Mitochondrial Division Inhibitor Research Articles

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis. We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages. We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

MitoQ alleviates LPS-mediated acute lung injury through regulating Nrf2/Drp1 pathway

Lipopolysaccharide (LPS) has been known to cause alveolar epithelial cell (AEC) apoptosis and barrier breakdown that characterize acute lung injury (ALI) and acute respiratory distress syndrome. We aimed to investigate whether mitoquinone (MitoQ), a mitochondria-targeted antioxidant, could alleviate LPS-induced AEC damage in ALI and its underlying mechanisms. In vitro studies in AEC A549 cell line, we noted that LPS could induce dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, AEC apoptosis and barrier breakdown, which could be reversed with MitoQ and mitochondrial division inhibitor 1 treatment. Moreover, the protective role of MitoQ was attenuated with Drp1 overexpression. Nuclear factor E2-related factor 2 (Nrf2) downregulation could block the effect of MitoQ by decreasing the expression of Nrf2 target genes in LPS-treated AEC, such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). Nrf2 gene knockdown in LPS-treated A549 cells prevented the protective effect of MitoQ from decreasing Drp1-mediated mitochondrial fission, AEC apoptosis and barrier breakdown. The lung protective effect of MitoQ by regulating the Drp1-mediated mitochondrial fission, AEC apoptosis and barrier breakdown was further confirmed in vivo with LPS-induced ALI mouse model. Additionally, the protective effect of MitoQ was inhibited by Nrf2 inhibitor ML385. We therefore conclude that MitoQ exerts ALI-protective effects by preventing Nrf2/Drp1-mediated mitochondrial fission, AEC apoptosis as well as barrier breakdown.

High glucose induces Drp1-mediated mitochondrial fission via the Orai1 calcium channel to participate in diabetic cardiomyocyte hypertrophy

Mitochondrial dysfunction and impaired Ca2+ handling are involved in the development of diabetic cardiomyopathy (DCM). Dynamic relative protein 1 (Drp1) regulates mitochondrial fission by changing its level of phosphorylation, and the Orai1 (Ca2+ release-activated calcium channel protein 1) calcium channel is important for the increase in Ca2+ entry into cardiomyocytes. We aimed to explore the mechanism of Drp1 and Orai1 in cardiomyocyte hypertrophy caused by high glucose (HG). We found that Zucker diabetic fat rats induced by administration of a high-fat diet develop cardiac hypertrophy and impaired cardiac function, accompanied by the activation of mitochondrial dynamics and calcium handling pathway-related proteins. Moreover, HG induces cardiomyocyte hypertrophy, accompanied by abnormal mitochondrial morphology and function, and increased Orai1-mediated Ca2+ influx. Mechanistically, the Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1) prevents cardiomyocyte hypertrophy induced by HG by reducing phosphorylation of Drp1 at serine 616 (S616) and increasing phosphorylation at S637. Inhibition of Orai1 with single guide RNA (sgOrai1) or an inhibitor (BTP2) not only suppressed Drp1 activity and calmodulin-binding catalytic subunit A (CnA) and phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) expression but also alleviated mitochondrial dysfunction and cardiomyocyte hypertrophy caused by HG. In addition, the CnA inhibitor cyclosporin A and p-ERK1/2 inhibitor U0126 improved HG-induced cardiomyocyte hypertrophy by promoting and inhibiting phosphorylation of Drp1 at S637 and S616, respectively. In summary, we identified Drp1 as a downstream target of Orai1-mediated Ca2+ entry, via activation by p-ERK1/2-mediated phosphorylation at S616 or CnA-mediated dephosphorylation at S637 in DCM. Thus, the Orai1–Drp1 axis is a novel target for treating DCM.

Mitochondrial Fission-Mediated Lung Development in Newborn Rats With Hyperoxia-Induced Bronchopulmonary Dysplasia With Pulmonary Hypertension.

Background: Bronchopulmonary dysplasia (BPD) is the most common chronic respiratory disease in premature infants. Oxygen inhalation and mechanical ventilation are common treatments, which can cause hyperoxia-induced lung injury, but the underlying mechanism is not yet understood. Mitochondrial fission is essential for mitochondrial homeostasis. The objective of this study was to determine whether mitochondrial fission (dynamin-related protein 1, Drp1) is an important mediator of hyperoxia lung injury in rats.Methods: The animal model of BPD was induced with high oxygen (80–85% O2). Pulmonary histological changes were observed by hematoxylin-eosin (HE) staining. Pulmonary microvessels were observed by immunofluorescence staining of von Willebrand Factor (vWF). Protein expression levels of Drp1 and p-Drp1 (Ser616) were observed using Western Blot. We used echocardiography to measure pulmonary artery acceleration time (PAT), pulmonary vascular resistance index (PVRi), peak flow velocity of the pulmonary artery (PFVP), pulmonary arteriovenous diameter, and pulmonary vein peak velocity. Mitochondrial division inhibitor-1 (Mdivi-1) was used as an inhibitor of Drp1, and administered through intraperitoneal injection (25 mg/kg).Results: Pulmonary artery resistance of the hyperoxide-induced neonatal rat model of BPD increased after it entered normoxic convalescence. During the critical stage of alveolar development in neonatal rats exposed to high oxygen levels for an extended period, the expression and phosphorylation of Drp1 increased in lung tissues. When Drp1 expression was inhibited, small pulmonary vessel development improved and PH was relieved.Conclusion: Our study shows that excessive mitochondrial fission is an important mediator of hyperoxia-induced pulmonary vascular injury, and inhibition of mitochondrial fission may be a useful treatment for hyperoxia-induced related pulmonary diseases.

Regulation of mitochondrial dynamics in 2-methoxyestradiol-mediated osteosarcoma cell death

Osteosarcoma (OS) is one of the most malignant tumors of childhood and adolescence. Research on mitochondrial dynamics (fusion/fission) and biogenesis has received much attention in last few years, as they are crucial for death of cancer cells. Specifically, it was shown that increased expression of the cytoplasmic dynamin-related protein 1 (Drp1) triggers mitochondrial fission (division), which activates BAX and downstream intrinsic apoptosis, effectively inhibiting OS growth. In the presented study, human OS cells (metastatic 143B OS cell line) were incubated with 2-methoxyestradiol (2-ME) at both physiologically and pharmacologically relevant concentrations. Cell viability was determined by the MTT assay. Confocal microscopy and western blot methods were applied to examine changes in Drp1 and BAX protein levels. Mitochondrial Division Inhibitor 1, MDIVI-1, was used in the study to further examine the role of Drp1 in 2-ME-mediated mechanism of action. To determine quantitative and qualitative changes in mitochondria, electron microscopy was used. 2-ME at all used concentrations increased mitochondrial fission and induced autophagy in OS cells. At the concentration of 1 µM 2-ME increased the area density of mitochondria in OS cells. Subsequent, upregulated expression of Drp1 and BAX proteins by 2-ME strongly suggests the activation of the intrinsic apoptosis pathway. We further observed 2-ME-mediated regulation of glycolytic state of OS cells. Therefore, we suggest that changes of mitochondrial dynamics may represent a novel mechanism of anticancer action of 2-ME. This finding may open new approaches to improve the efficacy of chemotherapy in the treatment of OS, however, it has to be confirmed by in vivo studies.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Attenuate Oxygen-Glucose Deprivation/Reperfusion-Induced Microglial Pyroptosis by Promoting FOXO3a-Dependent Mitophagy.

Background Mesenchymal stem cell-derived exosomes (MSC-exos) have been recognized as a promising therapeutic strategy for neonatal hypoxic-ischemic brain damage (HIBD). Recently, microglial pyroptosis was shown to play a vital role in the progression of neonatal HIBD. However, whether MSC-exos improve HIBD by regulating microglial pyroptosis remains unknown. Methods Exosomes were isolated from human umbilical cord mesenchymal stem cells (huMSCs) and identified by transmission electron microscopy (TEM), western blot, and nanoparticle tracking analysis (NTA). BV-2 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to induce microglial ischemia/reperfusion (I/R) in vitro. CCK-8, ELISA, western blot, and Hoechst 33342/PI double staining were performed to detect the pyroptosis of BV-2 cells. Conditioned medium (CM) from BV-2 cells exposed to different treatments was used to investigate its effect on neuronal injury. Moreover, 3-methyladenine (3-MA) and mitochondrial division inhibitor-1 (mdi-1) were used to verify the involvement of mitophagy in the protection of MSC-exos against OGD/R-induced pyroptosis in BV-2 cells. Finally, FOXO3a siRNA was used to investigate the involvement of FOXO3a in MSC-exo-induced mitophagy and pyroptosis inhibition. Results Exosomes from huMSCs were successfully extracted. In OGD/R-exposed BV-2 cells, MSC-exos increased cell viability and decreased the expression of NLRP3, cleaved caspase-1, and GSDMD-N as well as the release of IL-1β and IL-18. Compared with CM from OGD/R-exposed BV-2 cells treated with PBS, CM from OGD/R-exposed BV-2 cells treated with MSC-exos significantly increased the viability of SH-SY5Y cells and decreased LDH release. MSC-exos also increased the expression of TOM20 and COX IV in OGD/R-exposed BV-2 cells. Additionally, 3-MA and mdi-1 attenuated the inhibition of pyroptosis with MSC-exo treatment. Furthermore, FOXO3a siRNA partially abolished the neuroprotective effect of MSC-exos and attenuated mitophagy and pyroptosis inhibition induced by MSC-exo treatment. Conclusions Our findings demonstrated that MSC-exos increased FOXO3a expression to enhance mitophagy, therefore protecting microglia from I/R-induced pyroptosis and alleviating subsequent neuronal injury.

Zika Virus-Induced Neuronal Apoptosis via Increased Mitochondrial Fragmentation.

The 2015 to 2016 outbreak of Zika virus (ZIKV) infections in the Americas coincided with a dramatic increase in neurodevelopmental abnormalities, including fetal microcephaly, in newborns born to infected women. In this study, we observed mitochondrial fragmentation and disrupted mitochondrial membrane potential after 24 h of ZIKV infection in human neural stem cells and the SNB-19 glioblastoma cell line. The severity of these changes correlated with the amount of ZIKV proteins expressed in infected cells. ZIKV infection also decreased the levels of mitofusin 2, which modulates mitochondria fusion. Mitochondrial division inhibitor 1 (Mdivi-1), a small molecule inhibiting mitochondria fission, ameliorated mitochondria disruptions and reduced cell death in ZIKV-infected cells. Collectively, this study suggests that abnormal mitochondrial fragmentation contributes to ZIKV-induced neuronal cell death; rebalancing mitochondrial dynamics of fission-fusion could be a therapeutic strategy for drug development to treat ZIKV-mediated neuronal apoptosis.

Repeated hypoglycemia remodels neural inputs and disrupts mitochondrial function to blunt glucose-inhibited GHRH neuron responsiveness

Hypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counterregulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to these blunted effects are only poorly understood. Here, we report, with ISH, IHC, and the tissue-clearing capability of iDISCO+, that growth hormone releasing hormone (GHRH) neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivation in vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show that low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production, and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule mitochondrial division inhibitor-1, we preserved hypoglycemia sensitivity in vitro and in vivo. Our findings present possible mechanisms for the blunting of the CRR, significantly broaden our understanding of the structure of GHRH neurons, and reveal that mitochondrial dynamics play an important role in HAAF. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent HAAF in patients with diabetes.

Crohn's Disease Pathobiont Adherent-Invasive E coli Disrupts Epithelial Mitochondrial Networks With Implications for Gut Permeability.

Background & AimsAdherent-invasive Escherichia coli are implicated in inflammatory bowel disease, and mitochondrial dysfunction has been observed in biopsy specimens from patients with inflammatory bowel disease. As a novel aspect of adherent-invasive E coli–epithelial interaction, we hypothesized that E coli (strain LF82) would elicit substantial disruption of epithelial mitochondrial form and function.MethodsMonolayers of human colon-derived epithelial cell lines were exposed to E coli–LF82 or commensal E coli and RNA sequence analysis, mitochondrial function (adenosine triphosphate synthesis) and dynamics (mitochondrial network imaging, immunoblotting for fission and fusion proteins), and epithelial permeability (transepithelial resistance, flux of fluorescein isothiocyanate–dextran and bacteria) were assessed.ResultsE coli–LF82 significantly affected epithelial expression of ∼8600 genes, many relating to mitochondrial function. E coli–LF82–infected epithelia showed swollen mitochondria, reduced mitochondrial membrane potential and adenosine triphosphate, and fragmentation of the mitochondrial network: events not observed with dead E coli–LF82, medium from bacterial cultures, or control E coli. Treatment with Mitochondrial Division Inhibitor 1 (Mdivi1, inhibits dynamin-related peptide 1, guanosine triphosphatase principally responsible for mitochondrial fission) or P110 (prevents dynamin-related peptide 1 binding to mitochondrial fission 1 protein) partially reduced E coli–LF82–induced mitochondrial fragmentation in the short term. E coli–LF82–infected epithelia showed loss of the long isoform of optic atrophy factor 1, which mediates mitochondrial fusion. Mitochondrial Division Inhibitor 1 reduced the magnitude of E coli–LF82–induced increased transepithelial flux of fluorescein isothiocyanate dextran. By 8 hours after infection, increased cytosolic cytochrome C and DNA fragmentation were apparent without evidence of caspase-3 or apoptosis inducing factor activation.ConclusionsEpithelial mitochondrial fragmentation caused by E coli–LF82 could be targeted to maintain cellular homeostasis and mitigate infection-induced loss of epithelial barrier function. Data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession numbers GSE154121 and GSE154122 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154121).

Dynamic-related protein 1 inhibitor eases epileptic seizures and can regulate equilibrative nucleoside transporter 1 expression

BackgroundDynamic-related protein 1 (Drp1) is a key protein involved in the regulation of mitochondrial fission, and it could affect the dynamic balance of mitochondria and appears to be protective against neuronal injury in epileptic seizures. Equilibrative nucleoside transporter 1 (ENT1) is expressed and functional in the mitochondrial membrane that equilibrates adenosine concentration across membranes. Whether Drp1 participates in the pathogenesis of epileptic seizures via regulating function of ENT1 remains unclear.MethodsIn the present study, we used pilocarpine to induce status epilepticus (SE) in rats, and we used mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor to Drp1, to suppress mitochondrial fission in pilocarpine-induced SE model. Mdivi-1administered by intraperitoneal injection before SE induction, and the latency to firstepileptic seizure and the number of epileptic seizures was thereafter observed. The distribution of Drp1 was detected by immunofluorescence, and the expression patterns of Drp1 and ENT1 were detected by Western blot. Furthermore, the mitochondrial ultrastructure of neurons in the hippocampal CA1 region was observed by transmission electron microscopy.ResultsWe found that Drp1 was expressed mainly in neurons and Drp1 expression was significantly upregulated in the hippocampal and temporal neocortex tissues at 6 h and 24 h after induction of SE. Mitochondrial fission inhibitor 1 attenuated epileptic seizures after induction of SE, reduced mitochondrial damage and ENT1 expression.ConclusionsThese data indicate that Drp1 is upregulated in hippocampus and temporal neocortex after pilocarpine-induced SE and the inhibition of Drp1 may lead to potential therapeutic target for SE by regulating ENT1 after pilocarpine-induced SE.

Drp1-mediated mitochondrial fission induced autophagy attenuates cell apoptosis caused by 3-chlorpropane-1,2-diol in HEK293 cells

3-chlorpropane-1,2-diol (3-MCPD) is a heat-induced food process contaminant that threatens human health. As the primary target organ, the morphological and functional impairment of kidney and the related mechanism such as apoptosis and mitochondrial dysfunction were observed. However, the precise molecular mechanism remains largely unclear. This study aimed to explore the important role of mitochondrial fission and autophagy in the 3-MCPD-caused apoptosis of human embryonic kidney 293 (HEK293) cells. The results showed that blockage of dynamin-related protein-1 (Drp1) by mitochondrial division inhibitor 1 (Mdivi-1, 15 μM) apparently restored 3-MCPD-induced mitochondrial dysfunction, accompanied by prevented the collapse of mitochondrial membrane potential and ATP depletion, and suppressed the occurrence of autophagy. Induction of autophagy occurred following 2.5–10 mM 3-MCPD treatment for 24 h via AMPK mediated mTOR signaling pathway. Meanwhile, enhancement of autophagy by pretreatment with rapamycin (1 nM) alleviated the loss of cell viability and apoptosis induced by 3-MCPD whereas suppression of autophagy by 3-methyladenine (1 mM) further accelerated apoptosis, which was modulated through the mitochondria-dependent apoptotic pathway. Taking together, this study provides novel insights into the 3-MCPD-induced apoptosis in HEK293 cells and reveals that autophagy has potential as an effective intervention strategy for the treatment of 3-MCPD-induced nephrotoxicity.

Mdivi-1 attenuates oxidative stress and exerts vascular protection in ischemic/hypoxic injury by a mechanism independent of Drp1 GTPase activity

Vascular dysfunctions such as vascular hyporeactivity following ischemic/hypoxic injury are a major cause of death in injured patients. In this study, we showed that treatment with mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor of dynamin-related protein 1 (Drp1), significantly improved vascular reactivity in ischemic rats by attenuating oxidative stress. The antioxidative effects of Mdivi-1 were relatively Drp1-independent, and possibly due to an increase in the levels of the antioxidant enzymes, SOD1 and catalase, as well as to enhanced Nrf2 expression. In addition, we found that while Mdivi-1 had little effect on Drp1 GTPase activity in vascular smooth muscle cells, it inhibited hypoxia-induced Drp1 phosphorylation at Ser-616, reducing excessive mitochondrial fission and slightly enhancing mitochondrial fusion. These effects possibly contributed to vascular protection at an early stage of ischemic/hypoxic injury. Finally, Mdivi-1 stabilized hemodynamics, increased vital organ perfusion, and improved rat survival after ischemic/hypoxic injury, proving a promising therapeutic agent for ischemic/hypoxic injury.

Pleiotropic effects of mdivi-1 in altering mitochondrial dynamics, respiration, and autophagy in cardiomyocytes

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion events to adapt to changes in the cellular environment. Aberrant mitochondrial fission has been associated with several types of cardiovascular dysfunction; inhibition of pathologically aberrant mitochondrial fission has been shown to be cardioprotective. Pathological fission is mediated by the excessive activation of GTPase dynamin-related protein 1 (Drp1), making it an attractive therapeutic target in numerous cardiovascular diseases. Mitochondrial division inhibitor (mdivi-1) is widely used small molecule reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate injury. The purpose of our study was to understand the pleiotropic effects of mdivi-1 on mitochondrial dynamics, mitochondrial respiration, electron transport activities, and macro-autophagy. In this study, we found that mdivi-1 treatment decreased Drp1 expression, proteolytically cleaved L-OPA1, and altered the expression of OXPHOS complex proteins, resulting in increased superoxide production. The altered expression of OXPHOS complex proteins may be directly associated with decreased Drp1 expression, as Drp1 siRNA knockdown in cardiomyocytes showed similar effects. Results from an autophagy flux assay showed that mdivi-1 induced impaired autophagy flux that could be restored by Atg7 overexpression, suggesting that mdivi-1 mediated inhibition of macro-autophagy in cardiomyocytes. Treatment with mdivi-1 resulted in increased expression of p62, which is required for Atg7 overexpression-induced rescue of mdivi-1-mediated impaired autophagy flux. In addition, mdivi-1-dependent proteolytic processing of L-OPA1 was associated with increased mitochondrial superoxide production and altered expression of mitochondrial serine/proteases. Overall, the novel pleiotropic effect of mdivi-1 in cardiomyocytes included proteolytically cleaved L-OPA1, altered expression of OXPHOS complex proteins, and increased superoxide production, which together resulted in defects in mitochondrial respiration and inhibition of macro-autophagy.

Drp1-dependent mitochondrial fission contributes to Cr(VI)-induced mitophagy and hepatotoxicity

Hexavalent chromium [Cr(VI)] is seriously harmful to ecosystems and living organisms due to its strong toxicity. Role of dynamin-related protein 1 (Drp1) and Drp1-associated mitochondrial fragmentation in mitophagy and cytotoxicity after Cr(VI) exposure has not been clarified so far. We confirmed that Cr(VI) caused mitochondrial fission by up-regulating Drp1 expression and enhancing Drp1 mitochondrial translocation. By applying the intracellular Ca2+ antagonist BAPTA-AM and mitochondrial Ca2+ antagonist Ru360, we demonstrated that Cr(VI)-induced excessive mitochondrial fission was in a Ca2+-Drp1 dependent manner. The administration of Drp1 siRNA significantly suppressed the overactivation of mitophagy in Cr(VI)-induced hepatotoxicity. The specific Drp1 inhibitor mitochondrial division inhibitor-1 (Mdivi-1) blocked the overactive mitophagy and subsequently ameliorated hepatotoxicity caused by Cr(VI) in vivo. We reached the conclusion that Drp1-dependent mitochondrial fission contributes to Cr(VI)-induced mitophagy and hepatotoxicity, which may provide experimental basis for the study of chromium-associated toxicity, especially for the prevention of health damage in chromium-exposed population.

Mitochondrial division inhibitor 1 (mdivi-1) increases oxidative capacity and contractile stress generated by engineered skeletal muscle.

In skeletal muscle fibers, mitochondria are densely packed adjacent to myofibrils because adenosine triphosphate (ATP) is needed to fuel sarcomere shortening. However, despite this close physical and biochemical relationship, the effects of mitochondrial dynamics on skeletal muscle contractility are poorly understood. In this study, we analyzed the effects of Mitochondrial Division Inhibitor 1 (mdivi-1), an inhibitor of mitochondrial fission, on the structure and function of both mitochondria and myofibrils in skeletal muscle tissues engineered on micromolded gelatin hydrogels. Treatment with mdivi-1 did not alter myotube morphology, but did increase the mitochondrial turbidity and oxidative capacity, consistent with reduced mitochondrial fission. Mdivi-1 also significantly increased basal, twitch, and tetanus stresses, as measured using the Muscular Thin Film (MTF) assay. Finally, mdivi-1 increased sarcomere length, potentially due to mdivi-1-induced changes in mitochondrial volume and compression of myofibrils. Together, these results suggest that mdivi-1 increases contractile stress generation, which may be caused by an increase in maximal respiration and/or sarcomere length due to increased volume of individual mitochondria. These data reinforce that mitochondria have both biochemical and biomechanical roles in skeletal muscle and that mitochondrial dynamics can be manipulated to alter muscle contractility.

Inhibiting Drp1-Mediated Mitochondrial Fission Attenuates High-Fat Diet Induced Skeletal Muscle Insulin Resistance

Dynamin-related protein 1 (Drp1) is a critical mediator of mitochondrial fission. Recent studies have reported increased Drp1 activation in obese skeletal muscle, which is associated with reduced mitochondrial function and insulin sensitivity. PURPOSE: To determine if inhibiting Drp1-mediated mitochondrial fission by a pharmacologic inhibitor attenuates skeletal muscle insulin resistance induced by a high-fat diet (HFD) in mice. METHODS: 6-week old male C57BL/6J mice (n=9/group) were assigned to either a HFD (45% Fat) or low-fat diet (LFD, 10% Fat) group for a total of 5-weeks. A subgroup of HFD-fed mice received intraperitoneal injections of Mitochondrial Division Inhibitor 1 (MDIVI-1) (20 mg/kg) while the other mice received saline every other day for the last week of diet intervention. A glucose tolerance test was performed after 4 hours of fasting. Twenty-four hours after the final injection, quadriceps and gastrocnemius muscles were collected for further analysis. H2O2 levels were detected using Amplex Red Hydrogen Peroxide kit. Insulin signaling and protein markers of mitochondrial dynamics were measured by immunoblot analysis. RESULTS: HFD significantly increased glucose area under the curve (AUC) than LFD mice (19074 ± 2137 vs. 10726 ± 1254; p < 0.05), but MDIVI-1 treatment attenuated glucose AUC in HFD-fed mice (15354 ± 1278). MDIVI-1 treatment reduced Drp1 expression in mitochondrial fraction from HFD-fed mice when compared to the saline-treated counterparts (0.003 ± 0.001 vs. 0.012 ± 0.003; p < 0.05). The mitochondrial fraction Drp1 expression was positively associated with glucose AUC (r = 0.497, p = 0.042). Insulin-stimulated Aktser473 phosphorylation was reduced in insulin-stimulated skeletal muscle from HFD-fed mice compared to mice fed with LFD (0.70 ± 0.22 vs. 2.21 ± 0.39; p < 0.05), but no difference was found between HFD-fed mice treated with MDIVI-1 and LFD-fed mice. HFD had a significant increase in H2O2 than LFD-fed mice (0.351 ± 0.046 ± 0.186 ± 0.043; p < 0.05), while MDIVI-1 treatment reduced H2O2 production.in HFD-fed mice (0.351 ± 0.046 vs. 0.215 ± 0.364; p < 0.05) CONCLUSION: These data suggest that inhibiting Drp1-mediated mitochondrial fission attenuates skeletal muscle insulin resistance and improves whole-body glucose homeostasis in mice fed by HFD.

Drp-1 as Potential Therapeutic Target for Lipopolysaccharide-Induced Vascular Hyperpermeability.

Mitochondria-dependent apoptotic signaling has a critical role in the pathogenesis of vascular hyperpermeability (VH). Dynamin-related protein-1- (Drp-1-) mediated mitochondrial fission plays an important role in mitochondrial homeostasis. In the present study, we studied the involvement of Drp-1 in resistance to VH induced by lipopolysaccharide (LPS). To establish the model of LPS-induced VH, LPS at 15 mg/kg was injected into rats in vivo and rat pulmonary microvascular endothelial cells were exposed to 500 ng/ml LPS in vitro. We found that depletion of Drp-1 remarkedly exacerbated the mitochondria-dependent apoptosis induced by LPS, as evidenced by reduced apoptosis, mitochondrial membrane potential (MMP) depolarization, and activation of caspase-3 and caspase-9. Increased FITC-dextran flux indicated endothelial barrier disruption. In addition, overexpression of Drp-1 prevented LPS-induced endothelial hyperpermeability and upregulated mitophagy, as evidenced by the loss of mitochondrial mass and increased PINK1 expression and mitochondrial Parkin. However, the mitophagy inhibitor, 3-Methyladenine, blocked these protective effects of Drp-1. Furthermore, inhibition of Drp-1 using mitochondrial division inhibitor 1 markedly inhibited LPS-induced mitophagy and aggravated LPS-induced VH, as shown by increased FITC-dextran extravasation. These findings implied that Drp-1 strengthens resistance to mitochondria-dependent apoptosis by regulating mitophagy, suggesting Drp-1 as a possible therapeutic target in LPS-induced VH.

Phosphoglycerate Mutase 5 Knockdown Alleviates Neuronal Injury After Traumatic Brain Injury Through Drp1-Mediated Mitochondrial Dysfunction.

Aims: Traumatic brain injury (TBI) is a major cause of disability and death, and a better understanding of the underlying mechanisms of mitochondrial dysfunction will provide important targets for preventing damage from neuronal insults. Phosphoglycerate mutase 5 (PGAM5) is localized to the mitochondrial outer-inner membrane contact sites, and the PGAM5-Drp1 pathway is involved in mitochondrial dysfunction and cell death. The purpose of this project was to evaluate the effects of PGAM5 on neuronal injury and mitochondrial dysfunction. Results: PGAM5 was overexpressed in mice subjected to TBI and in primary cortical neurons injured by mechanical equiaxial stretching. PGAM5 deficiency alleviated neuroinflammation, blocked Parkin, PINK1, and Drp1 translocation to mitochondria and abnormal phosphorylation of Drp1, mitochondrial ultrastructural changes, and nerve malfunction in TBI mouse model. PGAM5-shRNA (short hairpin RNA) reduced Drp1 translocation and activation, including dephosphorylation of p-Drp1 on Ser622 (human Drp1 Ser616) and phosphorylation of Drp1 on Ser643 (human Drp1 Ser637). The levels of inflammatory cytokines, the degree of mitochondrial impairment (mitochondrial membrane potential, ADP/ATP, AMP/ADP, antioxidant capacity), and neuronal injury in stretch-induced primary cortical neurons were reduced by blocking expression of PGAM5. The inhibition of PGAM5 is neuroprotective via attenuation of Drp1 activation, similar to that achieved by mitochondrial division inhibitor-1 (Mdivi1)-mediated Drp1 inhibition. Innovation and Conclusion: Our findings demonstrate the critical role of PGAM5 in progression of neuronal injury from TBI via Drp1 activation (dephosphorylation of p-Drp1 on Ser622 and phosphorylation of Drp1 on Ser643)-mediated mitochondrial dysfunction. The data may open a window for developing new drugs to prevent the neuropathology of TBI.

Melatonin attenuates vascular calcification by inhibiting mitochondria fission via an AMPK/Drp1 signalling pathway.

Mitochondrial fission plays a role in cardiovascular calcification. Melatonin has previously been shown to protect against cardiovascular disease, so this study sought to explore whether it attenuates vascular calcification by regulating mitochondrial fission via the AMP‐activated protein kinase/dynamin‐related protein 1 (AMPK/Drp1) signalling pathway. The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualized by Alizarin red staining, while calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure the expression of runt‐related transcription factor 2 (Runx2), Drp1 and cleaved caspase 3. Melatonin markedly reduced calcium deposition and ALP activity. Runx2 and cleaved caspase 3 were down‐regulated, Drp1 was reduced in response to melatonin, and this was accompanied by decreased apoptosis. Melatonin also reduced levels of mitochondrial superoxide, reversed β‐glycerophosphate (β‐GP)‐induced ΔΨm dissipation and decreased mitochondrial fragmentation. The effects of melatonin in β‐GP‐treated VSMCs were similar to those of mitochondrial division inhibitor 1. Melatonin significantly activated the expression of AMPK and decreased Drp1 expression. Treatment with compound C ablated the observed benefits of melatonin treatment. These findings indicate that melatonin protects VSMCs against calcification by inhibiting mitochondrial fission via the AMPK/Drp1 pathway.

Involvement of Senescence and Mitochondrial Fission in Endothelial Cell Pro-Inflammatory Phenotype Induced by Angiotensin II.

Angiotensin II (AngII) has a crucial role in cardiovascular pathologies, including endothelial inflammation and premature vascular aging. However, the precise molecular mechanism underlying aging-related endothelial inflammation induced by AngII remains elusive. Here, we have tested a hypothesis in cultured rat aortic endothelial cells (ECs) that the removal of AngII-induced senescent cells, preservation of proteostasis, or inhibition of mitochondrial fission attenuates the pro-inflammatory EC phenotype. AngII stimulation in ECs resulted in cellular senescence assessed by senescence-associated β galactosidase activity. The number of β galactosidase-positive ECs induced by AngII was attenuated by treatment with a senolytic drug ABT737 or the chemical chaperone 4-phenylbutyrate. Monocyte adhesion assay revealed that the pro-inflammatory phenotype in ECs induced by AngII was alleviated by these treatments. AngII stimulation also increased mitochondrial fission in ECs, which was mitigated by mitochondrial division inhibitor-1. Pretreatment with mitochondrial division inhibitor-1 attenuated AngII-induced senescence and monocyte adhesion in ECs. These findings suggest that mitochondrial fission and endoplasmic reticulum stress have causative roles in endothelial senescence-associated inflammatory phenotype induced by AngII exposure, thus providing potential therapeutic targets in age-related cardiovascular diseases.