Inhibition of the proteasome preserves Mitofusin-2 and mitochondrial integrity,protecting cardiomyocytes during ischemia-reperfusion injury

Cardiomyocyte loss is the main cause of myocardial dysfunction following an ischemia-reperfusion (IR) injury. Mitochondrial dysfunction and altered mitochondrial network dynamics play central roles in cardiomyocyte death. Proteasome inhibition is cardioprotective in the setting of IR; however, the mechanisms underlying this protection are not well-understood. Several proteins that regulate mitochondrial dynamics and energy metabolism, including Mitofusin-2 (Mfn2), are degraded by the proteasome. The aim of this study was to evaluate whether proteasome inhibition can protect cardiomyocytes from IR damage by maintaining Mfn2 levels and preserving mitochondrial network integrity. Using ex vivo Langendorff-perfused rat hearts and in vitro neonatal rat ventricular myocytes, we showed that the proteasome inhibitor MG132 reduced IR-induced cardiomyocyte death. Moreover, MG132 preserved mitochondrial mass, prevented mitochondrial network fragmentation, and abolished IR-induced reductions in Mfn2 levels in heart tissue and cultured cardiomyocytes. Interestingly, Mfn2 overexpression also prevented cardiomyocyte death. This effect was apparently specific to Mfn2, as overexpression of Miro1, another protein implicated in mitochondrial dynamics, did not confer the same protection. Our results suggest that proteasome inhibition protects cardiomyocytes from IR damage. This effect could be partly mediated by preservation of Mfn2 and therefore mitochondrial integrity.     

Berberine Inhibits Doxorubicin-Triggered Cardiomyocyte Apoptosis via Attenuating Mitochondrial Dysfunction and Increasing Bcl-2 Expression

Cardiomyocyte apoptosis is an important event in doxorubicin (DOX)-induced cardiac injury. The aim of the present study was to investigate the protection of berberine (Ber) against DOX- triggered cardiomyocyte apoptosis in neonatal rat cardiomyocytes and rats. In neonatal rat cardiomyocytes, Ber attenuated DOX-induced cellular injury and apoptosis in a dose-dependent manner. However, Ber has no significant effect on viability of MCF-7 breast cancer cells treated with DOX. Ber reduced caspase-3 and caspase-9, but not caspase-8 activity in DOX-treated cardiomyocytes. Furthermore, Ber decreased adenosine monophosphate-activated protein kinase α (AMPKα) and p53 phosphorylation at 2 h, cytosolic cytochrome c and mitochondrial Bax levels and increased Bcl-2 level at 6 h in DOX-stimulated cardiomyocytes. Pretreatment with compound C, an AMPK inhibitor, also suppressed p53 phosphorylation and apoptosis in DOX-treated cardiomyocytes. DOX stimulation for 30 min led to a loss of mitochondrial membrane potential and a rise in the AMP/ATP ratio. Ber markedly reduced DOX-induced mitochondrial membrane potential loss and an increase in the AMP/ATP ratio at 1 h and 2 h post DOX exposure. In in vivo experiments, Ber significantly improved survival, increased stroke volume and attenuated myocardial injury in DOX-challenged rats. TUNEL and Western blot assays showed that Ber not only decreased myocardial apoptosis, caspase-3 activation, AMPKα and p53 phosphorylation, but also increased Bcl-2 expression in myocardium of rats exposed to DOX for 84 h. These findings indicate that Ber attenuates DOX-induced cardiomyocyte apoptosis via protecting mitochondria, inhibiting an increase in the AMP/ATP ratio and AMPKα phosphorylation as well as elevating Bcl-2 expression, which offer a novel mechanism responsible for protection of Ber against DOX-induced cardiomyopathy.     

Evidences for the mechanism of Shenmai injection antagonizing doxorubicin-induced cardiotoxicity

BackgroundDoxorubicin (DOX) is a widely used antitumor drug. However, its clinical application is limited for its serious cardiotoxicity. The mechanism of DOX-induced cardiotoxicity is attributed to the increasing of cell stress in cardiomyocytes, then following autophagic and apoptotic responses. Our previous studies have demonstrated the protective effect of Shenmai injection (SMI) on DOX-induced cardiotoxicity via regulation of inflammatory mediators for releasing cell stress.PurposeTo further investigate whether SMI attenuates the DOX-induced cell stress in cardiomyocytes, we explored the mechanism underlying cell stress as related to Jun N-terminal kinase (JNK) activity and the regulation of autophagic flux to determine the mechanism by which SMI antagonizes DOX-induced cardiotoxicity.Study designThe DOX-induced cardiotoxicity model of autophagic cell death was established in vitro to disclose the protected effects of SMI on oxidative stress, autophagic flux and JNK signaling pathway. Then the autophagic mechanism of SMI antagonizing DOX cardiotoxicity was validated in vivo.ResultsSMI was able to reduce the DOX-induced cardiomyocyte apoptosis associated with inhibition of activation of the JNK pathway and the accumulation of reactive oxygen species (ROS). Besides, SMI antagonized DOX cardiotoxicity, regulated cardiomyocytes homeostasis by restoring DOX-induced cardiomyocytes autophagy. Under specific circumstances, SMI depressed autophagic process by reducing the Beclin 1-Bcl-2 complex dissociation which was activated by DOX via stimulating the JNK signaling pathway. At the same time, SMI regulated lysosomal pH to restore the autophagic flux which was blocked by DOX in cardiomyocytes.ConclusionSMI regulates cardiomyocytes apoptosis and autophagy by controlling JNK signaling pathway, blocking DOX-induced apoptotic pathway and autophagy formation. SMI was also found to play a key role in restoring autophagic flux for counteracting DOX-damaged cardiomyocyte homeostasis.     

Ferritin heavy chain as main mediator of preventive effect of metformin against mitochondrial damage induced by doxorubicin in cardiomyocytes

The efficacy of doxorubicin (DOX) as an antitumor agent is greatly limited by the induction of cardiomyopathy, which results from mitochondrial dysfunction and iron-catalyzed oxidative stress in the cardiomyocyte. Metformin (MET) has been seen to have a protective effect against the oxidative stress induced by DOX in cardiomyocytes through its modulation of ferritin heavy chain (FHC), the main iron-storage protein. This study aimed to assess the involvement of FHC as a pivotal molecule in the mitochondrial protection offered by MET against DOX cardiotoxicity. The addition of DOX to adult mouse cardiomyocytes (HL-1 cell line) increased the cytosolic and mitochondrial free iron pools in a time-dependent manner. Simultaneously, DOX inhibited complex I activity and ATP generation and induced the loss of mitochondrial membrane potential. The mitochondrial dysfunction induced by DOX was associated with the release of cytochrome c to the cytosol, the activation of caspase 3, and DNA fragmentation. The loss of iron homeostasis, mitochondrial dysfunction, and apoptosis induced by DOX were prevented by treatment with MET 24 h before the addition of DOX. The involvement of FHC and NF-κB was determined through siRNA-mediated knockdown. Interestingly, the presilencing of FHC or NF-κB with specific siRNAs blocked the protective effect induced by MET against DOX cardiotoxicity. These findings were confirmed in isolated primary neonatal rat cardiomyocytes. In conclusion, these results deepen our knowledge of the protective action of MET against DOX-induced cardiotoxicity and suggest that therapeutic strategies based on FHC modulation could protect cardiomyocytes from the mitochondrial damage induced by DOX by restoring iron homeostasis.     

Sirtuin-3 (SIRT3) Protein Attenuates Doxorubicin-induced Oxidative Stress and Improves Mitochondrial Respiration in H9c2 Cardiomyocytes

Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction.     

Haemin attenuates intermittent hypoxia‐induced cardiac injury via inhibiting mitochondrial fission

Obstructive sleep apnoea (OSA) characterized by intermittent hypoxia (IH) is closely associated with cardiovascular diseases. IH confers cardiac injury via accelerating cardiomyocyte apoptosis, whereas the underlying mechanism has remained largely enigmatic. This study aimed to explore the potential mechanisms involved in the IH‐induced cardiac damage performed with the IH‐exposed cell and animal models and to investigate the protective effects of haemin, a potent haeme oxygenase‐1 (HO‐1) activator, on the cardiac injury induced by IH. Neonatal rat cardiomyocyte (NRC) was treated with or without haemin before IH exposure. Eighteen male Sprague‐Dawley (SD) rats were randomized into three groups: control group, IH group (PBS, ip) and IH + haemin group (haemin, 4 mg/kg, ip). The cardiac function was determined by echocardiography. Mitochondrial fission was evaluated by Mitotracker staining. The mitochondrial dynamics‐related proteins (mitochondrial fusion protein, Mfn2; mitochondrial fission protein, Drp1) were determined by Western blot. The apoptosis of cardiomyocytes and heart sections was examined by TUNEL. IH regulated mitochondrial dynamics‐related proteins (decreased Mfn2 and increased Drp1 expressions, respectively), thereby leading to mitochondrial fragmentation and cell apoptosis in cardiomyocytes in vitro and in vivo, while haemin‐induced HO‐1 up‐regulation attenuated IH‐induced mitochondrial fragmentation and cell apoptosis. Moreover, IH resulted in left ventricular hypertrophy and impaired contractile function in vivo, while haemin ameliorated IH‐induced cardiac dysfunction. This study demonstrates that pharmacological activation of HO‐1 pathway protects against IH‐induced cardiac dysfunction and myocardial fibrosis through the inhibition of mitochondrial fission and cell apoptosis.     

Resveratrol prevents doxorubicin cardiotoxicity through mitochondrial stabilization and the Sirt1 pathway

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, its incidence of cardiotoxicity compromises its therapeutic index. DOX-induced heart failure is thought to be caused by reduction/oxidation cycling of DOX to generate oxidative stress and cardiomyocyte cell death. Resveratrol (RV), a stilbene found in red wine, has been reported to play a cardioprotective role in diseases associated with oxidative stress. The objective of this study was to test the ability of RV to protect against DOX-induced cardiomyocyte death. We hypothesized that RV protects cardiomyocytes from DOX-induced oxidative stress and subsequent cell death through changes in mitochondrial function. DOX induced a rapid increase in reactive oxygen species (ROS) production in cardiac cell mitochondria, which was inhibited by pretreatment with RV, most likely owing to an increase in MnSOD activity. This effect of RV caused additional polarization of the mitochondria in the absence and presence of DOX to increase mitochondrial function. RV pretreatment also prevented DOX-induced cardiomyocyte death. The protective ability of RV against DOX was abolished when Sirt1 was inhibited by nicotinamide. Our data suggest that RV protects against DOX-induced oxidative stress through changes in mitochondrial function, specifically the Sirt1 pathway leading to cardiac cell survival.     

MicroRNA-532-3p regulates mitochondrial fission through targeting apoptosis repressor with caspase recruitment domain in doxorubicin cardiotoxicity

Doxorubicin (DOX) is a wide-spectrum antitumor drug, but its clinical application is limited by its cardiotoxicity. However, the mechanisms underlying DOX-induced cardiomyopathy remain mostly unclear. Here we observed that apoptosis repressor with caspase recruitment domain (ARC) was downregulated in mouse heart and cardiomyocytes upon DOX treatment. Furthermore, enforced expression of ARC attenuated DOX-induced cardiomyocyte mitochondrial fission and apoptosis. ARC transgenic mice demonstrated reduced cardiotoxicity upon DOX administration. DOX-induced mitochondrial fission required the activity of dynamin-related protein 1 (Drp1). In elucidating the molecular mechanism by which ARC was downregulated upon DOX treatment, miR-532-3p was found to directly target ARC and participated in DOX-induced mitochondrial fission and apoptosis. MiR-532-3p was not involved in DOX-induced apoptosis in cancer cells. Taken together, these findings provide novel evidence that miR-532-3p and ARC constitute an antiapoptotic pathway that regulates DOX cardiotoxicity. Therefore, the development of new therapeutic strategies based on ARC and miR-532-3p is promising for overcoming the cardiotoxicity of chemotherapy for cancer therapy.Doxorubicin (DOX) is one of the most widely used anticancer drugs. A large number of patients treated with DOX develop carditoxicity, which can lead to congestive heart failure.^{1, 2} The exact mechanisms of DOX cardiotoxicity remain unclear. Multiple mechanisms by which DOX-induced irreversible myocaridial injures have been proposed, including reactive oxygen species (ROS) production, lipid peroxidation, mitochondrial impairment, intracellular calcium dysregulation and direct suppression of heart-special gene expression.^{3, 4, 5} Most of these mechanisms ultimately result in the activation of apoptotic signaling, leading to progressive loss of cardiac myocytes.² Considering the limited capacity for proliferation, it is essential to understand the molecular signaling underlying DOX-induced cardiomyocyte death in order to establish interventions that can effectively prevent cardiac cell loss and, thereby, preserve cardiac function.The heart has evolutionarily developed a highly expressed antiapoptotic protein, apoptosis repressor with caspase recruitment domain (ARC).⁶ ARC was initially discovered as an endogenous apoptosis inhibitor in postmitotic cells, such as cardiomyocytes, skeletal muscle cells and neurons. It can antagonize both intrinsic and extrinsic apoptosis signaling pathways.^{6, 7, 8} ARC has a role in maintaining physiological cardiac function. Under pathological conditions, ARC is downregulated, which may contribute to the occurrence of many heart diseases, such as hypertrophy, heart failure and cardiac infraction.^{9, 10} ARC-deficient mice exerted significantly accelerated cardiomyopathy under conditions of cardiac ischemia or pressure overload.¹¹ Our previous work showed heart of cardiac-special ARC transgenic mice were more resistant to ischemia injury and hypertrophic stimuli.¹² As for DOX cardiotoxicity, it was reported that ARC was downregulated in cardiomyocytes exposed to DOX and led to a significant induction of apoptosis.⁵ Enforced expression of ARC dramatically increased the resistance of cardiomyocytes to undergo apoptotic cell death following DOX administration.⁵ But the mechanism by which ARC was downregulated during this process is largely unknown. Our previous work has also proved that highly expressed ARC contributed to cancer cell resistance to chemotherapy by targeting the mitochondrial fission machinery.¹³ However, whether ARC inhibits mitochondrial fission in cardiomyocytes upon DOX treatment remains to be investigated.MicroRNAs (miRNAs) are a class of small non-coding RNAs and negative regulators of target genes by altering mRNA translation or stability.¹⁴ MiRNAs functionally participated in a wide variety of physiological or pathological processes.¹⁵ MiRNAs can regulate cardiac function such as the conductance of electrical signal, heart muscle contraction, heart growth and morphogenesis.¹⁶ Manipulation of miRNA can be developed to therapeutic approaches. However, it is not yet clear whether miRNAs are involved in the regulation of DOX cardiotoxicity. In our previous study, we have demonstrated that miR-185 negatively regulated ARC in gastric cancer cells.¹⁷ However, whether miRNAs can regulate ARC in DOX cardiotoxicity is unknown.In this study, we focused on the function of ARC in DOX cardiotoxicity both in cardiomyocytes and mice hearts. We found that ARC was downregulated in cardiomyocytes administered DOX. Enforced expression of ARC inhibited DOX-induced mitochondrial fission and apoptosis in cardiomyocytes. Heart-special ARC transgenic mice exhibited reduced cardiotoxicity. Besides, miR-532-3p could sensitize cardiomyocytes to DOX-induced mitochondrial fission and apoptosis through negatively regulating ARC expression. Our results identified a novel regulatory pathway for apoptosis involving miR-532-3p–ARC and possibly provide a valuable insight to protect against DOX cardiotoxicity during cancer chemical therapy.     

MFN1介导的线粒体融合在心肌细胞凋亡中的作用研究

目的:探讨线粒体融合关键蛋白MFN1介导的线粒体融合在调控心肌细胞凋亡中的作用。方法:通过si RNA降低体外培养H9C2心肌细胞中MFN1的表达后,采用Western blot检测线粒体细胞色素c(Cyto c)释放及其下游凋亡效应分子Caspase9与Caspase3活性,流式细胞术检测细胞内活性氧(ROS)的产生情况,流式细胞术检测细胞凋亡的情况。结果:干扰MFN1可显著促进H9C2心肌细胞内细胞色素c由线粒体释放至胞浆,促进Caspase9与Caspase3的激活,增加细胞内活性氧ROS产生并提高细胞凋亡率(均P0.05)。结论:MFN1介导的线粒体融合可保护心肌细胞凋亡,其机制可能与抑制ROS产生与细胞色素C释放有关。     

N-3 polyunsaturated fatty acids decrease levels of doxorubicin-induced reactive oxygen species in cardiomyocytes -- involvement of uncoupling protein UCP2

Background

Use of the chemotherapeutic drug doxorubicin (DOX) is associated with serious cardiotoxicity, as it increases levels of reactive oxygen species (ROS). N-3 polyunsaturated fatty acid dietary supplements can be of benefit to patients undergoing cancer therapy. The aims of this study were to determine whether DOX-induced cardiotoxicity is related to mitochondrial uncoupling proteins and whether eicosapentaenoic acid (EPA, C20:5 n-3) or docosahexaenoic acid (DHA, C22:6 n-3) affects DOX-induced cardiomyocyte toxicity.

Results

Treatment of H9C2 cells with DOX resulted in decreased cell viability and UCP2 expression. Treatment with 100 μM EPA or 50 μM DHA for 24 h resulted in a maximal mitochondria concentration of these fatty acids and increased UCP2 expression. Pretreatment with 100 μM EPA or 50 μM DHA prevented the DOX-induced decrease in UCP2 mRNA and protein levels, but these effects were not seen with EPA or DHA and DOX cotreatment. In addition, the DOX-induced increase in ROS production and subsequent mitochondrial membrane potential change (∆ψ) were significantly attenuated by pretreatment with EPA or DHA.

Conclusion

EPA or DHA pre-treatment inhibits the DOX-induced decrease in UCP2 expression, increase in ROS production, and subsequent mitochondrial membrane potential change that contribute to the cardiotoxicity of DOX.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0101-3) contains supplementary material, which is available to authorized users.     

Mitochondrial ROS‐induced ERK1/2 activation and HSF2‐mediated AT1R upregulation are required for doxorubicin‐induced cardiotoxicity

Doxorubicin (DOX), one useful chemotherapeutic agent, is limited in clinical use because of its serious cardiotoxicity. Growing evidence suggests that angiotensin receptor blockers (ARBs) have cardioprotective effects in DOX‐induced cardiomyopathy. However, the detailed mechanisms underlying the action of ARBs on the prevention of DOX‐induced cardiomyocyte cell death have yet to be investigated. Our results showed that angiotensin II receptor type I (AT₁R) plays a critical role in DOX‐induced cardiomyocyte apoptosis. We found that MAPK signaling pathways, especially ERK1/2, participated in modulating AT₁R gene expression through DOX‐induced mitochondrial ROS release. These results showed that several potential heat shock binding elements (HSE), which can be recognized by heat shock factors (HSFs), located at the AT₁R promoter region. HSF2 markedly translocated from the cytoplasm to the nucleus when cardiomyocytes were damaged by DOX. Furthermore, the DNA binding activity of HSF2 was enhanced by DOX via deSUMOylation. Overexpression of HSF2 enhanced DOX‐induced cardiomyocyte cell death as well. Taken together, we found that DOX induced mitochondrial ROS release to activate ERK‐mediated HSF2 nuclear translocation and AT₁R upregulation causing DOX‐damaged heart failure in vitro and in vivo.     

Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species

Doxorubicin (DOX) is a broad spectrum anthracycline antibiotic used to treat a variety of cancers. Redox activation of DOX to form reactive oxygen species has been implicated in DOX-induced cardiotoxicity. In this work we investigated DOX-induced apoptosis in cultured bovine aortic endothelial cells and cardiomyocytes isolated from adult rat heart. Exposure of bovine aortic endothelial cells or myocytes to submicromolar levels of DOX induced significant apoptosis as measured by DNA fragmentation and terminal deoxynucleotidyltransferase-mediated nick-end labeling assays. Pretreatment of cells with 100 microm nitrone spin traps, N-tert-butyl-alpha-phenylnitrone (PBN) or alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) dramatically inhibited DOX-induced apoptosis. Ebselen (20-50 microm), a glutathione peroxidase mimetic, also significantly inhibited apoptosis. DOX (0.5-1 microm) inactivated mitochondrial complex I by a superoxide-dependent mechanism. PBN (100 microm), POBN (100 microm), and ebselen (50 microm) restored complex I activity. These compounds also inhibited DOX-induced caspase-3 activation and cytochrome c release. PBN and ebselen also restored glutathione levels in DOX-treated cells. We conclude that nitrone spin traps and ebselen inhibit the DOX-induced apoptotic signaling mechanism and that this antiapoptotic mechanism may be linked in part to the inhibition in formation or scavenging of hydrogen peroxide. Therapeutic strategies to mitigate DOX cardiotoxicity should be reexamined in light of these emerging antiapoptotic mechanisms of antioxidants.     

Attenuation of doxorubicin-induced contractile and mitochondrial dysfunction in mouse heart by cellular glutathione peroxidase

The cardiac toxicity of doxorubicin (DOX), a potent anticancer anthracycline antibiotic, is believed to be mediated through the generation of reactive oxygen species (ROS) in cardiomyocytes. This study aims to determine the function of cellular glutathione peroxidase (Gpx1), which is located in both mitochondria and cytosol, in defense against DOX-induced cardiomyopathy using a line of transgenic mice with cardiac overexpression of Gpx1. The Gpx1-overexpressing hearts were markedly more resistant than nontransgenic hearts to DOX-induced acute functional derangements, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impairs mitochondrial function of nontransgenic hearts as evident in a decreased rate of NAD-linked State 3 respiration, presumably a result of inactivation of complex I activity. This is associated with increases in the rates of NAD- and FAD-linked State 4 respiration and declines in P/O ratio, suggesting that the electron transfer and oxidative phosphorylation are uncoupled in these mitochondrial samples. These functional deficits of mitochondria could be largely prevented by Gpx1 overexpression. Taken together, these studies provide new evidence to further support the role of ROS, particularly H(2)O(2) and/or fatty acid hydroperoxides, in causing contractile and mitochondrial dysfunction in mouse hearts acutely exposed to DOX.     

Manipulation of Cardiac Phosphatidylinositol 3-Kinase (PI3K)/Akt Signaling by Apoptosis Regulator through Modulating IAP Expression (ARIA) Regulates Cardiomyocyte Death during Doxorubicin-induced Cardiomyopathy

PI3K/Akt signaling plays an important role in the regulation of cardiomyocyte death machinery, which can cause stress-induced cardiac dysfunction. Here, we report that apoptosis regulator through modulating IAP expression (ARIA), a recently identified transmembrane protein, regulates the cardiac PI3K/Akt signaling and thus modifies the progression of doxorubicin (DOX)-induced cardiomyopathy. ARIA is highly expressed in the mouse heart relative to other tissues, and it is also expressed in isolated rat cardiomyocytes. The stable expression of ARIA in H9c2 cardiac muscle cells increased the levels of membrane-associated PTEN and subsequently reduced the PI3K/Akt signaling and the downstream phosphorylation of Bad, a proapoptotic BH3-only protein. When challenged with DOX, ARIA-expressing H9c2 cells exhibited enhanced apoptosis, which was reversed by the siRNA-mediated silencing of Bad. ARIA-deficient mice exhibited normal heart morphology and function. However, DOX-induced cardiac dysfunction was significantly ameliorated in conjunction with reduced cardiomyocyte death and cardiac fibrosis in ARIA-deficient mice. Phosphorylation of Akt and Bad was substantially enhanced in the heart of ARIA-deficient mice even after treatment with DOX. Moreover, repressing the PI3K by cardiomyocyte-specific expression of dominant-negative PI3K (p110α) abolished the cardioprotective effects of ARIA deletion. Notably, targeted activation of ARIA in cardiomyocytes but not in endothelial cells reduced the cardiac PI3K/Akt signaling and exacerbated the DOX-induced cardiac dysfunction. These studies, therefore, revealed a previously undescribed mode of manipulating cardiac PI3K/Akt signaling by ARIA, thus identifying ARIA as an attractive new target for the prevention of stress-induced myocardial dysfunction.     

MitoQ protects dopaminergic neurons in a 6-OHDA induced PD model by enhancing Mfn2-dependent mitochondrial fusion via activation of PGC-1α

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra compacta (SNc). Although mitochondrial dysfunction is the critical factor in the pathogenesis of PD, the underlying molecular mechanisms are not well understood, and as a result, effective medical interventions are lacking. Mitochondrial fission and fusion play important roles in the maintenance of mitochondrial function and cell viability. Here, we investigated the effects of MitoQ, a mitochondria-targeted antioxidant, in 6-hydroxydopamine (6-OHDA)-induced in vitro and in vivo PD models. We observed that 6-OHDA enhanced mitochondrial fission by decreasing the expression of Mfn1, Mfn2 and OPA1 as well as by increasing the expression of Drp1 in the dopaminergic (DA) cell line SN4741. Notably, MitoQ treatment particularly upregulated the Mfn2 protein and mRNA levels and promoted mitochondrial fusion in the presence of 6-OHDA in a Mfn2-dependent manner. In addition, MitoQ also stabilized mitochondrial morphology and function in the presence of 6-OHDA, which further suppressed the formation of reactive oxygen species (ROS), as well as ameliorated mitochondrial fragmentation and cellular apoptosis. Moreover, the activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) was attributed to the upregulation of Mfn2 induced by MitoQ. Consistent with these findings, administration of MitoQ in 6-OHDA-treated mice significantly rescued the decrease of Mfn2 expression and the loss of DA neurons in the SNc. Taken together, our findings suggest that MitoQ protects DA neurons in a 6-OHDA induced PD model by activating PGC-1α to enhance Mfn2-dependent mitochondrial fusion.     

Tomato-oleoresin supplement prevents doxorubicin-induced cardiac myocyte oxidative DNA damage in rats

Doxorubicin (DOX) is an efficient chemotherapeutic agent used against several types of tumors; however, its use is limited due to severe cardiotoxicity. Since it is accepted that reactive oxygen species are involved in DOX-induced cardiotoxicity, antioxidant agents have been used to attenuate its side effects. To determine tomato-oleoresin protection against cardiac oxidative DNA damage induced by DOX, we distributed Wistar male rats in control (C), lycopene (L), DOX (D) and DOX+lycopene (DL) groups. They received corn oil (C, D) or tomato-oleoresin (5mg/kg body wt. day) (L, DL) by gavage for a 7-week period. They also received saline (C, L) or DOX (4mg/kg body wt.) (D, DL) intraperitoneally at the 3rd, 4th, 5th, and at 6th week. Lycopene absorption was checked by HPLC. Cardiac oxidative DNA damage was evaluated by the alkaline Comet assay using formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (endo III). Cardiomyocyte levels of SBs, SBs FPG and SBs Endo III were higher in rats from D when compared to other groups. DNA damage levels in cardiomyocytes from DL were not different when compared to C and L groups. The viability of cardiomyocytes from D or DL was lower than C or L groups (p<0.01). Lycopene levels (mean+/-S.D.nmol/kg) in saponified hearts were similar between L (47.43+/-11.78) and DL (49.85+/-16.24) groups. Our results showed: (1) lycopene absorption was confirmed by its cardiac levels; (2) DOX-induced oxidative DNA damage in cardiomyocyte; (3) tomato-oleoresin supplementation protected against cardiomyocyte oxidative DNA damage.