Potent Inducer Of Autophagy Research Articles

Transmembrane Protein 208: A Novel ER-Localized Protein That Regulates Autophagy and ER Stress

Autophagy and endoplasmic reticulum (ER) stress are both tightly regulated cellular processes that play central roles in various physiological and pathological conditions. Recent reports have indicated that ER stress is a potent inducer of autophagy. However, little is known about the underlying molecular link between the two processes. Here we report a novel human protein, transmembrane protein 208 (TMEM208) that can regulate both autophagy and ER stress. When overexpressed, TMEM208 impaired autophagy as characterized by the decrease of the accumulation of LC3-II, decreased degradation of autophagic substrates, and reduced expression of critical effectors and vital molecules of the ER stress and autophagy processes. In contrast, knockdown of the TMEM208 gene promoted autophagy, as demonstrated by the increase of LC3-II, increased degradation of autophagic substrates, and enhanced expression levels for genes key in the ER stress and autophagic processes. Taken together, our results reveal that this novel ER-located protein regulates both ER stress and autophagy, and represents a possible link between the two different cellular processes.

Abstract 2087: A novel Btk tyrosine kinase inhibitor, CTN06, as a potential candidate agent for breast cancer treatment.

Abstract Backgrounds: Btk family kinases are non-receptor tyrosine kinases which are involved in the regulation of diverse cellular processes such as growth, survival and migration. Btk up-regulation has been shown to contribute to oncogenesis of myeloid malignacies. However its functions in solid tumors have not been explored due to its prominent expression in B cells. In this study, we present the first evidence that Btk overexpression contributes to breast carcinogenesis. Material and Methods: CTN06-treated and BTK silenced cells were subjected to an Affimetrix human Genome U133A 2.0 microarray analysis for a global examination of Btk's functions in epithelial carcinogenesis. Signaling cascades which were altered by CTN06 treatment include STAT3, mTOR, S6K and Wnt/β-catenin. In vitro proliferation, migration/invasion assays were employed to demonstrate CTN06-mediated anti-cancer effects. RNA interference experiments were performed to show Btk's contribution to breast carcinogenesis and metastasis. Finally, xenograft mouse model was used to demonstrate the effects of CTN06 and the down-regulation of Btk on breast tumorigenesis. Results: Our preliminary data indicates that Btk is aberrantly expressed in various breast cancer cell lines and tissues but particularly in cells of invasive phenotype. These findings prompted us to further explore the role of Btk in breast cancer carcinogenesis. Using available structural models, our team has developed a collection of small molecule inhibitors for Btk and shown that one of them, CTN06, prominently inhibited the growth and migration of different breast cancer cell lines. Interestingly, CTN06 also appeared to be a potent autophagy inducer in the highly invasive and triple-negative MDA-MB-231 cells. Based on these premises and Btk's participation in several signaling cascades such as Src, PI3K, PLCγ and PLC, we hypothesize that Btk overexpression confers a growth and/or survival advantage for breast cancer cells; Btk inhibitors (CTN06) should effectively suppress breast cancer development by negatively modulating Btk-related networks and be evaluated for clinical use. We have obtained promising results not only in vitro cell line models but also in vivo animal xenograft experiments to demonstrate its efficacy and safety profile. Conclusions: We demonstrated for the first time that Btk overexpression is detected in breast cancer cells particularly in metastatic MDA-MB-231 cells and Btk-specific inhibitor CTN06 effectively suppressed both tumor growth and metastasis via negatively modulating the aforementioned signaling cascades in vitro and in vivo. Citation Format: Chih-Ming Su, Chin-Nien Chuang, Alexander Wu, Liang-Shun Wang, Wei-Hwa Lee, Chih-Hsiung Wu, Kit Lam, Hsing-Jien Kung, Chi-Tai Yeh. A novel Btk tyrosine kinase inhibitor, CTN06, as a potential candidate agent for breast cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2087. doi:10.1158/1538-7445.AM2013-2087

Beneficial lessons from viruses

To thwart the antimicrobial responses of their hosts, pathogens have evolved diverse mechanisms, including autophagy. Knowledge of such mechanisms has now led to a pro-autophagy peptide that may be of therapeutic value. See Article p.201 Autophagy is an essential degradation pathway that eliminates damaged proteins and organelles in cells and also protects against infection by diverse pathogens, including viruses. In this study, Beth Levine and colleagues construct a cell-permeable peptide, Tat-beclin 1, derived from part of an essential autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice, and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1. The Tat-beclin 1 peptide binds to the Golgi-associated plant pathogenesis-related protein 1 (GAPR-1), which functions as a negative regulator of autophagy. These results suggest that this beclin 1-derived autophagy-inducing peptide has potential for the prevention and treatment of a broad range of human diseases.

Identification of a candidate therapeutic autophagy-inducing peptide

The lysosomal degradation pathway of autophagy has a crucial role in defence against infection, neurodegenerative disorders, cancer and ageing. Accordingly, agents that induce autophagy may have broad therapeutic applications. One approach to developing such agents is to exploit autophagy manipulation strategies used by microbial virulence factors. Here we show that a peptide, Tat-beclin 1-derived from a region of the autophagy protein, beclin 1, which binds human immunodeficiency virus (HIV)-1 Nef-is a potent inducer of autophagy, and interacts with a newly identified negative regulator of autophagy, GAPR-1 (also called GLIPR2). Tat-beclin 1 decreases the accumulation of polyglutamine expansion protein aggregates and the replication of several pathogens (including HIV-1) in vitro, and reduces mortality in mice infected with chikungunya or West Nile virus. Thus, through the characterization of a domain of beclin 1 that interacts with HIV-1 Nef, we have developed an autophagy-inducing peptide that has potential efficacy in the treatment of human diseases.

BNIP3 Is Downregulated in Myelodysplastic Syndromes and Acute Myeloid Leukemia and Is a Potential Molecular Target for Decitabine

AbstractAbstract 1708In myelodysplastic syndromes (MDS), changes in the balance between pro- and anti-apoptotic proteins are associated with disease progression and evolution towards acute myeloid leukemia (AML). BNIP3 is a pro-apoptotic protein, member of the Bcl-2 superfamily, associated with the pathogenesis of many diseases, including cancer. More recently, BNIP3 was identified as a potent inducer of autophagy. In primary leukemia cells, BNIP3 expression has been shown to be reduced due to epigenetic modifications; however, there is a lack of studies of BNIP3 in MDS. The aim of this study was to characterize BNIP3 mRNA expression levels and DNA methylation status of its 5’ CpG island in bone marrow cells from MDS and AML patients, healthy donors and in leukemia hematopoietic cell lines. We also investigated the effects of BNIP3 silencing upon decitabine (DAC) treatment in U937 cells regarding colony formation, apoptosis and autophagy. Bone marrow aspirates were obtained from 21 healthy donors, 55 patients with MDS and 44 patients with AML at diagnosis. MDS patients were grouped into low-risk and high-risk groups according to FAB (RA/RARS=30; RAEB/RAEBt=25), WHO (RCUD/RCMD/RARS/del5/=29; RAEB1/RAEB2=17) and IPSS (low/INT-1=38; INT2/high=16). The study was approved by the ethics committee and informed written consent was provided. Gene expression was evaluated by qPCR and DNA methylation was performed by Combined Bisulfite Restriction Analysis. BNIP3 knockdown was performed in U937 cells with specific shRNA-expressing lentiviral vector. ShRNA encoding no specific sequence was used as control. Colony formation was carried out in semisolid methylcellulose medium. Apoptosis and autophagy were accessed by flow cytometry. All assays were performed in lentiviral transduced cells treated or not with 5μM DAC for 72 hours. Mann-Whitney and Student’s t-test were used for statistical analyses. We observed a significant decrease in BNIP3 mRNA expression in total bone marrow cells from AML and MDS compared to healthy donors (AML= 0.52 [5.27-0.00]; MDS=0.52 [5.25-0.02] versus healthy donors=1.09 [6.04-0.18]; P<0.05). BNIP3 expression did not differ between low-risk and high-risk MDS patients according to FAB and WHO classifications, and IPSS. Methylation of BNIP3 promoter was detected in 19% (4/21; 2/14 low-risk and 2/7 high-risk) MDS and 26% (4/15) AML patients, but not in any of the 6 healthy donors evaluated. BNIP3 expression was detected in all myeloid cell lines studied (K562, HL60, U937 and P39 cells). The lowest expression was found in U937, which presented methylation of BNIP3 promoter. Interestingly, in U937 cells, DAC treatment resulted in upregulation of BNIP3 expression (3.7-fold), total inhibition of colony formation, increased apoptosis and autophagy (50% for both). The effect of DAC on cell apoptosis was reduced by BNIP3 silencing; apoptosis was reduced by 48%±4.3 in shBNIP3 cells compared to 59%±5.7 in shControl cells (p<0.05). Autophagy was not modulated by DAC treatment in BNIP3 silenced cells. In bone marrow cells from MDS and AML patients, the low frequency of methylated BNIP3 promoter suggests that DNA methylation is not the single reason for the decreased BNIP3 expression in these patients. Other mechanisms, such as histone deacetylation, may be involved in aberrant BNIP3 expression. DAC treatment in BNIP3 silenced cells had no effect on autophagy, suggesting that other mechanisms besides increasing BNIP3 expression is involved in this cell function. On the other hand, DAC treatment in U937 cells was able to induce apoptosis, especially in cells expressing BNIP3, supporting the hypothesis that BNIP3 methylation is a molecular target for DAC. Disclosures:No relevant conflicts of interest to declare.

Cell death in HeLa cells upon imperatorin and cisplatin treatment

There is growing evidence that commonly applied chemotherapy regimens can be improved by introducing new, specific, active and low side-effect drugs, or by combining substances to obtain the required clinical effect. The aim of the present study was to investigate the effects of imperatorin and cisplatin, applied separately or in combination, on apoptosis, necrosis and autophagy induction in the human cervical carcinoma cell line(HeLa). Imperatorin appeared to be a potent autophagy inducer, rather than a necrotic or apoptotic one. In contrast, cisplatin induced mainly apoptosis and necrosis after 6 h and 24 h, while longer incubation resulted only in necrosis induction. When HeLa cells were incubated with both drugs, autophagy appeared most frequently,although to a smaller extent than that observed after imperatorin administered alone. At the molecular level, autophagy was correlated with the presence of the cleaved form of microtubule-associated protein 1 light chain LC3 - LC3II. It was also accompanied by the inhibition of heat shock proteins Hsp27 and Hsp72 expression.Our results indicate that imperatorin alone, or in combination with cisplatin, is mainly an autopahgy inducer in HeLa cells.

Cell death in HeLa cells upon imperatorin and cisplatin treatment

There is growing evidence that commonly applied chemotherapy regimens can be improved by introducing new, specific, active and low side-effect drugs, or by combining substances to obtain the required clinical effect. The aim of the present study was to investigate the effects of imperatorin and cisplatin, applied separately or in combination, on apoptosis, necrosis and autophagy induction in the human cervical carcinoma cell line (HeLa). Imperatorin appeared to be a potent autophagy inducer, rather than a necrotic or apoptotic one. In contrast, cisplatin induced mainly apoptosis and necrosis after 6 h and 24 h, while longer incubation resulted only in necrosis induction. When HeLa cells were incubated with both drugs, autophagy appeared most frequently, although to a smaller extent than that observed after imperatorin administered alone. At the molecular level, autophagy was correlated with the presence of the cleaved form of microtubule-associated protein 1 light chain LC3 — LC3II. It was also accompanied by the inhibition of heat shock proteins Hsp27 and Hsp72 expression. Our results indicate that imperatorin alone, or in combination with cisplatin, is mainly an autopahgy inducer in HeLa cells.

Caloric Restriction Enhances Cardiomyocyte Autophagy to Improve Energy Status and Ameliorates Left Ventricular Remodeling in the Postinfarction Heart

We previously reported that cardiomyocyte autophagy is an innate mechanism which prevents postinfarction cardiac remodeling. In this study, we examined effect of caloric restriction (CR), a potent inducer of autophagy, on postinfarction cardiac remodeling. Myocardial infarction (MI) was induced in mice. After one week of MI, mice were randomly divided into the following four groups. The control group was fed ad libitum (100%). Others were fed restricted diet respectively 80%, 60%, and 40% of the control mice's total calorie.

Death à la carte

Keywords: : European Workshop on Cell Deathcell deathBcl-2 familydeath receptor signalingpattern-recognition receptorsTNFinflammationautophagymetabolism

Caspase activation regulates the extracellular export of autophagic vacuoles

The endothelium plays a central role in the regulation of vascular wall cellularity and tone by secreting an array of mediators of importance in intercellular communication. Nutrient deprivation of human endothelial cells (EC) evokes unconventional forms of secretion leading to the release of nanovesicles distinct from apoptotic bodies and bearing markers of multivesicular bodies (MVB). Nutrient deficiency is also a potent inducer of autophagy and vesicular transport pathways can be assisted by autophagy. Nutrient deficiency induced a significant and rapid increase in autophagic features, as imaged by electron microscopy and immunoblotting analysis of LC3-II/LC3-I ratios. Increased autophagic flux was confirmed by exposing serum-starved cells to bafilomycin A1. Induction of autophagy was followed by indices of an apoptotic response, as assessed by microscopy and poly (ADP-ribose) polymerase cleavage in absence of cell membrane permeabilization indicative of necrosis. Pan-caspase inhibition with ZVAD-FMK did not prevent the development of autophagy but negatively impacted autophagic vacuole (AV) maturation. Adopting a multidimensional proteomics approach with validation by immunoblotting, we determined that nutrient-deprived EC released AV components (LC3I, LC3-II, ATG16L1 and LAMP2) whereas pan-caspase inhibition with ZVAD-FMK blocked AV release. Similarly, nutrient deprivation in aortic murine EC isolated from CASP3/caspase 3-deficient mice induced an autophagic response in absence of apoptosis and failed to prompt LC3 release. Collectively, the present results demonstrate the release of autophagic components by nutrient-deprived apoptotic human cells in absence of cell membrane permeabilization. These results also identify caspase-3 as a novel regulator of AV release.

Antitumor effect of arsenic trioxide in human K562 and K562/ADM cells by autophagy

Arsenic trioxide (As2O3) has been reported to have potent antitumor effects in vitro and in vivo by inducing cell death via cell cycle arrest and apoptosis in leukemia cells, but the mechanisms of As2O3-mediated cell death are not fully understood. In this study, we provided in vitro evidence that As2O3 was a potent inducer of autophagy in leukemia K562 and its drug-resistant line K562/ADM cells. As2O3 significantly activated autophagic cell death (programmed cell death type II) in leukemia cell lines. Numerous large cytoplasmic inclusions, abundant autophagic vacuoles, phagocytizing cytoplasm and organelles were observed in As2O3-treated cells using electron microscope. MDC-labeled autophagic vacuoles were observed by fluorescent inverted phase contrast microscopy and the enhanced MDC fluorescent staining was detected by flow cytometry in As2O3-treated cells. Furthermore, real-time quantitative RT-PCR revealed that the expression levels of Beclin-1 and LC3 genes, which play key roles in autophagy, increased in As2O3 treated samples than in controls, indicating that autophagy can potentially be involved in the antitumor properties of As2O3. The expression level of Bcl-2 gene, an anti-apoptotic molecule, decreased in As2O3 treated samples than in controls, suggesting that Bcl-2 may be involved in accumulating Beclin-1 and triggering autophagic cell death in As2O3-treated leukemia cells. Western blotting also showed that As2O3 up-regulated Beclin-1. Altogether, our data provide direct evidence that autophagic cell death is critical for the effects of As2O3 on acute myelogenous leukemia cells.

Old Antibiotics Target TB with a New Trick

Autophagy is now recognized as a cellular defense mechanism that can restrict the growth of Mycobacterium tuberculosis (Mtb). In this issue of Cell Host & Microbe, Kim etal. (2012) demonstrate that antibiotics routinely used to treat Mtb infection elicit a host autophagy response critical for bacterial clearance.

Methylthioninium chloride (methylene blue) induces autophagy and attenuates tauopathy in vitro and in vivo

More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubule-binding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy. Further analysis of the signaling pathways induced by MB suggested a mode of action similar to rapamycin. Results were recapitulated in a transgenic mouse model of tauopathy administered MB orally at three different doses for two weeks. These data support the use of this drug as a therapeutic agent in neurodegenerative diseases.

Acetylation: A Novel Link between Double-Strand Break Repair and Autophagy

Histone deacetylase (HDAC) inhibitors are clinically relevant because they are used as anticancer drugs. Recent evidence sheds light on an intriguing connection among the DNA damage response (DDR), protein acetylation, and autophagy. HDAC inhibitors have been shown to counteract key steps in the cellular response to double-strand break formation by affecting checkpoint activation, homologous recombination-mediated repair of DNA lesions, and stability of crucial enzymes involved in resection of DNA ends. The degradation of the resection factors depends on autophagy, which plays a detrimental role when cells are in a hyperacetylated state and experience treatment with radiomimetic anticancer drugs. Future work will be required to further investigate the mechanisms underlying the link between acetylation, autophagy, and the DDR, as well as the significance of mTORC1 inhibitors, which are potent inducers of autophagy that are now used in cancer treatment.

Sertindole, a Potent Antagonist at Dopamine D&lt;sub&gt;2&lt;/sub&gt; Receptors, Induces Autophagy by Increasing Reactive Oxygen Species in SH-SY5Y Neuroblastoma Cells

Autophagy is associated with cell survival and cell death. Autophagy is implicated in the pathophysiology of various human diseases. In order to identify autophagy regulatory molecules, we screened a chemical drug library in SH-SY5Y cells and selected Sertindole as a potent autophagy inducer. Sertindole was developed as an antipsychotic drug for Schizophrenia. Sertindole treatment highly induced the formation of autophagosomes as well as LC3 conversion. Subsequently, Sertindole-induced autophagy was efficiently suppressed by down regulation of ATG5. Sertindole also increased reactive oxygen species (ROS) production, which contributes to autophagy-associated cell death in neuroblastoma cells. ROS scavengers such as N-acetylcysteine and Trolox suppressed not only ROS generation but also autophagy activation by Sertindole. These results suggest Sertindole induces autophagy and autophagy-associated cell death by ROS production in neuroblastoma cells.

Abstract 9424: Caloric Restriction Augments Cardiomyocyte Autophagy to Mitigate Postinfarction Cardiac Remodeling and Heart Failure

We previously reported that cardiomyocyte autophagy is an innate mechanism that protects against progression of postinfarction cardiac remodeling. In the present study, we examined effect of caloric restriction (CR), a potent inducer of autophagy, on post infarction cardiac remodeling. Myocardial infarction (MI) was induced in mice by ligating the left coronary artery. After one week of MI, mice were randomly divided into the following four groups. The control group was fed ad libitum (100%). Others were fed restricted diet: 80%, 60%, and 40% of the total calorie which was taken by control mice. The water was provided ad libitum. Three weeks later, we performed echocardiography and catheter to evaluate the left ventricular (LV) function. There was no mouse that died through the period. The body weight was decreased in CR groups (post/pre ratio: 1.08±0.01 in control, 0.83±0.01 in 80% CR, 0.76±0.01 in 60% CR, 0.70±0.01 in 40% CR, p&lt;0.001). However, there was no difference in heart/body weight ratio. The dilatation and hypofunction of the LV were apparent in the control group: left ventricular diastolic dimension (LVDd) = 4.9±0.1 mm; left ventricular ejection fraction (LVEF) = 27±3.3%; and dP/dT = 4677±277 mmHg/s. However, both dilatation and hypofunction were significantly mitigated in groups undergoing CR: in 80%, 60%, and 40% CR, respectively, LVDd = 4.0±0.1 mm, 3.4±0.2 mm and 4.0±0.1 mm; LVEF = 36±2.8%, 51±4.3% and 43±1.5%; and dP/dT = 6849±221 mmHg/s, 7758±638 mmHg/s and 6923±553 mmHg/s, among which the 60% CR group showed the strongest therapeutic effect on postinfarction cardiac remodeling (control vs. 60% CR: p&lt;0.001). Cardiomyocyte autophagy was markedly activated in CR groups as evidenced by the overexpressed microtubule-associated protein 1 light chain 3-II (LC3-II) and specific ultrastructural findings including autophagosome formation. In conclusion, caloric restriction enhances cardiomyocyte autophagy and mitigates postinfarction cardiac remodeling and dysfunction; the optimal caloric restriction is suggested to be 60%. These findings suggest an important clinical implication that a dietary protocol can be a preventive strategy against progression of postinfarction left ventricular remodeling and heart failure.

Expression of ER stress and autophagy‐related molecules in human non‐small cell lung cancer and premalignant lesions

Stress that impairs endoplasmic reticulum (ER) function leads to an accumulation of unfolded or misfolded proteins in the ER (ER stress). Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles, which emerging data indicate that ER stress is also a potent inducer of autophagy. ER stress and autophagy are involved in human cancer. We examined the expression of ER stress-related proteins [GRP78 and C/EBP homologous protein (CHOP)] and autophagic proteins (Beclin-1 and LC3) in non-small cell lung carcinomas (NSCLCs), bronchioloalveolar carcinomas (BACs) and atypical adenomatous hyperplasias (AAHs) to understand their role in the NSCLC pathogenesis. The expression of GRP78 and CHOP, Beclin-1 and LC3 were analyzed using immunohistochemistry on tissue sections from 133 NSCLC (69 squamous cell carcinomas, 56 adenocarcinomas (AC) and eight other NSCLCs), 21 BAC and 9 AAH. Expression of GRP78 and Beclin-1 was correlated with low tumor stage (p < 0.001 and p = 0.019, respectively) and longer survival (p = 0.007 and p <0.001, respectively) by Kaplan-Meier analysis. However, CHOP was correlated with high tumor stage (p = 0.038) and shorter survival (p = 0.012). Expression of GRP78 and Beclin-1 was positively correlated (p = 0.006). Our study showed that the expression of GRP78, CHOP, Beclin-1 and LC3 in lung cancer and its relation with clinicopathologic factors and patients survival. These results suggest that GRP78, CHOP and Beclin-1 may play an important role in tumorigenesis of lung AC and may serve as new prognostic indicators for outcome of the patients with NSCLC.

CAMP induces autophagy via a novel pathway involving ERK, cyclin E and Beclin 1

Autophagy plays an important role in cellular remodelling during differentiation and development, however little is known about its regulation in stem cells. Here we show that cAMP, a well-known differentiation factor for mesenchymal stem cells (MSCs), is also a potent inducer of autophagy in these cells. We have previously shown that activation of the cAMP-signaling pathway inhibits proliferation of MSCs despite induction of the cell cycle component cyclin E. Here, we demonstrate a critical role of cyclin E in the induction of autophagy. Our data suggest a model in which cAMP-signaling via ERK-mediated induction of cyclin E leads to enhanced perinuclear recruitment of Beclin 1 and formation of autophagosomes. Given the roles of deregulated autophagy in neurodegenerative disorders and cAMP as a neurogenic inducer, identification of this novel autophagocytic pathway may provide new targets for intervention against neurological disorders.

Mitochondrial autophagy by Bnip3 involves Drp1-mediated mitochondrial fission and recruitment of Parkin in cardiac myocytes

The Bcl2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) is an atypical BH3-only protein that is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of mitochondrial autophagy, and in this study we have investigated the mechanisms by which Bnip3 induces autophagy in cardiac myocytes. We found that Bnip3 induced mitochondrial translocation of dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission in adult myocytes. Drp1-mediated mitochondrial fission correlated with increased autophagy, and inhibition of Drp1 reduced Bnip3-mediated autophagy. Overexpression of Drp1K38E, a dominant negative of Drp1, or mitofusin 1 prevented mitochondrial fission and autophagy by Bnip3. Also, inhibition of mitochondrial fission or autophagy resulted in increased death of myocytes overexpressing Bnip3. Moreover, Bnip3 promoted translocation of the E3 ubiquitin ligase Parkin to mitochondria, which was prevented in the presence of a Drp1 inhibitor. Interestingly, induction of autophagy by Bnip3 was reduced in Parkin-deficient myocytes. Thus our data suggest that induction of autophagy in response to Bnip3 is a protective response activated by the cell that involves Drp1-mediated mitochondrial fission and recruitment of Parkin.

Caloric Restriction Enhances Cardiomyocyte Autophagy and Mitigates Postinfarction Cardiac Remodeling and Dysfunction

We previously reported that cardiomyocyte autophagy is an innate mechanism that protects against progression of postinfarction cardiac remodeling. In the present study, we examined effect of caloric restriction (CR), a potent inducer of autophagy, on post infarction cardiac remodeling. Myocardial infarction (MI) was induced in mice by ligating the left coronary artery. After one week of MI, mice were randomly divided into the following four groups. The control group was fed ad libitum (100%). Others were fed restricted diet respectively 80%, 60%, and 40% of the total calorie which was taken by control mice.