Mitochondrial Division Inhibitor Mdivi-1 Research Articles

The mitochondrial division inhibitor Mdivi-1 protected organ function and extended the treatment window in rats with uncontrolled haemorrhagic shock.

To elucidate whether the application of the mitochondrial division inhibitor Mdivi-1 can protect organ function and prolong the treatment window for traumatic hemorrhagic shock. Before definitive haemostasis treatment, Mdivi-1 (0.25 mg/kg, 0.5 mg/kg and 1 mg/kg) was administered to uncontrolled haemorrhagic shock (UHS) model rats. Lactate Ringer's solution plus hydroxyethyl starch (130/0.4) was used as a control. The effects of Mdivi-1 on blood loss, fluid demand, survival time, vital organ function, myocardial mitochondrial structure, and mitochondrial function of the heart, liver, kidney and intestine, and oxidative stress at 1 hour after hypotensive resuscitation (50-60 mmHg) were investigated. In addition, we investigated the effect of varying doses of Mdivi-1 on the maintenance time of hypotensive resuscitation without definitive haemostasis and the beneficial effect of Mdivi-1 after prolonging the duration of hypotensive resuscitation to 2 hours. Compared to conventional resuscitative fluid, Mdivi-1 significantly reduced blood loss and fluid demand, improved important organ functions during hypotensive resuscitation, improved animal survival and reduced the incidence of early death. Mdivi-1 significantly alleviated oxidative stress injury, reduced mitochondrial damage and restored myocardial mitochondrial structure and mitochondrial function of the heart, liver, kidney and intestine. In addition, Mdivi-1 increased the maintenance time of hypotensive resuscitation and improved rat survival after the duration of hypotensive resuscitation was prolonged to 2 h. Mdivi-1 significantly prolonged the treatment window for traumatic hemorrhagic shock to 2 hours in UHS model rats. The underlying mechanism may be that Mdivi-1 inhibits excessive mitochondrial fission and oxidative stress and improves the structure and function of mitochondria.

SIRT4 Protects Müller Glial Cells Against Apoptosis by Mediating Mitochondrial Dynamics and Oxidative Stress.

SIRT4 is a member of the sirtuin family, which is related to mitochondrial function and possesses antioxidant and regulatory redox effects. Currently, the roles of SIRT4 in retinal Müller glial cells, oxidative stress, and mitochondrial function are still unclear. We confirmed, by immunofluorescence staining, that SIRT4 is located mainly in the mitochondria of retinal Müller glial cells. Using flow cytometry and Western blotting, we analyzed cell apoptosis, intracellular reactive oxygen species (ROS) levels, apoptotic and proapoptotic proteins, mitochondrial dynamics-related proteins, and mitochondrial morphology and number after the overexpression and downregulation of SIRT4 in rMC-1 cells. Neither the upregulation nor the downregulation of SIRT4 alone affected apoptosis. SIRT4 overexpression reduced intracellular ROS, reduced the BAX/BCL2 protein ratio, and increased the L-OPA/S-OPA1 ratio and the levels of the mitochondrial fusion protein MFN2 and the mitochondrial cleavage protein FIS1, increasing mitochondrial fusion. SIRT4 downregulation had the opposite effect. Mitochondria tend to divide after serum starvation for 24 h, and SIRT4 downregulation increases mitochondrial fragmentation and oxidative stress, leading to aggravated cell damage. The mitochondrial division inhibitor Mdivi-1 reduced oxidative stress levels and thus reduced cell damage caused by serum starvation. The overexpression of SIRT4 in rMC-1 cells reduced mitochondrial fragmentation caused by serum starvation, leading to mitochondrial fusion and reduced expression of cleaved caspase-3, thus alleviating the cellular damage caused by oxidative stress. Thus, we speculate that SIRT4 may protect retinal Müller glial cells against apoptosis by mediating mitochondrial dynamics and oxidative stress.

QiDongNing induces lung cancer cell apoptosis via triggering P53/DRP1-mediated mitochondrial fission.

Non-small-cell lung cancer (NSCLC) is a major cause of worldwide cancer death, posing a challenge for effective treatment. Our previous findings showed that Chinese herbal medicine (CHM) QiDongNing (QDN) could upregulate the expression of p53 and trigger cell apoptosis in NSCLC. Here, our objective was to investigate the mechanisms of QDN-induced apoptosis enhancement. We chose A549 and NCI-H460 cells for validation invitro, and LLC cells were applied to form a subcutaneous transplantation tumour model for validation in more depth. Our findings indicated that QDN inhibited multiple biological behaviours, including cell proliferation, cloning, migration, invasion and induction of apoptosis. We further discovered that QDN increased the pro-apoptotic BAX while inhibiting the anti-apoptotic Bcl2. QDN therapy led to a decline in adenosine triphosphate (ATP) and a rise in reactive oxygen species (ROS). Furthermore, QDN elevated the levels of the tumour suppressor p53 and the mitochondrial division factor DRP1 and FIS1, and decreased the mitochondrial fusion molecules MFN1, MFN2, and OPA1. The results were further verified by rescue experiments, the p53 inhibitor Pifithrin-α and the mitochondrial division inhibitor Mdivi1 partially inhibited QDN-induced apoptosis and mitochondrial dysfunction, whereas overexpression of p53 rather increased the efficacy of the therapy. Additionally, QDN inhibited tumour growth with acceptable safety invivo. In conclusion, QDN induced apoptosis via triggering p53/DRP1-mediated mitochondrial fission in NSCLC cells.

Decreased mitophagy aggravates benign prostatic hyperplasia in aged mice through DRP1 and estrogen receptor α

Benign prostatic hyperplasia (BPH) is an age-related disease, whose etiology largely remains unclear. The regulation of mitophagy plays a key role in aging and associated diseases, however, its function in BPH has not been studied. Although the expression of the androgen receptor is primarily implicated in BPH, the estrogen receptor (ER) has been reported to be involved in the development of BPH by mediating the proliferation of prostate cells. Here, we studied the involvement of mitophagy and ERs in spontaneous BPH in aging mice and investigated their functions. To identify the activation of mitophagy and expression of ERs, 8-week, 12-month, and 24-month-old mice were used. Mice were treated with mitochondrial division inhibitor mdivi-1, a dynamin-related protein 1 (Drp1) inhibitor, to examine the expression of mitophagy-related proteins and the development of BPH. In addition, prostate stromal cells were treated with an ER antagonist to investigate the regulation of mitophagy following the expression of ERs. With aging, the Drp1 and phosphorylation of parkin reduce. Electron microscopy revealed reduced mitochondrial fission and mitophagy. In addition, the expression of androgen receptor was decreased and that of ERα was increased in aged mice with BPH. Treatment with mdivi-1 exacerbated BPH and increased cell proliferation. In addition, blockade of ERα increased mitophagy and decreased cell proliferation. In conclusion, mitophagy is reduced with aging during the development of BPH. We speculate that spontaneous BPH progresses through the reduction in the expression of ERα in aged mice by downregulating mitophagy.

Hypoxia promotes pulmonary vascular remodeling via HIF-1α to regulate mitochondrial dynamics.

BackgroundIncreasing research suggests that mitochondrial defect plays a major role in pulmonary hypertension (PH) pathogenesis. Mitochondrial dynamics and quality control have a central role in the maintenance of the cell proliferation and apoptosis balance. However, the molecular mechanism underlying of this balance is still unknown.MethodsTo clarify the biological effects of hypoxic air exposure and hypoxia-inducible factor-1α (HIF-1α) on pulmonary arterial smooth muscle cell (PASMC) and pulmonary arterial hypertension rats, the cells were cultured in a hypoxic chamber under oxygen concentrations. Cell viability, reactive oxygen species level, cell death, mitochondrial morphology, mitochondrial membrane potential, mitochondrial function and mitochondrial biosynthesis, as well as fission-and fusion-related proteins, were measured under hypoxic conditions. In addition, rats were maintained under hypoxic conditions, and the right ventricular systolic pressure, right ventricular hypertrophy index and right ventricular weight/body weight ratio were examined and recorded. Further, we assessed the role of HIF-1α in the development and progression of PH using HIF-1α gene knockdown using small interfering RNA transfection. Mdivi-1 treatment was performed before hypoxia to inhibit dynamin-related protein 1 (Drp1).ResultsWe found that HIF-1α expression was increased during hypoxia, which was crucial for hypoxia-induced mitochondrial dysfunction and hypoxia-stimulated PASMCs proliferation and apoptosis. We also found that targeting mitochondrial fission Drp1 by mitochondrial division inhibitor Mdivi-1 was effective in PH model rats. The results showed that mitochondrial dynamics were involved in the pulmonary vascular remodeling under hypoxia in vivo and in vitro. Furthermore, HIF-1α also modulated mitochondrial dynamics in pulmonary vascular remodeling under hypoxia through directly regulating the expression of Drp1.ConclusionsIn conclusion, our data suggests that abnormal mitochondrial dynamics could be a marker for the early diagnosis of PH and monitoring disease progression. Further research is needed to study the signaling pathways that govern mitochondrial fission/fusion in PH.

Abstract 408: Defective Mitochondrial Dynamics Contribute to Cardiac Contractile Dysfunction in Desminopathy

Rational: Desmin plays a critical role in the maintenance of the structural and mechanical integrity of the sarcomere. Mutation in desmin causes disorganization of contractile apparatus leading to cardiomyopathy. Cardiomyocyte-specific expression of mutated desmin (a seven amino acid deletion R172-E178, D7-Des Tg) causes accumulations of toxic protein aggregates leading to cardiac contractile dysfunction. Whether these alterations result in maladaptive changes in mitochondrial dynamics and contribute to mitochondrial respiratory defects resulting in cardiomyopathies remains obscure. Objective: In this study, we investigated whether altered mitochondrial dynamics play a causative role in the development of cardiac contractile dysfunction and cardiomyocyte death in D7-Des Tg mouse model of desminopathy. Methods and results: D7-Des Tg hearts showed accumulation of desmin aggregates resulting in disruption of the desmin network, development of cardiomyocyte hypertrophy, and contractile dysfunction. Ultrastructural analysis by electron microscopy showed highly perturbed mitochondrial spatial organization and mitochondrial fragmentation in D7-Des Tg mice. D7-Des Tg mice hearts showed increased mtDNA copy number, increased expression of mitochondrial fission regulatory proteins, defects in mitochondrial respiration and activation of apoptotic cell death. Adenoviral-mediated expression of D7-Des in cardiomyocytes also showed increased mitochondrial fission, expression dependent inhibition of mitochondrial respiration and activation of cellular toxicity. Inhibition of mitochondrial fission by mitochondrial division inhibitor mdivi-1 significantly improved mitochondrial respiration and inhibited cellular toxicity associated with D7-Des expression in cardiomyocytes. Conclusion: Aberrant mitochondrial fission results in mitochondrial respiratory defects and apoptotic cell death in a D7-Des Tg hearts. Inhibition of aberrant mitochondrial fission using mitochondrial division inhibitor significantly preserved mitochondrial function and decreased apoptotic cell death. Therefore, our study shows that maladaptive aberrant mitochondrial fission causes desminopathy-associated cellular dysfunction.

Drp1-dependent mitochondrial fission mediates osteogenic dysfunction in inflammation through elevated production of reactive oxygen species.

Although previous studies have implicated pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), to be detrimental for osteogenic activity, the related regulatory mechanisms are not yet fully validated. Since mitochondria host several essential metabolic processes and play a pivotal role in cellular functions, whether and how mitochondrial function contributes to inflammation-induced bone destruction needs further exploration. Our findings revealed that TNF-α impaired osteoblast function, including decreased mRNA levels of osteogenic markers, suppressed ALP expression and activity, and compromised cellular viability. Moreover, increased reactive oxygen species (ROS)-mediated oxidative stress in the TNF-α-treated group enhanced excessive mitochondrial fragmentation and disrupted mitochondrial function. However, treatment with antioxidant N-acetyl cysteine (NAC) or mitochondrial division inhibitor Mdivi-1 protected the cells from these adverse phenomena. These findings provide new insights into the role of the Drp1-dependent mitochondrial pathway in the osteogenic dysfunction during inflammation, indicating that this pathway may be a target for the development of new therapeutic approaches for the prevention and treatment of inflammation-induced bone destruction.

The mitochondrial division inhibitor Mdivi-1 rescues mammalian neurons from anesthetic-induced cytotoxicity.

BackgroundConcerns have risen regarding the potential side effects of clinical exposure of the pediatric population to inhalational anesthetics, and how they might impact cognitive, learning, and memory functions. However, neither the mechanisms of anesthetic cytotoxicity, nor potential protective strategies, have yet been fully explored. In this study, we examined whether two of the most commonly used inhalational anesthetics, sevoflurane and desflurane, affect neuronal viability and synaptic network assembly between cultured rat cortical neurons.ResultsPrimary rat cortical neuron cultures were exposed to equipotent sevoflurane or desflurane for 1 hour. Neuron viability, synaptic protein expression, mitochondrial morphology, and neurite growth were assayed with immunostaining and confocal microscopy techniques. The effects of anesthetics on the functional development of neural networks were evaluated with whole-cell patch clamp recordings of spontaneous synaptic currents. Our results demonstrate that an acute exposure to sevoflurane and desflurane inhibits the development of neurite processes, impacts the mitochondria, and compromises synaptic proteins - concomitant with a reduction in synaptic function in mature networks. Interestingly, pretreatment of neurons with a mitochondrial division inhibitor (Mdivi-1) not only protected mitochondria integrity but also played a protective role against anesthetic-induced structural and functional neurotoxicity.ConclusionsWe show that Mdivi-1 likely plays a protective role against certain harmful effects of general anesthetics on primary rat neuronal cultures. In addition, Mdivi-1 alone plays a direct role in enhancing growth and modulating synaptic activity. This study highlights the importance of further study into possible protective agents against anesthetic neurotoxicity.

The mitochondrial division inhibitor mdivi-1 attenuates spinal cord ischemia–reperfusion injury both in vitro and in vivo: Involvement of BK channels

Mitochondrial division inhibitor (mdivi-1), a selective inhibitor of a mitochondrial fission protein dynamin-related protein 1 (Drp1), has been shown to exert protective effects in heart and cerebral ischemia–reperfusion models. The present study was designed to investigate the beneficial effects of mdivi-1 against spinal cord ischemia–reperfusion (SCIR) injury and its associated mechanisms. SCIR injury was induced by glutamate treatment in cultured spinal cord neurons and by descending thoracic aorta occlusion for 20min in rats. We found that mdivi-1 (10μM) significantly attenuated glutamate induced neuronal injury and apoptosis in spinal cord neurons. This neuroprotective effect was accompanied by decreased expression of oxidative stress markers, inhibited mitochondrial dysfunction and preserved activities of antioxidant enzymes. In addition, mdivi-1 significantly increased the expression of the large-conductance Ca2+- and voltage-activated K+ (BK) channels, and blocking BK channels by paxilline partly ablated mdivi-1 induced protection. The in vivo experiments showed that mdivi-1 treatment (1mg/kg) overtly mitigated SCIR injury induced spinal cord edema and neurological dysfunction with no organ-related toxicity in rats. Moreover, mdivi-1 increased the expression of BK channels in spinal cord tissues, and paxilline pretreatment nullified mdivi-1 induced protection after SCIR injury in rats. Thus, mdivi-1 may be an effective therapeutic agent for SCIR injury via activation of BK channels as well as reduction of oxidative stress, mitochondrial dysfunction and neuronal apoptosis.This article is part of a Special Issue entitled SI: Spinal cord injury.

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes.A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission.Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of mitochondrial fission; and (2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.

Perturbations in Mitochondrial Dynamics Induced by Human Mutant PINK1 Can Be Rescued by the Mitochondrial Division Inhibitor mdivi-1

Mutations in the mitochondrial encoded protein PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive Parkinson disease (PD). In mammalian cells, mutant PINK1 has been reported to promote fission or inhibit fusion in mitochondria; however, the mechanism by which this process occurs remains elusive. Using an ecdysone-inducible expression system in mammalian dopaminergic neuronal cells, we report here that human mutant PINK1 (L347P and W437X) mediates an overall fission effect by increasing the ratio of mitochondrial fission over fusion proteins, leading to excessive dysfunctional fragmented mitochondria. Knocking down endogenous Pink1 produces similar effects. In contrast, overexpressing human wild type PINK1 produces a pro-fusion effect by increasing the ratio of mitochondrial fusion/fission proteins without resulting in functionally compromised mitochondria. Parkin knockdown blocks the imbalance in fission/fusion proteins. Furthermore, overexpressing parkin and ubiquitin increases degradation of the mitochondrial fission hFis1 protein, suggesting PINK1 and parkin maintain proper mitochondrial function and integrity via the fission/fusion machinery. Through genetic manipulations and treatment with the small molecule mitochondrial division inhibitor (mdivi-1), which inhibits DLP1/Drp1, both structural and functional mitochondrial defects induced by mutant PINK1 were attenuated, highlighting a potential novel therapeutic avenue for Parkinson disease.