RETRACTED: miR-146a inhibits mitochondrial dysfunction and myocardial infarction by targeting cyclophilin D
2021; Cell Press; Volume: 23; Linguagem: Inglês
10.1016/j.omtn.2021.01.034
ISSN2162-2531
AutoresQiang Su, Yuli Xu, Ruping Cai, Rixin Dai, Xiheng Yang, Yang Liu, Binghui Kong,
Tópico(s)Circular RNAs in diseases
ResumoIncreasing evidence suggests that mitochondrial microRNAs (miRNAs) are implicated in the pathogenesis of cardiovascular diseases; however, their roles in ischemic heart disease remain unclear. Herein, we demonstrate that miR-146a is enriched in the mitochondrial fraction of cardiomyocytes, and its level significantly decreases after ischemic reperfusion (I/R) challenge. Cardiomyocyte-specific knockout of miR-146a aggravated myocardial infarction, apoptosis, and cardiac dysfunction induced by the I/R injury. Overexpression of miR-146a suppressed anoxia/reoxygenation-induced cardiomyocyte apoptosis by inhibiting the mitochondria-dependent apoptotic pathway and increasing the Bcl-2/Bax ratio. miR-146a overexpression also blocked mitochondrial permeability transition pore opening and attenuated the loss of mitochondrial membrane potential and cytochrome c leakage; meanwhile, miR-146a knockdown elicited the opposite effects. Additionally, miR-146a overexpression decreased cyclophilin D protein, not mRNA, expression. The luciferase reporter assay revealed that miR-146a binds to the coding sequence of the cyclophilin D gene. Restoration of cyclophilin D reversed the inhibitory action of miR-146a on cardiomyocyte apoptosis. Furthermore, cardiomyocyte-specific cyclophilin D deletion completely abolished the exacerbation of myocardial infarction and apoptosis observed in miR-146a cardiomyocyte-deficient mice. Collectively, these findings demonstrate that nuclear miR-146a translocates into the mitochondria and regulates mitochondrial function and cardiomyocyte apoptosis. Our study unveils a novel role for miR-146a in ischemic heart disease. Increasing evidence suggests that mitochondrial microRNAs (miRNAs) are implicated in the pathogenesis of cardiovascular diseases; however, their roles in ischemic heart disease remain unclear. Herein, we demonstrate that miR-146a is enriched in the mitochondrial fraction of cardiomyocytes, and its level significantly decreases after ischemic reperfusion (I/R) challenge. Cardiomyocyte-specific knockout of miR-146a aggravated myocardial infarction, apoptosis, and cardiac dysfunction induced by the I/R injury. Overexpression of miR-146a suppressed anoxia/reoxygenation-induced cardiomyocyte apoptosis by inhibiting the mitochondria-dependent apoptotic pathway and increasing the Bcl-2/Bax ratio. miR-146a overexpression also blocked mitochondrial permeability transition pore opening and attenuated the loss of mitochondrial membrane potential and cytochrome c leakage; meanwhile, miR-146a knockdown elicited the opposite effects. Additionally, miR-146a overexpression decreased cyclophilin D protein, not mRNA, expression. The luciferase reporter assay revealed that miR-146a binds to the coding sequence of the cyclophilin D gene. Restoration of cyclophilin D reversed the inhibitory action of miR-146a on cardiomyocyte apoptosis. Furthermore, cardiomyocyte-specific cyclophilin D deletion completely abolished the exacerbation of myocardial infarction and apoptosis observed in miR-146a cardiomyocyte-deficient mice. Collectively, these findings demonstrate that nuclear miR-146a translocates into the mitochondria and regulates mitochondrial function and cardiomyocyte apoptosis. Our study unveils a novel role for miR-146a in ischemic heart disease. IntroductionMyocardial infarction, which is characterized by prolonged ischemia of the coronary arteries, remains the leading cardiovascular cause of death worldwide.1Kuhn T.C. Knobel J. Burkert-Rettenmaier S. Li X. Meyer I.S. Jungmann A. Sicklinger F. Backs J. Lasitschka F. Muller O.J. et al.Secretome Analysis of Cardiomyocytes Identifies PCSK6 (Proprotein Convertase Subtilisin/Kexin Type 6) as a Novel Player in Cardiac Remodeling After Myocardial Infarction.Circulation. 2020; 141: 1628-1644Crossref PubMed Scopus (26) Google Scholar Blood flow restoration to the ischemic myocardium is currently the most timely and effective strategy to improve the outcome of patients with acute myocardial infarction.2Cannon 3rd, R.O. Mechanisms, management and future directions for reperfusion injury after acute myocardial infarction.Nat. Clin. Pract. Cardiovasc. Med. 2005; 2: 88-94Crossref PubMed Scopus (84) Google Scholar However, such restoration often leads to ischemic reperfusion (I/R) injury.3Muzumdar R.H. Huffman D.M. Calvert J.W. Jha S. Weinberg Y. Cui L. Nemkal A. Atzmon G. Klein L. Gundewar S. et al.Acute humanin therapy attenuates myocardial ischemia and reperfusion injury in mice.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1940-1948Crossref PubMed Scopus (113) Google Scholar Apoptosis of the terminally differentiated cardiomyocytes has been documented as an important pathogenic factor involved in cardiac I/R injury.4Yan K. An T. Zhai M. Huang Y. Wang Q. Wang Y. Zhang R. Wang T. Liu J. Zhang Y. et al.Mitochondrial miR-762 regulates apoptosis and myocardial infarction by impairing ND2.Cell Death Dis. 2019; 10: 500Crossref PubMed Scopus (44) Google Scholar Evidence has accumulated showing that the suppression of cardiomyocyte apoptosis is beneficial for decreasing infarction size and improving myocardial contractile dysfunction after reperfusion, suggesting that this manipulation may be a useful approach for protection against I/R injury.4Yan K. An T. Zhai M. Huang Y. Wang Q. Wang Y. Zhang R. Wang T. Liu J. Zhang Y. et al.Mitochondrial miR-762 regulates apoptosis and myocardial infarction by impairing ND2.Cell Death Dis. 2019; 10: 500Crossref PubMed Scopus (44) Google Scholar,5Zhu J. Yao K. Wang Q. Guo J. Shi H. Ma L. Liu H. Gao W. Zou Y. Ge J. Ischemic Postconditioning-Regulated miR-499 Protects the Rat Heart Against Ischemia/Reperfusion Injury by Inhibiting Apoptosis through PDCD4.Cell. Physiol. Biochem. 2016; 39: 2364-2380Crossref PubMed Scopus (54) Google Scholar Nevertheless, the fundamental mechanisms underlying cardiomyocyte apoptosis are poorly understood.A large body of evidence has indicated that mitochondrial dysfunction is associated with the development of many diseases, including cardiovascular disorders.4Yan K. An T. Zhai M. Huang Y. Wang Q. Wang Y. Zhang R. Wang T. Liu J. Zhang Y. et al.Mitochondrial miR-762 regulates apoptosis and myocardial infarction by impairing ND2.Cell Death Dis. 2019; 10: 500Crossref PubMed Scopus (44) Google Scholar,6Liu X.Y. Zhang F.R. Shang J.Y. Liu Y.Y. Lv X.F. Yuan J.N. Zhang T.T. Li K. Lin X.C. Liu X. et al.Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity.Cell Death Dis. 2018; 9: 610Crossref PubMed Scopus (21) Google Scholar,7Das S. Ferlito M. Kent O.A. Fox-Talbot K. Wang R. Liu D. Raghavachari N. Yang Y. Wheelan S.J. Murphy E. Steenbergen C. Nuclear miRNA regulates the mitochondrial genome in the heart.Circ. Res. 2012; 110: 1596-1603Crossref PubMed Scopus (241) Google Scholar Cardiomyocytes contain many mitochondria; hence, mitochondria-dependent apoptosis affects cardiomyocyte survival.4Yan K. An T. Zhai M. Huang Y. Wang Q. Wang Y. Zhang R. Wang T. Liu J. Zhang Y. et al.Mitochondrial miR-762 regulates apoptosis and myocardial infarction by impairing ND2.Cell Death Dis. 2019; 10: 500Crossref PubMed Scopus (44) Google Scholar,8Gustafsson A.B. Gottlieb R.A. Heart mitochondria: gates of life and death.Cardiovasc. Res. 2008; 77: 334-343Crossref PubMed Scopus (304) Google Scholar The mechanisms by which mitochondria-dependent apoptosis is regulated include the cyclophilin D-mitochondrial permeability transition pore (mPTP) opening, subsequent disruption of mitochondrial membrane potential (MMP), cytochrome c release, and caspase activation, which collectively lead to mitochondrial dysfunction.9Grimm S. Brdiczka D. The permeability transition pore in cell death.Apoptosis. 2007; 12: 841-855Crossref PubMed Scopus (154) Google Scholar Therefore, the maintenance of mitochondrial structure and homeostasis is a promising strategy for the amelioration of cardiomyocyte apoptosis and cardiac injury.7Das S. Ferlito M. Kent O.A. Fox-Talbot K. Wang R. Liu D. Raghavachari N. Yang Y. Wheelan S.J. Murphy E. Steenbergen C. Nuclear miRNA regulates the mitochondrial genome in the heart.Circ. Res. 2012; 110: 1596-1603Crossref PubMed Scopus (241) Google Scholar,8Gustafsson A.B. Gottlieb R.A. Heart mitochondria: gates of life and death.Cardiovasc. Res. 2008; 77: 334-343Crossref PubMed Scopus (304) Google Scholar,10Song R. Hu X.Q. Zhang L. Mitochondrial MiRNA in Cardiovascular Function and Disease.Cells. 2019; 8: 1475Crossref Scopus (29) Google ScholarMicroRNAs (miRNAs) are a class of non-coding 12- to 23-nucleotide RNAs that regulate gene expression at the posttranscriptional level.4Yan K. An T. Zhai M. Huang Y. Wang Q. Wang Y. Zhang R. Wang T. Liu J. Zhang Y. et al.Mitochondrial miR-762 regulates apoptosis and myocardial infarction by impairing ND2.Cell Death Dis. 2019; 10: 500Crossref PubMed Scopus (44) Google Scholar, 5Zhu J. Yao K. Wang Q. Guo J. Shi H. Ma L. Liu H. Gao W. Zou Y. Ge J. Ischemic Postconditioning-Regulated miR-499 Protects the Rat Heart Against Ischemia/Reperfusion Injury by Inhibiting Apoptosis through PDCD4.Cell. Physiol. Biochem. 2016; 39: 2364-2380Crossref PubMed Scopus (54) Google Scholar, 6Liu X.Y. Zhang F.R. Shang J.Y. Liu Y.Y. Lv X.F. Yuan J.N. Zhang T.T. Li K. Lin X.C. Liu X. et al.Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity.Cell Death Dis. 2018; 9: 610Crossref PubMed Scopus (21) Google Scholar miRNAs have been found to play roles in cardiac dysfunctions, such as heart failure, cardiac hypertrophy, and ischemic injury.4Yan K. An T. Zhai M. Huang Y. Wang Q. Wang Y. Zhang R. Wang T. Liu J. Zhang Y. et al.Mitochondrial miR-762 regulates apoptosis and myocardial infarction by impairing ND2.Cell Death Dis. 2019; 10: 500Crossref PubMed Scopus (44) Google Scholar,5Zhu J. Yao K. Wang Q. Guo J. Shi H. Ma L. Liu H. Gao W. Zou Y. Ge J. Ischemic Postconditioning-Regulated miR-499 Protects the Rat Heart Against Ischemia/Reperfusion Injury by Inhibiting Apoptosis through PDCD4.Cell. Physiol. Biochem. 2016; 39: 2364-2380Crossref PubMed Scopus (54) Google Scholar,7Das S. Ferlito M. Kent O.A. Fox-Talbot K. Wang R. Liu D. Raghavachari N. Yang Y. Wheelan S.J. Murphy E. Steenbergen C. Nuclear miRNA regulates the mitochondrial genome in the heart.Circ. Res. 2012; 110: 1596-1603Crossref PubMed Scopus (241) Google Scholar,10Song R. Hu X.Q. Zhang L. Mitochondrial MiRNA in Cardiovascular Function and Disease.Cells. 2019; 8: 1475Crossref Scopus (29) Google Scholar miR-146 was initially identified as a critical regulator of hematopoiesis, inflammation, and immunity.11Taganov K.D. Boldin M.P. Chang K.J. Baltimore D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.Proc. Natl. Acad. Sci. USA. 2006; 103: 12481-12486Crossref PubMed Scopus (3453) Google Scholar However, more recently it was shown to also be associated with the development of cardiovascular diseases, including doxorubicin cardiotoxicity, diabetic cardiomyopathy, and atherosclerosis.12Pan J.A. Tang Y. Yu J.Y. Zhang H. Zhang J.F. Wang C.Q. Gu J. miR-146a attenuates apoptosis and modulates autophagy by targeting TAF9b/P53 pathway in doxorubicin-induced cardiotoxicity.Cell Death Dis. 2019; 10: 668Crossref PubMed Scopus (38) Google Scholar, 13Feng B. Chen S. Gordon A.D. Chakrabarti S. miR-146a mediates inflammatory changes and fibrosis in the heart in diabetes.J. Mol. Cell. Cardiol. 2017; 105: 70-76Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Li K. Ching D. Luk F.S. Raffai R.L. Apolipoprotein E enhances microRNA-146a in monocytes and macrophages to suppress nuclear factor-κB-driven inflammation and atherosclerosis.Circ. Res. 2015; 117: e1-e11Crossref PubMed Scopus (142) Google Scholar Interestingly, emerging evidence suggests the presence and regulatory roles of miRNAs in the mitochondria.4Yan K. An T. Zhai M. Huang Y. Wang Q. Wang Y. Zhang R. Wang T. Liu J. Zhang Y. et al.Mitochondrial miR-762 regulates apoptosis and myocardial infarction by impairing ND2.Cell Death Dis. 2019; 10: 500Crossref PubMed Scopus (44) Google Scholar,7Das S. Ferlito M. Kent O.A. Fox-Talbot K. Wang R. Liu D. Raghavachari N. Yang Y. Wheelan S.J. Murphy E. Steenbergen C. Nuclear miRNA regulates the mitochondrial genome in the heart.Circ. Res. 2012; 110: 1596-1603Crossref PubMed Scopus (241) Google Scholar,10Song R. Hu X.Q. Zhang L. Mitochondrial MiRNA in Cardiovascular Function and Disease.Cells. 2019; 8: 1475Crossref Scopus (29) Google Scholar In the present study, we for the first time demonstrate that miR-146a localizes to the mitochondria, where its level dramatically changes after an I/R episode. Although the influence of miR-146a, mediated by a conventional miRNA-regulated pathway, on I/R injury had been proposed previously, our findings reveal a novel role and mechanism of action for mitochondrial miR-146 in cardiac I/R injury. Herein, we demonstrate that mitochondrial miR-146a is a key regulator of mitochondrial dysfunction, cardiomyocyte apoptosis, and heart ischemic disease.ResultsMitochondrial miR-146a is downregulated in the myocardium under I/R conditionsMicroarray analysis of the heart mitochondrial fraction identified 15 miRNAs that were differentially expressed in the tissues of sham-operated control mice and mice that underwent I/R. Among the 15 dysregulated miRNAs, miR-535-5p, miR-330, miR-146a, miR-181a, and miR-34a were significantly downregulated. The difference of miR-146a levels was the most pronounced compared to the changes in other mitochondrial miRNAs (Table S3). This was further confirmed by qRT-PCR, in which mitochondrial 12S rRNA was used as an internal control (Figure 1A). Thus, we decided to focus on the role of miR-146a, which is conserved between mice and humans (Figure S1A), in cardiac I/R injury. We found that miR-146a was enriched primarily in the mitochondrial fraction compared to its level in the cytosolic fraction. I/R challenge significantly reduced miR-146a level in the mitochondria but not in the cytosol (Figure 1B). In line with the in vivo results, miR-146a level in cardiomyocytes was decreased upon anoxia/reoxygenation (A/R) treatment in a time-dependent manner (Figure S1B). We also observed that A/R challenge led to a significant reduction in intracellular ATP levels (Figure S1C), which is a hallmark of mitochondrial dysfunction. Interestingly, the miR-146a level was positively correlated with ATP level, with a correlation coefficient of 0.8581 (Figure S1D), suggesting that mitochondrial miR-146a may play a role in mitochondrial dysfunction during cardiac I/R injury. Moreover, qRT-PCR results revealed that the abundance of miR-146a in the mitochondrial fraction was similar to that in the whole heart, suggesting that miR-146a is predominantly localized in the mitochondria (Figure 1C). Fluorescence in situ hybridization confirmed that miR-146a was present in the mitochondria of cardiomyocytes (Figure 1D).Ago2 has been shown to localize in the mitochondria and play an important role in the translocation of miRNAs into the mitochondria.4Yan K. An T. Zhai M. Huang Y. Wang Q. Wang Y. Zhang R. Wang T. Liu J. Zhang Y. et al.Mitochondrial miR-762 regulates apoptosis and myocardial infarction by impairing ND2.Cell Death Dis. 2019; 10: 500Crossref PubMed Scopus (44) Google Scholar,7Das S. Ferlito M. Kent O.A. Fox-Talbot K. Wang R. Liu D. Raghavachari N. Yang Y. Wheelan S.J. Murphy E. Steenbergen C. Nuclear miRNA regulates the mitochondrial genome in the heart.Circ. Res. 2012; 110: 1596-1603Crossref PubMed Scopus (241) Google Scholar Additionally, dicer, which generates miRNA maturation from primary miRNA, is present exclusively in the cytoplasm, not in mitochondria.7Das S. Ferlito M. Kent O.A. Fox-Talbot K. Wang R. Liu D. Raghavachari N. Yang Y. Wheelan S.J. Murphy E. Steenbergen C. Nuclear miRNA regulates the mitochondrial genome in the heart.Circ. Res. 2012; 110: 1596-1603Crossref PubMed Scopus (241) Google Scholar As expected, we observed that Ago2 was expressed both in cytosolic and mitochondrial fractions of cardiomyocytes, while dicer was largely absent from mitochondria (Figure S2). Moreover, the RNA-binding protein immunoprecipitation (RIP) experiment showed that miR-146a in the mitochondrial fraction was co-immunoprecipitated with Ago2, not dicer (Figure 1E), confirming mitochondrial localization of miR-146a. To investigate miR-146a biogenesis, we examined the presence of pre-miR-146a in the whole heart, as well as in the nuclear, cytosolic, and mitochondrial fractions. Results show that minimal levels of pre-miR-146a were observed in the nuclear and mitochondrial fractions compared with that in the total heart homogenate (Figure 1F). This indicates that miR-146a biosynthesis occurs in a conventional manner, with miR-146a translocating into the mitochondria following maturation in the cytosol.Cardiomyocyte-specific knockout of miR-146a exacerbates cardiac I/R injuryTo investigate the association between the downregulation of miR-146a and I/R-induced myocardial injury, we performed I/R surgery in miR-146a cardiomyocyte-specific knockout mice (Figure S3). As shown in Figure 2A, no infarct was observed in the myocardium of miR-146af/f mice and miR-146aCKO mice under sham conditions. Mice that underwent I/R revealed an apparent infarct, which was significantly aggravated by cardiomyocyte-specific deficiency of miR-146a (Figure 2B). The release of lactate dehydrogenase (LDH), a marker for cardiac injury, was expectedly increased after I/R injury. Knockout of miR-146a further increased the level of LDH (Figure 2C). Echocardiography examination showed that the impairment of cardiac function was exacerbated in miR-146aCKO mice, as evidenced by lower values of ejection fraction (EF) and fractional shortening (FS) (Figures 2D and 2E). Cardiomyocyte apoptosis is an important contributor to myocardial dysfunction and failure. Transferase dUTP nick end labeling (TUNEL) staining revealed no significant difference in the number of cardiomyocytes undergoing apoptosis in the myocardium between miR-146af/f and miR-146aCKO mice under sham conditions. I/R stimulation markedly increased the number of apoptotic cells, and this increase was further enhanced by cardiomyocyte-specific knockout of miR-146a (Figures 2F and 2G).Figure 2Deficiency of miR-146a in cardiomyocytes exacerbates I/R injuryShow full caption(A) Cardiomyocyte-specific miR-146a knockout mice (146aCKO) and their control littermates (146af/f) received I/R (30 min ischemia and 4 h reperfusion) or sham treatment. Representative images of myocardial tissue sections are shown. (B) Myocardial infarct size expressed as the ratio of infarcted area (IF) to the area at risk (AAR) (IF/AAR). ∗∗p < 0.01 versus sham 146af/f; ##p < 0.01 versus sham 146aCKO, n = 6 per group. (C) Lactate dehydrogenase (LDH) level in serum was measured by a commercial kit. ∗∗p < 0.01 versus sham 146af/f; ##p < 0.01 versus sham 146aCKO, n = 12–16 per group. (D and E) Echocardiographic analysis of left ventricular ejection fraction (EF) (D) and left ventricular fractional shortening (FS) (E) at the end of 24 h reperfusion. ∗∗p < 0.01 versus sham 146af/f; ##p < 0.01 versus sham 146aCKO, n = 8–10 per group. (F) Representative images of TUNEL and α-actinin staining of myocardial tissue sections after 30 min myocardial ischemia followed by 4 h reperfusion. Green TUNEL staining represents apoptotic cells, cardiomyocytes are labeled with an anti-α-actinin antibody showing red color, and the nuclei are stained blue by 4′,6-diamidino-2-phenylindole (DAPI). (G) Quantification of apoptotic cardiomyocytes in each group. ∗∗p < 0.01 versus sham 146af/f; ##p < 0.01 versus sham 146aCKO, n = 7 per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)miR-146a upregulation ameliorates A/R-induced cardiomyocyte apoptosis in vitroWe next examined whether the regulatory role of miR-146a in cardiomyocyte apoptosis was also evident in vitro, under A/R conditions. The annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay showed that the ratio of apoptotic cells did not change after overexpression or knockdown of miR-146a, as compared with that in the relevant negative control experiment. A/R treatment markedly enhanced the fraction of apoptotic cardiomyocytes, which was inhibited by miR-146a upregulation yet further augmented by miR-146a knockdown (Figures 3A and 3B ). In addition, TUNEL staining also demonstrated that overexpression of miR-146a markedly attenuated apoptosis of cardiomyocytes induced by A/R, whereas downregulation of miR-146a was associated with enhanced cell apoptosis (Figures 3C and 3D). Given the alterations of miR-146a in the mitochondria, we explored whether miR-146a affects the mitochondria-dependent apoptotic pathway and cardiomyocyte survival. The anti-apoptotic Bcl-2 protein family and the pro-apoptotic Bax protein from the same family are essential for the initiation of mitochondrial apoptosis. Lower expression of Bcl-2 and higher expression of Bax were found in A/R-treated cardiomyocytes compared with the respective levels in untreated cells. The Bcl-2/Bax ratio, which determines the fate of cell survival, was significantly decreased by A/R. miR-146a upregulation reversed A/R-induced decrease in the Bcl-2/Bax ratio by increasing Bcl-2 expression and attenuating Bax expression. However, miR-146a downregulation further augmented the effects of A/R on Bcl-2 and Bax expression levels and the Bcl-2/Bax ratio (Figures 3E and 3F).Figure 3miR-146a mediates cardiomyocyte apoptosis induced by anoxia/reoxygenation (A/R) treatmentShow full caption(A and B) Cardiomyocytes were transfected with miR-146a mimics (146a-m, 50 nmol/L, A), miR-146a inhibitor (146a-i, 50 nmol/L, B), or their corresponding negative control sequences (NC-m or NC-i) for 24 h before A/R treatment for another 48 h. Cardiomyocyte apoptosis was determined by annexin V-FITC/propidium iodide (PI) staining followed by flow cytometry. (C and D) TUNEL (green) and DAPI (blue) staining of cardiomyocytes treated with miR-146a mimics (C) or miR-146a inhibitor (D) under A/R conditions. (E and F) Changes in expression levels of Bcl-2 and Bax proteins in cardiomyocytes treated with miR-146a mimics (E) or miR-146a inhibitor (F) after A/R stimulation. Representative western blot images are shown in leftmost panels. Bar charts represent densitometric analysis data of relative levels of Bcl-2, Bax, and Bcl-2/Bax ratio. ∗∗p < 0.01 versus NC-m control or NC-i control; ##p < 0.01 versus NC-m A/R or NC-i A/R, n = 6–8.View Large Image Figure ViewerDownload Hi-res image Download (PPT)miR-146a attenuates mitochondrial dysfunction under A/R conditionsThe loss of MMP leading to mitochondrial dysfunction is considered as the predominant consequence of the imbalance between Bcl-2 and Bax expression levels. We thus determined the effects of miR-146a on MMP in cardiomyocytes by using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benza-midazolocarbocyanin iodide (JC-1) staining. Upon A/R stimulation, the green fluorescence of JC-1 significantly increased, whereas the red fluorescence decreased, resulting in an elevation in the green/red fluorescence ratio. This suggests that A/R led to the decrease of MMP. Upregulation of miR-146a significantly inhibited A/R-induced reduction of MMP. The opposite result was observed in miR-146a inhibitor-treated cells (Figure 4A). Mitochondrial membrane depolarization could induce mPTP opening and subsequently lead to cytochrome c release from the mitochondria to the cytosol. Confocal microscopy showed that miR-146a overexpression inhibited A/R-induced decrease in fluorescence of calcein-AM, a fluorescent dye that is a sensitive indicator of mPTP opening, suggesting that miR-146a prevented the opening of mPTP (Figure 4B). In contrast, the reduction of calcein-AM fluorescence was further potentiated by miR-146a downregulation (Figure 4C). Moreover, A/R treatment increased cytochrome c protein expression in the cytosol; however, it decreased its expression in the mitochondria, indicating transient release of cytochrome c. The translocation of cytochrome c from the mitochondria to the cytosol was abrogated by miR-146a mimics yet promoted by the miR-146a inhibitor (Figures 4D and 4E). Furthermore, activation of caspases, a downstream event of cytochrome c release, was also induced by A/R stimulation, as evidenced by increased cleavage of caspase-9, caspase-3, and PARP. miR-146a overexpression inhibited, whereas miR-146a inhibition enhanced the activation of caspase-9, caspase-3, and PARP (Figure S4). Taken together, these data indicate that miR-146a ameliorates cardiomyocyte apoptosis at least partially via inhibiting the mitochondria-dependent apoptotic pathway.Figure 4miR-146a ameliorates A/R-induced mitochondrial dysfunction in cardiomyocytesShow full caption(A) Cardiomyocytes were treated with miR-146a mimics or miR-146a inhibitor for 24 h prior to A/R stimulation for 48 h and then with AngII (10 μM) for another 24 h. Mitochondrial membrane potential was measured using JC-1 staining. Representative images of JC-1-derived fluorescence in cardiomyocytes are shown in the left panel. JC-1 aggregate image displays red fluorescence, JC-1 monomer image displays green fluorescence, and the merged image combines red and green fluorescence images. Quantitative analysis of the green/red fluorescence ratio is provided in the right panel. (B and C) Opening of mitochondrial permeability transition pore in cardiomyocytes treated with miR-146a mimics (B) or miR-146a inhibitor (C) under A/R conditions was detected by calcein-AM staining. (D and E) Cytochrome c protein expression in the cytosol and mitochondria was determined by western blot. Transfection with miR-146a mimics inhibited the release of cytochrome c into the cytosol (D), whereas miR-146a inhibitor promoted the release of cytochrome c (E). Mitochondrial marker protein Tom20 was used as an internal control. ∗∗p < 0.01 versus NC-m control or NC-i control; ##p < 0.01 versus NC-m A/R or NC-i A/R, n = 6–8.View Large Image Figure ViewerDownload Hi-res image Download (PPT)miR-146a regulates cyclophilin D expressionTo understand how miR-146a regulates mitochondrial dysfunction, we tested whether miR-146a influenced the expression of the mPTP complex, including voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and cyclophilin D. qRT-PCR showed that overexpression of miR-146a did not alter mRNA levels of Vdac1, Slc25a4, and Ppif genes encoding VDAC, ANT, and cyclophilin D, respectively, in mitochondria of cardiomyocytes (Figure 5A). We then examined expression levels of these proteins in response to transfection with miR-146a mimics and found that only cyclophilin D expression was markedly decreased in cardiomyocyte mitochondria by miR-146a upregulation (Figure 5B). Notably, the other miRNAs that were downregulated upon cardiac I/R injury, as determined via microarray analysis, such as miR-535-5p, miR-330, miR-181a, and miR-34a, did not affect cyclophilin D expression (Figure S5). In addition, overexpression of miR-146a almost completely abolished A/R-induced increase in cyclophilin D expression (Figure 5C). In contrast, miR-146a knockdown significantly increased cyclophilin D protein expression; however, it did not alter its mRNA level (Figures 5D and 5E). Furthermore, RNAhybird program showed that the cyclophilin D gene coding sequence (CDS) contains a potential binding site for miR-146a (Figure 5F). Therefore, we examined the effect of miR-146a on cyclophilin D translation by using the luciferase assay. We constructed wild-type CDSs of cyclophilin D containing the binding site of miR-146a or its mutant version and cloned them into the luciferase vector. The luciferase reporter assay revealed that miR-146a decreased the translation of wild-type cyclophilin D-CDS; however, it had no distinct effect on the mutant cyclophilin D-CDS (Figure 5G). Further, the RIP results confirmed that miR-146a binds cyclophilin D in mitochondria of cardiomyocytes (Figure 5H). In addition, overexpression of miR-146a inhibited the expression of the wild-type cyclophilin D-CDS; however, it did not exert any inhibitory effect on the mutant cyclophilin D-CDS, suggesting that inhibition by miR-146a of cyclophilin D expression is binding site-dependent (Figure S6). Collectively, these results demonstrate that cyclophilin D is a specific target of miR-146a in mitochondria of cardiomyocytes.Figure 5miR-146a regulates cyclophilin D expressionShow full caption(A) qRT-PCR analysis of mRNA expression of the Vdac1, Slc25a4, and Ppif genes encoding voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and cyclophilin D (cyclophilin D), respectively, in cardiomyocyte mitochondria after transfection with miR-146a mimics for 48 h (n = 5). (B) miR-146a upregulation decreased cyclophilin D protein expression in mitochondria; however, it had no distinct effect on expression levels of VDAC and ANT. ∗∗p < 0.01 versus control, n = 6. (C) Cardiomyocytes were pretreated with miR-146a mimics before A/R stimulation. Western blot analysis of cyclophilin D protein expression in the mitochondrial fraction is shown. ∗∗p < 0.01 versus control; ##p < 0.01 versus A/R, n = 6. (D and E) Cyclophilin D mRNA (D) and protein (E) ex
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