Mitochondrial Fission Protein Drp1 Research Articles

Protective role of mesenchymal stem cells and mesenchymal stem cell-derived exosomes in cigarette smoke-induced mitochondrial dysfunction in mice

BackgroundCigarette smoke (CS)-induced lung inflammation and Chronic Obstructive Pulmonary disease (COPD) involves mitochondrial dysfunction. Mesenchymal stem cells (MSC) and MSC-derived exosomes (EXO) are reported to show therapeutic effects in many animal models of inflammation and injury. In the present study, we determined the role of MSC and EXO intervention in CS-induced lung inflammation with a focus on mitochondrial dysfunction. MethodsEXO were characterized using Western blot for exosomal markers, tunable resistive pulse sensing by qNano and transmission electron microscopy (TEM). Mitochondrial reporter mice (mt-Keima and mito-QC) were exposed to air or CS for 10 days. mt-Keima mice were treated with intraperitoneal injections of MSC or EXO or MSC and EXO (MSC + EXO) for 10 days. Total cell counts, differential cell counts were performed using automated cell counter and flow cytometry respectively. Further, the levels of pro-inflammatory mediators in bronchoalveolar lavage (BAL) fluid were measured using ELISA. Western blot analysis, quantitative PCR, confocal microscopy were used in the current study to determine the effects in the lungs of CS exposed mice. Seahorse flux analyzer was used to measure the oxidative-phosphorylation (OXPHOS) in the BEAS2B cells and BEAS2B - mMSC co-culture experiments. ResultsCS exposure increased the inflammatory cellular infiltrations in the lungs of the mt-Keima mice. MSC + EXO treatment showed protection compared to individual treatments (MSC or EXO alone). There were no changes in the mitophagy proteins like PINK1 and Parkin, which was also found using the mito-QC mice. CS exposure led to significant increase in the mitochondrial fission protein DRP1 and other DAMPs pathway mediators like S100A4 and S100A8, HMGB1, RAGE and AGE. MSC + EXO treatment increased the gene expression of (fusion genes) mfn1, mfn2 and opa1. Additionally, the rhot1 gene expression was increased in MSC + EXO treatment group compared to Air- and CS exposed groups. BEAS2B-mMSC co-cultures showed protective response against the CSE-altered mitochondrial respiration parameters, confirming the beneficial effect of MSC towards human bronchial lung epithelial cells. ConclusionCS affects some of early mitochondrial genes involved in the fission/fusion process, enhancing the damage response along with altered cytokine levels. MSC + EXO combination treatment showed their protective effects. MSC + EXO combination treatment may act against these early events caused by CS exposure owing to its anti-inflammatory and other mitochondrial transfer mechanisms.

Roux-en-Y gastric bypass surgery restores insulin-mediated glucose partitioning and mitochondrial dynamics in primary myotubes from severely obese humans.

Background/Objectives:Impaired insulin-mediated glucose partitioning is an intrinsic metabolic defect in skeletal muscle from severely obese humans (BMI ≥ 40 kg/m2). Roux-en-Y gastric bypass (RYGB) surgery has been shown to improve glucose metabolism in severely obese humans. The purpose of the study was to determine the effects of RYGB surgery on glucose partitioning, mitochondrial network morphology, and markers of mitochondrial dynamics skeletal muscle from severely obese humans.Subject/Methods:Human skeletal muscle cells were isolated from muscle biopsies obtained from RYGB patients (BMI = 48.0 ± 2.1, n=7) prior to, 1-month and 7-months following surgery and lean control subjects (BMI = 22.4 ± 1.1, n=7). Complete glucose oxidation, non-oxidized glycolysis rates, mitochondrial respiratory capacity, mitochondrial network morphology and regulatory proteins of mitochondrial dynamics were determined in differentiated human myotubes.Results:Myotubes derived from severely obese humans exhibited enhanced glucose oxidation (13.5%; 95%CI [7.6, 19.4], P = 0.043) and reduced non-oxidized glycolysis (−1.3%; 95%CI [−11.1, 8.6]) in response to insulin stimulation at 7-months after RYGB when compared to the pre-surgery state (−0.6%; 95%CI [−5.2, 4.0] and 19.5%; 95%CI [4.0, 35.0], P =0.006), and were not different from the lean controls (16.7%; 95%CI [11.8, 21.5] and 1.9%; 95%CI [−1.6, 5.4], respectively). Further, number of fragmented mitochondria and Drp1(Ser616) phosphorylation and were trended to reduced/reduced (0.0104, 95%CI [0.0085, 0.0126], P = 0.091 and 0.0085, 95%CI [0.0068, 0.0102], P = 0.05) in myotubes derived from severely obese humans at 7-months after RYGB surgery in comparison to the pre-surgery state. Finally, Drp1(Ser616) phosphorylation was negatively correlated with insulin-stimulated glucose oxidation (r = −0.49, P = 0.037).Conclusion/Interpretation:These data indicate that an intrinsic metabolic defect of glucose partitioning in skeletal muscle from severely obese humans is restored by RYGB surgery. The restoration of glucose partitioning may be regulated through reduced mitochondrial fission protein Drp1 phosphorylation.

Abstract 549: Cardiac Aging is Associated With Impaired Mitophagy and Formation of Megamitochondria

Aging is associated with increased risk of developing cardiovascular disease. Currently, the molecular mechanisms contributing to the cardiac aging process and the resulting pathogenesis are unclear. Here, we have characterized changes in mitochondria and mitochondrial autophagy (mitophagy) in aging mouse hearts. To evaluate structural and functional changes that occur with cardiac aging, we compared young (4 months) and old (24 month) mice. We found that aging led to cardiac hypertrophy as characterized by increased left ventricular (LV) mass index and heart weight/body weight ratio as well as elevated myosin heavy chain 7 mRNA levels. Although echocardiography analysis showed no detectable changes in systolic function, the aged mice showed evidence of diastolic dysfunction as there was a significant decrease in the E/A ratio. We also found increased levels of fibrosis and inflammation in the aged hearts. Evaluation of autophagy in aged hearts revealed impaired autophagic flux, as indicated by accumulation of the autophagy substrate p62. Treatment of mice with the mTOR inhibitor Rapamycin, led to the expected increase in LC3II-positive autophagosomes in young hearts but not in the aged hearts. Parkin is an E3 ubiquitin ligase involved in labeling mitochondria for mitophagy by ubiquitinating proteins on the outer mitochondrial membrane. We found a significant increase in Parkin at the mitochondria in aged hearts which was accompanied with a significant increase in mitochondrial protein ubiquitination and p62 levels. However, LC3II protein levels in the mitochondrial fraction did not increase. This suggests that although mitochondria have been labeled for mitophagy, they are not eliminated due to decreased formation of autophagosomes. Finally, ultrastructural analysis of hearts showed the presence of enlarged mitochondria in aged hearts. Although we observed no changes in mitochondrial fusion proteins Mfn1/2, we found decreased activation of the mitochondrial fission protein Drp1. Overall, these data suggest that which might contribute to the development of age-related cardiac pathologies.

Altered mitochondrial network morphology and regulatory proteins in mitochondrial quality control in myotubes from severely obese humans with or without type 2 diabetes

Healthy mitochondrial networks are maintained via balanced integration of mitochondrial quality control processes (biogenesis, fusion, fission, and mitophagy). The purpose of this study was to investigate the effects of severe obesity and type 2 diabetes (T2D) on mitochondrial network morphology and expression of proteins regulating mitochondrial quality control processes in cultured human myotubes. Primary human skeletal muscle cells were isolated from biopsies from lean, severely obese nondiabetic individuals and severely obese type 2 diabetic individuals (n = 8-9/group) and were differentiated to myotubes. Mitochondrial network morphology was determined in live cells via confocal microscopy and protein markers of mitochondrial quality control were measured by immunoblotting. Myotubes from severely obese nondiabetic and type 2 diabetic humans exhibited fragmented mitochondrial networks (P < 0.05). Mitochondrial fission protein Drp1 (Ser616) phosphorylation was higher in myotubes from severely obese nondiabetic humans when compared with the lean controls (P < 0.05), while mitophagy protein Parkin expression was lower in myotubes from severely obese individuals with T2D in comparison to the other groups (P < 0.05). These data suggest that regulatory proteins in mitochondrial quality control processes, specifically mitochondrial fission protein Drp1 (Ser616) phosphorylation and mitophagy protein Parkin, are intrinsically dysregulated at cellular level in skeletal muscle from severely obese nondiabetic and type 2 diabetic humans, respectively. These differentially expressed mitochondrial quality control proteins may play a role in mitochondrial fragmentation evident in skeletal muscle from severely obese and type 2 diabetic humans. Novelty Mitochondrial network morphology and mitochondrial quality control proteins are intrinsically dysregulated in skeletal muscle cells from severely obese humans with or without T2D.

Amyloid Beta and Phosphorylated Tau-Induced Defective Autophagy and Mitophagy in Alzheimer's Disease.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by memory loss and multiple cognitive impairments. Several decades of intense research have revealed that multiple cellular changes are implicated in the development and progression of AD, including mitochondrial damage, synaptic dysfunction, amyloid beta (Aβ) formation and accumulation, hyperphosphorylated tau (P-Tau) formation and accumulation, deregulated microRNAs, synaptic damage, and neuronal loss in patients with AD. Among these, mitochondrial dysfunction and synaptic damage are early events in the disease process. Recent research also revealed that Aβ and P-Tau-induced defective autophagy and mitophagy are prominent events in AD pathogenesis. Age-dependent increased levels of Aβ and P-Tau reduced levels of several autophagy and mitophagy proteins. In addition, abnormal interactions between (1) Aβ and mitochondrial fission protein Drp1; (2) P-Tau and Drp1; and (3) Aβ and PINK1/parkin lead to an inability to clear damaged mitochondria and other cellular debris from neurons. These events occur selectively in affected AD neurons. The purpose of our article is to highlight recent developments of a Aβ and P-Tau-induced defective autophagy and mitophagy in AD. This article also summarizes several aspects of mitochondrial dysfunction, including abnormal mitochondrial dynamics (increased fission and reduced fusion), defective mitochondrial biogenesis, reduced ATP, increased free radicals and lipid peroxidation, and decreased cytochrome c oxidase (COX) activity and calcium dyshomeostasis in AD pathogenesis. Our article also discusses how reduced levels of Drp1, Aβ, and P-Tau can enhance the clearance of damaged mitochondria and other cellular debris by autophagy and mitophagy mechanisms.

The role of DRP1 in ropivacaine-induced mitochondrial dysfunction and neurotoxicity

Ropivacaine is a commonly used local anaesthetic, but its side effects remain largely unknown. In the present study, we investigated the side effects of ropivacaine in human neuronal SH-5Y5Y cells. We show that 0.5% and 1% ropivacaine could cause fission-like mitochondrial morphological changes. Ropivacaine exclusively induces mitochondrial fission protein DRP1, generation of ROS and causes mitochondrial dysfunction including decreasing mitochondrial membrane potential, the activity of cytochrome C oxidase and ATP production. The side effects of ropivacaine appear to be dependent on DRP1 expression as silencing of DRP1 in neuronal cells abolishes ropivacaine-induced morphological changes and mitochondrial dysfunction. Silencing of DRP1 prevents ropivacaine-induced cellular LDH release and cell death. Moreover, DRP1-deficient neuronal cells are resistant to ropivacaine-induced apoptosis and silencing of DRP1 rescues the activity of cytochrome C oxidase and cellular ATP production. Collectively, our data indicate that imbalances in mitochondrial dynamics, mitochondrial dysfunction and cell death resulting from ropivacaine are all dependent on DRP1 expression. Our study provides valuable data to assess the safety of ropivacaine.

Therapeutic Telomerase Reactivation for the Prevention of Doxorubicin‐Induced Cardiotoxicity

BackgroundDoxorubicin (DOX) is one of the most potent chemotherapy agents. Its clinical use however, is restricted due to the severe risk of cardiotoxicity, presumably attributed to high levels of reactive oxygen species (ROS) leading to cardiomyocyte death. Telomerase prevents cell death through maintaining telomeres. In addition, telomerase has non‐canonical, anti‐apoptotic and pro‐survival traits, likely owing to detoxification of ROS. However, telomerase is silenced in adult murine and human hearts.PurposeTo test whether pharmacological reactivation of telomerase may be effective as a therapeutic strategy to prevent doxorubicin‐induced cardiotoxicity.Methods and ResultsWe used a AAV9‐based gene therapy approach to reinstate telomerase expression in cardiomyocytes in a mouse model of chronic DOX‐induced cardiotoxicity. Telomerase treated mice presented with preserved heart weights in comparison to AAV‐empty controls after DOX treatment. Histopathological analyses revealed that this rescue was accompanied by preserved cardiomyocyte size and reduced apoptosis rates. This was also reflected by improved cardiac function parameters determined by echocardiography and Millar catheterization. At the molecular level we observed altered mitochondria morphology and reduced mitochondrial fission in response to telomerase expression. In line, Western blotting post telomerase reactivation revealed lower levels of the mitochondrial fission protein Drp1, which plays a known role in cardioprotection. Complementary in vitro experiments confirmed anti‐apoptotic effects of telomerase in DOX treated human induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes.ConclusionOur data suggest that cardiac‐specific telomerase reactivation could be a novel strategy for the prevention of doxorubicin‐induced cardiotoxicity in cancer survivors.Support or Funding InformationThis work was supported by the DFG (BA5631/2‐1 to CB) and ERA‐CVD Network (EXPERT grant to TT).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial Fission Is Required for Blue Light-Induced Apoptosis and Mitophagy in Retinal Neuronal R28 Cells.

Light emitting diodes (LEDs) are widely used to provide illumination due to their low energy requirements and high brightness. However, the LED spectrum contains an intense blue light component which is phototoxic to the retina. Recently, it has been reported that blue light may directly impinge on mitochondrial function in retinal ganglion cells (RGCs). Mitochondria are high dynamic organelles that undergo frequent fission and fusion events. The aim of our study was to elucidate the role of mitochondrial dynamics in blue light-induced damage in retinal neuronal R28 cells. We found that exposure to blue light (450 nm, 1000 lx) for up to 12 h significantly up-regulated the expression of mitochondrial fission protein Drp1, while down-regulating the expression of mitochondrial fusion protein Mfn2 in cells. Mitochondrial fission was simultaneously stimulated by blue light irradiation. In addition, exposure to blue light increased the production of reactive oxygen species (ROS), disrupted mitochondrial membrane potential (MMP), and induced apoptosis in R28 cells. Notably, Drp1 inhibitor Mdivi-1 and Drp1 RNAi not only attenuated blue light-induced mitochondrial fission, but also alleviated blue light-induced ROS production, MMP disruption and apoptosis in cells. Compared with Mdivi-1 and Drp1 RNAi, the antioxidant N-acetyl-L-cysteine (NAC) only slightly inhibited mitochondrial fission, while significantly alleviating apoptosis after blue light exposure. Moreover, we examined markers for mitophagy, which is responsible for the clearance of dysfunctional mitochondria. It was found that blue light stimulated the conversion of LC3B-I to LC3B-II as well as the expression of PINK1 in R28 cells. Mdivi-1 or Drp1 RNAi efficiently inhibited the blue light-induced expression of PINK1 and co-localization of LC3 with mitochondria. Thus, our data suggest that mitochondrial fission is required for blue light-induced mitochondrial dysfunction and apoptosis in RGCs.

Mitochondrial network structure homeostasis and cell death

Mitochondria are the major cellular energy‐producing organelles and intracellular source of reactive oxygen species. These organelles are responsible for driving cell life and death through mitochondrial network structure homeostasis, which is determined by a balance of fission and fusion. Recent advances revealed that a number of components of the fission and fusion machinery, including dynamin‐related protein 1 (Drp1), mitofusin1/2 (Mfn1/2) and Optic atrophy 1 (OPA1), that have been implicated in mitochondrial shape changes are indispensible for autophagy, apoptosis and necroptosis. Drp1 is the main regulator of mitochondrial fission and has become a key point of contention. The controversy focuses on whether Drp1 is directly involved in the regulation of cell death and, if involved, whether is it a stimulator or a negative regulator of cell death. Here, we examine the relevance of the homeostasis of the mitochondrial network structure in 3 different types of cell death, including autophagy, apoptosis and necroptosis. Furthermore, a variety of cancers often exhibit a fragmented mitochondrial phenotype. Thus, the fragmented ratio can reflect tumor progression that predicts prognosis and therapeutic response. In addition, we investigate whether the targeting of the mitochondrial fission protein Drp1 could be a novel therapeutic approach.

Effect of Ginkgo biloba extract (EGb50) on mitochondrial function in SH-SY5Y cells after hypoxia/reoxygenation injury

Ischemic cerebrovascular disease and cerebral ischemia/reperfusion injury threaten the health of human being. We studied the protective effect of Ginkgo biloba extract 50 (EGb50) on the mitochondrial function in SH-SY5Y cells after hypoxia/reoxygenation (H/R) injury and explored its mechanisms, so as to provide new ideas for studies on the treatment for ischemic cerebrovascular disease. We established the H/R injury model in SH-SY5Y cells after administrating EGb50. Subsequently, the mitochondrial membrane potential and the concentration of intracellular Ca²⁺ were measured by flow cytometer. The levels of optic atrophy1 (Opa1) and dynamin-like protein 1 (Drp1) were evaluated by immunofluorescence and western blot. The results showed that the mitochondrial membrane potential was decreased and the level of intracellular Ca²⁺ was increased after H/R injury. Moreover, the expression of mitochondrial fusion protein Opa1 was decreased, while the expression of mitochondrial fission protein Drp1 was increased. However, EGb50 significantly increased the mitochondrial membrane potential and suppressed the level of intracellular Ca²⁺. In addition, EGb50 increased the expression of Opa1 and decreased the expression of Drp1. The results demonstrated that EGb50 has a neuroprotective effect on SH-SY5Y cells after H/R injury, and could improve the energy metabolism and mitochondrial function. The underlying mechanisms may be associated with the regulation of mitochondrial fusion and fission, which provided data support for the treatment of ischemic cerebrovascular disease with EGb50.

Mitochondrial Dynamics in Rat Heart Induced by 5-Fluorouracil.

BackgroundPatients treated with 5-FU can develop rare but potentially severe cardiac effects, including cardiomyopathy, angina pectoris, ventricular tachycardia, heart failure, acute myocardial infarction, and cardiogenic shock. The specific pathologies and mechanisms are not fully understood. Research found that mitochondrial dynamics are widely detected in many angiocardiopathies. Therefore, in the present study we studied the mitochondrial damage and explored the role of mitochondrial fusion/fission proteins on myocardium of rats treated with 5-fluorouracil (5-FU).Material/MethodsThirty male SD rats were randomly divided into 3 groups with 10 rats in each group: (1) control group, (2) low 5-FU group (25 mg/kg), (3) high 5-FU group (50 mg/kg). The animals received intraperitoneal injection for 5 consecutive days. We assessed alterations in mitochondrial morphology, ATP content, mitochondrial membrane potential, and mitochondria fusion/fission proteins expression in hearts of rats receiving intraperitoneal injection with different doses of 5-FU.Results5-FU intraperitoneal injection induced ultra-structural damage in hearts, such as mitochondrial swelling, cristae disorder, and vacuolization. These changes were accompanied by decreases of mitochondrial membrane potential. The low dose of 5-FU led to a slight increase in ATP content. However, the high 5-FU dose caused a more significant reduction compared with the control group. Furthermore, 5-FU intraperitoneal injection significantly increased specific mitochondrial fission proteins (Drp1 and Fis1) and decreased mitochondrial fusion proteins (Opa1, Mfn1, and Mfn2) in rat hearts. However, no changes in cardiac structure and function were detected by echocardiogram. The high dose caused more damage to mitochondrial function than the low dose.ConclusionsMitochondrial damage is a potentially important mechanism and early indicator for 5-FU-induced cardiovascular disease.

Neuronal Mitochondrial Dysfunction Activates the Integrated Stress Response to Induce Fibroblast Growth Factor 21

SummaryStress adaptation is essential for neuronal health. While the fundamental role of mitochondria in neuronal development has been demonstrated, it is still not clear how adult neurons respond to alterations in mitochondrial function and how neurons sense, signal, and respond to dysfunction of mitochondria and their interacting organelles. Here, we show that neuron-specific, inducible in vivo ablation of the mitochondrial fission protein Drp1 causes ER stress, resulting in activation of the integrated stress response to culminate in neuronal expression of the cytokine Fgf21. Neuron-derived Fgf21 induction occurs also in murine models of tauopathy and prion disease, highlighting the potential of this cytokine as an early biomarker for latent neurodegenerative conditions.

Chronic cerebral hypoperfusion accelerates Alzheimer’s disease pathology with the change of mitochondrial fission and fusion proteins expression in a novel mouse model

Mitochondrial dynamically undergo massive fusion and fission events to continuously maintain their function in cells. Although an impaired balance of mitochondrial fission and fusion was reported in in-vitro and in-vivo Alzheimer’s disease (AD) model, changes of mitochondrial fission and fusion proteins have not been reported in AD with chronic cerebral hypoperfusion (HP) as an etiological factor related to the development of elder AD. To clarify the impacts of HP on mitochondrial fission and fusion, related oxidative stress in the pathogenesis of AD, and protective effect of galantamine, the novel AD with HP mouse model (APP23 + HP) was applied in this project. Compared with APP23 mice, APP23 + HP mice greatly enhanced the number of Aβ oligomer-positive/phosphorylated tau (pTau) cells, the expression of mitochondrial fission proteins (Drp1 and Fis1), and decreased the expression of mitochondrial fusion proteins (Opa1 and Mfn1) in the cerebral cortex (CTX) and thalamus (TH) at 12 month (M) of age. Moreover, the expression of peroxidation products (4-HNE and 8-OHdG) showed a significant increase in CTX and TH of APP23 + HP mice at 12 M. However, above neuropathological characteristics were retrieved by galantamine (Gal) treatment, detected through immunohistochemical analyses. The present study demonstrates that cerebral HP shifted the balance in mitochondrial morphology from fusion to fission with increasing Aβ oligomer/pTau accumulations in APP23 mice, and such neuropathologic processes were strongly attenuated by Gal treatment.

Redox Regulation of Mitochondrial Fission Protein Drp1 by Protein Disulfide Isomerase Limits Endothelial Senescence

In BriefKim et al. demonstrate a molecular link between PDIA1 and Drp1 oxidoreduction, which protects against mitochondrial fragmentation and ROS elevation, limiting endothelial senescence. This study provides insights into restoring endothelial PDIA1 function or targeting Drp1 Cys oxidation as potential therapeutic strategies for treating diabetes-associated vascular and metabolic diseases.

Mitochondrial Fission Is Required for Angiotensin II-Induced Cardiomyocyte Apoptosis Mediated by a Sirt1-p53 Signaling Pathway.

Hypertension-induced cardiac apoptosis is a major contributor to early-stage heart-failure. Our previous studies have found that p53-mediated mitochondrial fission is involved in aldosterone-induced podocyte apoptosis. However, it is not clear that whether p53-induced mitochondrial fission is critical for hypertensive Angiotensin II (AngII)-induced cardiomyocyte apoptosis. In this study, we found that inhibition of the mitochondrial fission protein Drp1 (dynamin-related protein 1) by Mdivi-1 prevented cardiomyocyte apoptosis and cardiac remodeling in SHRs. In vitro we found that treatment of cultured neonatal rat cardiomyocytes with AngII induced Drp1 expression, mitochondrial fission, and apoptosis. Knockdown of Drp1 inhibited AngII-induced mitochondrial fission and cardiomyocyte apoptosis. Furthermore, AngII induced p53 acetylation. Knockdown of p53 inhibited AngII-induced Drp1 expression, mitochondrial fission, and cardiomyocyte apoptosis. Besides, we found that Sirt1 was able to reverse AngII-induced p53 acetylation and its binding to the Drp1 promoter, which subsequently inhibited mitochondrial fission and eventually attenuated cardiomyocyte apoptosis. Collectively, these results suggest that AngII degrades Sirt1 to increase p53 acetylation, which induces Drp1 expression and eventually results in cardiomyocyte apoptosis. Sirt1/p53/Drp1dependent mitochondrial fission may be a valuable therapeutic target for hypertension induced heart failure.

Mitochondrial fission protein Drp1 inhibition promotes cardiac mesodermal differentiation of human pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or Mdivi-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with Mdivi-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.

Mitochondrial fission contributes to heat-induced oxidative stress in skeletal muscle but not hyperthermia in mice

AimsWe have previously demonstrated in vitro that heat-induced skeletal muscle damage is associated with an increase in dynamin-related protein 1 (Drp1)-mediated mitochondrial fission and no change in mitochondrial fusion. In this study, we investigated the in vivo effects of mitochondrial fission inhibition on heat-induced oxidative skeletal muscle injury and hyperthermic response in mice. Main methodsCore body temperatures of mice pre-treated with vehicle or Mdivi-1 were recorded by radio telemetry during heat exposure. Tissue samples were obtained immediately following heat exposure. Key findingsWe found that heat exposure caused increased mitochondrial fragmentation and mitochondrial fission protein Drp1 expression, whereas had no effect on the mitochondrial fusion-related proteins mitofusin 1, mitofusin 2 and OPA1 in mouse gastrocnemius muscles. Two groups of mice with a similar high level of heat-induced hyperthermia were allowed to recover for at least one week and subsequently treated with Mdivi-1 and vehicle, respectively. Neither Mdivi-1 nor vehicle altered the hyperthermic responses of mice during heat exposure. However, Mdivi-1 significantly reduced mitochondrial fragmentation and Drp1, reactive oxygen species levels and apoptotic responses in mouse gastrocnemius muscles following heat exposure compared with vehicle. SignificanceThese results suggest that Drp1-mediated mitochondrial fission plays a role in heat-induced oxidative stress in skeletal muscle, but not in hyperthermic response in mice.

Mitochondrial fission as a driver of stemness in tumor cells: mDIVI1 inhibits mitochondrial function, cell migration and cancer stem cell (CSC) signalling.

Mitochondria are dynamic organelles frequently undergoing fission and fusion events to maintain their integrity, bioenergetics and spatial distribution, which is fundamental to the processes of cell survival. Disruption in mitochondrial dynamics plays a role in cancer. Therefore, proteins involved in regulating mitochondrial dynamics are potential targets for treatment. mDIVI1 is an inhibitor of the mitochondrial fission protein DRP1, which induces i) mitochondrial oxidative stress and ii) effectively reduces mitochondrial metabolism. We show here that mDIVI1 is able to inhibit 3D tumorsphere forming capacity, cell migration and stemness-related signalling in breast cancer cells, indicating that mDIVI1 can potentially be used for the therapeutic elimination of cancer stem cells (CSCs).

Chronic dietary creatine enhances hippocampal-dependent spatial memory, bioenergetics, and levels of plasticity-related proteins associated with NF-κB.

The brain has a high demand for energy, of which creatine (Cr) is an important regulator. Studies document neurocognitive benefits of oral Cr in mammals, yet little is known regarding their physiological basis. This study investigated the effects of Cr supplementation (3%, w/w) on hippocampal function in male C57BL/6 mice, including spatial learning and memory in the Morris water maze and oxygen consumption rates from isolated mitochondria in real time. Levels of transcription factors and related proteins (CREB, Egr1, and IκB to indicate NF-κB activity), proteins implicated in cognition (CaMKII, PSD-95, and Egr2), and mitochondrial proteins (electron transport chain Complex I, mitochondrial fission protein Drp1) were probed with Western blotting. Dietary Cr decreased escape latency/time to locate the platform (P < 0.05) and increased the time spent in the target quadrant (P < 0.01) in the Morris water maze. This was accompanied by increased coupled respiration (P < 0.05) in isolated hippocampal mitochondria. Protein levels of CaMKII, PSD-95, and Complex 1 were increased in Cr-fed mice, whereas IκB was decreased. These data demonstrate that dietary supplementation with Cr can improve learning, memory, and mitochondrial function and have important implications for the treatment of diseases affecting memory and energy homeostasis.

Mitochondrial Morphology Controls Hematopoietic Stem Cell Self-Renewal and Confers Them Divisional Memory

Hematopoietic stem cells (HSC) have extensive regenerative potential but limited self-renewal ability. The repopulation potential is gradually lost with HSC divisions and during stress hematopoiesis. Hence, we hypothesize that activation or inactivation of signaling pathways or other mechanisms, inherently occurring during HSC activation into cycle, drives their progressive functional decline. To address this, we used single cell RNA-sequencing and compared the transcriptome of HSCs [Lin-c-Kit+Sca-1+CD48-CD150+] that is expressed upon exit from quiescence and the one that is inherited by daughter cells upon HSC division. We compared HSCs with full self-renewal activity to HSCs that have lost self-renewal activity [i.e. before (NT-HSC) and after transplantation (T-HSC), respectively]. Analysis of the data was performed using unsupervised methods, namely iterative clustering and guide-gene selection (ICGS), [Olsson et al, Nature, 2016] and principal component (PCA) analysis. Each revealed that genes categorized in mitochondrial organization were dramatically upregulated in activated NT-HSC and remained highly expressed in their daughter cells. Remarkably, ICGS revealed at least 2 distinct regulatory states within T-HSC; one that clustered within NT-HSC, and another one that was clearly separated from NT-HSC. The latter was exclusively comprised of daughter T-HSCs, and quite unexpectedly was demarcated by a lack of expression of mitochondrial genes along with changes in expression of genes related to cytoskeleton organization. Using EGFP-mitochondria reporter mice; which labels mitochondria regardless of their membrane potential and activated state, we found that NT-HSC had 4-fold higher mitochondrial content than progenitors. High resolution confocal imaging indicated that mitochondria were punctuated and the network was dispersed in the entire cell volume in NT-HSC. However, in T-HSCs, mitochondria formed one or a few larger aggregates. Mitochondrial membrane potential was also severely reduced in T-HSC in vivo, indicating that HSC accumulate dysfunctional mitochondria after replicative stress during stress hematopoiesis. During cell division, in vitro, mitochondria were unequally distributed to daughter cells of single T-HSC division [one daughter cell had more aggregated mitochondria], whereas mitochondria were equally partitioned during division of NT-HSC, suggesting that HSC lose mitochondrial quality control after replicative stress. Further, mitochondria aligned along the microtubule network in NT-HSC but not in T-HSC. Mitochondrial morphology and distribution are maintained through regulated cycles of fission and fusion, which is essential for maintaining a functional organelle and for equal mitochondrial inheritance during division. We found that the mitochondrial fission protein Drp1, which normally accumulated and wrapped around mitochondria in NT-HSC during cell activation, failed to do so in T-HSCs, perhaps due to impaired mitochondrial-cytoskeleton association. Genetic loss of Drp1 caused severe mitochondrial aggregation in HSCs, and impaired HSC self-renewal and repopulation ability in competitive transplant studies, indicating that accumulation of aggregated and dysfunctional mitochondria causes HSC functional defects. To understand whether HSCs accumulated abnormal mitochondrial morphology with divisional history during homeostatic conditions, we used the GFP label-retaining H2B mouse model. Remarkably, mitochondria were well dispersed and homogeneously distributed in GFP labeled-retaining HSCs (i.e. HSC that had not divided), while HSCs with history of cell division (no GFP) had larger and more compact aggregates. Hence, once HSCs exit from quiescence, they irreversibly accumulate dysfunctional mitochondria over the course of divisions due to lack of mitochondrial quality control mechanisms, which drives gradual heterogeneity and loss of self-renewal ability. Thus, HSCs use mitochondrial architecture to remember their divisions, in turn affecting their self-renewal ability. Therefore, drugs or pharmacological inhibitors targeting mitochondrial organization or its quality can be new therapeutic approach to improve HSC function following transplantation. DisclosuresNo relevant conflicts of interest to declare.