Mitochondria-targeted Antioxidant Mito-TEMPO Research Articles

Isoliquiritigenin attenuates glutamate-induced mitochondrial fission via calcineurin-mediated Drp1 dephosphorylation in HT22 hippocampal neuron cells

Numerous studies suggest that glutamate toxicity is a major contributor to neuronal dysfunction and death in several neurodegenerative diseases. In our previous study, isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis showed neuroprotective effects against neuronal cell death mediated by intracellular reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential. However, the mechanisms by which ISL protects against glutamate-induced oxidative stress are unknown. In the present study, we focused on the cellular and molecular mechanisms underlying the inhibition of ROS production and induction of mitochondrial dysfunction by ISL in glutamate-stimulated HT22 mouse hippocampal neuron cells. The results revealed that ISL inhibited glutamate-induced mitochondrial ROS production and decline of glutathione levels and ATP generation in HT22 cells. Interestingly, we discovered that ISL prevents glutamate-induced mitochondrial fission by inhibiting the dephosphorylation of Drp1 at the serine 637 residue, which is a regulatory factor of mitochondrial dynamics, and both a S637D mutation of Drp1, which resulted in a phosphorylation-mimetic form of Drp1 at Ser637, and mitochondria-targeted antioxidant Mito-TEMPO inhibited glutamate-induced mitochondrial fission. Furthermore, ISL also prevented the increase of intracellular calcium accompanied by activation of calcineurin, which is a key regulator of dephosphorylation of Drp1 (Ser637), in glutamate-treated HT22 cells. Taken together, our results demonstrated that ISL protects against glutamate-induced mitochondrial fission by inhibiting the increase of mitochondrial ROS and intracellular calcium, which are accompanied by dephosphorylation of Drp1 (Ser637), and consequently attenuates glutamate-induced neuronal cell death. Therefore, these findings suggest that ISL exhibits the potential for protection against glutamate toxicity. These results may contribute to the development of new drugs and novel strategies for the treatment of neurodegenerative disorders related to glutamate toxicity.

Aging related functional and structural changes in the heart and aorta: MitoTEMPO improves aged-cardiovascular performance.

Aging in humans represents declining in cardio-protective systems, however its mechanisms are not known yet. We aimed to analyse how aging affects key mechanisms responsible for contractile dysfunction via comparing the improperly synchrony between electrical and mechanical activities in male aged-rats (24-month old) comparison to those of adult-rats (6-month old). We determined significantly increased systemic oxidative stress with decreased antioxidant capacity, clear insulin resistance and hypertrophy in aged-rats with normal fasting blood glucose. We also determined significantly high level of reactive oxygen species, ROS production in fluorescent dye chloromethyl-2',7'-dichlorodihydrofluoroscein diacetate (DCFDA) loaded isolated cardiomyocytes from aged-rats, confirming the increased oxidative stress in these hearts. In situ electrocardiograms, ECGs presented significant prolongations in RR- and QT-intervals in the aged-rats. Invasive hemodynamic measurements demonstrated marked increases in the heart rate and mean arterial pressure and decreases in the ejection-fraction and preload-recruitable stroke-work, together with depressed contraction and relaxation activities in aortic rings. In light and electron microscopy examinations in aged-rats, significant increases in muscle fibre radius and amount of collagen fibres were detected in the heart as well as markedly flattened and partial local splitting in elastic lamellas in the aorta, besides irregularly clustered mitochondria and lysosomes around the myofilaments in cardiomyocytes. MitoTEMPO treatment of tissue samples and cardiomyocytes from aged-rats for 1-h induced significant structural improvements. In the second part of our study, we have shown that mitochondria-targeted antioxidant MitoTEMPO antagonized all alterations in the heart samples as well as penylephrine-induced contractile and acetylcholine-induced relaxation responses of aged-rat aortic rings. Overall, the present data strongly support the important role of mitochondrial oxidative stress in the development of aged-related insufficiencies and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in aging-associated complications.

IDH2 deficiency accelerates skin pigmentation in mice via enhancing melanogenesis

Melanogenesis is a complex biosynthetic pathway regulated by multiple agents, which are involved in the production, transport, and release of melanin. Melanin has diverse roles, including determination of visible skin color and photoprotection. Studies indicate that melanin synthesis is tightly linked to the interaction between melanocytes and keratinocytes. α-melanocyte-stimulating hormone (α-MSH) is known as a trigger that enhances melanin biosynthesis in melanocytes through paracrine effects. Accumulated reactive oxygen species (ROS) in skin affects both keratinocytes and melanocytes by causing DNA damage, which eventually leads to the stimulation of α-MSH production. Mitochondria are one of the main sources of ROS in the skin and play a central role in modulating redox-dependent cellular processes such as metabolism and apoptosis. Therefore, mitochondrial dysfunction may serve as a key for the pathogenesis of skin melanogenesis. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) is a key enzyme that regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury through the generation of NADPH. Downregulation of IDH2 expression resulted in an increase in oxidative DNA damage in mice skin through ROS-dependent ATM-mediated p53 signaling. IDH2 deficiency also promoted pigmentation on the dorsal skin of mice, as evident from the elevated levels of melanin synthesis markers. Furthermore, pretreatment with mitochondria-targeted antioxidant mito-TEMPO alleviated oxidative DNA damage and melanogenesis induced by IDH2 deficiency both in vitro and in vivo. Together, our findings highlight the role of IDH2 in skin melanogenesis in association with mitochondrial ROS and suggest unique therapeutic strategies for the prevention of skin pigmentation.

MROS-TXNIP axis activates NLRP3 inflammasome to mediate renal injury during ischemic AKI.

Ischemia/reperfusion (I/R) is a critical risk factor for acute kidney injury (AKI). Recent studies provided evidence that tubular epithelial cells (TEC)-associated inflammation aggravates kidney injury and impairs tissue repair after I/R injury. Here we demonstrated that the Nod-like receptor protein 3 (NLRP3) inflammasome is activated by mitochondrial reactive oxygen species (mROS) during I/R injury via direct interactions between the inflammasome and thioredoxin-interacting protein (TXNIP). Firstly, we found that NLRP3 inflammasome activation was induced by I/R injury, peaking at day 3 after reperfusion. Consistent with this observation, NLRP3 deletion significantly attenuated I/R-induced kidney damage and markers of inflammasome activation. Then, we observed mitochondrial dysfunction, characterized by ultrastructural changes and cytochrome C (Cyt c) redistribution. Mitochondria-targeted antioxidant MitoTEMPO prevented mROS overproduction and the decline in mitochondrial membrane potential (MMP) in vitro. MitoTEMPO treatment also inhibited NLRP3 inflammasome activation and co-localization of NLRP3 and TXNIP after simulated ischemia/reperfusion (SI/R) injury. Finally, we transfected HK-2 cells with TXNIP siRNA to explore the role of TXNIP in mROS-induced NLRP3 inflammasome activation. We found that TXNIP siRNA significantly inhibited NLRP3 inflammasome activation. These results demonstrate that NLRP3 inflammasome is activated through the mROS-TXNIP-NLRP3 pathway and provide a potential therapeutic target in ischemic AKI.

Traumatic Brain Injury Impairs Myogenic Constriction of Cerebral Arteries: Role of Mitochondria-Derived H2O2 and TRPV4-Dependent Activation of BKca Channels.

Traumatic brain injury (TBI) impairs autoregulation of cerebral blood flow, which contributes to the development of secondary brain injury, increasing mortality of patients. Impairment of pressure-induced myogenic constriction of cerebral arteries plays a critical role in autoregulatory dysfunction; however, the underlying cellular and molecular mechanisms are not well understood. To determine the role of mitochondria-derived H2O2 and large-conductance calcium-activated potassium channels (BKCa) in myogenic autoregulatory dysfunction, middle cerebral arteries (MCAs) were isolated from rats with severe weight drop-impact acceleration brain injury. We found that 24 h post-TBI MCAs exhibited impaired myogenic constriction, which was restored by treatment with a mitochondria-targeted antioxidant (mitoTEMPO), by scavenging of H2O2 (polyethylene glycol [PEG]-catalase) and by blocking both BKCa channels (paxilline) and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels (HC 067047). Further, exogenous administration of H2O2 elicited significant dilation of MCAs, which was inhibited by blocking either BKCa or TRPV4 channels. Vasodilation induced by the TRPV4 agonist GSK1016790A was inhibited by paxilline. In cultured vascular smooth muscle cells H2O2 activated BKCa currents, which were inhibited by blockade of TRPV4 channels. Collectively, our results suggest that after TBI, excessive mitochondria-derived H2O2 activates BKCa channels via a TRPV4-dependent pathway in the vascular smooth muscle cells, which impairs pressure-induced constriction of cerebral arteries. Future studies should elucidate the therapeutic potential of pharmacological targeting of this pathway in TBI, to restore autoregulatory function in order to prevent secondary brain damage and decrease mortality.

Palmitic Acid-Induced Podocyte Apoptosis via the Reactive Oxygen Species-Dependent Mitochondrial Pathway

Background/Aims: Chronic kidney disease (CKD) is often accompanied by hyperlipidemia, which accelerates progression of the disease. Podocyte injury can lead to dysfunction of the glomerular filtration barrier, which is associated with proteinuria, a risk marker for the progression of CKD. Our previous studies demonstrated that palmitic acid (PA) can induce podocyte apoptosis; however, the underlying mechanisms are unclear. In the present study, we investigated the specific molecular mechanisms of PA-induced apoptosis in cultured podocytes. Methods: We cultured mouse podocytes and treated them with PA. Then, cell viability was measured using the Cell Counting Kit-8 colorimetric assay, lipid uptake was assessed by Oil Red O staining and boron-dipyrromethene staining, apoptosis was measured by flow cytometry, mitochondrial injury was assessed by JC-1 staining and transmission electron microscopy, and mitochondrial production of reactive oxygen species (ROS) was evaluated by fluorescence microscopy using the MitoSOX Red reagent. The effects of PA on the mitochondria-mediated caspase activation pathway were investigated by examining the expression of caspase-8, cleaved caspase-9, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), Bax, Bid, cytochrome c, and Fas-associated protein with death domain (FADD) using western blotting. The translocation of Bax and cytochrome c were detected by immunofluorescence. Results: PA treatment significantly increased lipid accumulation and induced podocyte apoptosis. We investigated whether the two primary apoptosis signaling pathways (death receptor-mediated pathway and mitochondria-mediated pathway) were involved in the execution of PA-induced podocyte apoptosis, and found that the levels of FADD, caspase-8, and Bid did not significantly change during this process. Meanwhile, PA treatment induced an increase in Bax protein expression and a decrease in Bcl-2 protein expression, with Bax translocation to the mitochondria. Furthermore, PA treatment induced mitochondrial impairment, and triggered the release of cytochrome c from the mitochondria to cytosol, with a concomitant dose-dependent increase in the levels of cleaved caspase-9, cleaved caspase-3, and PARP. Meanwhile, PA treatment increased mitochondrial production of ROS, and the mitochondria-targeted antioxidant mitoTEMPO significantly ameliorated PA-induced podocyte apoptosis. Conclusion: Our findings indicated that PA induced caspase-dependent podocyte apoptosis through the mitochondrial pathway, and mitochondrial ROS production participated in this process, thus potentially contributing to podocyte injury

Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality

Human spermatozoa cryopreservation is an important means of assisted reproductive technology and male fertility preservation. Although this technique is particularly useful, sperm cryopreservation significantly reduces the quality of spermatozoa after freezing and thawing. The objective of the study is to examine the efficacy of mitochondria-targeted antioxidant MitoTEMPO in improving sperm quality during semen cryopreservation processes. Semen samples were collected and cryopreserved in extenders containing different concentrations (0.0, 0.5, 5, 50, and 500 μM) of MitoTEMPO. Sperm motility, viability, membrane integrity, mitochondrial membrane potential and antioxidant activities were measured and analyzed. The results showed that the addition of MitoTEMPO (5–50 μM) significantly improved post-thaw sperm motility, viability, membrane integrity and mitochondrial membrane potential (P < .05). Meanwhile, antioxidant enzymes activities were enhanced and MDA content were decreased in the group supplemented with MitoTEMPO. In conclusion, mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality and antioxidant enzymes profile.

Protective effects of resveratrol on mitochondrial function in the hippocampus improves inflammation-induced depressive-like behavior

Growing evidence suggests that inflammatory processes may be involved in depressive disorders. Inflammation is known to induce mitochondrial dysfunction in the nervous system. However, whether mitochondrial dysfunction is involved in the occurrence of inflammation-induced depressive-like behavior remains to be investigated. The present study aims to firstly, clarify whether mitochondrial dysfunction contributes to lipopolysaccharide (LPS)-induced depression-like behavior in mice and secondly, determine whether the anti-oxidant resveratrol alleviates inflammation-induced depressive-like behavior through the prevention of mitochondrial dysfunction in the hippocampus. We found that the administration of LPS led to mitochondrial oxidative stress and dysfunction as evidenced by increased mitochondrial superoxide production and decreased mitochondrial membrane potential and ATP production in the hippocampus. These effects were attenuated by intracerebroventricular (ICV) Injection of the mitochondria-targeted antioxidant Mito-TEMPO. LPS-treated mice displayed depressive-like behaviors as evidenced by reduced sucrose preference, increased immobility time and decreased struggling time in the forced swimming test. Both Mito-TEMPO and resveratrol could significantly improve the LPS-induced depressive-like behaviors. In contrast, ICV Injection of rotenone, the mitochondrial respiratory chain inhibitor, induced mitochondrial oxidative stress and dysfunction in the hippocampus, and resulted in depressive-like behaviors. Moreover, resveratrol alleviated the LPS-induced apoptosis of hippocampal cells. The antidepressant action of resveratrol was accomplished through the interruption of mitochondrial oxidative stress and the prevention of cell apoptosis in the hippocampus. These findings support the potential for resveratrol as a possible pharmacological agent for depression treatment in the future.

Mitochondria-Targeted Antioxidant Mito-Tempo Protects Against Aldosterone-Induced Renal Injury In Vivo

Background/Aims: Growing evidence suggests mitochondrial dysfunction (MtD) and the Nlrp3 inflammasome play critical roles in chronic kidney disease (CKD) progression. We previously reported that Aldosterone (Aldo)-induced renal injury in vitro is directly caused by mitochondrial reactive oxygen species (mtROS)-mediated activation of the Nlrp3 inflammasome. Here we aimed to determine whether a mitochondria-targeted antioxidant (Mito-Tempo) could prevent Aldo-induced kidney damage in vivo. Methods: C57BL/6J mice were treated with Aldo and/or Mito-Tempo (or ethanol as a control) for 4 weeks. Renal injury was evaluated by Periodic Acid-Schiff reagent or Masson’s trichrome staining and electron microscopy. ROS were measured by DCFDA fluorescence and ELISA. MtD was determined by real-time PCR and electron microscopy. Activation of the Nlrp3 inflammasome and endoplasmic reticulum stress (ERS) was detected via western blot. Results: Compared with control mice, Aldo-infused mice showed impaired renal function, increased mtROS production and MtD, Nlrp3 inflammasome activation, and elevated ERS. We showed administration of Mito-Tempo significantly improved renal function and MtD, and reduced Nlrp3 inflammasome activation and ERS in vivo. Conclusion: Mitochondria-targeted antioxidants may attenuate Aldo-infused renal injury by inhibiting MtD, the Nlrp3 inflammasome, and ERS in vivo. Therefore, targeting mtROS might be an effective strategy for preventing CKD.

Resolution of mitochondrial oxidant stress improves aged-cardiovascular performance.

Senescence is a major factor that increases oxidative stress in mitochondria, which contributes toward the pathogenesis of heart disease. However, the effect of antioxidant therapy on cardiac mitochondria in aged-cardiac performance remains elusive. We postulated that the mitochondrial targeting of superoxide scavenging would have benefits in the aged heart. Generation of superoxide in the mitochondria and nicotinamide adenine dinucleotide phosphate oxidase activity increased in the heart of old mice compared with that in young mice. In old mice treated with a mitochondria-targeted antioxidant MitoTEMPO (180 µg/kg/day, 28 days) co-infusion using a subcutaneously implanted minipump, levels of superoxide in the mitochondria and nicotinamide adenine dinucleotide phosphate oxidase activity as well as hydrogen peroxide decreased markedly in cardiomyocytes. Treatment with MitoTEMPO in old mice improved the systolic and diastolic function assessed by echocardiography. Endothelium-dependent vasodilation in isolated coronary arteries and endothelial nitric-oxide synthase phosphorylation were impaired in old mice compared with that in young mice and were improved by MitoTEMPO treatment. Mitochondria from the old mice myocardium showed lower rates of complex I-dependent and II-dependent respiration compared with that from young mice. Supplementation of MitoTEMPO in old mice improved the respiration rates and efficiency of ATP generation in mitochondria to a level similar to that of young mice. Resolution of oxidative stress in mitochondria by MitoTEMPO in old mice restored cardiac function and the capacity of coronary vasodilation to the same magnitude observed in young mice. An antioxidant strategy targeting mitochondria could have a therapeutic benefit in heart disease with senescence.

Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons

Mitochondrial defects including excess reactive oxygen species (ROS) production and compromised ATP generation are featured pathology in Alzheimer's disease (AD). Amyloid beta (Aβ)-mediated mitochondrial ROS overproduction disrupts intra-neuronal Redox balance, in turn exacerbating mitochondrial dysfunction leading to neuronal injury. Previous studies have found the beneficial effects of mitochondria-targeted antioxidants in preventing mitochondrial dysfunction and neuronal injury in AD animal and cell models, suggesting that mitochondrial ROS scavengers hold promise for the treatment of this neurological disorder. In this study, we have determined that mitotempo, a novel mitochondria-targeted antioxidant protects mitochondrial function from the toxicity of Aβ in primary cultured neurons. Our results showed that Aβ-promoted mitochondrial superoxide production and neuronal lipid oxidation were significantly suppressed by the application of mitotempo. Moreover, mitotempo also demonstrated protective effects on mitochondrial bioenergetics evidenced by preserved mitochondrial membrane potential, cytochrome c oxidase activity as well as ATP production. In addition, the Aβ-induced mitochondrial DNA (mtDNA) depletion and decreased expression levels of mtDNA replication-related DNA polymerase gamma (DNA pol γ) and Twinkle were substantially mitigated by mitotempo. Therefore, our study suggests that elimination of excess mitochondrial ROS rescues mitochondrial function in Aβ-insulted neruons; and mitotempo has the potential to be a promising therapeutic agent to protect mitochondrial and neuronal function in AD.

Mitochondria-targeted antioxidant Mito-Tempo protects against acetaminophen hepatotoxicity.

Acetaminophen (APAP) hepatotoxicity is characterized by an extensive mitochondrial oxidant stress. However, its importance as a drug target has not been clarified. To investigate this, fasted C57BL/6J mice were treated with 300mg/kg APAP and the mitochondria-targeted antioxidant Mito-Tempo (MT) was given 1.5h later. APAP caused severe liver injury in mice, as indicated by the increase in plasma ALT activities and centrilobular necrosis. MT dose-dependently reduced the injury. Importantly, MT did not affect APAP-protein adducts formation, glutathione depletion or c-jun N-terminal kinase activation and its mitochondrial translocation. In contrast, hepatic glutathione disulfide and peroxynitrite formation were dose-dependently reduced by MT, indicating its effective mitochondrial oxidant stress scavenging capacity. Consequently, mitochondrial translocation of Bax and release of mitochondrial intermembrane proteins such as apoptosis-inducing factor were prevented, and nuclear DNA fragmentation was eliminated. To demonstrate the importance of mitochondria-specific antioxidant property of MT, we compared its efficacy with Tempo, which has the same pharmacological mode of action as MT but lacks the mitochondria targeting moiety. In contrast to the dramatic protection by MT, the same molar dose of Tempo did not significantly reduce APAP hepatotoxicity. In contrast, even a 3h post-treatment with MT reduced 70% of the injury, and the combination of MT with N-acetylcysteine (NAC) provided superior protection than NAC alone. We conclude that MT protects against APAP overdose in mice by attenuating the mitochondrial oxidant stress and preventing peroxynitrite formation and the subsequent mitochondrial dysfunction. MT is a promising therapeutic agent for APAP overdose patients.

Delphinidin prevents high glucose-induced cell proliferation and collagen synthesis by inhibition of NOX-1 and mitochondrial superoxide in mesangial cells

This study examined the effect of delphinidin on high glucose-induced cell proliferation and collagen synthesis in mesangial cells. Glucose dose-dependently (5.6–25 mM) increased cell proliferation and collagen I and IV mRNA levels, whereas pretreatment with delphinidin (50 μM) prevented cell proliferation and the increased collagen mRNA levels induced by high glucose (25 mM). High glucose increased reactive oxygen species (ROS) generation, and this was suppressed by pretreating delphinidin or the antioxidant N-acetyl cysteine. NADPH oxidase (NOX) 1 was upregulated by high glucose, but pretreatment with delphinidin abrogated this upregulation. Increased mitochondrial superoxide by 25 mM glucose was also suppressed by delphinidin. The NOX inhibitor apocynin and mitochondria-targeted antioxidant Mito TEMPO inhibited ROS generation and cell proliferation induced by high glucose. Phosphorylation of extracellular signal regulated kinase (ERK)1/2 was increased by high glucose, which was suppressed by delphinidin, apocynin or Mito TEMPO. Furthermore, PD98059 (an ERK1/2 inhibitor) prevented the high glucose-induced cell proliferation and increased collagen mRNA levels. Transforming growth factor (TGF)-β protein levels were elevated by high glucose, and pretreatment with delphinidin or PD98059 prevented this augmentation. These results suggest that delphinidin prevents high glucose-induced cell proliferation and collagen synthesis by inhibition of NOX-1 and mitochondrial superoxide in mesangial cells.

Transient Cerebral Ischemia Promotes Brain Mitochondrial Dysfunction and Exacerbates Cognitive Impairments in Young 5xFAD Mice.

Alzheimer's disease (AD) is heterogeneous and multifactorial neurological disorder; and the risk factors of AD still remain elusive. Recent studies have highlighted the role of vascular factors in promoting the progression of AD and have suggested that ischemic events increase the incidence of AD. However, the detailed mechanisms linking ischemic insult to the progression of AD is still largely undetermined. In this study, we have established a transient cerebral ischemia model on young 5xFAD mice and their non-transgenic (nonTg) littermates by the transient occlusion of bilateral common carotid arteries. We have found that transient cerebral ischemia significantly exacerbates brain mitochondrial dysfunction including mitochondrial respiration deficits, oxidative stress as well as suppressed levels of mitochondrial fusion proteins including optic atrophy 1 (OPA1) and mitofusin 2 (MFN2) in young 5xFAD mice resulting in aggravated spatial learning and memory. Intriguingly, transient cerebral ischemia did not induce elevation in the levels of cortical or mitochondrial Amyloid beta (Aβ)1-40 or 1–42 levels in 5xFAD mice. In addition, the glucose- and oxygen-deprivation-induced apoptotic neuronal death in Aβ-treated neurons was significantly mitigated by mitochondria-targeted antioxidant mitotempo which suppresses mitochondrial superoxide levels. Therefore, the simplest interpretation of our results is that young 5xFAD mice with pre-existing AD-like mitochondrial dysfunction are more susceptible to the effects of transient cerebral ischemia; and ischemic events may exacerbate dementia and worsen the outcome of AD patients by exacerbating mitochondrial dysfunction.

Therapeutic inhibition of mitochondrial reactive oxygen species with mito-TEMPO reduces diabetic cardiomyopathy

AimsThe mitochondria are important sources of reactive oxygen species (ROS) in the heart. Mitochondrial ROS production has been implicated in the pathogenesis of diabetic cardiomyopathy, suggesting that therapeutic strategies specifically targeting mitochondrial ROS may have benefit in this disease. We investigated the therapeutic effects of mitochondria-targeted antioxidant mito-TEMPO on diabetic cardiomyopathy. MethodsThe mitochondria-targeted antioxidant mito-TEMPO was administrated after diabetes onset in a mouse model of streptozotocin-induced type-1 diabetes and type-2 diabetic db/db mice. Cardiac adverse changes were analyzed and myocardial function assessed. Cultured adult cardiomyocytes were stimulated with high glucose, and mitochondrial superoxide generation and cell death were measured. ResultsIncubation with high glucose increased mitochondria superoxide generation in cultured cardiomyocytes, which was prevented by mito-TEMPO. Co-incubation with mito-TEMPO abrogated high glucose-induced cell death. Mitochondrial ROS generation, and intracellular oxidative stress levels were induced in both type-1 and type-2 diabetic mouse hearts. Daily injection of mito-TEMPO for 30 days inhibited mitochondrial ROS generation, prevented intracellular oxidative stress levels, decreased apoptosis and reduced myocardial hypertrophy in diabetic hearts, leading to improvement of myocardial function in both type-1 and type-2 diabetic mice. Incubation with mito-TEMPO or inhibition of Nox2-containing NADPH oxidase prevented oxidative stress levels and cell death in high glucose-stimulated cardiomyocytes. Mechanistic study revealed that the protective effects of mito-TEMPO were associated with down-regulation of ERK1/2 phosphorylation. ConclusionsTherapeutic inhibition of mitochondrial ROS by mito-TEMPO reduced adverse cardiac changes and mitigated myocardial dysfunction in diabetic mice. Thus, mitochondria-targeted antioxidants may be an effective therapy for diabetic cardiac complications.

Abstract 13631: Therapeutic Inhibition of Mitochondrial Reactive Oxygen Species With Mito-tempo Reduces Diabetic Cardiomyopathy

Introduction: The mitochondria are important sources of reactive oxygen species (ROS) in the heart. Mitochondrial ROS production and subsequent oxidative damage have been implicated in the pathogenesis of diabetic cardiomyopathy. We hypothesized that therapeutic strategies specifically targeting mitochondrial ROS may have benefit in diabetic cardiomyopathy. Hypothesis: We hypothesized that therapeutic inhibition of mitochondrial ROS with mito-TEMPO reduces diabetic cardiomyopathy. Methods: The mitochondria-targeted antioxidant mito-TEMPO was administrated after diabetes onset in a mouse model of streptozotocin (STZ)-induced type-1 diabetes and type-2 diabetic db/db mice. Cardiac adverse changes were analyzed and myocardial function assessed. Cultured adult cardiomyocytes were stimulated with high glucose, and single mitochondrial superoxide generation and cell death were measured. Results: Incubation with high glucose increased mitochondria superoxide generation in cultured adult cardiomyocytes, which was prevented by mito-TEMPO. Co-incubation with mito-TEMPO also abrogated high glucose-induced cell death. Mitochondrial ROS generation, and intracellular ROS formation and oxidative damage were induced in both type-1 and type-2 diabetic mouse hearts. Daily injection of mito-TEMPO for 30 days inhibited mitochondrial ROS generation, prevented intracellular ROS formation and oxidative damage, decreased apoptosis and reduced myocardial hypertrophy in diabetic hearts without change of blood glucose, leading to improvement of myocardial function in both type-1 and type-2 diabetic mice. Mechanistic study revealed that the protective effects of mito-TMEPO were associated with down-regulation of ERK1/2 phosphorylation in diabetic hearts and high glucose-stimulated cardiomyocytes. Conclusions: Therapeutic inhibition of mitochondrial ROS by mitochondria-targeted antioxidant mito-TEMPO reduced adverse cardiac changes and mitigated myocardial dysfunction in both type-1 and type-2 diabetic mice. Thus, mitochondria-targeted antioxidants may be an effective therapy for diabetic cardiac complications.

Critical role of c-jun N-terminal protein kinase in promoting mitochondrial dysfunction and acute liver injury

The mechanism by which c-Jun N-terminal protein kinase (JNK) promotes tissue injury is poorly understood. Thus we aimed at studying the roles of JNK and its phospho-target proteins in mouse models of acute liver injury. Young male mice were exposed to a single dose of CCl4 (50mg/kg, IP) and euthanized at different time points. Liver histology, blood alanine aminotransferase, and other enzyme activities were measured in CCl4-exposed mice without or with the highly-specific JNK inhibitors. Phosphoproteins were purified from control or CCl4-exposed mice and analyzed by differential mass-spectrometry followed by further characterizations of immunoprecipitation and activity measurements. JNK was activated within 1h while liver damage was maximal at 24h post-CCl4 injection. Markedly increased phosphorylation of many mitochondrial proteins was observed between 1 and 8h following CCl4 exposure. Pretreatment with the selective JNK inhibitor SU3327 or the mitochondria-targeted antioxidant mito-TEMPO markedly reduced the levels of p-JNK, mitochondrial phosphoproteins and liver damage in CCl4-exposed mice. Differential proteomic analysis identified many phosphorylated mitochondrial proteins involved in anti-oxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. Aldehyde dehydrogenase, NADH-ubiquinone oxidoreductase, and α-ketoglutarate dehydrogenase were phosphorylated in CCl4-exposed mice but dephosphorylated after SU3327 pretreatment. Consistently, the suppressed activities of these enzymes were restored by SU3327 pretreatment in CCl4-exposed mice. These data provide a novel mechanism by which JNK, rapidly activated by CCl4, promotes mitochondrial dysfunction and acute hepatotoxicity through robust phosphorylation of numerous mitochondrial proteins.

Abstract 243: The E3 Ubiquitin Ligase Parkin Mediates Clearance of Damaged Mitochondria via Two Distinct Pathways

Damaged mitochondria release reactive oxygen species and pro-death factors which can lead to loss of cardiac myocytes. To protect against such damage, myocytes have developed several mechanisms of quality control that act both on the protein and organelle levels. We have previously identified the E3 ubiquitin ligase Parkin as an important regulator of mitochondrial clearance via autophagy in the myocardium. Here, we report that Parkin can also mediate clearance of mitochondria via the endosomal-lysosomal pathway. We found that Parkin promotes clearance of damaged mitochondria in both wild type (WT) and autophagy-deficient Atg5 knockout mouse embryonic fibroblasts (MEFs) treated with the mitochondria uncoupler FCCP. Mitochondrial damage leads to rapid activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. We further observed increased activation of Rab5, a protein involved in early endosome formation, in both WT and Atg5-/- MEFs after treatment with FCCP. In addition, we observed sequestration of damaged mitochondria in Rab5+ and Rab7+ early and late endosomes, respectively. Mitochondria also colocalized with Lamp2+ vesicles in Atg5-/- MEFs indicating that the mitochondria are ultimately being delivered to the lysosomes for degradation. Overexpression of Rab5S34N, a dominant negative of Rab5, reduces FCCP-mediated clearance and increases cell death in Atg5-/- MEFs. Pharmacological inhibition of the endosomal-lysosomal pathway also results in increased FCCP-mediated cell death. Furthermore, we confirmed that FCCP treatment or simulated ischemia reperfusion exposure induces Rab5 activation with subsequent mitochondrial sequestration in early endosomes in neonatal myocytes. Interestingly, the activation of Rab5 is abrogated in the presence of the mitochondrial targeted antioxidant Mito-Tempo, suggesting that mitochondrial ROS is involved in the activation the endosomal pathway. Mitochondrial clearance via this pathway is also dependent on Parkin, as FCCP treatment fails to activate Rab5 and induce mitochondrial clearance in both WT and Atg5-/- MEFS in the absence of Parkin. Thus, our data suggest that Parkin can mediate clearance of damaged mitochondria via two distinct pathways in cells.

Preclinical evaluation of the mitochondria‐targeted antioxidant MitoTEMPO on the renal microcirculation in a pediatric model of sepsis‐induced renal injury

Microcirculatory failure, mitochondrial dysfunction and oxidant generation are thought to be key events during sepsis that lead to multi-organ failure. Acute kidney injury is a leading cause of mortality in septic children. We developed a clinically relevant cecal ligation and puncture (CLP) sepsis model in rat pups 17-19 days old to identify and evaluate new therapeutic approaches. Analysis of the renal microcirculation using intravital video microscopy at 18 h post CLP or sham surgery showed a significant decrease in the percentage of continuously flowing capillaries (from 75% to 40%) and an increase in capillaries with no flow (from 5% to 30%). Microcirculatory failure was associated with a 2-fold increase in mitochondrial superoxide generation in renal tubules. We then performed dose-response studies to evaluate the effectiveness of the mitochondria-targeted antioxidant MitoTEMPO to reduce mitochondria superoxide and to improve renal microcirculation. MitoTEMPO was administered at doses of 1, 3, or 10 mg/kg (ip) at the time of CLP or sham surgery. Superoxide and perfusion were measured at 18 h. MitoTEMPO produced dose-dependent decreases in mitochondrial superoxide and increases in microcirculatory perfusion with 3 mg/kg being the lowest most efficacious dose to preserve the renal microcirculation. These findings suggest a previously unrecognized link between mitochondrial oxidants in the renal tubule epithelium and the regulation of microvascular perfusion. The protective effect of MitoTEMPO in the kidney suggests that agents like it should be evaluated further for their effectiveness at reducing sepsis-induced multi-organ failure. Supported by T32 GM106999 and AHA PRE20450050.

Cellular redox status determines sensitivity to BNIP3-mediated cell death in cardiac myocytes.

The atypical BH3-only protein Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) is an important regulator of hypoxia-mediated cell death. Interestingly, the susceptibility to BNIP3-mediated cell death differs between cells. In this study we examined whether there are mechanistic differences in BNIP3-mediated cell death between neonatal and adult cardiac myocytes. We discovered that BNIP3 is a potent inducer of cell death in neonatal myocytes, whereas adult myocytes are remarkably resistant to BNIP3. When exploring the potential underlying basis for the resistance, we discovered that adult myocytes express significantly higher levels of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) than neonatal myocytes. Overexpression of MnSOD confers resistance to BNIP3-mediated cell death in neonatal myocytes. In contrast, the presence of a pharmacological MnSOD inhibitor, 2-methoxyestradiol, results in increased sensitivity to BNIP3-mediated cell death in adult myocytes. Cotreatment with the mitochondria-targeted antioxidant MitoTEMPO or the MnSOD mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride abrogates the increased cell death by 2-methoxyestradiol. Moreover, increased oxidative stress also restores the ability of BNIP3 to induce cell death in adult myocytes. Taken together, these data indicate that redox status determines cell susceptibility to BNIP3-mediated cell death. These findings are clinically relevant, given that pediatric hearts are known to be more vulnerable than the adult heart to ischemic injury. Our studies provide important insight into why pediatric hearts are more sensitive to ischemic injury and may help in the clinical management of childhood heart disease.