Function And Mechanism Of MiR-199a-5p For Endoplasmic Reticulum Stress In Cardiomyocytes During Chronic Hypoxia

Objective:Chronic hypoxia is an important pathophysiological change in patients with cyanotic congenital heart defect(CCHD) or long-term hypoxic-ischemic heart disease. During the continuing hypoxic process, the cardiac tissue gradually attuned to hypoxia through a series of physiological and biochemical regulatory process, and the cardiac failure rarely occurs in these patients, who can still survive for a period of time. Exploring the underlying mechanism by which hearts adapt to long-term hypoxia is helpful for improving clinic cardioprotection, especially during perioperative period of CCHD. Our department have already observed the endoplasmic reticulum stress(ERS) and enhanced unfolded protein response(UPR) process in myocardial samples from chronic hypoxic CCHD patients. The levels of 78-kD glucose-regulated protein(GRP78), a major indicator and molecular chaperone of UPR, and the ER stress sensors, the activating transcription factor 6(ATF6) were higher in CCHD patients, which played a protective role for hypoxic heart. Meanwhile, our team also found enhanced signal transducer and activator of transcription 3(STAT3) signaling in CCHD, which is beneficial for myocardial protection during hypoxia. Based on the earlier research, the present study aims to determine whether changes of miR-199a-5p, an microRNA expressed in abundance in the heart, play an important role in regulating UPR process in hypoxic heart. Moreover, the mechanism whether STAT3 signaling affects miR-199a-5p levels was also studied.Method:Part I: We collected the myocardiac tissue of CCHD patients and acyanotic congenital heart defects(ACHD) patients for observing ATF6 and GRP78 expression as well as mi R-199a-5p levels. These gene and protein expression levels were analyzed by Western Blot and RT-qPCR technologies. The correlation between them was also studied.Part II: We established an in-vitro model of culturing human cardiomyocytes(HCMs) under both normoxic and hypoxic conditions. The mi R-199a-5p mimic and inhibitor were transfected into HCM cells to change the miR-199a-5p levels. The cell apoptosis was detected by flow cytometry and The expression of ATF6, GRP78 and CHOP were analyzed by Western Blot. The direct targeting action of miR-199a-5p to the ATF6 and GRP78 3′-UTR binding sites was studied by luciferase assay.Part III: The ratio of cardiac pSTAT3/STAT3 expression as well as interleukin(IL)-6 and IL-11 levels in cardiac tissue homogenate of CCHD and ACHD patients were observed. The in-vivo model of hypoxic mouse(under a hypobaric chamber with 50% oxygen content of the normal atmosphere) with/without injection of S3I-201, a STAT3-DNA binding inhibitor, were used to study whether STAT3 signal activation affect miR-199a-5p expression. The ChIP assay was performed to detect the interaction of pSTAT3 on miR-199 a gene promotors. The effect of S3I-201 and miR-199a-5p antagomir on downstream ATF6, GRP78 and apoptosis indicator CHOP were also observed.Results:1. An correlation between miR-199a-5p levels and expression levels of GRP78 and ATF6 in CCHD hearts was found.Compared with ACHD control group, the levels of miR-199a-5p profoundly decreased in cardiac tissue of CCHD individuals undergoing chronic hypoxia. Meanwhile, the CCHD group exhibited a significant increase in GRP78 and ATF6 protein and mRNA levels. Moreover, there is a strong linear association between decreased miR-199a-5p levels and the up-regulated expression of its potential targets GRP78 and ATF6, as well as a correlation between miR-199a-5p levels and oxygen saturation that could distinguish CCHD patients from ACHD patients.2. miR-199a-5p affects ER stress-related apoptosis by direct targeting GRP78 and ATF6It was found that moderate in-vitro hypoxia(3% O2), but not severe hypoxia(1% O2) was in accordance with the actual UPR process and cardiac status observed in CCHD patients. The results also indicate that moderate and persistent hypoxia in CCHD patients is enough to maintain miR-199a-5p at a much lower level.The miR-199a-5p mimic transfection significantly accentuated hypoxic induction of ATF6 and GRP78 gene expression in HCMs, as well as increased apoptotic rates and CHOP expression. In contrast, the miR-199a-5p inhibitor, increased ATF6 and GRP78 expression in hypoxic HCMs and inhibited apoptosis.Using luciferase reporter plasmid that containing the wild type or mutant sequences of ATF6 and GRP78 3’-UTR, we found that the miR-199a-5p mimic can down-regulate the relative luciferase activity both in HEK293 T cells and in HCM cells, which indicated that mi R-199a-5p can control ATF6 and GRP78 expression by directly targeting their 3’UTRs. These targeting effect was more powerful in HCM cells than HEK293 T cells, and hypoxia could significantly change the targeting action of miR-199a-5p on 3’UTRs of ATF6 and GRP78 in HCMs.3. The activation of STAT3 signaling promotes down-regulation of miR-199a-5p in hypoxic cardiomyocytesIt was demonstrated that pSTAT3/STAT3 protein expression ratio significantly increased in CCHD myocardial specimens compared with ACHD, which maybe resulted from increased IL-6 and IL-11 levels. A significant negative correlation between myocardial miR-199a-5p expression levels and IL-6 or IL-11 levels was observed in CCHD patients.By conducting experiments in hypoxic mice as well as by intervening STAT3 activation by S3I-201, It was observed that the miR-199a-5p levels decreased markedly in cardiac tissues of hypoxic mice for 7 and 21 hypoxic days. The miR-199a-5p came from private mi R-199a-1 and miR-199a-2. It was found that hypoxia mainly affect the miR-199a-2 expression. The injection of pSTAT3 inhibitor S3I-201 obviously reversed the decreasing of myocardial miR-199a-5p levels and pri-miR-199a-2 expression in hypoxic mice. The ChiP assay demonstrated that pSTAT3 can bind to miR-199a-2 gene promotors, and this binding was more significant in hearts of hypoxic mice.It was also observed that pSTAT3 inhibitor in hypoxic mice prevented the up-regulating expression of miR-199a-5p-target genes, ATF6 and GRP78, and caused increasing CHOP expression in hypoxic mice. And these change can be recovered by injection miR-199a-5p antagomir.Conclusions:In summary, our results demonstrated that chronic hypoxia could down-regulated mi R-199a-5p levels through activating STAT3 pathway. Decreased miR-199a-5p promoted expression of GRP78 and ATF6, the two major ER stress sensors and UPR effectors, thus benefiting UPR process and the protection of cardiocytes against ERS-related apoptosis. This is a novel adaptation mechanism of cardiac myocytes to chronic hypoxia. We propose that mi R-199a-5p could be a valuable diagnostic and therapeutic target for patients of CCHD and other hypoxic-ischemic heart disease.