Regulation Of Mitochondrial Fusion Research Articles

Resveratrol alleviates hyperglycemia-induced cardiomyocyte hypertrophy by maintaining mitochondrial homeostasis via enhancing SIRT1 expression

To investigate whether resveratrol alleviates hyperglycemia-induced cardiomyocyte hypertrophy by enhancing the expression of silent information regulation 2 homolog 1 (SIRT1) to maintain mitochondrial homeostasis. Rat cardiomyocytes H9c2 cells with or without lentivirus-mediated mRNA interference of SIRT1 were cultured in high glucose (HG) and treated with resveratrol for 72 h. The changes in superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, reactive oxygen species (ROS) level, and relative surface of the cells were examined, and the mRNA expressions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and protein expressions of SIRT1, mitochondrial fusion related proteins optic atrophy protein 1 (OPA1) and mitofusin 2, mitochondrial division related proteins dynamin-related protein 1 (DRP1) and fission protein 1 (FIS1), and mitophagy-related proteins BNIP3L and LC3 were detected using RT-qPCR and Western blotting. HG exposure significantly decreased SOD activity, increased MDA content, ROS production, relative cell surface, and the mRNA expressions of ANF and BNP in the cardiomyocytes; the protein expressions of SIRT1, OPA1, mitofusin 2 and BNIP3L and LC3-Ⅱ/LC3-Ⅰ ratio were all decreased and the protein expressions of DRP1 and FIS1 increased in HG-exposed cells (P<0.01). All these changes in HG-exposed cardiomyocytes were significantly alleviated by treatment with resveratrol (P<0.05). The protective effects of resveratrol against HG exposure in the cardiomyocytes were obviously attenuated by transfection of the cells with si-SIRT1 (P<0.05). Resveratrol inhibits hyperglycemia-induced cardiomyocyte hypertrophy by reducing oxidative stress, the mechanisms of which involve enhancement of SIRT1 protein expression, regulation of mitochondrial fusion and division balance, and promoting BNIP3L-mediated mitophagy to maintain mitochondrial homeostasis in the cells.

ACC1 Inhibition Enhances Treg Gvhd Treatment Efficacy through Regulation of Mitochondrial Fusion and Elongation

Regulatory T-cells (Treg) play a critical role in preventing autoimmune and alloimmune reactions. Clinical trials demonstrated that Treg adoptive transfer significantly reduced but did not uniformly eliminate grades II-IV graft-vs-host disease (GVHD). Treg more dominantly rely on fatty acid oxidation (FAO) as a fuel source, in contrast to glycolytic conventional T-cells. We hypothesized that increasing FAO would augment Treg efficacy by increasing potency and persistence, thereby ultimately decreasing the Treg cell number required to enhance Treg-mediated GVHD reduction. The FAO and fatty acid synthesis (FAS) pathways compete for acetyl-CoA use. Acetyl-CoA carboxylase-1 (ACC1) skews acetyl-CoA toward FAS and results in downstream inhibition of CPT1a, the rate-limiting enzyme in FAO. ACC1 deletion or inhibition skews naive CD4 T-cell toward FAO, favoring Treg over Th17 differentiation. To precisely determine the role of ACC1 on in vivo Treg-mediated suppression of GVHD, we tested the effects of a 2-hour treatment of murineTreg with ND630, a pharmacological inhibitor of ACC1 currently in clinical trials for hepatic steatosis, or Treg-specific ACC1 knockout (KO). ACC1 inhibition or KO increased in vitro Treg suppressive function, and when infused into mice with established bronchiolitis obliterans (BOS) and chronic GVHD (cGVHD), ACC1KO Treg were more potent than wild-type Treg in reversing BOS pulmonary resistance (FigA), a finding consistent in each of 3 replicate transplants. Mechanistically, both ND630 treated Treg and ACC1KO Treg demonstrated an increased oxygen consumption rate (OCR), a surrogate for mitochondrial oxidative phosphorylation (OXPHOS), a critical source of ATP for Treg. This was associated with an increase in uptake of BoDipyC16, a synthetic fatty acid, suggesting exogenous fatty acids may drive the increase in OXPHOS through FAO. IACS, an OXPHOS inhibitor, abrogated OCR augmentation and precluded increased suppressive function after ND630 treatment, indicating a critical role for OXPHOS in driving Treg suppressor function. To directly evaluate effects on Treg mitochondria, where OXPHOS occurs, Treg were stained with MitoTracker dyes that measure mitochondrial mass and membrane potential, two key surrogate measurements for mitochondrial function. Compared to controls, ND630 treated and ACC1KO Treg each showed increased mitochondrial mass and membrane potential, as well as enhanced mitochondrial polarization (membrane potential to mass ratio), which is required for full Treg functionality. Expansion and scanning electron microscopic analysis of Treg mitochondria revealed enhanced mitochondrial fusion and elongation after ND630 treatment ( FigB). This mitochondrial structural change boosts OXPHOS, consistent with a direct effect of ACC1 inhibition/KO on mitochondrial fitness. In light of these mitochondrial morphology changes, we hypothesized that directly modifying the mitochondria itself, without inhibiting ACC1, might result in augmented Treg function. Consistent with this hypothesis, a 2-hour treatment with the combination of M1 (mitochondrial fusion promoter) and mDIVI1 (mitochondrial fission inhibitor), significantly enhanced Treg function in vitro, and enhanced Treg OCR to a similar degree as ND630. Electron microscopy confirmed that M1/mDIVI1 treatment resulted in a similar degree of mitochondrial fusion as ND630 treatment. Similar to ACC1KO Treg, M1/mDIVI1 treated Treg were more potent than control Treg in reversing established cGVHD. To further explore mitochondrial fusion effects on Treg function, we used siRNA to knockdown the critical mitochondrial fusion protein mitofusin 1 (MFN1). A 50% decrease in MFN1 protein significantly decreased Treg in vitro functionality and OCR, and treatment of MFN1 knockdown Treg with ND630 had no functional or metabolic effect (FigC), pointing to mitochondrial fusion as a critical component of the mechanism for ACC1 inhibition/KO. In summary, ACC1 inhibition or deletion amplifies murine Treg potency resulting in superior metabolic fitness, and greater efficacy in treating cGVHD mice with established BOS, in part through enhancing mitochondrial fusion. Since ND630 is already under investigation in patients, ACC1 inhibition with ND630 could be readily translatable for clinical trials with a goal of utilizing Tregs with inhibited or deleted ACC1 to improve GVHD therapy.

SIRT4 is a regulator of human skeletal muscle fatty acid metabolism influencing inner and outer mitochondrial membrane-mediated fusion

ObjectiveThe mitochondrial phenotype, governed by the balance of fusion-fission, is a key determinant of energy metabolism. The inner and outer mitochondrial membrane (IMM) fusion proteins optic atrophy 1 (OPA1) and Mitofusin 1 and 2 (Mfn1/2) play an important role in this process. Recent evidence also shows that Sirtuin 4 (SIRT4), located within the mitochondria, is involved in the regulation of fatty acid oxidation. The purpose of this study was to determine if SIRT4 expression regulates inner and outer mitochondrial-mediated fusion and substrate utilization within differentiated human skeletal muscle cells (HSkMC). Material and methodsSIRT4 expression was knocked down using small interfering RNA (siRNA) transfection in differentiated HSkMC. Following knockdown, mitochondrial respiration was determined by high-resolution respirometry (HRR) using the Oroboros Oxygraph O2k. Live cell confocal microscopy, quantified using the Mitochondrial Network Analysis (MiNA) toolset, was used to examine mitochondrial morphological change. This was further examined through the measurement of key metabolic and mitochondrial morphological regulators (mRNA and protein) induced by knockdown. ResultsSIRT4 knockdown resulted in a significant decrease in LEAK respiration, potentially explained by a decrease in ANT1 protein expression. Knockdown further increased oxidative phosphorylation and protein expression of key regulators of fatty acid metabolism. Quantitative analysis of live confocal imaging of fluorescently labelled mitochondria following SIRT4 knockdown supported the role SIRT4 plays in the regulation of mitochondrial morphology, as emphasized by an increase in mitochondrial network branches and junctions. Measurement of key regulators of mitochondrial dynamics illustrated a significant increase in mitochondrial fusion proteins Mfn1, OPA1 respectively, indicative of an increase in mitochondrial size. ConclusionsThis study provides evidence of a direct relationship between the mitochondrial phenotype and substrate oxidation in HSkMC. We identify SIRT4 as a key protagonist of energy metabolism via its regulation of IMM and OMM fusion proteins, OPA1 and Mfn1. SIRT4 knockdown increases mitochondrial capacity to oxidize fatty acids, decreasing LEAK respiration and further increasing mitochondrial elongation via its regulation of mitochondrial fusion.

PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics

Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.

MFN2 deficiency promotes cardiac response to hypobaric hypoxia by reprogramming cardiomyocyte metabolism.

Under hypobaric hypoxia (HH), the heart triggers various defense mechanisms including metabolic remodeling against lack of oxygen. Mitofusin 2 (MFN2), located at the mitochondrial outer membrane, is closely involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the role of MFN2 in cardiac response to HH has not been explored. Loss- and gain-of-function approaches were used to investigate the role of MFN2 in cardiac response to HH. In vitro, the function of MFN2 in the contraction of primary neonatal rat cardiomyocytes under hypoxia was examined. Non-targeted metabolomics and mitochondrial respiration analyses, as well as functional experiments were performed to explore underlying molecular mechanisms. Our data demonstrated that, following 4 weeks of HH, cardiac-specific MFN2 knockout (MFN2 cKO) mice exhibited significantly better cardiac function than control mice. Moreover, restoring the expression of MFN2 clearly inhibited the cardiac response to HH in MFN2 cKO mice. Importantly, MFN2 knockout significantly improved cardiac metabolic reprogramming during HH, resulting in reduced capacity for fatty acid oxidation (FAO) and oxidative phosphorylation, and increased glycolysis and ATP production. In vitro data showed that down-regulation of MFN2 promoted cardiomyocyte contractility under hypoxia. Interestingly, increased FAO through palmitate treatment decreased contractility of cardiomyocyte with MFN2 knockdown under hypoxia. Furthermore, treatment with mdivi-1, an inhibitor of mitochondrial fission, disrupted HH-induced metabolic reprogramming and subsequently promoted cardiac dysfunction in MFN2-knockout hearts. Our findings provide the first evidence that down-regulation of MFN2 preserves cardiac function in chronic HH by promoting cardiac metabolic reprogramming.

MFN2-mediated mitochondrial fusion facilitates acute hypobaric hypoxia-induced cardiac dysfunction by increasing glucose catabolism and ROS production

BackgroundRapid ascent to high-altitude environment which is characterized by acute hypobaric hypoxia (HH) may increase the risk of cardiac dysfunction. However, the potential regulatory mechanisms and prevention strategies for acute HH-induced cardiac dysfunction have not been fully clarified. Mitofusin 2 (MFN2) is highly expressed in the heart and is involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the significance of MFN2 in the heart under acute HH has not been investigated. Methods and resultsOur study revealed that MFN2 upregulation in hearts of mice during acute HH led to cardiac dysfunction. In vitro experiments showed that the decrease in oxygen concentration induced upregulation of MFN2, impairing cardiomyocyte contractility and increasing the risk of QT prolongation. Additionally, acute HH-induced MFN2 upregulation promoted glucose catabolism and led to excessive mitochondrial reactive oxygen species (ROS) production in cardiomyocytes, ultimately resulting in decreased mitochondrial function. Furthermore, co-immunoprecipitation (co-IP) and mass spectrometry analyses indicated that MFN2 interacted with the NADH-ubiquinone oxidoreductase 23 kDa subunit (NDUFS8). Specifically, acute HH-induced MFN2 upregulation increased NDUFS8-dependent complex I activity. ConclusionsTaken together, our studies provide the first direct evidence that MFN2 upregulation exacerbates acute HH-induced cardiac dysfunction by increasing glucose catabolism and ROS production. General significanceOur studies indicate that MFN2 may be a promising therapeutic target for cardiac dysfunction under acute HH.

MIRO-1 interacts with VDAC-1 to regulate mitochondrial membrane potential in Caenorhabditis elegans.

Precise regulation of mitochondrial fusion and fission is essential for cellular activity and animal development. Imbalances between these processes can lead to fragmentation and loss of normal membrane potential in individual mitochondria. In this study, we show that MIRO-1 is stochastically elevated in individual fragmented mitochondria and is required for maintaining mitochondrial membrane potential. We further observe a higher level of membrane potential in fragmented mitochondria in fzo-1 mutants and wounded animals. Moreover, MIRO-1 interacts with VDAC-1, a crucial mitochondrial ion channel located in the outer mitochondrial membrane, and this interaction depends on the residues E473 of MIRO-1 and K163 of VDAC-1. The E473G point mutation disrupts their interaction, resulting in a reduction of the mitochondrial membrane potential. Our findings suggest that MIRO-1 regulates membrane potential and maintains mitochondrial activity and animal health by interacting with VDAC-1. This study provides insight into the mechanisms underlying the stochastic maintenance of membrane potential in fragmented mitochondria.

High-fat diet causes mitochondrial damage and downregulation of mitofusin-2 and optic atrophy-1 in multiple organs.

High-fat consumption promotes the development of obesity, which is associated with various chronic illnesses. Mitochondria are the energy factories of eukaryotic cells, maintaining self-stability through a fine-tuned quality-control network. In the present study, we evaluated high-fat diet (HFD)-induced changes in mitochondrial ultrastructure and dynamics protein expression in multiple organs. C57BL/6J male mice were fed HFD or normal diet (ND) for 24 weeks. Compared with ND-fed mice, HFD-fed mice exhibited increased body weight, cardiomyocyte enlargement, pulmonary fibrosis, hepatic steatosis, renal and splenic structural abnormalities. The cellular apoptosis of the heart, liver, and kidney increased. Cellular lipid droplet deposition and mitochondrial deformations were observed. The proteins related to mitochondrial biogenesis (TFAM), fission (DRP1), autophagy (LC3 and LC3-II: LC3-I ratio), and mitophagy (PINK1) presented different changes in different organs. The mitochondrial fusion regulators mitofusin-2 (MFN2) and optic atrophy-1 (OPA1) were consistently downregulated in multiple organs, even the spleen. TOMM20 and ATP5A protein were enhanced in the heart, skeletal muscle, and spleen, and attenuated in the kidney. These results indicated that high-fat feeding caused pathological changes in multiple organs, accompanied by mitochondrial ultrastructural damage, and MFN2 and OPA1 downregulation. The mitochondrial fusion proteins may become promising targets and/or markers for treating metabolic disease.

ALDH2 attenuates ischemia and reperfusion injury through regulation of mitochondrial fusion and fission by PI3K/AKT/mTOR pathway in diabetic cardiomyopathy

The function of mitochondrial fusion and fission is one of the important factors causing ischemia-reperfusion (I/R) injury in diabetic myocardium. Aldehyde dehydrogenase 2 (ALDH2) is abundantly expressed in heart, which involved in the regulation of cellular energy metabolism and stress response. However, the mechanism of ALDH2 regulating mitochondrial fusion and fission in diabetic myocardial I/R injury has not been elucidated. In the present study, we found that the expression of ALDH2 was downregulated in rat diabetic myocardial I/R model. Functionally, the activation of ALDH2 resulted in the improvement of cardiac hemodynamic parameters and myocardial injury, which were abolished by the treatment of Daidzin, a specific inhibitor of ALDH2. In H9C2 cardiomyocyte hypoxia-reoxygenation model, ALDH2 regulated the dynamic balance of mitochondrial fusion and fission and maintained mitochondrial morphology stability. Meanwhile, ALDH2 reduced mitochondrial ROS levels, and apoptotic protein expression in cardiomyocytes, which was associated with the upregulation of phosphorylation (p-PI3KTyr458, p-AKTSer473, p-mTOR). Moreover, ALDH2 suppressed the mitoPTP opening through reducing 4-HNE. Therefore, our results demonstrated that ALDH2 alleviated the ischemia and reperfusion injury in diabetic cardiomyopathy through inhibition of mitoPTP opening and activation of PI3K/AKT/mTOR pathway.

A Germline‐Specific Regulator of Mitochondrial Fusion is Required for Maintenance and Differentiation of Germline Stem and Progenitor Cells (Adv. Sci. 36/2022)

A Germline‐Specific Regulator of Mitochondrial Fusion is Required for Maintenance and Differentiation of Germline Stem and Progenitor Cells (Adv. Sci. 36/2022)

PGC-1α Affects Epileptic Seizures by Regulating Mitochondrial Fusion in Epileptic Rats.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), regulated by AMPK, is an important regulator of mitochondrial fusion. At present, whether the AMPK/PGC-1α signaling pathway regulates mitochondrial dynamics in epileptic rats is still unknown. Adult male Sprague-Dawley (SD) rats were randomly divided into fourgroups: the control group (0.9% saline, n = 5), the EP groups (lithium-pilocarpine was used to induce epilepsy, and tissues were harvested at 6 and 24h, every time point, n = 5), the EP + Compound C group (the specific inhibitor of PGC-1α, 15mg/kg in 2% DMSO, n = 5), and the EP + DMSO group (0.9% saline + 2% DMSO, n = 5). To investigate whether PGC-1α participates in seizures by regulating the expression of mitofusin1/2(MFN1/2)in rats. In this study, the behavioral results indicate that the seizure susceptibility of the rats to epilepsy was increased when the expression of PGC-1α was inhibited. Subsequently, Western blot results suggested that the expression level of both MFN1 and MFN2 in the hippocampus was higher at 6 and 24h after an epileptic seizure. Besides, the expression of PGC-1α and MFN2 was significantly decreased in the hippocampus when the epileptic rats were treated with Compound C. Furthermore, the immunofluorescence analysis of the localization of MFN1/2 and PGC-1α showed that MFN1/2 was mainly expressed in neurons but not astrocytes in the hippocampus and cerebral cortex of rats. Meanwhile, PGC-1α colocalized with the excitatory post-synaptic marker PSD95, suggesting that PGC-1α may regulate the seizure susceptibility of the rats by mediating excitatory post-synaptic signaling. The AMPK/PGC-1α signaling pathway may play an important role in the lithium-pilocarpine-induced epileptic rat model by mediating the expression of fusion proteins.

A Germline-Specific Regulator of Mitochondrial Fusion is Required for Maintenance and Differentiation of Germline Stem and Progenitor Cells.

Maintenance and differentiation of germline stem and progenitor cells (GSPCs) is important for sexual reproduction. Here, the authors identify zebrafish pld6 as a novel germline-specific gene by cross-analyzing different RNA sequencing results, and find that pld6 knockout mutants develop exclusively into infertile males. In pld6 mutants, GSPCs fail to differentiate and undergo apoptosis, leading to masculinization and infertility. Mitochondrial fusion in pld6-depleted GSPCs is severely impaired, and the mutants exhibit defects in piRNA biogenesis and transposon suppression. Overall, this work uncovers zebrafish Pld6 as a novel germline-specific regulator of mitochondrial fusion, and highlights its essential role in the maintenance and differentiation of GSPCs as well as gonadal development and gametogenesis.

Abstract P3088: The Histone Lysine Methyltransferase SMYD1a Protects Heart From Ischemic Injury By Regulating OPA1 Mediated Cristae Remodeling And Supercomplex Formation

The histone lysine methyltransferase SMYD1 has been shown to be critical for embryonic cardiac development and in maintaining cardiomyocyte homeostasis in adult mice. Subsequently, we reported that loss of Smyd1 in the adult mouse myocardium leads to progressive cardiac hypertrophy and heart failure, which is accompanied with downregulation of mitochondrial proteins involved in oxidative phosphorylation, including Ppargc1a , and reduction of mitochondrial respiration capacity. To build upon these results and evaluate if SMYD1a can attenuate disease-induced remodeling in an animal model, we generated transgenic mice which inducibly express SMYD1a (the human ortholog) in cardiomyocytes and subjected them to permanent occlusion (PO) of the LAD. This lead to &gt;50% reduction in infarct size and preserved cardiac function, as compared to littermate controls. Additionally, we demonstrated that under physiological conditions SMYD1a maintains metabolic homeostasis by regulating expression of Ppargc1a and its downstream targets, including components of the electron transport chain. Our molecular analysis shows that observed protection from ischemic injury results from enhanced mitochondrial respiration through Complex I and II as well as increased ATP production. This is associated with increased mitochondria cristae, and formation and stabilization of respiratory chain supercomplexes within the cristae. These changes in cristae structure occur concomitant with enhanced OPA1 expression, a major regulator of mitochondrial fusion and cristae morphology. Through this work we have established that OPA1 is a novel, functionally important downstream target of SMYD1a by which cardiomyocytes upregulate energy efficiency, protecting them from ischemic injury. These results also highlight SMYD1a as the only known epigenetic regulator of cristae morphology and provide broad implications for understanding the epigenetic mechanisms driving cardiac metabolism. Ultimately this work has identified a novel signaling pathway by which cardiomyocytes regulate energy efficiency, protecting them from ischemic injury.

Regulation of mitochondrial fusion and mitophagy by intra-tumoral delivery of membrane-fused mitochondria or Midiv-1 enhances sensitivity to doxorubicin in triple-negative breast cancer

Increasing mitochondrial fusion by intra-tumoral grafting of membrane-fused mitochondria created with Pep-1 conjugation (P-Mito) contributes to breast cancer treatment, but it needs to be validated. Using mitochondrial division inhibitor-1 (Mdivi-1, Mdi) to disturb mitochondrial dynamics, we showed that the antitumor action of P-Mito in a mouse model of triple-negative breast cancer depends upon mitochondrial fusion and that Mdi treatment alone is ineffective. P-Mito significantly enhanced Doxorubicin (Dox) sensitivity by inducing mitochondrial fusion and mitophagy, and the same efficiency was also achieved with Mdi by inhibiting mitophagy. Cell death was induced via the p53 pathway and AIF nuclear translocation in the case of P-Mito, versus the caspase-dependent pathway for Mdi. Notably, both mitochondrial treatments reduced oxidative stress and blood vessel density of xenograft tumors, especially P-Mito, which was accompanied by inhibition of nuclear factor kappa-B activation. Furthermore, through enrichment analysis, four microRNAs in serum microvesicles induced by P-Mito caused expression of predicted targets via the PI3K-Akt pathway, and significantly impacted regulation of nuclear processes and myeloid cell differentiation. Clustering of gene-sets implicated a major steroid catabolic network. This study showed diverse roles of mitochondria in breast cancer and revealed effective adjuvant therapy targeting mitochondrial fusion and mitophagy.

Impact of exercise training on mitochondrial dynamics during ischemia-reperfusion

Mitochondrial fission is a key trigger of cardiac ischemia-reperfusion injuries (IR) injuries. Despite exercise training (ExTr) is known as an efficient strategy to protect the heart, its impact on the dynamic of the mitochondrial network remain to be investigated. To evaluate the impact of ExTr on the process of mitochondrial fission during IR. Isolated hearts from ExTr or Sedentary (Sed) rats were subjected to IR. Hearts samples were collected after 10 min of reperfusion for biochemical assays or electron microscopic analysis. Finally, DHR123 was used to detect ROS production on isolated cardiomyocytes treated or not with the mitochondrial fission inhibitor Mdivi1. We confirmed on isolated rat hearts that ExTr reduced heart sensitivity to IR. This was associated with lower ROS production evaluated by DHE fluorescence and higher mitochondria Ca2+ retention capacities. We next confirmed that IR was associated with mitochondrial fission as well as the translocation and activation of the Dynamin related protein 1 (Drp1), known as the central regulator of mitochondrial fission. Interestingly, ExTr limited both the activation of Drp1 and the fission of mitochondria. We also reported that ExTr tended to increase the level of Mfn2, known as a central regulator of mitochondrial fusion. Finally, on isolated cardiomyocytes stimulated with H2O2 to mimic IR, we reported that Mdivi1 impact the level of ROS production only in Sed cells. This confirms that mitochondrial fission was more pronounced in Sed cells and contributes to increased ROS production during IR. Altogether, our results support the hypothesis that ExTr is able to modulate the dynamic of the mitochondrial network during IR, which could participate to exercise-induced cardioprotection.

SS31 Alleviates Pressure Overload-Induced Heart Failure Caused by Sirt3-Mediated Mitochondrial Fusion

Heart failure caused by pressure overload is one of the leading causes of heart failure worldwide, but its pathological origin remains poorly understood. It remains critical to discover and find new improvements and treatments for pressure overload-induced heart failure. According to previous studies, mitochondrial dysfunction and myocardial interstitial fibrosis are important mechanisms for the development of heart failure. The oligopeptide Szeto-Schiller Compound 31 (SS31) can specifically interact with the inner mitochondrial membrane and affect the integrity of the inner mitochondrial membrane. Whether SS31 alleviates pressure overload-induced heart failure through the regulation of mitochondrial fusion has not yet been confirmed. We established a pressure-overloaded heart failure mouse model through TAC surgery and found that SS31 can significantly improve cardiac function, reduce myocardial interstitial fibrosis, and increase the expression of optic atrophy-associated protein 1 (OPA1), a key protein in mitochondrial fusion. Interestingly, the role of SS31 in improving heart failure and reducing fibrosis is inseparable from the presence of sirtuin3 (Sirt3). We found that in Sirt3KO mice and fibroblasts, the effects of SS31 on improving heart failure and improving fibroblast transdifferentiation were disappeared. Likewise, Sirt3 has direct interactions with proteins critical for mitochondrial fission and fusion. We found that SS31 failed to increase OPA1 expression in both Sirt3KO mice and fibroblasts. Thus, SS31 can alleviate pressure overload-induced heart failure through Sirt3-mediated mitochondrial fusion. This study provides new directions and drug options for the clinical treatment of heart failure caused by pressure overload.

MFN2 knockdown promotes osteogenic differentiation of iPSC-MSCs through aerobic glycolysis mediated by the Wnt/\u03b2-catenin signaling pathway

BackgroundMitofusin-2 (MFN2) is a kind of GTPase that participates in the regulation of mitochondrial fusion, which is related to a variety of physiological and pathological processes, including energy metabolism, cell differentiation, and embryonic development. However, it remains unclear whether MFN2 is involved in the metabolism and osteogenic differentiation of mesenchymal stem cells (MSCs).MethodsMFN2 knockdown (MFN2-KD) and MFN2-overexpressing (MFN2-OE) induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) were constructed by lentivirus. The commercial kits were utilized to detect the glycolysis and oxidative phosphorylation (OXPHOS) rate. Flow cytometry, Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), RNA-seq, immunofluorescence, and immunoprecipitation were employed for phenotype and molecular mechanism assessment.ResultsWe demonstrated that MFN2 and Wnt/β-catenin signaling pathway regulated glycolysis of iPSC-MSCs. The lack of MFN2 promoted the osteogenic differentiation of iPSC-MSCs, and aerobic glycolysis in the presence of sufficient oxygen, which increased glucose consumption and lactic acid production, as well as the glycolytic enzyme activity and gene expression. Inhibiting the Wnt/β-catenin signaling pathway normalized the enhanced glycolytic rate and osteogenic differentiation of MFN2-KD iPSC-MSCs. MFN2-OE iPSC-MSCs displayed the opposite phenotype.ConclusionsDownregulating MFN2 promotes osteogenic differentiation of iPSC-MSCs through aerobic glycolysis mediated by the Wnt/β-catenin signaling pathway. Our research reveals the new function of MFN2 in regulating the osteogenic differentiation and energy metabolism of MSCs, which will provide a new therapeutic target and theoretical basis for alveolar bone repair and periodontal regenerative treatment.

NR4A1 Promotes LPS-Induced Acute Lung Injury through Inhibition of Opa1-Mediated Mitochondrial Fusion and Activation of PGAM5-Related Necroptosis.

Mitochondrial dysfunction and necroptosis have been perceived as the primary molecular mechanisms underscoring acute lung injury. Meanwhile, nuclear receptor subfamily 4 group A member 1 (NR4A1) is considered a regulator of inflammation-related endothelial injury in lung tissue although the downstream molecular events remain elusive. In this study, we employed NR4A1−/− mice to decipher the role of NR4A1 in the onset and progression of acute lung injury with a focus on mitochondrial damage and necroptosis. Our results demonstrated that NR4A1 was significantly upregulated in lipopolysaccharide- (LPS-) treated lung tissues. Knockout of NR4A1 overtly improved lung tissue morphology, inhibited inflammation, and reduced oxidative stress in LPS-treated lung tissue. A cell signaling study suggested that NR4A1 deletion repressed levels of PGAM5 and attenuated LPS-mediated necroptosis in primary murine alveolar epithelial type II (ATII) cells, the effects of which were mitigated by PGAM5 overexpression. Moreover, LPS-mediated mitochondrial injury including mitochondrial membrane potential collapse and mitochondrial oxidative stress was drastically improved by NR4A1 deletion. Furthermore, NR4A1 deletion preserved mitochondrial homeostasis through activation of Opa1-related mitochondrial fusion. Silencing of Opa1 triggered mitochondrial dysfunction in NR4A1-deleted ATII cells. Taken together, our data identified NR4A1 as a novel regulator of LPS-related acute lung injury through regulation of mitochondrial fusion and necroptosis, indicating therapeutic promises of targeting NR4A1 in the treatment of acute lung injury in clinical practice.

Schizosaccharomyces pombe Fzo1 is subjected to the ubiquitin-proteasome-mediated degradation during the stationary phase.

Mitochondria are highly dynamic organelles that undergo fission and fusion to adapt to the metabolic needs of the cell. Mitofusins are dynamin-like GTPases that play a key role in the regulation of mitochondrial fusion and metabolism. In Saccharomyces cerevisiae, mitofusin Fzo1 levels are controlled by post-translational ubiquitination and degradation. However, it is not clear whether the levels of the Schizosaccharomyces pombe mitofusin Fzo1 are similarly regulated. In this study, we examined the expression S. pombe Fzo1 during normal growth. We showed that Fzo1 protein levels but not mRNA expression levels were reduced during the stationary phase. The protein was stabilized by the proteasome inhibitor bortezomib. Disruption of ubc8 encoding a ubiquitin-conjugating enzyme and rsv2 encoding an S. pombe homolog of S. cerevisiae RPN4 known for activating the expression of genes required for proteasomal biogenesis suppresses the proteasomal degradation of Fzo1 during the stationary phase. Overexpression of fzo1 prevents its degradation. Our results suggest that like S. pombe Fzo1 expression is not regulated by transcription but rather by proteolytic degradation during the stationary phase. Our findings also suggest that although S. cerevisiae and S. pombe Fzo1 proteins are regulated by ubiquitin-proteasomal degradation, different ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3) are involved in their degradation.

MiR-125b Disrupts Mitochondrial Dynamics via Targeting Mitofusin 1 in Cisplatin-Induced Acute Kidney Injury

Background: Mitochondria are dynamic organelles whose structure are maintained by continuous fusion and fission. During acute kidney injury (AKI) progression, mitochondrial fission in renal tubular cells was elevated, characterized by mitochondrial fragmentation. It is tightly associated with mitochondrial dysfunction, which has been proven as a critical mechanism responsible for AKI. However, the initiating factor for the disruption of mitochondrial dynamics in AKI was not well understood. Objectives: To explore the molecular mechanisms of mitochondrial disorders and kidney damage. Methods: We established cisplatin-induced AKI model in C57BL/6 mice and proximal tubular cells, and detected the expression of miR-125b by qPCR. Then we delivered miR-125b antagomir after cisplatin treatment in mice via hydrodynamic-based gene transfer technique. Subsequently, we performed luciferase reporter and immunoblotting ­assays to prove miR-125b could directly modulate mitofusin1 (MFN1) expression. We also tested the role of miR-125b in mitochondrial and renal injury through immunofluorescent staining, qPCR, and immunoblotting assays. Results: miR-125b levels were induced in cisplatin-challenged mice and cultured tubular cells. Anti-miR-125b could effectively alleviate cisplatin-induced mitochondrial fragmentation and kidney injury both in vitro and in vivo. Furthermore, miR-125b could directly regulate MFN1, which is a key regulator of mitochondrial fusion. Our study indicated that miR-125b is upregulated during cisplatin-induced AKI. Inhibition of miR-125b may suppress mitochondrial and renal damage through upregulating MFN1. This study suggests that miR-125b could be a potential therapeutic target in AKI.