Doxorubicin-induced cardiomyocyte apoptosis: Role of mitofusin 2

BackgroundDoxorubicin (DOX) is an anti-tumor agent that is widely used in clinical setting for cancer treatment. The application of the DOX, however, is limited by its cardiac toxicity which can induce heart failure through an undefined mechanism. Mitofusin 2 (Mfn2) is a mitochondrial GTPase fusion protein that is located on the outer membrane of mitochondria (OMM). The Mfn2 plays an important role in mitochondrial fusion and fission. The aim of this study is to identify the role of the Mfn2 in DOX-induced cardiomyocyte apoptosis.MethodsCultured neonatal rat cardiomyocytes were used in this study. Mfn2 expression in cardiomyocytes was determined after the cardiomyocytes were challenged with DOX. Cardiomyocyte mitochondrial fission, mitochondrial reactive oxygen species (ROS) production was assessed with mitochondrial fragmentation and MitoSOX fluorescence probe, respectively. Cardiomyocyte apoptosis was determined with caspase3 activity and TUNEL staining.ResultsChallenging of the cardiomyocytes with DOX resulted in increasing in cardiomyocyte oxidative stress and apoptosis. In addition, levels of Mfn2 in cardiomyocytes were decreased after the cells were challenged with DOX which was associated with increased mitochondrial fission (fragmentation) and mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 attenuated the DOX-induced increase in mitochondrial fission and prevented cardiomyocyte mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 or pretreatment of cardiomyocytes with an anti-oxidant, Mito-tempo, also prevented the DOX-induced cardiomyocyte apoptosis.ConclusionOur results indicate that DOX results in a decreased cardiomyocyte Mfn2 expression which promotes mitochondrial fission and ROS production further leads to cardiomyocyte apoptosis.     

MicroRNA-15b enhances hypoxia/reoxygenation-induced apoptosis of cardiomyocytes via a mitochondrial apoptotic pathway

Myocardial ischemia reperfusion (I/R) can induce altered expression of microRNAs (miRNAs). The miRNAs—miR-15a, miR-15b and miR-16 have been shown to play a role in apoptosis, although not in cardiac-related models. We investigated the roles of miR-15b in hypoxia/reoxygenation (H/R)-induced apoptosis of cardiomyocytes. Quantitative real time polymerase chain reaction results showed that the expression of miR-15a and miR-15b were up-regulated in Sprague–Dawley rat hearts subjected to I/R. Expression levels of miR-15b increased more than four fold above basal levels. Similar results were obtained for cardiomyocytes exposed to H/R. Recombinant adenoviral vectors were generated to explore the functional role of miR-15b in cultured cardiomyocytes exposed to H/R. Overexpression of miR-15b enhanced cell apoptosis and the loss of mitochondrial membrane potential, as determined by flow cytometric analysis. Conversely, down-regulated expression was cytoprotective. The effects of miR-15b can by mimicked by Bcl-2 short-interfering RNAs. The inhibition of miR-15b increased expression levels of the Bcl-2 protein without affecting Bcl-2 mRNA levels, suppressed the release of mitochondrial cytochrome c to the cytosol and decreased the activities of caspase-3 and 9. It is possible that miR-15b is the upstream regulator of a mitochondrial signaling pathway for H/R induced apoptosis.     

Novel MicroRNA-455-3p and its protective effects against abnormal APP processing and amyloid beta toxicity in Alzheimer's disease

The purpose of our study is to understand the protective role of miR-455-3p against abnormal amyloid precursor protein (APP) processing, amyloid beta (Aβ) formation, defective mitochondrial biogenesis/dynamics and synaptic damage in AD progression. In-silico analysis of miR-455-3p has identified the APP gene as a putative target. Using mutant APP cells, miR-455-3p construct, biochemical and molecular assays, immunofluorescence and transmission electron microscopy (TEM) analyses, we studied the protective effects of miR-455-3p on – 1) APP regulation, amyloid beta (Aβ)(1–40) & (1–42) levels, mitochondrial biogenesis & dynamics; 3) synaptic activities and 4) cell viability & apoptosis. Our luciferase reporter assay confirmed the binding of miR-455-3p at the 3’UTR of APP gene. Immunoblot, sandwich ELISA and immunostaining analyses revealed that the reduced levels of the mutant APP, Aβ(1–40) & Aβ(1–42), and C99 by miR-455-3p. We also found the reduced levels of mRNA and proteins of mitochondrial biogenesis (PGC1α, NRF1, NRF2, and TFAM) and synaptic genes (synaptophysin and PSD95) in mutant APP cells; on the other hand, mutant APP cells that express miR-455-3p showed increased mRNA and protein levels of biogenesis and synaptic genes. Additionally, expression of mitochondrial fission proteins (DRP1 and FIS1) were decreased while the fusion proteins (OPA1, Mfn1 and Mfn2) were increased by miR-455-3p. Our TEM analysis showed a decrease in mitochondria number and an increase in the size of mitochondrial length in mutant APP cells transfected with miR-455-3p. Based on these observations, we cautiously conclude that miR-455-3p regulate APP processing and protective against mutant APP-induced mitochondrial and synaptic abnormalities in AD.     

MicroRNA-140 attenuates myocardial ischemia-reperfusion injury through suppressing mitochondria-mediated apoptosis by targeting YES1

Myocardial ischemia-reperfusion (I/R) injury is thought to have its detrimental role in coronary heart disease (CHD), which is considered as the foremost cause of death all over the world. However, molecular mechanism in the progression of myocardial I/R injury is still unclear. The goal of this study was to investigate the expression and function of microRNA-140 (miR-140) in the process of myocardial I/R injury. The miR-140 expression level was analyzed in the myocardium with I/R injury and control myocardium using quantitative real-time polymerase chain reaction. Then the relation between the level of miR-140 and YES proto-oncogene 1 (YES1) was also investigated via luciferase reporter assay. Assessment of myocardial infarct size measurement of serum myocardial enzymes and electron microscopy analysis were used for analyzing the effect of miR-140 on myocardial I/R injury. We also used Western blot analysis to examine the expression levels of the mitochondrial fission–related proteins, Drp1 and Fis1. miR-140 is downregulated, and YES1 is upregulated after myocardial I/R injury. Overexpression of miR-140 could reduce the increase related to myocardial I/R injury in infarct size and myocardial enzymes, and it also could inhibit the expression of proteins related to mitochondrial morphology and myocardial I/R-induced mitochondrial apoptosis by targeting YES1. Taken together, these findings may provide a novel insight into the molecular mechanism of miR-140 and YES1 in the progression of myocardial I/R injury. MiR-140 might become a promising therapeutic target for treating myocardial I/R injury.     

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.     

Mitochondrial dynamic alterations regulate melanoma cell progression

Research on mitochondrial fusion and fission (mitochondrial dynamics) has gained much attention in recent years, as it is important for understanding many biological processes, including the maintenance of mitochondrial functions, apoptosis, and cancer. The rate of mitochondrial biosynthesis and degradation can affect various aspects of tumor progression. However, the role of mitochondrial dynamics in melanoma progression remains controversial and requires a mechanistic understanding to target the altered metabolism of cancer cells. Therefore, in our study, we disrupted mitochondrial fission with mdivi-1, the reported inhibitor of dynamin related protein 1 (Drp1), and knocked down Drp1 and Mfn2 to evaluate the effects of mitochondrial dynamic alterations on melanoma cell progression. Our confocal study results showed that mitochondrial fission was inhibited both in mdivi-1 and in Drp1 knockdown cells and, in parallel, mitochondrial fusion was induced. We also found that mitochondrial fission inhibition by mdivi-1 induced cell death in melanoma cells. However, silencing Drp1 and Mfn2 did not affect cell viability, but enhanced melanoma cell migration. We further show that dysregulated mitochondrial fusion by Mfn2 knockdowns suppressed the oxygen consumption rate of melanoma cells. Together, our findings suggest that mitochondrial dynamic alterations regulate melanoma cell migration and progression.     

Two mitofusin proteins, mammalian homologues of FZO, with distinct functions are both required for mitochondrial fusion

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. Although these morphologic changes are considered crucial for cellular functions, the underlying mechanisms remain elusive, especially in mammalian cells. We characterized two rat mitochondrial outer membrane proteins, Mfn1 and Mfn2, with distinct tissue expressions, that are homologous to Drosophila Fzo, a GTPase involved in mitochondrial fusion. Expression of the GTPase-domain mutant of Mfn2 (Mfn2(K109T)) in HeLa cells induced mitochondrial fragmentation in which Mfn2(K109T) localized at the restricted domains. Immuno-electronmicroscopy revealed that Mfn2(K109T) was concentrated at the contact domains between adjacent mitochondria, suggesting that fusion of the outer membrane was arrested at some intermediate step. Mfn1 expression induced highly connected tubular network structures depending on the functional GTPase domain. The Mfn1-induced tubular networks were suppressed by co-expression with Mfn2. In vivo depletion of either isoform by RNA interference revealed that both are required to maintain normal mitochondrial morphology. The fusion of differentially-labeled mitochondria in HeLa cells subjected to depletion of either Mfn isoform and subsequent cell fusion by hemagglutinating virus of Japan revealed that both proteins have distinct functions in mitochondrial fusion. We conclude that the two Mfn isoforms cooperate in mitochondrial fusion in mammalian cells.     

miR-361-regulated prohibitin inhibits mitochondrial fission and apoptosis and protects heart from ischemia injury

Cardiovascular disease remains the leading cause of morbidity and mortality worldwide. Emerging evidences suggest that the abnormal mitochondrial fission participates in pathogenesis of cardiac diseases, including myocardial infarction (MI) and heart failure. However, the molecular components regulating mitochondrial network in the heart remain largely unidentified. Here we report that miR-361 and prohibitin 1 (PHB1) constitute an axis that regulates mitochondrial fission and apoptosis. The results show that PHB1 attenuates mitochondrial fission and apoptosis in response to hydrogen peroxide treatment in cardiomyocytes. Cardiac-specific PHB1 transgenic mice show reduced mitochondrial fission and myocardial infarction sizes after myocardial infarction surgery. MiR-361 is responsible for the dysfunction of PHB1 and suppresses the translation of PHB1. Knockdown of miR-361 reduces mitochondrial fission and apoptosis in vivo and in vitro. MiR-361 cardiac-specific transgenic mice represent elevated mitochondrial fission and myocardial infarction sizes upon myocardial ischemia injury. This study identifies a novel signaling pathway composed of miR-361 and PHB1 that regulates mitochondrial fission program and apoptosis. This discovery will shed new light on the therapy of myocardial infarction and heart failure.The heart drives the blood flow in the body and it has a large requirement of energy. Mitochondria meet the high energy demand of the heart by consistently providing large amounts of ATP through oxidative phosphorylation. Thus, mitochondrial malfunction is tightly related to cardiac diseases and contributes to cardiomyocyte injury, cardiomyopathy and heart failure. Mitochondria morphology is also associated with the function. Mitochondria constantly undergo fission and fusion. Fission leads to the formation of small round mitochondria and promotes cell apoptosis,^{1, 2, 3, 4, 5, 6, 7} whereas fusion results in mitochondria elongation and have a protective role in cardiomyocytes maintenance.⁸ The above findings strongly suggest that mitochondrial fission and fusion machinery is important for cardiac function. In addition, unveiling the mechanism of mitochondrial network regulation will provide a novel therapeutic strategy for heart failure.The mitochondrial prohibitin complex is a macromolecular structure at the inner mitochondrial membrane that is composed of prohibitin 1 (PHB1) and prohibitin 2 subunits.⁹ These two proteins comprise an evolutionary conserved and ubiquitously expressed family of membrane proteins and are implicated in several important cellular processes such as mitochondrial biogenesis and function, cell proliferation, replicative senescence, and cell death.^{10, 11} The first mammalian PHB1 was identified as a potential tumor suppressor with anti-proliferative activity.¹² Recent findings suggest that PHB1 has an important role in regulating mitochondrial morphology. Loss of PHB1 results in accumulation of fragmented mitochondria in MEFs and HeLa cells.^{13, 14} However, it is not yet clear whether PHB1 participates in the regulation of mitochondrial dynamics in cardiomyocytes.MicroRNAs (miRNAs) are a class of short single-stranded non-coding endogenous RNAs and act as negative regulators of gene expression by inhibiting mRNA translation or promoting mRNA degradation.^{15, 16} Although the function of miRNAs has been widely studied in apoptosis, development, differentiation and proliferation, few works have been focused on miRNAs in the mitochondrial network regulation. It has been reported that miR-30b targets to p53 and inhibits mitochondrial fission.¹⁷ In addition, other miRNAs also affect the function of mitochondria by targeting to mitochondrial calcium uniporter.¹⁸ The study of miRNA function in mitochondria may shed new light on the machinery that underlies mitochondrial regulation.This study unveils that PHB1 is involved in the regulation of mitochondrial network in cardiomyocytes. PHB1 inhibits mitochondrial fission and apoptosis in cardiomyocytes. In addition, PHB1 transgenic mice exhibit a reduced myocardial infarction sizes upon myocardial ischemia injury in vivo. In searching for the mechanism by which PHB1 is downregulated under pathologic condition, we identify miR-361 participates in the suppression of PHB1 translation. MiR-361 initiates mitochondrial fission, apoptosis and myocardial infarction through downregulating PHB1. Our results reveal a novel mitochondrial regulating model, which is composed of miR-361 and PHB1. Modulation of their levels may represent a novel approach for interventional treatment of myocardial infarction and heart failure.     

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.     

The mitochondrial ubiquitin ligase plays an anti‐apoptotic role in cardiomyocytes by regulating mitochondrial fission

Apoptosis plays a critical role in the development of myocardial infarction. Cardiomyocytes are enriched with mitochondria and excessive mitochondrial fission can trigger cellular apoptosis. Recently, the mitochondrial ubiquitin ligase (MITOL), localized in the mitochondrial outer membrane, was reported to play an important role in the regulation of mitochondrial dynamics and apoptosis. However, the underlying mechanism of its action remains uncertain. The present study was aimed at uncovering the role of MITOL in the regulation of cardiomyocyte apoptosis. Our results showed that MITOL expression was up‐regulated in cardiomyocytes in response to apoptotic stimulation. Mitochondrial ubiquitin ligase overexpression blocked dynamin‐related protein 1 accumulation in the mitochondria, and attenuated the mitochondrial fission induced by hydrogen peroxide. Conversely, MITOL knockdown sensitized cardiomyocytes to undergo mitochondrial fission, resulting in subsequent apoptosis. These findings suggest that MITOL plays a protective role against apoptosis in cardiomyocytes, and may serve as a potential therapeutic target for apoptosis‐related cardiac diseases.     

Mitofusin 2 protects cerebellar granule neurons against injury-induced cell death

Of the GTPases involved in the regulation of the fusion machinery, mitofusin 2 (Mfn2) plays an important role in the nervous system as point mutations of this isoform are associated with Charcot Marie Tooth neuropathy. Here, we investigate whether Mfn2 plays a role in the regulation of neuronal injury. We first examine mitochondrial dynamics following different modes of injury in cerebellar granule neurons. We demonstrate that neurons exposed to DNA damage or oxidative stress exhibit extensive mitochondrial fission, an early event preceding neuronal loss. The extent of mitochondrial fragmentation and remodeling is variable and depends on the mode and the severity of the death stimuli. Interestingly, whereas mitofusin 2 loss of function significantly induces cell death in the absence of any cell death stimuli, expression of mitofusin 2 prevents cell death following DNA damage, oxidative stress, and K+ deprivation induced apoptosis. More importantly, whereas wild-type Mfn2 and the hydrolysis-deficient mutant of Mfn2 (Mfn2(RasG12V)) function equally to promote fusion and lengthening of mitochondria, the activated Mfn2(RasG12V) mutant shows a significant increase in the protection of neurons against cell death and release of proapoptotic factor cytochrome c. These findings highlight a signaling role for Mfn2 in the regulation of apoptosis that extends beyond its role in mitochondrial fusion.     

Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins

Mitochondria are dynamic organelles that undergo continual fusion and fission to maintain their morphology and functions, but the mechanism involved is still not clear. Here, we investigated the effect of mitochondrial oxidative stress triggered by high-fluence low-power laser irradiation (HF-LPLI) on mitochondrial dynamics in human lung adenocarcinoma cells (ASTC-a-1) and African green monkey SV40-transformed kidney fibroblast cells (COS-7). Upon HF-LPLI-triggered oxidative stress, mitochondria displayed a fragmented structure, which was abolished by exposure to dehydroascorbic acid, a reactive oxygen species scavenger, indicating that oxidative stress can induce mitochondrial fragmentation. Further study revealed that HF-LPLI caused mitochondrial fragmentation by inhibiting fusion and enhancing fission. Mitochondrial translocation of the profission protein dynamin-related protein 1 (Drp1) was observed following HF-LPLI, demonstrating apoptosis-related activation of Drp1. Notably, overexpression of Drp1 increased mitochondrial fragmentation and promoted HF-LPLI-induced apoptosis through promoting cytochrome c release and caspase-9 activation, whereas overexpression of mitofusin 2 (Mfn2), a profusion protein, caused the opposite effects. Also, neither Drp1 overexpression nor Mfn2 overexpression affected mitochondrial reactive oxygen species generation, mitochondrial depolarization, or Bax activation. We conclude that mitochondrial oxidative stress mediated through Drp1 and Mfn2 causes an imbalance in mitochondrial fission-fusion, resulting in mitochondrial fragmentation, which contributes to mitochondrial and cell dysfunction.     

microRNA-340-5p inhibits hypoxia/reoxygenation-induced apoptosis and oxidative stress in cardiomyocytes by regulating the Act1/NF-κB pathway

MicroRNAs (miRNAs) have been reported to play critical roles in the occurrence, progression, and treatment of many cardiovascular diseases. However, the molecular mechanism by which miRNA regulates target gene expression in ischemia-reperfusion (I/R) injury in acute myocardial infarction (AMI) is not entirely clear. MiR-340-5p was reported to be downregulated in acute ischemic stroke. However, it still remains unknown whether miR-340-5p is mediated in the pathogenesis process of I/R injury after AMI. In the present study, male C57BL/6 J mice and H9C2 cardiomyocytes were used as experimental models. Real-time polymerase chain reaction analysis, Western blot analysis, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling immunofluorescence staining assay were conducted to examine related indicators in the study. We confirmed that the expression of miR-340-5p is downregulated after I/R in AMI mice and hypoxia/reperfusion (H/R)-induced cardiomyocytes. miR-340-5p could inhibit apoptosis and oxidative stress in H/R-induced H9C2 cells via downregulating activator 1 (Act1). The inhibiting action of miR-340-5p on H/R-induced apoptosis and oxidative stress in cardiomyocytes was partially reversed after Act1 overexpression. Moreover, the results showed that the NF-κB pathway may be mediated in the role of miR-340-5p on H/R-induced cardiomyocyte apoptosis and oxidative stress. We demonstrated that upregulation of miR-340-5p suppresses apoptosis and oxidative stress induced by H/R in H9C2 cells by inhibiting Act1. Therapeutic strategies that target miR-340-5p, Act1, and the NF-κB pathway could be beneficial for the treatment of I/R injury after AMI.