Promotion Of Mitochondrial Fusion Research Articles

Dysregulation of mitochondrial dynamics and mitophagy are involved in high-fat diet-induced steroidogenesis inhibition

Male obesity is a pandemic health issue and can disrupt testicular steroidogenesis. Here, we explored the mechanism by which a high-fat diet (HFD) induced steroidogenic inhibition. As expected, HFD induced lipid droplet accumulation and reduced the expression of StAR, P450scc, and 3β-HSD, three steroidogenic enzymes, in mouse testes. Palmitic acid (PA), a saturated fatty acid usually used to trigger lipotoxicity in vitro, induced greater accumulation of lipid droplets and the downregulation of steroidogenic enzymes in TM3 cells. Mechanistically, both HFD and PA disturbed mitochondrial fusion/fission dynamics and then induced mitochondrial dysfunction and mitophagy inhibition in mouse Leydig cells. Additionally, mitochondrial fusion promoter M1 attenuated PA-induced imbalance of mitochondrial dynamics, mitophagy inhibition, mitochondrial reactive oxygen species (ROS) production, and mitochondrial dysfunction in TM3 cells. Mitofusin 2 (Mfn2) knock-down further aggravated the PA-induced imbalance of mitochondrial dynamics, mitochondrial ROS production, and mitochondrial dysfunction in TM3 cells. Importantly, M1 rescued PA-induced downregulation of steroidogenic enzymes, whereas Mfn2 knock-down further aggravated PA-induced downregulation of steroidogenic enzymes in TM3 cells. Overall, our results provide laboratory evidence that mitochondrial dysfunction and mitophagy inhibition caused by dysregulation of mitochondrial fusion may be involved in HFD-induced steroidogenesis inhibition in mouse Leydig cells.

METTL3-mediated NDUFB5 m6A modification promotes cell migration and mitochondrial respiration to promote the wound healing of diabetic foot ulcer

BackgroundDiabetic foot ulcer (DFU) is the most devastating complication of diabetes mellitus (DM) and plays a major role in disability and death in DM patients. NADH: ubiquinone oxidoreductase subunit B5 (NDUFB5) plays an important role in maintaining mitochondrial respiration, but whether it is involved in regulating the progression of advanced glycation end products (AGEs)-mediated DFU is still unclear.MethodsFirstly, the role of AGEs on cell viability, migration, and mitochondrial respiration in human umbilical vein endothelial cells (HUVECs) was explored in vitro. Next, NDUFB5 expression was detected in human samples and AGEs-treated HUVECs, and NDUFB5’s effect on AGEs-induced HUVECs injury and skin wound in diabetic mice was further clarified. In addition, the role of m6A modification mediated by methyltransferase-like 3 (METTL3) in regulating NDUFB5 expression and AGEs-induced HUVECs injury was investigated.ResultsNDUFB5 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs, whereas mitochondrial fusion promoter M1 facilitated cell viability, migration, and mitochondrial oxiadative respiration in NDUFB5 knockdown HUVECs. Meanwhile, NDUFB5 promotes skin wound healing in diabetic mice. Besides, METTL3-mediated m6A modification and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced NDUFB5 expression in HUVECs. Furthermore, METTL3 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs by increasing NDUFB5.ConclusionMETTL3-mediated NDUFB5 m6A modification inhibits AGEs-induced cell injury in HUVECs. METTL3 and NDUFB5 might serve as potential targets for DFU therapy in the future.

Triphenyl phosphate induces cardiotoxicity through myocardial fibrosis mediated by apoptosis and mitophagy of cardiomyocyte in mice

Triphenyl phosphate (TPHP) is an organophosphorus flame retardant, but its cardiac toxicity has not been adequately investigated. Therefore, in the current study, the effect of TPHP on the heart and the underlying mechanism involved was evaluated. C57BL/6 J mice were administered TPHP (0, 5, and 50 mg/kg/day) for 30 days. In addition, H9c2 cells were treated with three various concentrations (0, 50, and 150 μM) of TPHP, with and without the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine or the mitochondrial fusion promoter M1. TPHP caused cardiac fibrosis and increased the levels of CK-MB and LDH in the serum. TPHP increased the levels of ROS, malondialdehyde (MDA), and decreased the level of superoxide dismutase (SOD) and Glutathione peroxidase (GSH-Px). Furthermore, TPHP caused mitochondrial damage, and induced fusion and fission disorders that contributed to mitophagy in both the heart of C57BL/6 J mice and H9c2 cells. Transcriptome analysis showed that TPHP induced up- or down-regulated expression of various genes in myocardial tissue and revealed enriched apoptosis pathways. It was also found that TPHP could remarkably increase the expression levels of Bax, cleaved Caspase-9, cleaved Caspase-3, and decreased Bcl-2, thereby causing apoptosis in H9c2 cells. Taken together, the results suggested that TPHP promoted mitophagy through mitochondria fusion dysfunction resulting from oxidative stress, leading to fibrosis by inducing myocardial apoptosis.

Dl-3-n-butylphthalide attenuates cerebral ischemia/reperfusion injury in mice through AMPK-mediated mitochondrial fusion.

Introduction: NBP is a compound isolated from celery seeds, which was approved by the National Medical Products Administration in 2002 for clinical treatment of ischemic stroke. However, in brain ischemia/reperfusion (I/R) injury, the related research on mitochondrial dynamics and its mechanism of action of NBP still need to be further studied. The aim of this study was to assess NBP on cerebral pathology in ischemic stroke in vivo, with a specific focus on the molecular mechanisms of how NBP promotes mitochondrial fusion. Methods: Male C57BL/6 mice were utilized in this study and were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). Pre-ischemia, NBP was administered through intraperitoneal (i.p.) injection for 7days. Results: Our findings demonstrated that NBP effectively reduced infarct volume, improved neurological dysfunction, enhanced cerebral blood flow, and promoted mitochondrial fusion in mice subjected to MCAO/R. More importantly, the pro-fusion effects of NBP were found to be linked to the activation of AMPK/Mfn1 pathway, and with the activation of neurological function, which was partially eliminated by inhibitors of AMPK. Discussion: Our results revealed that NBP is a novel mitochondrial fusion promoter in protecting against ischemic stroke through the AMPK-mediated Mfn1. These findings contribute to the understanding of novel mechanisms involved in the protection of neurological function following NBP treatment for ischemic stroke.

Mitochondrial Fusion Promoter Given During Ischemia Has Greater Neuroprotective Efficacy Than When Given at Onset of Reperfusion in Rats with Cardiac Ischemia/Reperfusion Injury.

Cardiac ischemia/reperfusion (I/R) injury has been shown to impose deleterious effects not only on the heart but also on the brain. Our previous study demonstrated that pretreatment with a mitochondrial fusion promoter (M1) provided central neuroprotective effects following cardiac I/R injury. To investigate the effects of M1 given during the ischemic phase and M1 given at the beginning of reperfusion on brain pathologies following cardiac I/R. Male Wistar rats were randomly divided into either a sham operation (n = 6) or cardiac I/R injury (n = 18) group. Rats with cardiac I/R injury were then randomly divided into 3 subgroups: 1) Control, 2) M1 treatment during cardiac ischemia (2 mg/kg, intravenous (i.v.)), and 3) M1 treatment at the beginning of reperfusion (2 mg/kg, i.v.). After euthanasia, the brain of each rat was removed for further analysis. Cardiac I/R injury caused brain mitochondrial dynamic imbalance, brain mitochondrial dysfunction, brain apoptosis, microglial dysmorphology, brain inflammation, tau hyperphosphorylation, and synaptic dysplasticity. M1 treatment at both time points effectively improved these parameters. M1 given during the ischemic phase had greater efficacy with regard to preventing brain mitochondrial dysfunction and suppressing brain inflammation, when compared to M1 given at the beginning of reperfusion. Our findings suggest that treatment with this mitochondrial fusion promoter prevents mitochondrial dynamic imbalance in the brain of rats with cardiac I/R injury, thereby attenuating brain pathologies. Interestingly, giving the mitochondrial fusion promoter during the ischemic phase exerted greater neuroprotection than if given at the beginning of reperfusion.

In vitro and in vivo studies on the effect of a mitochondrial fusion promoter on Leydig cell integrity and function.

Background: The interstitial testicular Leydig cells are responsible for the production of testosterone, which functionally deteriorate with normal aging. Decreased expression of mitochondrial steroidogenic interactome proteins and diminished mitochondrial function in aging Leydig cells suggest that mitochondrial dynamics play a role in maintaining adequate levels of testosterone. Optic atrophy 1 (OPA1) protein regulates mitochondrial dynamics and cristae formation in many cell types. Previous studies showed that increasing OPA1 expression in dysfunctional Leydig cells restored mitochondrial function and recovered androgen production to levels found in healthy Leydig cells. These findings suggested that mitochondrial dynamics may be a promising target to ameliorate diminished testosterone levels in aging males. Methods: We used twelve-month-old rats to explore the relationship between mitochondrial dynamics and Leydig cell function. Isolated Leydig cells from aged rats were treated ex vivo with the cell-permeable mitochondrial fusion promoter 4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl)hydrazono)ethyl) phenol (mitochondrial fusion promoter M1), which enhances mitochondrial tubular network formation. In parallel, rats were treated with 2mg/kg/day M1 for 6weeks before Leydig cells were isolated. Results: Ex vivo M1-treated cells showed enhanced mitochondrial tubular network formation by transmission electron microscopy, enhanced Leydig cell mitochondrial integrity, improved mitochondrial function, and higher testosterone biosynthesis compared to controls. However, in vivo treatment of aged rats with M1 not only failed to re-establish testosterone levels to that of young rats, it also led to further reduction of testosterone levels and increased apoptosis, suggesting M1 toxicity in the testis. The in vivo M1 toxicity seemed to be tissue-specific, however. Conclusion: Promoting mitochondrial fusion may be one approach to enhancing cell health and wellbeing with aging, but more investigations are warranted. Our findings suggest that fusion promoters could potentially enhance the productivity of aged Leydig cells when carefully regulated.

Pharmacologically targeted mitochondrial fission/fusion performs neuroprotection against trastuzumab‐induced cognitive deficits in rats via suppressing mitochondrial dysfunction and oxidative stress

AbstractBackgroundTrastuzumab (TRZ) has been utilized primarily as a chemotherapeutic treatment for advanced human epidermal growth factor receptor type 2‐expressing malignant tumors. Regardless, TRZ‐associated neurotoxicity has emerged as an adverse clinical complication of oncologic therapy by increasing oxidative stress and damaging mitochondrial function. As the neuroprotective effects of mitochondrial fission inhibitor (MDV) and fusion promoter (M1) have been illustrated for a variety of neurological pathologies, the anti‐neurotoxic mechanisms of MDV and M1 against TRZ are unknown.MethodTwenty male Wistar rats were randomly divided into either the TRZ group (4 mg/kg/day for 7 days, I.P., n = 15) or the control group (CON, n = 5). In the TRZ group, rats also received one of the following co‐treatments: 1) vehicle (VEH, n = 5), 2) MDV (1.2 mg/kg/day, n = 5), or 3) M1 (2 mg/kg/day, n = 5) via I.P. injection for 7 days. Subsequently, novel object location (NOL) and novel object recognition (NOR) tests for long‐term learning and memory were performed. Then, the brain tissue was used for determining mitochondrial functions, and the blood serum was also used to measure the oxidative stress levels.ResultTRZ (the VEH group) increased brain mitochondrial dysfunction and systemic oxidative stress via increasing reactive oxygen species (ROS) production, mitochondrial depolarization, mitochondrial swelling, and serum malondialdehyde levels, respectively, when compared to the CON group (p<0.05, Figure 1). TRZ (the VEH group) impaired long‐term learning and memory, as indicated by the attenuated percentage preference index of NOL and NOR tests, compared to the CON group (p<0.05, Figure 1). MDV and M1 co‐therapies exerted neuroprotection by rescuing brain mitochondrial function and systemic oxidative stress, resulting in the restoration of long‐term cognitive parameters (p<0.05, Figure 1).ConclusionTargeting brain mitochondrial dynamics could be an effective remedy against TRZ‐induced cognitive impairment in rats via suppressing mitochondrial dysfunction and oxidative stress. Therefore, pharmacological inhibition of mitochondrial fission and promotion of mitochondrial fusion could be a novel anti‐neurotoxicity‐induced by TRZ.

Administration of mitochondrial fusion promoter during ischemia and at the onset of reperfusion exert neuroprotective effects in rats with cardiac ischemia/reperfusion injury

AbstractBackgroundAcute myocardial infarction (AMI) is one of the fetal cardiovascular diseases and a leading cause of death (1, 2). Our previous study demonstrated that pretreatment of mitochondrial fusion promoter (M1) provided neuroprotective effects on the brain following cardiac ischemia/reperfusion (I/R) injury (3). However, the effects of M1 given during ischemia and M1 given at the onset of reperfusion on brain pathologies following cardiac I/R have never been investigated.MethodSixteen male Wistar rats were randomly divided into either a sham‐operation (n = 8) or cardiac I/R (n = 24). In the cardiac I/R group, rats were then randomly divided into 3 subgroups, including the I/R operation with vehicle (0.9% sodium chloride solution for 15 minutes before I/R protocol, intravenous (i.v.) injection), the I/R operation with M1 administration during cardiac ischemia (2 mg/kg, 15 minutes after left anterior descending (LAD) coronary artery ligation, i.v. injection), and the I/R operation with M1 administration at the onset of reperfusion group (2 mg/kg at the beginning of reperfusion, i.v. injection). At the end of experimental protocol, rats were sacrificed and brains were obtained for further molecular investigation.ResultThe results demonstrated that cardiac I/R increased brain inflammation, caused microglial dysmorphology, caused brain mitochondrial dysfunction, increased tau hyperphosphorylation, and increased brain apoptosis (p<0.05, Figure 1). M1 given during ischemia and M1 given at the onset of reperfusion effectively attenuated brain pathologies following cardiac I/R by preserving microglial morphology, improving brain mitochondrial function, decreasing tau hyperphosphorylation, and decreasing brain apoptosis (p<0.05, Figure 1). Interestingly, M1 given during ischemia had better efficacy to suppressing brain inflammation, when compared to M1 given at the onset of reperfusion apoptosis (p<0.05, Figure 1).ConclusionThese findings suggest the administration of mitochondrial fusion promoter effectively decreased brain pathologies following cardiac I/R. The administration of mitochondrial fusion promoter during ischemia had greater suppression on brain inflammation than administration at the onset of reperfusion. The novel findings obtained from the present study provided the therapeutic strategies which can be translated into clinical settings for cardiac I/R injury.

Upregulation of mitochondrial fusion as potential cardioprotective strategies against trastuzumab-induced cardiotoxicity in rats

Abstract Background Trastuzumab (TRZ) is a commonly used chemotherapeutic drug for treating advanced malignancies that express human epidermal growth factor receptor type 2. However, TRZ has been linked with cardiotoxicity, which is an unfavourable clinical side effect of cancer treatment. Alterations in mitochondrial dynamic mechanisms, particularly reduced fusion, have been shown to be a key mechanistic insight into the progression of chemotherapy-induced cardiotoxicity. Despite the cardioprotective benefits of a mitochondrial fusion promoter (M1) that have been established in various cardiovascular settings, its ability to counteract the cardiotoxicity caused by TRZ remains unknown. Purpose To examine the effect of M1 treatment on mitochondrial fusion, myocardial oxidative stress, apoptosis, and cardiac injury biomarker in TRZ-induced cardiotoxicity rats. Methods Male Wistar rats (n=18) were divided into the TRZ group (4 mg/kg/day for 7 days, I.P., n=12) and the control group (CON, n=6). In the TRZ group, at the time of first TRZ injection all rats also received the intervention with either the vehicle injection (VEH, n=6) or M1 (M1, 2 mg/kg/day, n=6) via I.P. injection for 7 days. The left ventricular ejection fraction (LVEF) was measured using echocardiography, and troponin-I levels were determined using blood serum. Then, the cardiac tissue was processed to assess the levels of malondialdehyde (MDA) for oxidative stress, mitochondrial fusion, and apoptotic proteins. Results TRZ induced cardiotoxicity via downregulation of mitochondrial fusion along with upregulation of myocardial oxidative stress and apoptosis, as indicated by decreasing Mfn1 protein expression and increasing the levels of MDA and Bax to Bcl-2 ratio, respectively (Fig. 1A, B, C, and E). TRZ also showed markedly higher levels of serum Troponin-I (Fig. 1D) and impaired LV function (77% LVEF for TRZ vs. 89% LVEF for CON). M1 co-treatment promoted Mfn1-mediated mitochondrial fusion, resulting in a reduction in MDA-associated oxidative stress, apoptosis, Troponin-I, and cardiac dysfunction (16% improvement over the TRZ group) (Fig. 1A-E). Conclusion M1 effectively protected the heart against TRZ-induced cardiotoxicity by promoting mitochondrial fusion and preventing oxidative stress and apoptosis-mediated myocardial injury. These findings suggest that the pharmacological promotion of mitochondrial fusion could be a novel anti-cardiotoxicity strategy in TRZ-induced cardiotoxicity.

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.

SAT429 Progesterone Secretion And The Estrous Cycle In The Mouse Are Disrupted By Mitochondrial Fusion Promoter M1 Injection During Proestrus

Abstract Disclosure: Y.P. Budi: None. M. Hsu: None. Y. Lin: None. Y. Lee: None. H. Chiu: None. C. Chiu: None. Y. Jiang: None. In the female reproductive system, mitochondria play a crucial role in steroid hormone synthesis. Literature reports on changes in mitochondrial dynamics throughout the ovarian cycle, but its role in the ovarian cycle is still unknown. In this study, a mitochondrial fusion promotor, M1, was used to study the impact of mitochondrial dynamics in the female reproductive system. The estrus cycle of mice was monitored before, during, and after the M1 treatment to test the impact of M1 on ovarian functions. Our results showed that M1 treatment in mice leads to the disruptions of estrous cycles in vagina smears. To observe the acute effects of M1, serum hormones level was observed, and the decrease in serum LH and progesterone was recorded in the animal. Similarly, our ovarian tissue culture also indicates inhibitions of progesterone secretion and ovulations. Although no significant changes in mitochondrial networks were observed in the ovaries, there was significant up-regulation of mitochondrial respiratory complexes in M1 treatments through transcriptomic analysis. In contrast to the estrogen and steroid biosynthesis up-regulated in M1, the extracellular matrix molecules, remodeling enzymes, and adhesion signaling decreased. Collectively, our study provides novel targets to regulate the ovarian cycles through the mitochondria. However, more studies are still necessary to provide the functional connections between mitochondria and the female reproductive system. Presentation: Saturday, June 17, 2023

PM2.5 promotes pulmonary fibrosis by mitochondrial dysfunction.

Pulmonary fibrosis is known as an incurable lung disorder with irreversible progression of chronic injury, myofibroblast proliferation, extracellular matrix (ECM) accumulation, and tissue scarring. Atmospheric particulate matter 2.5 (PM2.5 ) is implicated as a risk factor of several diseases, especially lung diseases such as pulmonary fibrosis. The molecular mechanism which participates PM2.5 -induced pulmonary fibrosis in type II alveolar cells (AEII) has yet to be determined. Our results proved that short- and long-term exposure to PM2.5 significantly stimulated epithelial-mesenchymal transition (EMT) activity in AEII cells, according to, changes in gene signature analyzed by RNA-seq and cell morphology. Furthermore, Gene Ontology (GO) enrichment analysis also suggested that mitochondrial dysfunction was related to progression of pulmonary fibrosis in AEII after PM2.5 exposure. We observed a marked decline in mitochondria membrane potential (MMP), as well as fragmented mitochondria, in AEII cells exposed to PM2.5 , which suggests that energy metabolism is suppressed after PM2.5 exposure. We also confirmed that PM2.5 exposure could influence the expression levels of Mfn1, Mfn2, and Drp1 in AEII. Pretreatment of mitochondrial fusion promoter M1 was able to reverse mitochondrial dysfunction as well as EMT in AEII. These data suggested the key role of mitochondrial fragmentation in AEII, which was induced by PM2.5 exposure, and participated pathogenesis of pulmonary fibrosis. Finally, we investigated the response of lung tissue exposed to PM2.5 in vivo. The data indicated that the lung tissue exposed to PM2.5 obviously induced collagen accumulation. Moreover, IHC results revealed that PM2.5 enhanced Drp1 expression but suppressed Mfn1 and Mfn2 expression in lung tissue. The current study provides novel insight of pulmonary fibrosis caused by PM2.5 exposure.

Triphenyl phosphate induced apoptosis of mice testicular Leydig cells and TM3 cells through ROS-mediated mitochondrial fusion inhibition

Triphenyl phosphate (TPHP) is a widely used organophosphate flame retardant and has biological toxicity. Previous studies showed TPHP can restrain testosterone biosynthesis in Leydig cells, while the underlying mechanisms remain unclear. In this study, C57BL/6J male mice were exposed to 0, 5, 50, and 200 mg/kg B.W. of TPHP for 30 d by oral, as well as TM3 cells were treated with 0, 50, 100, and 200 μM of TPHP for 24 h. Results showed that TPHP induced testes damage, including spermatogenesis disorders and testosterone synthesis inhibition. Meanwhile, TPHP can cause apoptosis in testicular Leydig cells and TM3 cells, as evidenced by the increased apoptosis rate and decreased Bcl-2/Bax ratio. Moreover, TPHP disrupted mitochondrial ultrastructure of testicular Leydig cells and TM3 cells, reduced healthy mitochondria content and depressed mitochondrial membrane potential of TM3 cells, as well as inhibited mitochondrial fusion proteins mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and optic atrophy 1 (Opa1) expression, without effect on mitochondrial fission proteins dynamin-related protein 1 (Drp1) and fission 1 (Fis1) in testicular tissue and/or TM3 cells. Then, the mitochondrial fusion promoter M1 was used to pre-treat TPHP-exposed TM3 cells to determine the roles of mitochondrial fusion inhibition in TPHP-induced Leydig cells apoptosis. The results showed M1 pretreatment alleviated the above changes and further mitigated TM3 cells apoptosis and testosterone levels decreased, indicating TPHP induced TM3 cells apoptosis by inhibited mitochondrial fusion. Intriguingly, the intervention experiment of N-acetylcysteine (NAC) showed that TPHP-induced mitochondrial fusion inhibition is ROS dependent, because inhibition of ROS overproduction alleviated mitochondrial fusion inhibition, and subsequently relieved TPHP-induced apoptosis in TM3 cells. In summary, above data revealed that apoptosis is a specific mechanism for TPHP-induced male reproductive toxicity, and that ROS-mediated mitochondrial fusion inhibition is responsible for Leydig cells apoptosis caused by TPHP.

The injections of mitochondrial fusion promoter M1 during proestrus disrupt the progesterone secretion and the estrous cycle in the mouse

After ovulation, the mitochondrial enzyme CYP11A1 cleavage the cholesterol into pregnenolone for progesterone synthesis, suggesting that mitochondrial dynamics play a vital role in the female reproductive system. The changes in the mitochondria dynamics throughout the ovarian cycle have been reported in literature, but the correlation to its role in the ovarian cycle remains unclear. In this study, mitochondrial fusion promotor, M1, was used to study the impact of mitochondria dynamics in the female reproductive system. Our results showed that M1 treatment in mice can lead to the disruptions of estrous cycles in vagina smears. The decrease in serum LH was recorded in the animal. And the inhibitions of progesterone secretion and ovulations were observed in ovarian culture. Although no significant changes in mitochondrial networks were observed in the ovaries, significant up-regulation of mitochondrial respiratory complexes was revealed in M1 treatments through transcriptomic analysis. In contrast to the estrogen and steroid biosynthesis up-regulated in M1, the molecules of extracellular matrix, remodeling enzymes, and adhesion signalings were decreased. Collectively, our study provides novel targets to regulate the ovarian cycles through the mitochondria. However, more studies are still necessary to provide the functional connections between mitochondria and the female reproductive systems.

Mitochondrial fragmentation and donut formation enhance mitochondrial secretion to promote osteogenesis

Mitochondrial components have been abundantly detected in bone matrix, implying that they are somehow transported extracellularly to regulate osteogenesis. Here, we demonstrate that mitochondria and mitochondrial-derived vesicles (MDVs) are secreted from mature osteoblasts to promote differentiation of osteoprogenitors. We show that osteogenic induction stimulates mitochondrial fragmentation, donut formation, and secretion of mitochondria through CD38/cADPR signaling. Enhancing mitochondrial fission and donut formation through Opa1 knockdown or Fis1 overexpression increases mitochondrial secretion and accelerates osteogenesis. We also show that mitochondrial fusion promoter M1, which induces Opa1 expression, impedes osteogenesis, whereas osteoblast-specific Opa1 deletion increases bone mass. We further demonstrate that secreted mitochondria and MDVs enhance bone regeneration invivo. Our findings suggest that mitochondrial morphology in mature osteoblasts is adapted for extracellular secretion, and secreted mitochondria and MDVs are critical promoters of osteogenesis.

SIRT3 promotes metabolic maturation of human iPSC-derived cardiomyocytes via OPA1-controlled mitochondrial dynamics

The metabolic patterns and energetics of human induced pluripotent stem cell-derived cardiomyocytes (HiPSC-CMs) are much less than those of normal adult cardiomyocytes, which has limited their application in disease therapy and regenerative medicine. It has been demonstrated that SIRT3, a mitochondria-target deacetylase, controls mitochondrial metabolism in physiological and pathological conditions. In this research, We investigated the role and regulatory mechanism of SIRT3 in energy metabolism in HiPSC-CMs. We found that the expression of SIRT3 was increased during the differentiation and maturation of HiPSC-CMs. Knocking down SIRT3 impaired mitochondrial structure, mitochondrial respiration capacity, and fatty acid oxidation but enhanced glycolysis. However, honokiol, a pharmacological activator of SIRT3, improved the mitochondrial ultrastructure and energetics, and promoted oxidative phosphorylation in HiPSC-CMs. Furthermore, SIRT3 regulated the acetylation of OPA1, and the knockdown of OPA1 blocked the promotion of energy metabolism by honokiol, meanwhile, knocking down OPA1 impaired mitochondrial fusion, mitochondrial respiration capacity, and fatty acid oxidation which were reversed by M1 (a mitochondrial fusion promoter) in HiPSC-CMs. In summary, SIRT3 regulated energetics and promoted metabolism remodeling by targeting the OPA1-controlled mitochondrial dynamics in HiPSC-CMs, and targeting SIRT3 may have revelatory implications in the treatment of cardiovascular diseases and the application of HiPSC-CMs to regenerative medicine.

Mitochondrial dynamics, Leydig cell function, and age-related testosterone deficiency.

The mitochondrial translocator protein (18 kDa; TSPO) is a high-affinity cholesterol-binding protein that is an integral component of the cholesterol trafficking scaffold responsible for determining the rate of cholesterol import into the mitochondria for steroid biosynthesis. Previous studies have shown that TSPO declines in aging Leydig cells (LCs) and that its decline is associated with depressed circulating testosterone levels in aging rats. However, TSPO's role in the mechanistic decline in LC function is not fully understood. To address the role of TSPO depletion in LC function, we first examined mitochondrial quality in Tspo knockout mouse tumor MA-10 nG1 LCs compared to wild-type MA-10 cells. Tspo deletion caused a disruption in mitochondrial function and membrane dynamics. Increasing mitochondrial fusion via treatment with the mitochondrial fusion promoter M1 or by optic atrophy 1 (OPA1) overexpression resulted in the restoration of mitochondrial function and mitochondrial morphology as well as in steroid formation in TSPO-depleted nG1 LCs. LCs isolated from aged rats form less testosterone than LCs isolated from young rats. Treatment of aging LCs with M1 improved mitochondrial function and increased androgen formation, suggesting that aging LC dysfunction may stem from compromised mitochondrial dynamics caused by the age-dependent LC TSPO decline. These results, taken together, suggest that maintaining or enhancing mitochondrial fusion may provide therapeutic strategies to maintain or restore testosterone levels with aging.

Chronic mitochondrial fusion promotor as a novel pharmacological intervention to alleviate left ventricular dysfunction in rats with chronic myocardial infarction

Abstract Background Decreased cardiac mitochondrial fusion associated with increased mitochondrial dysfunction has been demonstrated in acute myocardial infarction pathology. However, whether this pathophysiologic process is associated with myocardial injury in chronic myocardial infarction (CMI) is still unclear. Recently, a novel pharmacological intervention to promote mitochondrial fusion using mitochondrial fusion promotor-M1 (M1) has been shown to exert cardioprotection against myocardial ischemia and reperfusion injury. However, the roles of M1 in CMI has never been investigated. Purpose We investigated the potential cardioprotective benefits of chronic treatment with mitochondrial fusion promotor-M1 in rats with chronic MI model. Methods Adult male Wistar rats were assigned to the sham group (n=5) and the CMI group (n=15), which was induced by a permanent left anterior descending coronary occlusion. After 1 week of operation, rats in the CMI group were randomly divided into 3 subgroups which was treated with one of the followings: 1) vehicle (3% DMSO/day, ip, CON, n=5), 2) enalapril (10 mg/kg/day, po, ENA, n=5), or 3) mitochondrial fusion promotor-M1 (2 mg/kg/day, ip, M1, n=5) once daily for 4 weeks. In the end, echocardiography for the left ventricular (LV) function was performed, and the heart was removed to determine the mitochondrial function and malondialdehyde (MDA) level for oxidative stress. Results CMI rats that received vehicle showed significantly impaired LV function and cardiac mitochondrial function as evidenced by a 60% reduction in LV ejection fraction (LVEF) along with increased mitochondrial reactive oxygen species (ROS) production, mitochondrial depolarization and mitochondrial swelling, respectively, when compared with the sham rats (Fig. 1A–D). The CMI rats also exhibited a higher cardiac tissue MDA level than those of sham rats, further indicating cardiac oxidative stress (Fig. 1E). Interestingly, chronic treatment with either enalapril or M1 effectively attenuated CMI-induced mitochondrial dysfunction and oxidative stress upregulation, thus increasing LVEF (27% improvement for both ENA and M1), when compared with the vehicle group (Fig. 1A). Conclusion Long-term promotion of mitochondrial fusion mitigated cardiac mitochondrial dysfunction and oxidative stress, eventually culminating in improved LV function in rats with CMI. These findings suggest that chronic modulation of mitochondrial fusion could be a promising pharmacological intervention to improve LV function in CMI. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1. The National Science and Technology Development Agency Thailand2. The Thailand Research Fund (RGJ)

Modulation of mitochondrial dynamics rescues cognitive function in rats with 'doxorubicin-induced chemobrain' via mitigation of mitochondrial dysfunction and neuroinflammation.

Doxorubicin (DOX), an effective, extensively used chemotherapeutic drug, can cause cognitive deterioration in cancer patients. The associated debilitating neurological sequelae are referred to as chemobrain. Our recent work demonstrated that Dox treatment resulted in an imbalance in mitochondrial dynamics, ultimately culminating in cognitive decline in rats. Therefore, in this study, we aim to explore the therapeutic efficacy of a pharmacological intervention, which modulates mitochondrial dynamics using a potent mitochondrial fission inhibitor (Mdivi-1) and mitochondrial fusion promoter (M1) against Dox-induced chemobrain. In the study, male Wistar rats were randomly assigned to receive either normal saline solution or six doses of Dox (3 mg·kg-1 ) via intraperitoneal injection. Then, the Dox-treated rats were intraperitoneally given either 1% DMSO as the vehicle, Mdivi-1 (1.2 mg·kg-1 ), M1 (2 mg·kg-1 ), or a combined treatment of Mdivi-1 and M1 for 30 consecutive days. Long-term learning and memory were evaluated using the novel object location task and novel object recognition task. Following euthanasia, the rat brains were dissected to enable further molecular investigation. We demonstrated that long-term treatment with mitochondrial dynamic modulators suppressed mitochondrial fission in the hippocampus following Dox treatment, leading to an improvement in brain homeostasis. Mitochondrial dynamic modulator treatments restored cognitive function in Dox-treated rats by attenuating neuroinflammation, decreasing oxidative stress, preserving synaptic integrity, reducing potential Alzheimer's related lesions, and mitigating both apoptosis and necroptosis following Dox administration. Together, our findings suggested that mitochondrial dynamics modulators protected against Dox-induced cognitive impairment by rebalancing mitochondrial homeostasis and attenuating both oxidative and inflammatory insults.

Pyrroloquinoline quinone (PQQ) protects mitochondrial function of HEI-OC1 cells under premature senescence

The aim of this study was to investigate the effects of pyrroloquinoline quinone (PQQ), an oxidoreductase cofactor, on the H2O2-induced premature senescence model in HEI-OC1 auditory cells and to elucidate its mechanism of action in vitro. Cells were treated with PQQ for 1 day before H2O2 (100 μM) exposure. Mitochondrial respiratory capacity was damaged in this premature senescence model but was restored in cells pretreated with PQQ (0.1 nM or 1.0 nM). A decrease in mitochondrial potential, the promotion of mitochondrial fusion and the accelerated movement of mitochondria were all observed in PQQ-pretreated cells. The protein expression of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) were significantly decreased under H2O2 exposure while they were increased with PQQ pretreatment, and PGC-1α acetylation was significantly decreased. In conclusion, PQQ has a protective effect on the premature senescence model of HEI-OC1 auditory cells and is associated with the SIRT1/PGC-1α signaling pathway, mitochondrial structure, and mitochondrial respiratory capacity.