-cost and high-precision. Based on the data models of tunnel networks, measurement networks and mobile miners, the global positioning method is divided into four steps: (1) calculate the real time personnel location in local areas using a location engine, and send it to the upper computer through the gateway; (2) correct any localization errors resulting from the underground tunnel environmental interference; (3) determine the global three-dimensional position by coordinate transformation; (4) estimate the personnel locations in the blind areas. A prototype system constructed to verify the positioning performance shows that the proposed positioning system has good reliability, scalability, and positioning performance. In particular, the static localization error of the positioning system is less than 2.4 m in the underground tunnel environment and the moving estimation error is below 4.5 m in the corridor environment. The system was operated continuously over three months without any failures

Excess protein O-GlcNAcylation links metabolic derangements to right ventricular dysfunction in pulmonary arterial hypertension

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

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

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-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 (RVH) [monocrotaline (MCT) and Sugen-5416 hypoxia (SuHx)] were compared with compensated RVH rats [pulmonary artery banding (PAB)]. Echocardiography and right heart catheterization were performed. Macrophages, atrial natriuretic peptide (ANP) 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) and MCC950 (an NLRP3 inhibitor). Macrophage-NLRP3 activity was evaluated in PAH-RVF patients. Measurements and Main Results: Macrophages, fibrosis, and ANP were increased in MCT and SuHx-RVs but not LVs or PAB rats. While MCT-RV macrophages were inflammatory, lung macrophages were anti-inflammatory. CCR2+ macrophages (monocyte-derived) were increased in MCT- and SuHx-RVs and highly expressed NLRP3. The macrophage-NLRP3 pathway was upregulated in PAH patients’ decompensated RVs. Cultured MCT-monocytes showed NLRP3 activation, and in co-culture 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 activation, and improved RV function without regressing pulmonary vascular disease. Conclusion: NLRP3-macrophage activation occurs in the decompensated RV in preclinical PAH models and PAH patients. Inhibiting GP130 or NLRP3 signaling improves RV function. The concept that PAH-RVF results from RV inflammation rather than solely from elevated RV afterload suggest a new therapeutic paradigm