Autophagy is required for the maintenance of cardiomyocytes homeostasis. However, the abnormal autophagy could lead to the development of heart failure. Autophagy is enhanced during myocardial ischemia/reperfusion; it remains to elucidate the molecular regulation of autophagy. We report here that miR-325, ARC and E2F1 constitute an axis that regulates autophagy. Our results showed that miR-325 expression is upregulated upon anoxia/reoxygenation and ischemia/reperfusion. Cardiomyocyte-specific overexpression of the miR-325 potentiates autophagic responses and myocardial infarct sizes, whereas knockdown of miR-325 inhibited autophagy and cell death. We searched for the downstream mediator of miR-325 and identified that ARC is a target of miR-325. ARC transgenic mice could attenuate autophagy and myocardial infarction sizes upon pressure-overload-induced heart failure, whereas ARC null mice exhibited an increased autophagic accumulation in the heart. The suppression of ARC by miR-325 led to its inability to repress autophagic program. In exploring the molecular mechanism by which miR-325 expression is regulated, our results revealed that the transcription factor E2F1 contributed to promote miR-325 expression. E2F1 null mice demonstrated reduced autophagy and myocardial infarction sizes upon ischemia/reperfusion. Our present study reveals a novel autophagic regulating model that is composed of E2F1, miR-325 and ARC. Modulation of their levels may provide a new approach for tackling cardiac failure.
Autophagy is a process of catabolism of cellular components including protein aggregates, organelles and viruses, and is necessary for the maintenance of cell homeostasis.1, 2, 3, 4 The morphological character of autophagy is the formation of autophagosome that encompasses cellular components and subsequently fuses with lysosomes. It finally degrades the inclusion into reusable amino acids and other materials.5, 6 However, the abnormal autophagy is related to a variety of pathological disorders including cancer, neurodegenerative and cardiovascular diseases.7, 8, 9, 10
MicroRNAs (miRNAs) are about 22-nucleotides long and act as negative regulators of gene expression.11, 12, 13, 14 Strikingly, a single microRNA is even able to modulate complex physiological or disease phenotypes by regulating the entire functional networks.12, 15, 16 They can bind to the 3′ untranslated region (3′UTR) of mRNAs, thereby inhibiting mRNA translation or promoting mRNA degradation.11 They participate in the regulation of a variety of cellular events such as apoptosis, proliferation, development, differentiation and cell conversion. For example, miR-200c strongly suppresses the ability of normal mammary stem cells to form mammary ducts and tumor formation.17 miR-31 uses multiple mechanisms to oppose the breast cancer metastasis.18 miR-208 regulates cardiac growth and gene expression in response to stress and hormonal signaling.14
Autophagy is regulated by a variety of factors in which the autophagy-related genes (ATGs) control the different stages of autophagy including initiation and elongation.1, 19 Beclin 1 is ATG6 and is required for the formation of the autophagasome in autophagy.20 LC3-I is ATG8, which is conjugated to the lipid phosphatidylethanolamine to form LC3-II that fuses with the inner and outer surfaces of the autophagosome.21 Intriguingly, apoptosis and autophagy have a cross-talk, and some proteins can regulate both autophagy and apoptosis. ATG12–ATG3 forms a complex that produces an expansion in mitochondrial mass and inhibits cell death mediated by mitochondrial pathways.22 Bcl-2 can inhibit apoptosis and autophagy.7, 23 Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis.24 Inhibition of autophagy leads to the occurrence of apoptosis.25
A few reports have shown that miRNAs are involved in the regulation of autophagy. miR-101 is a potent inhibitor of autophagy.26 miR-30a sensitizes tumor cells to cis-platinum via suppressing Beclin 1-mediated autophagy.27 miR-204 regulates autophagy through LC3-II.28 It remains largely unknown as to how autophagy is regulated by miRNAs.
Apoptosis repressor with caspase recruit domain (ARC) is a protein not only highly expressed in the heart and skeletal muscle29, 30 but also in a variety of cancers.31, 32 It was originally identified to be a caspase-inhibiting protein and can inhibit apoptosis induced by a variety of stimuli.30 Further evidence demonstrates that it also can bind to Bax33 and Puma,29 thereby quenching their apoptotic signal. It is not yet clear whether ARC can regulate autophagic program.
Our present study unveils that ARC is an antiautophagic protein. ARC knockout mice exhibit accelerated autophagy, whereas ARC transgenic mice can attenuate autophagy. In searching for the mechanism by which ARC is downregulated under autophagic condition, we identified miR-325 participates in the suppressing of ARC translation. miR-325 initiates autophagic program through targeting ARC. We further found that E2F1 can promote the expression of miR-325. miR-325 is a downstream mediator of E2F1 in autophagic cascades. Taken together, our findings provide novel evidence that miRNA can regulate autophagic program.
ARC regulates autophagy under the physiological condition
To understand the role of ARC in the machinery of autophagy, we employed ARC knockout mice. A characteristic of autophagy is the recruitment of the microtubule-associated protein 1 light chain 3 (LC3) to autophagic vesicles. Evidence for the increased autophagic activity was revealed by an augmentation in LC3-II levels in an ARC-deficient heart compared with wild-type mice. Noticeably, LC3 processing was not obviously detected in other tissues where ARC is not abundantly expressed (Figure 1a). These data indicate that ARC-deficient mice exhibited an autophagic response specific to the heart and not reflective of other global responses. The electron micrographic (EM) analysis of ventricular tissue was also used to identify double-membrane bound autophagic vesicles. ARC-deficient hearts demonstrated extensive vacuolization (Figure 1b). Taken together, these data indicate an increased autophagic accumulation upon ARC deficiency.
ARC inhibits autophagy under the pathological condition
We attempted to understand the role of ARC in autophagy under the pathological condition and observed that anoxia/reoxygenation (A/R) could reduce the level of ARC (Supplementary Figure 1A). During autophagy, LC3-II relocalizes to the autophagosomal membranes. Thus, the accumulation of GFP-LC3 puncta provides an effective way to detect autophagosomes. We found that A/R resulted in an increased GFP-LC3 punctuate structures (Supplementary Figure 1B). Enforced expression of ARC significantly reduced punctate accumulations of GFP-LC3 (Figure 2a) and LC3-II levels (Supplementary Figure 2A). To determine whether ARC can inhibit the formation of autophagosomes or promote the degradation, we measured p62 and observed that enforced expression of ARC caused an increase in p62 levels (Supplementary Figure 2B). Autophagic flux is considered to be a better indicator of autophagic activition. We measured the autophagic flux in the presence and absence of lysosomal inhibitor bafilomycin A1 (BFA), which inhibits fusion of autophagosome and lysosome. Our results showed that ARC reduced autophagosome abundance even in the presence of BFA (Figure 2b). In addition, enforced expression of ARC significantly reduced cell death induced by A/R (Figure 2c). The results in EM analysis show that ARC reduced autophagic vesicles in cardiomyocytes (Figure 2d). Taken together, these results suggest that ARC inhibits autophagy and cell death induced by A/R in cardiomyocytes. We tested whether ARC has a role in the pathogenesis of myocardial infarction. ARC transgenic mice could attenuate autophagy (Figures 2e and f) and myocardial infarction sizes (Figure 2g), as well as ameliorated myocardial function (Supplementary Figure 2C), in response to ischemia/reperfusion (I/R). These data suggest that ARC is able to inhibit autophagy and autophagic cell death in the heart.
We investigated the mechanism by which ARC regulates autophagy. Beclin 1 is a well-known key regulator of autophagy,34, 35 and recent studies have shown that Beclin 1 mediates the autophagic cell death in cardiomyocytes.36 Our results also showed that knockdown of Beclin 1 attenuated autophagy (Supplementary Figure 3A) and cell death (Supplementary Figure 3B). Overexpression of Beclin 1 sensitized cells to undergo death, and increased cell death was observed upon both A/R and Beclin 1 treatment in the presence of a lysosomal inhibitor bafilomycin A1 (Supplementary Figure 3C). These lines of evidence encouraged us to test whether there is a cross-talk between ARC and Beclin 1. We performed immunoprecipitation. The results showed that Beclin 1 was immunoprecipitated with an anti-ARC antibody, and the immunoprecipitated amount was decreased upon A/R condition (Figure 2h). Thus, it appears that Beclin 1 is a target of ARC in the cascade of autophagy. Knockdown of ATG5 also attenuated cell death induced by A/R (Supplementary Figure 3D) and Beclin 1 treatment (Supplementary Figure 3E), but ATG5 failed to co-immunoprecipitate with ARC (data not shown). It is reported that Bcl-2 could bind to Beclin 1 and inhibit Beclin 1-dependent autophagy.7 Thus, we explore whether ARC can affect Beclin 1 binding to Bcl-2. Our results showed that overexpression (Supplementary Figure 3F, upper panel) or knockdown (Supplementary Figure 3F, lower panel) of ARC doesn’t affect the levels of Bcl-2-immunoprecipitated Beclin 1.
To further test whether ARC can regulate autophagic cell death, we compared the effect of z-VAD-fmk (a pancaspase inhibitor) and ARC on autophagic cell death. Overexpression of Beclin 1 sensitized cells to undergo death, and ARC could inhibit this cell death. ARC effect is much more than that of z-VAD-fmk (Supplementary Figures 4A and B). Whereas, the caspase-3 activity were partly inhibited by ARC compared with z-VAD-fmk (Supplementary Figure 4C). In cardiomyocytes, we compared the effect of z-VAD-fmk alone, the autophagy inhibitor 3-methyladenine (3-MA) alone and their combination on cell death upon A/R. The inhibitory effect of ARC on cell death was more effective than z-VAD-fmk alone, 3-MA alone or their combination (Supplementary Figure 4D). In the animal model, we compared the effect of z-VAD-fmk alone, Beclin 1 siRNA alone and their combination with ARC transgenic mice on myocardial infarction. ARC transgenic mice exhibited a significant reduction in myocardial infarction size than the mice treated with z-VAD-fmk alone, Beclin 1 siRNA alone or their combination (Supplementary Figure 4E), suggesting that the effect of ARC is not solely on apoptosis and ARC can also regulate autophagic cell death. Taken together, these data suggest that the ARC can regulate autophagy in vitro and in vivo.
ARC is a target of miR-325
miRNA is able to suppress gene expression. To explore the underlying mechanism responsible for ARC downregulation, we tested whether miRNA participates in the regulation of ARC expression. miR-325 is expressed at a low level in the heart but was upregulated upon A/R (Supplementary Figure 5A) and I/R (Supplementary Figure 5B). We analyzed its potential targets using the online software of RNAhybrid and found that ARC is a potential target of miR-325 in mouse (Figure 3a), human and rat (Supplementary Figure 5C). We tested whether miR-325 is related to ARC expression. Enforced expression of miR-325 could reduce ARC levels (Figure 3b). Knockdown of miR-325 increased the ARC levels upon A/R (Figure 3c). Thus, it appears that miR-325 is a regulator of ARC. To understand whether miR-325 can directly target ARC, we prepared the luciferase constructs (Figure 3d). The luciferase assay revealed that miR-325 could suppress the translational activity of ARC (Figure 3e). The administration of miR-325 antagomir but not the antagomir negative control (antagomir-NC) could block the effect of miR-325. Introduction of mutations that disrupt base pairing between miR-325 and ARC rescued luciferase expression (Figure 3f) and ARC protein expression (Figure 3g). We also employed the target protector technology in which a target protector is able to disrupt the specific interaction of miRNA–mRNA pairs. We observed that the inhibitory effect of miR-325 on ARC was reduced in the presence of ARC target protector (Figure 3h). Furthermore, different lines (Figure 3i) of miR-325 transgenic mice (Supplementary Figure 6) demonstrated reduced levels of ARC in the animal model (Figure 3j). These data suggest that the inhibitory effect of miR-325 on ARC translation is specific.
miR-325 conveys the autophagic signal
We tested whether miR-325 has a functional role. In the cellular model, knockdown of miR-325 (Supplementary Figure 7A) reduced punctate accumulations of GFP-LC3 (Figure 4a) and induced an increase in p62 levels (Supplementary Figure 7B) upon A/R. Further, we measured the autophagic flux in the presence and absence of bafilomycin A1. The results showed that knockdown of miR-325 reduced autophagosome abundance even in the presence of BFA (Figure 4b). In addition, knockdown of miR-325 reduced cell death induced by A/R (Figure 4c) and autophagic vesicles in cardiomyocytes observed by EM analysis (Figure 4d). In the animal model, miR-325 transgenic mice potentiate autophagic responses revealed by EM analysis of ventricular tissue (Figure 4e), an increase in LC3-II levels (Figure 4f), an enlarged myocardial infarct sizes (Figure 4g) and pronounced cardiac dysfunction (Supplementary Figure 7C). These data suggest that miR-325 participates in mediating the signal for autophagy and cell death in the heart.
miR-325 exerts its autophagic effect through ARC
We explored how miR-325 exerts its effect on autophagy. The inhibitory effect of miR-325 on ARC expression encouraged us to test whether ARC is a mediator of miR-325. Although knockdown of miR-325 could inhibit autophagy and cell death, this effect was significantly abolished by knockdown of endogenous ARC (Figures 5a and b). To confirm the relationship between miR-325 and ARC in autophagy machinery, we employed the target protector technology. To this end, we produced ARC target protector and observed that autophagy and cell death was reduced in the presence of ARC target protector (Figures 5c and d). These data suggest that miR-325 targets ARC in the cascades of autophagy and cell death.
To better understand the relationship between miR-325 and ARC, we tested whether these two factors have an impact on the animal model. We produced miR-325 transgenic mice, miR-325/ARC-with UTR bitransgenic mice (325/ARC-UTR) and miR-325/ARC-without UTR bitransgenic mice (325/ARC) (Figure 5e). The 325/ARC but not 325/ARC-UTR bitransgenic mice exhibited a reduced autophagic response (Figure 5f) and myocardial infarct sizes (Figure 5g). Thus, ARC and miR-325 have a cross-talk in the animal model.
E2F1 regulates miR-325 expression
We asked how miR-325 expression is regulated under the pathological condition. To address this question, we analyzed the promoter region of mouse miR-325 and observed that there is an optimal consensus binding site of E2F1 (Figure 6a). In addition, there are binding sites in the promoter regions of human and rat miR-325 (Supplementary Figure 8A). We tested whether E2F1 can bind to the promoter region of miR-325. The ChIP assay revealed that E2F1 bound to the miR-325 promoter under the physiological condition. A/R treatment led to an increase in the association levels of E2F1 with miR-325 promoter (Figure 6b). The luciferase assay demonstrated that E2F1 could stimulate the activity of miR-325 wild-type promoter but not that of the mutant (Figure 6c). A/R induced an elevation in E2F1 expression levels (Supplementary Figure 8B) and miR-325 promoter activity (Figure 6d). Enforced expression of E2F1 led to an elevation of miR-325 levels (Figure 6e). Knockdown of E2F1 induced a decrease in miR-325 expression levels (Supplementary Figure 8C). E2F1 knockout mice exhibited a low level of miR-325 (Figure 6f). These data suggest that E2F1 can transcriptionally upregulate miR-325.
We investigated the role of E2F1 in autophagy in cardiomyocytes and myocardial infarction. Knockdown of E2F1 was able to attenuate autophagy and cell death upon A/R (Figure 6g, Supplementary Figure 8D). Enforced expression of E2F1 led to autophagy and cell death (Supplementary Figures 8E and F). E2F1 knockout mice demonstrated reduced autophagy revealed by LC3-II levels (Figure 6h) and myocardial infarction sizes (Figure 6i) upon ischemia/reperfusion. These data suggest that E2F1 participates in mediating the signal for autophagy and cell death in the heart.
Our present study revealed that miR-325 is integrated into the autophagic machinery. Further, we identified ARC was able to regulate the autophagic program. miR-325 provokes autophagy through repressing ARC. Our results demonstrate that miR-325 and ARC constitute an axis controlling autophagy and cell death, and target that this axis can bring beneficial effect.
Autophagy is a physiological and evolutionarily conserved phenomenon maintaining homeostatic functions like protein degradation and organelle turnover. Autophagy can have a cytoprotective role in cells under conditions of nutrient starvation.25 Yet accumulating data show that the autophagic machinery can also be recruited to kill cells under certain conditions generating a caspase-independent form of programmed cell death, named autophagic cell death. The abnormal autophagy is related to a variety of pathological disorders including cancer, neurodegenerative and cardiovascular diseases,7, 8, 9, 10 and the autophagic machinery may also be used for cell self-destruction.37 Consistent with these reports, our results suggested that inhibition of autophagy protects cell death induced by the myocardial I/R injury. Further investigation on how excessive autophagy affects cell signaling and growth under pathological condition will provide more targets to understand the basis of the diseases.
ARC has an important role in regulating the apoptotic pathway and inhibits apoptosis through different manners. It can interact with Fas, FADD and Bax,33, 38 inhibit cytochrome c release39 and maintain mitochondrial membrane potential.40, 41 These lines of evidence suggest that ARC is a critical factor for maintaining the cellular function. Our present work reveals that ARC participates in the regulation of autophagy to maintain the equilibrium between autophagic cell death and cell survival. In particular, we found that ARC-deficient mice exhibit more autophagy, suggesting that ARC is a physiological regulator of autophagy. Starvation is a physiologic inducer of autophagy, future studies can test whether ARC is able to influence autophagic program of starvation.
Other antiapoptotic proteins can regulate autophagic program. For example, Bcl-2 binds to Beclin 1 and disrupts its autophagy function. In the absence of Bcl-2 binding, Beclin 1 mutants induce excessive autophagy and promote cell death.7 Bcl-xL can interact with ATG5 thereby preventing cytochrome c release and caspase activation.24 There is a complex relationship between apoptosis and autophagy. Our present work shows that ARC can bind to Beclin 1 and may disrupt its autophagy function. The direct interaction of ARC with Beclin 1 is of particular interest with respect to the heart, because cardiomyocytes have the limited proliferative capability, and induction of autophagic cell death in cardiomyocytes may lead to pathophysiological disorders such as myocardial infarction and heart failure. ARC is highly expressed in cardiomyocytes. The ability of ARC to directly associate with Beclin 1 enables ARC to more efficiently control autophagic cell death so that the heart does not lose more cardiomyocytes under the physiological as well as pathological conditions. Our results may have important therapeutic implications for the employment of ARC in the treatment of autophagy-related diseases.
Growing evidence has shown that miRNA can regulate apoptosis. For example, miR-23a-27a-24-2 cluster induces caspase-dependent and -independent apoptosis.42 miR-101 and miR-29b repress the antiapoptotic protein Mcl-1.43 Also, other reports show that miRNAs can regulate the autophagy. For example, reduced levels of miR-23b increase levels of ATG12 and autophagy in pancreatic cancer cells.44 Angiotensin II induces downregulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy.45 Enforced expression of miR-155 can increase autophagic activity in human nasopharyngeal cancer and cervical cancer cells, whereas knockdown of miR-155 can inhibit hypoxia-induced autophagy.46 Autophagy is controlled by a complex interplay between the anti- and pro-autophagic proteins,47, 48, 49, 50 and more than 800 microRNAs have been identified.51 The function of miR-325 remains unknown. Our present work found that it can regulate autophagic program through targeting ARC protein. Accordingly, it can be speculated that the identification of miRNAs that regulate autophagic proteins may fill in the gap between unknown aspects of cell biology and the pathogenesis of diseases.
E2F1 is a member of E2F family that has most often been implicated in mediating apoptosis. It is reported that E2F1 expression is increased in response to myocardial ishemia/reperfusion injury, and ishemia/reperfusion injury is attenuated in E2F1 null hearts.52 In present work, we investigated the role of E2F1 in autophagy upon myocardial ishemia/reperfusion injury. E2F1 knockout mice demonstrated reduced autophagy and myocardial infarction sizes upon ischemia/reperfusion injury. E2F1 participates in the autophagic pathway by regulating the expression of miR-325. Modulation of their levels may provide a new approach for tackling myocardial infarction.
Materials and Methods
Cells culture, anoxia/reoxygenation, apoptosis and cell death assay
Details are in the Supplementary Methods.
Adenoviral constructions and infection
Mouse E2F1 cDNA and Beclin 1 were from Origene. The GPF-LC3 expression plasmid was from Cell Biolabs Inc. (San Diego, CA, USA). The adenoviruses harboring E2F1, Beclin 1 and GFP-LC3 were constructed using the Adeno-X expression system (Clontech, Mountain View, CA, USA). The adenoviruses harboring ARC or β-galactosidase (β-gal) are as we described elsewhere.53 To construct adenovirus encoding miR-325, the mature miR-325 sequence was cloned into the adenovirus system according to the manufacturer’s instructions.
Generation of cardiac-specific miR-325 transgenic mice, ARC transgenic mice with 3′UTR, ARC transgenic mice without 3′-UTR, ARC knockout mice and E2F1 knockout mice
Generation of cardiac-specific miR-325 and ARC transgenic mice with 3′UTR, ARC transgenic mice without 3′UTR, ARC and E2F1 knockout mice are described in the Supplementary Methods.
siRNA constructions of ARC, E2F1, Beclin 1 and ATG5
ARC siRNA sequence is 5′-IndexTermCTAGAAGCTGAAGCTACTG-3′; the scramble ARC siRNA sequence is 5′-IndexTermAGTCAGACTGAATGCAGTC-3′. E2F1 siRNA sequence is 5′-IndexTermATCTGACCACCAAACGCTT-3′; the scramble E2F1 siRNA sequence is 5′-IndexTermCACTCACGACTACGATCTA-3′. Beclin 1 siRNA sequence is 5′-IndexTermGATCCTGGACCGGGTCACC-3′; the scramble Beclin 1 siRNA sequence is 5′-IndexTermCCTAGGCTCACGTGACGCG-3′. ATG5 siRNA sequence is 5′-IndexTermGCACTTTCAGAAGGTTATG-3′; the scramble ATG5 siRNA sequence is 5′-IndexTermTATAGCGTAGCGTATGATC-3′. They were cloned into pSilencer adeno 1.0-CMV vector (Ambion, Grand Island, NY, USA) according to the manufacturer′s instructions.
Staining of autophagosomes, immunoblot, immunoprecipitation, autophagic flux
Details are in the Supplementary Methods.
Preparation of the luciferase construct of ARC 3′UTR and luciferase assay
ARC 3′UTR was amplified by PCR with primer sets: 5′-IndexTermAGGCCAGAATCCTTAGCTGTC-3′ and 5′-IndexTermCCTTCCTTCTTTCCTTCCTTC-3′. Mutations were generated with QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, Cambridge, MA, USA) and sequence confirmed. Wild type and mutated 3′UTRs were subcloned into the pGL3 vector (Promega, Madison, WI, USA) immediately downstream of the stop codon of the luciferase gene. Luciferase activity assay was performed as we described.54
Target protector preparation, transfection of antagomir, constructions of mouse miR-325 promoter and its mutant
Details are in the Supplementary Methods.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assay was performed as we described.54 In brief, cells were washed with PBS and incubated for 10 min with 1% formaldehyde at room temperature. The cross-linking was quenched with 0.1 M glycine for 5 min. Cells were washed twice with PBS and lysed for 1 h at 4 °C in a lysis buffer. The cell lysates were sonicated into chromatin fragments with an average length of 500 to 800 bp as assessed by agarose gel electrophoresis. The samples were precleared with Protein-A agarose (Roche, Mannheim, Germany) for 1 h at 4 °C on a rocking platform, and 5 μg specific antibodies were added and rocked for overnight at 4 °C. Immunoprecipitates were captured with 10% (vol/vol) Protein-A agarose for 4 h. Before use, Protein-A agarose was blocked twice at 4 °C with salmon sperm DNA (2 μg/ml) overnight. DNA fragments were purified with a QIAquick Spin Kit (Qiagen, Venlo, Netherlands). The purified DNA was used as a template and amplified with the following primer sets: for the analysis of E2F1 binding to the promoter region of miR-325, the oligonucleotides were as follows: forward: 5′- IndexTermGCAATACACAGGTTAAATGAC-3′; reverse: 5′-IndexTermGATAAGATCATTGTACTGTGG-3′.
Quantitative reverse transcription-polymerase chain reaction
Stem-loop quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for mature miR-325 was performed as described55 on an Applied Biosystems ABI Prism 7000 sequence detection system. Total RNA was extracted using Trizol reagent. After DNAse I (Takara, Japan) treatment, RNA was reverse transcribed with reverse transcriptase (ReverTra Ace, Toyobo). The results of qRT-PCR were normalized to that of U6. The sequences of U6 primers were forward: 5′-IndexTermGCTTCGGCAGCACATATACTAA-3′; reverse: 5′-IndexTermAACGCTTCACGAATTTGCGT-3′.
Transmission electron microscopy, ischemia/reperfusion (I/R), histology and echocardiographic assessment
Details are in the Supplementary Methods.
Data are expressed as the mean±S.E.M. of at least three independent experiments. We evaluated the data with Student’s t test. We used a one-way analysis of variance for multiple comparisons. A value of P<0.05 was considered significant.
Additional experimental procedures
These are described in the Supplementary Information.
apoptosis repressor with caspase recruitment domain
microtubule-associated protein 1 light chain 3
quantitative reverse transcription polymerase chain reaction
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This work was supported by National Natural Science Foundation of China (81270160, 31010103911), National Basic Research Program of China (973 Program, 2011CB965300), Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01030000).
The authors declare no conflict of interest.
Edited by D Rubinsztein
Supplementary Information accompanies this paper on Cell Death and Differentiation website
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Bo, L., Su-Ling, D., Fang, L. et al. Autophagic program is regulated by miR-325. Cell Death Differ 21, 967–977 (2014) doi:10.1038/cdd.2014.18
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