Published online before print April 20, 2009, doi: 10.1161/CIRCULATIONAHA.108.814145
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(Circulation. 2009;119:2357-2366.)
© 2009 American Heart Association, Inc.
Molecular Cardiology |
MicroRNA-320 Is Involved in the Regulation of Cardiac Ischemia/Reperfusion Injury by Targeting Heat-Shock Protein 20
From the Departments of Pharmacology and Cell Biophysics (X.R., J.W., X.W., J.Q., K.J., P.N., T.J.P., G.F.) and Environmental Health (M.A.S.), University of Cincinnati College of Medicine, Cincinnati, Ohio. Present address of J.W. is Bassett Research Institute, Mary Imogene Bassett Hospital, Cooperstown, NY.
Correspondence to Guo-Chang Fan, PhD, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0575. E-mail fangg{at}ucmail.uc.edu
Received August 11, 2008; accepted February 25, 2009.
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Abstract |
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Background— Recent studies have identified critical roles for microRNAs (miRNAs) in a variety of cellular processes, including regulation of cardiomyocyte death. However, the signature of miRNA expression and possible roles of miRNA in the ischemic heart have been less well studied.
Methods and Results— We performed miRNA arrays to detect the expression pattern of miRNAs in murine hearts subjected to ischemia/reperfusion (I/R) in vivo and ex vivo. Surprisingly, we found that only miR-320 expression was significantly decreased in the hearts on I/R in vivo and ex vivo. This was further confirmed by TaqMan real-time polymerase chain reaction. Gain-of-function and loss-of-function approaches were employed in cultured adult rat cardiomyocytes to investigate the functional roles of miR-320. Overexpression of miR-320 enhanced cardiomyocyte death and apoptosis, whereas knockdown was cytoprotective, on simulated I/R. Furthermore, transgenic mice with cardiac-specific overexpression of miR-320 revealed an increased extent of apoptosis and infarction size in the hearts on I/R in vivo and ex vivo relative to the wild-type controls. Conversely, in vivo treatment with antagomir-320 reduced infarction size relative to the administration of mutant antagomir-320 and saline controls. Using TargetScan software and proteomic analysis, we identified heat-shock protein 20 (Hsp20), a known cardioprotective protein, as an important candidate target for miR-320. This was validated experimentally by utilizing a luciferase/GFP reporter activity assay and examining the expression of Hsp20 on miR-320 overexpression and knockdown in cardiomyocytes.
Conclusions— Our data demonstrate that miR-320 is involved in the regulation of I/R-induced cardiac injury and dysfunction via antithetical regulation of Hsp20. Thus, miR-320 may constitute a new therapeutic target for ischemic heart diseases.
Key Words: apoptosis • ischemia • microRNA • myocardial infarction • reperfusion
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Introduction |
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More than 1 million Americans suffer from myocardial infarction every year.1 Both human autopsy data and evidence from rodent models of myocardial infarction indicate that most cell death happens by apoptosis during the initial 2 to 4 hours after coronary occlusion.2,3 Clinical treatment of myocardial infarction by thrombolytic therapy and revascularization by percutaneous coronary intervention or coronary artery bypass graft surgery are effective.1,3 However, given the health, economic, and personal burden caused by ischemic heart disease, research into additional treatment modalities is imperative. Furthermore, the molecular mechanisms that regulate gene expression during myocardial ischemia/reperfusion (I/R) are still not completely understood.
Clinical Perspective on p 2366
MicroRNAs (miRNAs) are a class of endogenous non–protein-coding RNAs comprising 
22 nucleotides.4–6 They regulate gene expression via RNA-induced silencing complexes, targeting them to mRNAs where they inhibit translation or direct destructive cleavage.4–6 Increasing evidence indicates the importance of miRNAs in the regulation of cardiac developmental and pathological processes.7–10 For example, inhibition of miR-133 was sufficient to induce cardiomegaly in vivo9; similarly, targeted deletion of miR-1-2 revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electric conduction, and cell cycle control.10 More recently, miR-1 and miR-133 were shown to produce opposing effects on oxidative stress–induced apoptosis in H9c2 cells, with miR-1 being proapoptotic and miR-133 being antiapoptotic.11 Van Rooij et al12 reported a signature pattern of stress-responsive miRNAs that could evoke cardiac hypertrophy and heart failure. Accordingly, miR-208 deficiency resulted in blunted hypertrophic and fibrotic responses to transverse aortic constriction.13 These results suggest that miRNAs have a fundamental role in the development of heart disease. However, the signature of miRNA expression and possible roles of miRNAs in myocardial infarction are less well studied.
In this study, we performed miRNA arrays to detect the expression pattern of miRNAs in murine hearts subjected to I/R in vivo and ex vivo. Surprisingly, we observed that only miR-320 expression was consistently decreased in murine hearts on I/R in vivo and ex vivo. Overexpression of miR-320 in cardiomyocytes resulted in increased sensitivity to I/R injury, whereas knockdown of endogenous miR-320 with the use of antisense methodology ex vivo and antagomir administration in vivo was cytoprotective. Using TargetScan software, proteomic analysis, and a luciferase/GFP reporter assay in vitro, we identified heat-shock protein 20 (Hsp20) as a real target for miR-320. Taken together, our findings implicate miR-320 as a potential therapeutic target for ischemic heart disease.
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Methods |
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An expanded Methods section is available in the online-only Data Supplement.
microRNA Extraction, miRNA Microarray, and Quantitative Real-time Polymerase Chain Reaction
miRNAs were isolated from mouse hearts (B6129SF2/JF2, 10 to 12 weeks old) after 24-hour reperfusion preceded by 30-minute ischemia, via left anterior descending (LAD) coronary artery occlusion, or from mouse hearts (FVB/N) subjected to 45-minute no-flow global ischemia and 2-hour reperfusion ex vivo, with the use of the mirVana miRNA isolation kit (Ambion, Inc, Austin, Tex), according to the manufacturer’s protocol. The concentration of RNA was determined by a NanoDrop ND-1000 Spectrophotometer (NanoDrop Tech, Rockland, Del). miRNA expression profiling was determined by miRNA microarray analysis with the use of the mouse miRNA array probes (mirVana miRNA Bioarrays version 2, Ambion, Inc) that include 266 mature mouse miRNAs. Dysregulated miRNAs were validated by using the mirVana quantitative real-time polymerase chain reaction (RT-PCR) miRNA Detection Kit TaqMan miRNA assays. Primer sets for these miRNAs, including control snoRNA412, were purchased from Ambion, Inc. Microarrays were performed in the Genomics and Microarray Laboratory, University of Cincinnati Medical Center. The data generated by GenePix Pro version 5.0 software were analyzed to identify differentially expressed miRNAs. Data normalization was performed in 2 separate steps for each microarray, as described previously (details are available in the online-only Data Supplement).14 All RT reactions, including no-template controls and RT minus controls, were run in triplicate in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems, Carlsbad, Calif). Relative expression was calculated by the comparative threshold cycle method, as described previously.15
Cell Culture and Construction of Adenoviral Vectors
Adult ventricular cardiomyocytes were isolated from 2-month-old male Sprague-Dawley rats (Harlan Laboratory, Indianapolis, Ind), as described previously.16 Primary miR-320 DNA was PCR amplified with the use of high-fidelity AccuPrime Taq DNA polymerase (Invitrogen, Carlsbad, Calif) from mouse genomic DNA. After sequencing, the amplified fragment (470 bp) was inserted under the CMV promoter into the AdEasy-1/Shuttle backbone, similar to our previous construction of adenoviral vectors.17 Antisense miR-320 adenovirus (named as AdasmiR-320) was generated by cloning the primary miR-320 DNA in the reverse orientation relative to the CMV promoter.
Generation of a miR-320 Transgenic Mouse Model
Transgenic mice were constructed by using a 470-bp DNA fragment containing murine primary miR-320 under the control of the 
-myosin heavy chain promoter. The expression levels of miR-320 were detected by Northern blot as described in Methods.
Global Ischemia Ex Vivo and Cardiac Injury Analysis
The cellular and functional responses to I/R were assessed in mice by using an isolated perfused heart model, as described previously.18
In Vivo Administration of Antagomir-320
Chemically modified antisense oligonucleotides (antagomir) have been used to inhibit microRNA expression in vivo.9,19–21 Antagomirs were synthesized by Dharmacon (www.dharmacon.com). Sequences are 5'-uscsgcccucucaacccagcusususus-Chol-3' (antagomir-320), 5'-uscsgcccucucaaccgcagascscsus-Chol-3' (antagomir-320 mutant as control). Lower-case letters represent 2'-O-methyl–modified oligonucleotides, subscript "s" represents a phosphorothioate linkage, "Chol" represents linked cholesterol, and underlined letters are a mutated seed sequence. Antagomir oligonucleotides were deprotected, desalted, and purified by high-performance liquid chromatography. FVB/N male mice (6 weeks old) received either antagomir-320 or mutant antagomir-320 at a dose of 80 mg/kg body weight or a comparable volume of saline (200 µL) through tail vein injection. Regional ischemia in vivo was performed at 3 days after treatment.
Additional Methods
An expanded Methods section containing details of simulated I/R treatment and cell survival assay, Northern blot detection of miR-320 expression, Western blot analysis, GFP repression experiments, and luciferase reporter assay for targeting Hsp20 3'UTRs is available in the online-only Data Supplement.
Statistical Analysis
All values are expressed as mean±SEM. Student t test was used for 2-group comparisons. Comparisons of parameters among 
3 groups were analyzed by 1-way ANOVA for single-factor or 2-way ANOVA for 2-factor variables with repeated measures, followed by Student t test with Bonferroni correction for multiple comparisons. Differences were considered statistically significant at a value of P<0.05.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Aberrant Expression of miRNAs in I/R Hearts
Successful ischemia after 30 minutes of LAD occlusion was confirmed by visual observation (cyanosis) and continuous ECG monitoring. After 24-hour reperfusion, the hearts were perfused with 1% TTC, followed by perfusion with 5% phthalo blue. As expected, the I/R group displayed significant cardiac infarction (infarction size, 21.7±2.3%; n=6), whereas the sham group showed no infarction (Figure 1A). To further evaluate cardiac injury, we measured cardiomyocyte apoptosis using 2 quantitative assays: terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining and an enzyme-linked immunosorbent assay–based nucleosome assay. As shown in Figure 1B, the proportion of transferase–mediated dUTP nick-end labeling–positive nuclei in the myocardium of mice subjected to I/R was significantly increased compared with the shams (7.6±1.5% versus 0.6±0.2%; P
0.01; n=6). Furthermore, DNA fragmentation, measured by the levels of mononucleosomes and oligonucleosomes, was significantly higher in the lysates of I/R hearts relative to the shams (Figure 1C). These data indicate that I/R in vivo induces cardiac injury.
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To determine the potential involvement of miRNAs in cardiac I/R injury, we used microarray analysis to determine miRNA levels in murine hearts after I/R in vivo (30-minute LAD occlusion followed by 24-hour reperfusion). We were excited to find that, compared with the sham group, only 6 of 640 probed miRNAs were differentially expressed in I/R hearts (P<0.01); 5 miRNAs were upregulated, and only miR-320 was downregulated (Figure 1D and Table). These results were further validated by TaqMan RT-PCR assay (Figure 1E). Notably, miR-7 expression was not detectable (Figure 1E), in agreement with its very low microarray intensity (Table). Furthermore, we extended our studies to an ex vivo model of no-flow global ischemia (45 minutes) followed by 2-hour reperfusion and found that miRNAs upregulated in vivo were not dysregulated in ex vivo I/R hearts compared with shams (online-only Data Supplement). This may be due to in vivo confounding effects, such as systemic circulation and a host of peripheral complications, activating different signaling pathways from ex vivo models, or may be related to the time points examined. However, miR-320 was consistently downregulated in ex vivo I/R hearts, suggesting that miR-320 is an I/R-related microRNA. Therefore, we chose miR-320 for further determination of its potential roles in I/R-induced cardiac injury.
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MiR-320 Sensitizes Adult Rat Cardiomyocytes to Simulated I/R-Induced Apoptosis
To investigate the functional significance of miR-320 in ischemic heart, gain-of-function and loss-of function approaches were employed in cultured adult rat cardiomyocytes. We generated 2 adenoviral vectors encoding primary miR-320 in the sense or antisense direction, designated as AdmiR-320 and AdasmiR-320, respectively (Figure 2A). After infection of the cardiomyocytes with these recombinant adenoviral vectors for 48 hours, we observed nearly 100% infection efficiency (Figure 2B). Importantly, no apparent morphological alterations or differences were found in the number of adherent cells and rod-shaped cells among the AdmiR-320–, AdasmiR-320–, and AdGFP-infected groups (Figure 2B). After 60 hours of adenoviral infection, TaqMan RT-PCR clearly showed overexpression of the exogenous miR-320 in AdmiR-320–infected cardiomyocytes, whereas endogenous miR-320 was successfully knocked down in AdasmiR-320 cells by 40% (Figure 2C). We next examined the effects of miR-320 on cell survival on 1-hour simulated ischemia, followed by 3-hour reperfusion. Cell viability analysis showed that ectopic expression of miR-320 reduced cell survival by 15%, whereas knockdown of endogenous miR-320 revealed opposite effects (ie, increased survival by 20%) relative to GFP cells (Figure 2D and 2E). Furthermore, on I/R, AdmiR-320–infected myocytes exhibited a significant increase in nuclear fragmentation compared with Ad.GFP-infected myocytes (48±4% versus 35±3%; Figure 2F). In contrast, infection with AdasmiR-320 significantly reduced the number of I/R-induced condensed nuclei (22±5%; Figure 2F). Consistently, the levels of DNA fragmentation in cell lysates on I/R were significantly higher in miR-320 cells. Conversely, asmiR-320 cells presented with reduced DNA fragmentation compared with the Ad.GFP group (Figure 2G). Taken together, these data suggest that miR-320 may play an important role in I/R-mediated cardiac injury, probably through regulation of the cell death/apoptosis process.
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Effects of miR-320 Overexpression on I/R Cardiac Injury In Vivo
To further elucidate the in vivo effects of miR-320 on I/R, we generated 6 transgenic mouse lines that carry the mouse primary miR-320 DNA under the control of the -myosin heavy chain mouse promoter (Figure 3A). All miR-320 transgenic mice were healthy and showed no apparent cardiac morphological or pathological abnormalities. Northern blot analysis (Figure 3B) revealed that miR-320 was successfully overexpressed in the transgenic hearts from lines 1, 5, and 6 (2- to 3-fold increases). We selected lines 5 and 6 for further I/R studies. These hearts were subjected in vivo to 30 minutes of myocardial ischemia, via coronary artery occlusion, followed by 24-hour reperfusion. We observed that the ratio of infarct-to-risk region was 39.4±3.5% in line 5 hearts and 44.5±2.4% in line 6 hearts (n=6 for each line) compared with 22.7±4.1% in wild-type (WT) hearts (n=8; P<0.001) under in vivo I/R conditions (Figure 3C and 3D), whereas the region at risk was not significantly different among groups (Figure 3E).
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Increased Cardiac Injury and Dysfunction in miR-320 Hearts on I/R Ex Vivo
To further examine the effects of increased expression of miR-320 on cardiac functional recovery during I/R, we used an isolated perfused heart preparation. Hearts from transgenic line 6 were stabilized for 30 minutes and subjected to 30 minutes of global ischemia and 1-hour reperfusion. During reperfusion, the transgenic hearts exhibited significantly depressed functional recovery compared with the WTs (Figure 4A through 4D), evidenced by the decreased rates of contraction (+dP/dt) and relaxation (–dP/dt) in miR-320 hearts during reperfusion relative to WT hearts (Figure 4A and 4B). Similarly, the left ventricular developed pressure recovered to 55±5% of preischemic values after 1-hour reperfusion in miR-320 hearts compared with 87±3% in WT hearts (Figure 4C). The left ventricular end-diastolic pressure was also markedly increased in the miR-320 hearts during reperfusion (Figure 4D) relative to WT hearts.
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To determine the degree of necrosis in these I/R hearts, we assessed the level of lactate dehydrogenase (LDH) released during the first 10 minutes of reperfusion after global ischemia. We observed that the total LDH was 3-fold higher in miR-320 hearts than in WTs (Figure 4E), demonstrating increased necrosis in miR-320–overexpressing hearts. Furthermore, heart lysates from a subset of experimental WT and transgenic hearts were assayed for DNA fragmentation with the use of a quantitative nucleosome assay. Transgenic hearts exhibited a 1.7-fold increase over WT hearts (Figure 4F), indicating that overexpression of miR-320 enhanced I/R-induced cardiac apoptosis.
MiR-320 Acts Directly at the Hsp20 3'UTR
To elucidate the potential mechanism of miR-320 in the regulation of cardiac I/R injury, we first identified the putative targets of miR-320 using computational predictions, as detailed at TargetScan (http://genes.mit.edu/targetscan/). Even though this analysis yielded 482 potential candidates, prediction does not consider the secondary structure of the target mRNA, which controls the accessibility of miRNA binding, suggesting that some predicted targets are not real. Given the major function of miRNAs as protein expression regulators, we searched the protein expression profile in I/R hearts. It is exciting that 1 study reported that the expression levels of only 12 proteins were altered in the murine heart proteome after I/R relative to sham hearts.22 After matching the proteomic data with the TargetScan result, we found that only Hsp20 (also named HspB6) was listed among the assumed targets for miR-320 in the murine heart (Figure 5A), and the near-perfect complementary base pairing is located at 193 to 211 bp of mouse Hsp20 3'UTR (Figure 5B). More interestingly, the seed sequence of Hsp20 3'UTR targeting to miR320 is highly conserved among the species of mouse, human, rat, and dog (Figure 5C), suggesting a critical role in their physiology.
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To validate whether miR-320 directly recognizes the 3'UTR of Hsp20, we cotransfected H9c2 cells with a construct (Figure 5D) containing the 3'UTR of Hsp20 fused downstream to the GFP coding sequence along with miR-320 or a negative control miRNA. Overexpression of miR-320 resulted in a marked reduction of the GFP fluorescence intensity (Figure 5E). This result was subsequently confirmed in both HEK-293 and H9c2 cells with a luciferase assay (Figure 5F through 5H). Cotransfection of miR-320 in H9c2 cells strongly inhibited the luciferase activity from the reporter construct containing the 3'UTR segment of Hsp20, whereas no effect was observed with a construct containing a mutated segment of Hsp20 3'UTR (seed sequence CAGCUUU was mutated to GACACAA; Figure 5H). This effect was specific because no change was seen in luciferase reporter activity when a negative control miRNA was cotransfected with either reporter construct (Figure 5H). Collectively, these data indicate that Hsp20 transcripts may represent a genuine target of miR-320.
MiR-320 Downregulates Hsp20 Protein In Vitro and In Vivo
To ascertain whether miR-320 regulates Hsp20 expression, we harvested adult rat cardiomyocytes 60 hours after infection with AdmiR-320 or AdasmiR-320. Although RT-PCR demonstrated that Hsp20 messenger RNA copy numbers were similar among these groups (data not shown), cardiomyocytes infected with AdmiR-320 had reduced Hsp20 protein by 30%, whereas knockdown of endogenous miR-320 by AdasmiR-320 infection increased the levels of Hsp20 protein by 60% (Figure 6A), indicating that miR-320 acts as a negative regulator of Hsp20 translation.
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In miR-320 transgenic hearts, Hsp20 levels were reduced by 21% in line 5 and 30% in line 6, respectively (Figure 6B). Furthermore, we examined the time course for both Hsp20 and miR-320 expression during I/R in vivo and ex vivo (Figure 6C through 6F). The expression levels of Hsp20 were significantly increased at the end of ischemia after 1-hour reperfusion and 24-hour reperfusion (in vivo), which was negatively correlative with the reduced expression of miR-320 in the hearts (in vivo; Figure 6D) and cardiomyocytes (ex vivo; Figure 6F). Taken together, these data indicate that miR-320 expression is correlated to Hsp20 levels in the heart, and miR-320 efficiently represses Hsp20 expression in vivo and ex vivo.
Knockdown of miR-320 Decreases the In Vivo Cardiac Infarction Size
To further evaluate the biological role of downregulation of miR-320 on myocardial infarction, we knocked down miR-320 via a single tail vein injection of cholesterol-modified antagomir-320 (80 mg/kg). Mutant antagomir-320 and saline were used as controls. Three days after administration of antagomir-320, TaqMan RT-PCR analysis showed a dramatic reduction of miR-320 expression in the heart tissue (Figure 7A). In contrast, mutant antagomir-320 had no effect on the expression level of miR-320 compared with the saline control (Figure 7A). These results indicate that antagomir-320 efficiently downregulates miR-320 expression in the heart, consistent with previous reports, with the use of antagomirs.9,19–21 In parallel, administration of antagomir-320, but not antagomir-320 mutant, was associated with greatly increased levels of Hsp20 in the hearts (Figure 7B). Furthermore, these hearts treated with antagomirs for 3 days were also subjected to a 30-minute coronary occlusion, followed by 24-hour reperfusion. We observed that the infarct size was greatly reduced in the antagomir-320–treated hearts (6.2±1.5% versus 16.9±3.8% and 20.8±4.3% area at risk in the antagomir-320 mutant–treated and saline-treated controls, respectively; P<0.05; Figure 7C). In addition, region at risk was not significantly different among groups (Figure 7C). Taken together, these results suggest that systemically or locally applied inhibitory miR-320 molecules (ie, antagomir-320) may protect the heart against I/R injury in vivo.
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Discussion |
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Cardiomyocyte death/apoptosis is a key cellular event in ischemic hearts.2 It is well established that multiple genes are aberrantly expressed in infarct hearts, which are responsible for cardiac remodeling after I/R.22 Because miRNAs are endogenous regulators of gene expression, it is reasonable to hypothesize that they may be involved in I/R-induced cardiac injury. We therefore, for the first time, applied a well-established mouse cardiac I/R model in vivo and ex vivo to determine the miRNA expression signature in ischemic hearts. We were excited to find that only miR-320 expression was consistently dysregulated in ischemic hearts in vivo and ex vivo, suggesting that miR-320 is an I/R-related microRNA in the murine heart. Furthermore, knockdown of endogenous miR-320 expression reduced cardiomyocyte death and apoptosis induced by simulated I/R, whereas overexpression of miR-320 increased sensitivity to I/R-triggered cell death. Thus, at the cellular level, both loss-of-function and gain-of function experiments indicate that miR-320 is a negative regulator of cardioprotection against I/R injury. These cellular effects were further confirmed in vivo with cardiac-specific overexpression of miR-320 mouse models and antagomir-320 treatment, in which miR-320 hearts were sensitive, whereas antagomir-320-treated hearts were resistant, to I/R-induced cardiac injury.
It should be noted that several studies have demonstrated aberrant expression of miR-320 in both animal heart hypertrophy and human heart disease.23–25 Expression of miR-320 was downregulated in murine hearts after aortic banding at day 7 but not at days 14 and 28.23 In end-stage human failing hearts, expression of miR-320 was increased by 3.4-fold.24 Ikeda et al25 examined the expression of 428 microRNAs in 67 human left ventricular samples including control, ischemic cardiomyopathy, dilated cardiomyopathy, and aortic stenosis diagnostic groups. They observed that miR-320 was upregulated in ischemic cardiomyopathy and aortic stenosis samples, but no significant alterations were noted in dilated cardiomyopathy samples.25 Collectively, these data suggest that miR-320 could be involved in the regulation of multiple independent pathophysiological processes in the heart, especially in the ischemic heart.
Although the results from computational miRNA target prediction algorithms revealed that miR-320 had 482 potential targets, previous proteomic data showed that 12 proteins were altered in the heart on I/R.22 Surprisingly, among 12 altered proteins, only Hsp20 was listed in miR-320 TargetScan results. When it is considered that proteomic approaches have restrictions because of technical limitations and different turnover times for proteins, there could be >12 proteins altered in I/R hearts compared with sham hearts. Therefore, it is impossible to exclude other potential targets of miR-320, which may also contribute to modulation of cardiac I/R injury. Indeed, a new proteomic approach with the use of stable isotope labeling with amino acids in cell culture (SILAC), which directly measures genomewide alterations in protein synthesis induced by miRNAs, indicated that overexpression of a miRNA in HeLa cells mildly downregulated thousands of proteins.26,27 In the present study, we validated that Hsp20 was 1 of the miR-320 targeted proteins in the heart. Furthermore, mouse Hsp20 3'UTR has 11 potential miRNA binding regions including a miR-320 binding site (Figure II in the online-only Data Supplement); however, the microarray intensity of miR-320 in the heart was far higher than those of 10 other targeted miRNAs (online-only Data Supplement). These results suggest that Hsp20 may be regulated to a great extent by miR-320 in the heart, which is an important addition to other possible mechanisms regulating Hsp20 protein synthesis such as transcription and protein degradation.
It is noteworthy that Hsp20, compared with other small heat-shock proteins, is mostly upregulated in animal hearts on ischemic conditions,22 exercise training,28 and rapid right ventricular pacing.29 This suggests that Hsp20 may play a major role in cellular stress resistance and development of tolerance as an adaptive response after exposure to various stimuli. Our data presented in this study indicate that downregulation of miR-320 might represent an important adaptive mechanism to upregulate the expression levels of Hsp20 during I/R because previous reports from our laboratory and others have shown that Hsp20 (1) protects hearts against I/R-, doxorubicin-, and chronic isoproterenol-induced apoptosis and remodeling18,30,31; (2) inhibits platelet aggregation32; (3) regulates activities of vasorelaxation33; and (4) enhances contractile function.18,30
In conclusion, our data suggest that dysregulation of miR-320 expression contributes to ischemic heart disease. Knockdown of endogenous miR-320 provides protection against I/R-induced cardiomyocyte death and apoptosis by targeting Hsp20, a well-studied cardioprotector. Future studies in which an inducible system is used will be helpful to identify the exact therapeutic role for miR-320 in ischemic heart disease. Because miRNAs often have numerous targets, it is important to further explore the target network of miR-320, which may be involved in maintaining cardiac output after infarction. This may lead to rational target selection for therapeutic intervention in patients suffering from heart disease.
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Acknowledgments |
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We are grateful to Dr Evangelia G. Kranias for helpful discussion of this manuscript.
Sources of Funding
This study was supported by National Institutes of Health grant HL-087861 (Dr Fan).
Disclosures
None.
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*The first 3 authors contributed equally to this article.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.814145/DC1.
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