Influences Of MicroRNA-497 On Cell Apoptosis- And Autophagy- Mediated Myocardial Anoxia/Reoxygenation Injury

Background and objectiveMyocardial infarction (MI) is a leading cause of morbidity and mortality worldwide in patients with coronary heart disease.Early reperfusion of the ischemic region by thrombolytic treatment or percutaneous coronary intervention,although effective for salvaging the damaged myocardium, can lead to additional injury such as cardiomyocyte death and loss of cardiac function which is known asischemia/ reperfusion (IR) injury.[1,2]The cellular mechanisms underlying IR-induced injury remain incompletely explored.MicroRNAs(miRNAs) are a class of small noncoding RNA molecules that negatively modulate gene and protein expression by promoting RNA degradation and inhibiting transcription through binding to the 3’untranslated regions of targeted mRNAs. MicroRNAs can modulate complex physiological or disease phenotypes,therefore play a crucial role in the regulation of biological functions. [3-6] Accumulating evidence indicates the importance of several miRNAs in the regulation of myocardial IR injury, [7-12]raising the possibility of making them potential therapeutic targets.Recent studies have shown that the miR-15 family plays a critical role in myocardial ischemia and heart failure.[13-15]The miR-15 family members including miR-15a, miR-15b, miR-16, miR-195, miR-424 and miR-497, show 5’-end sequence similarity and many common targets. [16,17]MiR-15 and miR-16 were reported to induce apoptosis by targeting Bcl-2.[18]Anti-miR chemistries targeting miR-15 in mice were reported to reduce myocardial infarct size, [15] while inhibition of either miR-15a or miR-16 enhanced post-ischemic neovascularization.[19]It has been demonstrated that miR-195 plays a pro-apoptotic role in cardiomyocytes. [20]However the exact function of miR-497 in cardiomyocyte or heart remains completely unknown.miR-497 is predicted to target anti-apoptosis gene Bcl-2 and autophagy gene microtubule-associated protein 1 light chain 3B(LC3B).In non-cardiomyocytes, miR-497 has been demonstrated to promote ischemic neuronal death by negatively regulating Bcl-2 and Bcl-w,[21]and play inhibitory roles in tumorigenesis as a tumor suppressor. [22,23]In human neuroblastoma cells,overexpression of miR-497 was shown to increase reactive oxygen species formation,disrupt mitochondrial membrane potential,and induce cytochrome C release. [24] Since both apoptosis and autophagy are deeply involved in myocardial IR injury,we designed this study to investigate the effects of miR-497 on the fate of cardiomyocytes in response to anoxia/reoxygenation (AR) or IR insult.Our data suggest that miR-497 is a new therapeutic target for myocardial IR injury.Experimental Approaches1. Mouse MI and IR modelMice (C57BL/6,male,weighed 20-25 g,aged 8-10 weeks) were subjected to make MI models or IR (ischemia/reperfusion) models.The left coronary artery (LCA) permanent ligation or ligation for 45min followed by 24h of reperfusion was carried out as we described previously.[25]MI models were divided into 3 groups:(1) Sham group;(2)MI 2w group;(3) MI 4w group.The MI mice were sacrificed2w or 4w for realtime PCR and western blotting.IR models were divided as follows:(1)Sham;(2)IR+Scramble;(3)IR+Ad-miR-497-sponge;(4)IR+vector;(5)IR+A d-miR-497;Advirus was directly injected into myocardium 3 days before the operation.The operated mice that survived for 24h were sacrificed for realtime PCR, 2%TTC staining analysis and paraffin section.2. Cell culture and transfectionNeonatal rat cardiomyocytes (NRCs) were isolated from 1-2-day-old Sprague-Dawley rats.Either specific mimic or Ad-miR-497-sponge was transfected to the cultured NRCs which induced over-or down-expression of miR-497, respectively.Double-stranded miR-497 mimic and the relative scrambled control RNA at a final concentration of 50 nM were introduced into the NRCs. Transfected cells were harvested at 36 h. The Ad-miR-497-sponge was directly transfected to the cultured NRCs (multiplicity of infection (MOI)= 10), and cells were harvested at 48h.3. Anoxia/reoxygenation in NRCs and cell viability assayImmediately after transfection of miR-497 mimic or sponge or vectot,NRCs were placed in either a low oxygen atmosphere or normoxia conditions.Normoxia condition was created in a nomoxia incubator with 21%O2 and 5%CO2 at 37℃.To induce anoxia,NRCs were incubated in a humidified environment at 37℃ in a 3-gas hypoxic chamber maintaining 5%CO2and 1%O2(oxygen was expelled by nitrogen) for various time periods(3-24h).After anoxia,NRCs were exposed for 2h of reoxygenation under normoxia condition.The cells that were not subjected to AR were incubated under normoxia for the same duration and served as control.The cells were harvested for the isolation of cellular RNA and protein.Cell viability of the cultured cardiomyocytes in response to various treatments was determined using MTT assay according to the manufacture’s instruction.4. Real-time PCR assay of microRNAsTotal RNA was extracted from NRCs or heart tissues using E.Z.N.ATM Total RNA Kit II (OMEGA,USA) in accordance with the manufacturer’s protocol.The concentration of RNA was measured with a NanoDrop Spectrophotometer.For reverse transcription and quantitative realtime polymerase chain reaction(PCR),10ng of total RNA per sample was used with the All-in-one TM miRNA Q-PCR Detection Kit. Specific primers for miR-15 family members were obtained from GeneCopoeia Inc.5. Apoptosis and autophagosome detectionNRCs were incubated in 35-mm Petri dishes.Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nickend labeling and Hoechst 33258 according to the manufacturer’s instructions.Autophagosomes in NRCs were labeled with MDC staining as described elsewhere.[26]After incubation with 50 nmol/L MDC in phosphate buffered saline(PBS) at 37℃ for 10 min,the NRCs were washed with PBS and fixed with 4% paraformaldehyde.The apoptosis nucleus and autophagic vacuoles were visualized with confocal microscope.Cultured NRCs or murine heart sections were prepared for transmission electron microscopy (TEM) to investigate the formation of autophagosome.Results1. Myocardial miR-497 expression was consistently downregulated in response to MI or ARReal-time quantitative PCR showed that miR-497 was expressed in the tissues ofheart, lung, kidney, liver and brain, and the expression levels were significantly higherin heart when compared with other tissues. In mice with MI, myocardial miR-497 expression was dynamically down-regulated from 1 day to 4 weeks. In cultured NRCs exposed to various time periods of anoxia, miR-497 expression was not changed for 3 or 6 h of anoxia but significantly decreased 24 h later (P< 0.001). Intriguingly, miR-497 expression was dramatically decreased in atime-dependent manner when NRCs were exposed to 2h of reoxygenation following3-24 h of anoxia. These findings indicatethat miR-497 is involved in myocardial ischemia and preferentially in IR injury,suggesting that miR-497 is an IR-related microRNA in the murine heart.Based onthese results, we chose 3h anoxia/2h reoxygenation for the following experiments.2. MiR-497 directly targets Bcl-2 and LC3BUsing bioinformatics algorithms, anti-apoptosis gene Bcl2, and autophagy gene LC3B, were predicted as putative targets of miR-497 (Figure 1D). Consistent with the miR-497 downregulation in infarcted heart, we noted that both Bcl-2 and LC3B-II protein levels were increased. In cardiomyocytes with AR insult, Bcl-2 protein level was decreased, while LC3B-Ⅱ and beclin-1 were increased.3. MiR-497 mimic induces cardiomyocyte apoptosis and autophagyTo characterize miR-497 function, we overexpressed synthetic mature miR-497(miR-497 mimics) in NRCs.50 nM of miR-497 mimics was identified to increase the expression of miR-497 by more than 2 folds. MiR-497 mimics significantly increased the apoptosis of NRCs even in the normoxia condition, as shown by Hoechst staining and TUNEL staining. In response to AR, the apoptosis was significantly enhanced by miR-497 mimics (P< 0.01). MDC is the specific marker for autolysosomes,we examined the effect of miR-497 mimics on the incorporation of MDC into NRCs in response to AR. As a result, miR-497 mimics significantly inhibited autophagosome formation in cardiomyocytes during AR.Similar results were obtained using TEM examination. These results indicate a pro-apoptosis and an anti-autophagic role of miR-497 in cardiomyocytes.4. Silencing of miR-497 improves cell survivalTo further examine the contribution of miR-497 to the regulation of AR injury, NRCs were transfected with the Ad-miR-497-sponge. A satisfactory infection efficiency of miR-497 sponge was obtained in cardiomyocytes. The Hoechst and TUNEL staining analyses showed that miR-497-sponge exerted noinfluence on cardiomyocytes under normoxia, but significantly decreased apoptosis in cardiomyocytes with AR insult (P< 0.05). Autophagy detected by MDC was significantly enhanced in miR-497 sponge-treated NRCs than in vector-treated cells. These results indicate that knockdown of miR-497 attenuates AR injury in cardiomyocytes.5. Effects of miR-497 expression change on protein expression related to autophagy and apoptosisIn cultured NRCs, Western blot analysis showed that miR-497 mimics significantly down-regulated protein expressions of Bcl2, LC3B-Ⅱ and beclin-1, and up-regulated Bax expression. miR-497 sponge exerted opposite effects and increased autophagic influx as determined by P62 downregulation. By using Ad-sh-beclin-1, lysosomal inhibitor Baf and autophagy inhibitor wortmannin, we further investigated whether the increase of LC3B-II by miR-497 sponge is affected by beclin 1. We noted that the upregulation of LC3B-II was enhanced by Baf and antagonized by sh-beclin-1 and Wor.6. MiR-497 regulates autophagy and apoptosis related signal proteins in the context of ARIn cultured neonatal rat cardiomyocytes exposed to AR, loss and gain function of miR-497 also exerted significant influence on protein expression of LC3B-II, beclin-1,Bcl-2 and Bax. We noted that miR-497 decreased Bcl-2, LC3B-Ⅱ and beclin-1 and increased Bax in cardiomyocytes in the presence of AR. Inhibiting lysosomal degradation with Baf increased beclin-1 and LC3B-II expression levels in the presence of miR-497 mimic or sponge. Addition of Baf to miR-497 mimic or sponge increased Bax expression, suggesting that inhibition of autophagic flux may promote apoptosis. MTT cell viability assay shows that inhibition of miR-497 increased cell survival in the context of AR.7. MiR-497 sponge reduces myocardial infarct size in miceTo elucidate the in vivo effects of miR-497 inhibition on IR injury,we directlyinjected Ad-miR-497-sponge or Ad-miR-497 into left ventricular myocardium of mice 3 days before the onset of IR procedure.As shown in Figure 6A,3 days after intra-myocardial injection,more than 50% adenovirus transduction efficiency was reached.Consistently, miR-497 levels were significantly decreased when the mice were subjected to ischemia for 45 min and reperfusion for 24h.Meanwhile,miR-497 levels were further reduced in Ad-miR-497-sponge-treated group but significantly increased in Ad-miR-497-treated group. Myocardial infarct size determined with TTC was significantly smaller in mice treated with Ad-miR-497-sponge than in mice receiving scramble vector infection, while myocardial infarct size was larger in Ad-miR-497-treated mice than in control vector-treated group. Similar to the results in cultured NRCs, treatment with Ad-miR-497-sponge enhanced cardiomyocyte autophagy and inhibited apoptosis in IR mice, while Ad-miR-497 exerted opposite effects.Conclusions1. In mice with MI and cultured NRCs exposed to anoxia/reoxygenation, miR-497 expression is dynamically down-regulated. And the targeting protein levelsof both Bcl-2 and LC3Bare correspondingly increased. These findings indicate that microRNA-497 expression is associated with the ischemia reperfusion injury.2. MiR-497 mimics significantly down-regulated protein expression of Bcl2, LC3B-Ⅱ and beclin-1, and up-regulated Bax expression. miR-497 sponge exerted opposite effects and increased autophagic influx.Inhibition of miR-497 increased cell survival in the context of AR.3. Inhibition of miR-497 limits myocardial infarct size in mice, suggesting that miR-497 is a novel therapeutic target for myocardial ischemiareperfusion injury.