miR-125a, miR-139 and miR-324 contribute to Urocortin protection against myocardial ischemiareperfusion injury

1 SCIentIFIC RepoRts | 7: 8898 | DOI:10.1038/s41598-017-09198-x www.nature.com/scientificreports miR-125a, miR-139 and miR-324 contribute to Urocortin protection against myocardial ischemiareperfusion injury Ignacio Díaz1, Eva Calderón-Sánchez1, Raquel Del Toro1, Javier Ávila-Médina1,2, Eva Sánchez de Rojas-de Pedro1, Alejandro Domínguez-Rodríguez1,2, Juan Antonio Rosado3, Abdelkrim Hmadcha 4, Antonio Ordóñez1 & Tarik Smani 1,2 Urocortin 1 and 2 (Ucn-1 and Ucn-2) have established protective actions against myocardial ischemiareperfusion (I/R) injuries. However, little is known about their role in posttranscriptional regulation in the process of cardioprotection. Herein, we investigated whether microRNAs play a role in urocortininduced cardioprotection. Administration of Ucn-1 and Ucn-2 at the beginning of reperfusion significantly restored cardiac function, as evidenced ex vivo in Langendorff-perfused rat hearts and in vivo in rat subjected to I/R. Experiments using microarray and qRT-PCR determined that the addition of Ucn-1 at reperfusion modulated the expression of several miRNAs with unknown role in cardiac protection. Ucn-1 enhanced the expression of miR-125a-3p, miR-324-3p; meanwhile it decreased miR-139-3p. Similarly, intravenous infusion of Ucn-2 in rat model of I/R mimicked the effect of Ucn-1 on miR-324-3p and miR-139-3p. The effect of Ucn-1 involves the activation of corticotropin-releasing factor receptor-2, Epac2 and ERK1/2. Moreover, the overexpression of miR-125a-3p, miR-324-3p and miR-139-3p promoted dysregulation of genes expression involved in cell death and apoptosis (BRCA1, BIM, STAT2), in cAMP and Ca2+ signaling (PDE4a, CASQ1), in cell stress (NFAT5, XBP1, MAP3K12) and in metabolism (CPT2, FoxO1, MTRF1, TAZ). Altogether, these data unveil a novel role of urocortin in myocardial protection, involving posttranscriptional regulation with miRNAs. Ischemia induced by the interruption of heart blood flow evokes significant cardiac myocytes damages. Paradoxically, the subsequent reperfusion also activates various injury responses and further tissue lesions what is known as ischemia and reperfusion (I/R) injuries1. Advances in the understanding of pathophysiological mechanisms of I/R injury has identified a number of possible therapeutic targets and cardioprotectors such as ligands that activate RISK (reperfusion injury salvage kinase) or SAFE (survival activating factor enhancement) pathways; metoprolol, beta1-selective blocker; or cyclosporine-A among others drugs as reviewed recently2. Urocortin isoforms (Ucn-1, Ucn-2, and Ucn-3) have emerged as potential therapeutic agonists that improve heart performance and protect it from I/R injuries3, 4. In the cardiovascular system, urocortin binds to a G-protein-coupled receptor called CRF-R2 (corticotropin releasing factor receptor-2), whose activation enhances cAMP intracellular concentration5, classically related to PKA activation. Now, it’s known that cAMP also activates Epac (exchange protein activated by cAMP), important regulator of several cAMP-regulated processes in heart6. We have previously shown that the addition of Ucn-1 improved intracellular Ca2+ concentration ([Ca2+]i) handling during I/R[7], and efficiently protected hearts from I/R damages by the modulation of apoptotic genes, CD40lg, Xiap and BAD, through the activation of Epac2 and ERK1/28. 1Grupo de Fisiopatología Cardiovascular, Instituto de Biomedicina de Sevilla-IBiS, Universidad de Sevilla/HUVR/Junta de Andalucía/CSIC, Seville, 41013, CIBERCV, Spain. 2Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, Seville, 41009, Spain. 3Departamento de Fisiología, Universidad de Extremadura, Caceres, 10071, Spain. 4 Center for Molecular Biology and Regenerative Medicine-CABIMER, Unversity of Pablo de Olavide-University of Seville-CSIC, Seville, Spain., Seville, 41010, CIBERDEM, Spain. Ignacio Díaz, Eva Calderón-Sánchez and Raquel Del Toro contributed equally to this work. Correspondence and requests for materials should be addressed to T.S. (email: [email protected]) Received: 21 April 2017 Accepted: 21 July 2017 Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 SCIentIFIC RepoRts | 7: 8898 | DOI:10.1038/s41598-017-09198-x Recently, microRNAs (miRNAs) have emerged as important regulators of cardiovascular physiopathological processes9. miRNAs are non-coding single-stranded small RNA molecules with typically 18–25 nucleotides, acting as negative regulators of gene expression by inhibiting the translation of mRNA and/or promoting mRNA degradation by complementary base-pairing with 3-untranslated regions (3-UTRs). miRNAs are involved in regulating cardiac function, such as contraction, heart growth and morphogenesis. Interestingly, several miRNAs modulate the expression of hundreds of key proteins involved in I/R injury and myocardial infarct10. For example, miR-21 is upregulated by ischemic postconditioning in mouse hearts, where it played a protective role through PTEN/Akt signaling pathway11. miR-133a and miR133b are implicated in cardioprotection during ischemic postconditioning12, and remote ischemic preconditioning13. In addition, miR-125b protected heart from I/R injury by the prevention of apoptotic signaling14, meanwhile miR-499 mediates cardiac protection against I/R injury by targeting the called programmed cell death 4 (PDCD4) during ischemia postconditioning15. In the present study, we explored the involvement of miRNAs in the regulation of urocortin-induced heart protection. We observed that Ucn-1 differentially regulates the expression of miR-125a-3p, miR-324-3p and miR- 139-3p whose overexpression modulates several genes associated with a wide range of heart functions as cell stress, metabolism, cell survival and apoptosis. Results Urocortin-1 applied at the onset of reperfusion recovers hemodynamic performances in perfused rats hearts. The protective effect of Ucn-1 on heart contractility was examined ex vivo in Langendorffperfused rat hearts submitted to I/R. First, Table1 shows that, in control hearts, the addition of 10 nM Ucn-1 increased significantly heart contractility examined by the maximum derivative of left ventricular pressure (dP/ dt) and the left ventricular pressure (LVP), meanwhile it decreased the left ventricular end diastolic pressure (LVEDP) and the coronary vascular resistances (VR). Next, Fig.1A shows that hearts exposed to 40 minutes of ischemia and 60 minutes of reperfusion (I/R) showed a significant decrease in dP/dt, indicating prominent loss in heart contractility. However, the administration of 10 nM Ucn-1, 5 minutes before reperfusion and maintained during 30 minutes, recovered completely heart’s contractility. The analysis of others hemodynamic parameters indicates that Ucn-1 improved the LVEDP (Fig.1B), and the VR (Fig.1C), whose progressive rise observed in reperfusion was prevented significantly by Ucn-1. Interestingly, the effects of Ucn-1 on heart performances were even maintained 60 minutes after washing out the peptide. Altogether, these data confirm that Ucn-1 addition at the onset of reperfusion recovers heart contractility, increases the ventricular diastolic loading and prevents coronary artery vasoconstriction, preserving effectively hearts’ performances. Urocortin-1 regulates differentially the expression of cardiac miRNA in perfused hearts. Once the cardioprotective effect of Ucn-1 was confirmed, we sought to determine whether Ucn-1 (10 nM) might regulate post-transcriptional processes involved in cardioprotection during I/R. We performed a miRNA array analysis in hearts’ samples taken 1 hour after reperfusion from Langendorff-perfused hearts undergoing the same protocol as in Fig.1. Table2 shows that Ucn-1 promoted differential expression of several miRNAs. 29 of them were up and 9 were downregulated based on the threshold of fold change >1.3. The expression of 20 of these miRNAs was significantly different at p < 0.05 as shown in Fig.2A. To validate microarray results, we further examined the expression of miRNAs using qRT-PCR in samples from Langendorff-perfused hearts undergoing the same protocol as in Fig.1. We focused on 6 miRNAs, miR- 30c-2, miR-29a-3p, miR-125a-3p, miR-139-3p, miR-320, and miR-324-3p, which role in cardioprotection is still unknown. As shown in Fig.2B, Ucn-1 upregulated significantly the expression of miR-125a-3p and miR-324-3p, meanwhile it downregulated the expression of miR-139-3p. Nevertheless, changes in the levels of miR-29a-3p, miR-30c-2, and miR-320 were not significantly affected by Ucn-1. Urocortin-1 modulates the expression of miR-125a-3p, miR-324-3p, and miR-139-3p in adult cardiac myocytes. To assess the endogenous expression of miR-125a-3p, miR-324-3p, and miR-139-3p, we treated isolated cardiac myocytes with different concentrations of Ucn-1 (2, 10 and 50 nM). Figure3 shows that the expression of miR-125a-3p and miR-324 are increased significantly upon treatment with Ucn-1. In contrast, the level of miR-139-3p did not change significantly in cardiac myocytes incubated with any of the used Ucn-1 doses. Next, we examined whether Ucn-1 may regulate the miRNAs expression in adult cardiac myocytes subjected to I/R protocol. We found that, after I/R, the expression of miR-125a-3p (Fig.4A) and miR-324-3p (Fig.4B) decreased, whereas the expression of miR-139-3p increased (Fig.4C). Interestingly, cells treatment with Ucn-1 (10 nM) reverted the effect of I/R. Ucn-1 enhanced significantly the expression of both miR-125a-3p (Fig.4A) Hemodynamics Paramaeters Control Ucn-1 (10 nM) LVP (mmHg) 75.4 ± 5.6 130.9 ± 3.3 LVEDP (mmHg) 10.4 ± 0.1 −4.6 ± 2.9 +dP/dt (%) 100 195.9 ± 16.6 VR (%) 100 78.0 ± 6.5 Table 1. Summary data of changes in hemodynamics parameters produced by 10 nM Ucn-1 in Langendorffperfused hearts. Left ventricular pressure (LVP), left ventricular end diastolic pressure (LVEDP), maximum derivative of left ventricular pressure (+dP/dt), and vascular resistance (VR) were evaluated 20 minutes after Ucn-1 addition in 6 hearts. www.nature.com/scientificreports/ 3 SCIentIFIC RepoRts | 7: 8898 | DOI:10.1038/s41598-017-09198-x and miR-324-3p (Fig.4B), meanwhile Ucn-1 downregulated miR-139-3p (Fig.4C), confirming the data obtained in perfused hearts (Fig.2B). Furthermore, as shown in Fig.4A–C, these regulations of miRNAs by Ucn-1 were prevented in cardiac myocytes pretreated with astressin (Ast, 0.5 µM), the specific antagonist of CRF-R2 receptor. Therefore, Ucn-1 regulated significantly the expression of miRNAs through a mechanism involving CRF-R2. Signaling pathway involved in urocortin-1 regulation of miRNAs. Recently, we have shown that ERK1/2 and Epac2 are involved in various cardioprotective effects of Ucn-18. Therefore, we examined the implication of ERK1/2 and Epac2 signaling pathway in Ucn-1 regulation of miR-125a-3p, miR-324-3p and miR-139-3p. Figure4D shows that Ucn-1 (10 nM) addition to isolated cardiac myocytes under I/R enhanced the phosphorylation of both ERK p42 and p44 isoforms, which was potently reduced by PD 098059 (5 µM), specific inhibitor of ERK1/28. Interestingly, cells pretreatment with PD 098059 prevented Ucn-1 upregulation of miR-125a-3p (Fig.4A) and miR-324-3p (Fig.4B), but it did not affect miR-139-3p (Fig.4C) expression levels during cells reperfusion. We further investigated Ucn-1 effect on Epac. Fig.4E shows that Ucn-1 significantly increased the expression of Epac2 under I/R, which was prevented completely in cells pretreated with astressin. Furthermore, Epac2 specific inhibition with ESI-05 (10 μM)16, blocked the effect of Ucn-1 on miR-324-3p (Fig.4B) and miR-139-3p (Fig.4C) regulation, but not miR-125a-3p expression (Fig.4A). In addition, we found that the incubation of cardiac myocytes with 8CPT (10 μM), specific agonist of Epac1 and 216, mimicked mostly the effect of Ucn-1. The administration of 8CPT prevented I/R-induced changes on the expression of miR-324-3p and miR-139-3p; meanwhile it did not affect the change induced by I/R of miR-125a-3p expression. Figure 1. Urocortin-1 recovers the hemodynamic function of Langendorff perfused rat hearts submitted to I/R. (A) Graph shows summary data of the contractility expressed as +dP/dt (%) in hearts subjected to ischemia/reperfusion protocol (I/R, full circle, n = 5), and in hearts treated with 10 nM Ucn-1 (+Ucn-1, open square, n = 5). Ucn-1 was applied 5 minutes before ischemia ends and during the first 30 minutes in reperfusion as indicated by area in grey. (B) Shows summary data of the left ventricular end diastolic pressure (LVEDP, mmHg) in the same conditions as in “A”. (C) Bar graph shows the changes in the mean of vascular resistances (VR, mmHg*min/ml). Values were taken 60 minute after reperfusion and were normalized to controls’ values. Data are means ± S.E.M. “*” and “**” indicate significance at p < 0.05 and p < 0.01 of I/R vs +Ucn-1. www.nature.com/scientificreports/ 4 SCIentIFIC RepoRts | 7: 8898 | DOI:10.1038/s41598-017-09198-x Altogether, these data suggest that Ucn-1 applied on the onset of reperfusion regulates differentially miR- 125a-3p, miR-324-3p and miR-139-3p through ERK1/2 and/or Epac2 signaling pathways. Urocortin-2 infusion protects heart from I/R injuries and regulates the expression of miR- NAs. To give more insight into the regulation of miRNAs by urocortin, we investigated its effect in vivo in rat experimental model of I/R provoked by transient LCA ligation. As Ucn-1 binds both to CRF-R1 and CRF-R2 expressed in the brain, in the heart and the peripheral organs; therefore, instead of Ucn-1, we infused Ucn-2 i.v. in tails’ rat 5 minutes before reperfusion to avoid its possible effects on central nervous system4. First, we confirmed that Ucn-2 and Ucn-1 have similar beneficial effects on contractility expressed as dP/dt and LVEDP in Langendorff-perfused heart (Supplement Fig.1). Second, we evaluated the infusion of Ucn-2 at 50 µg/Kg, 150 µg/Kg and 300 µg/Kg on cardiac contractility in I/R rat model. Figure5A shows that temporary ligation of LCA reduced significantly the contractile capacity of heart, as observed by M-Mode echocardiography 24 hours after surgery. As shown in Fig.5B the ejection fraction (EF) in I/R group was significantly reduced (50.8% ± 3.2) as compared with Sham (70.1% ± 2.8); whereas, EF recovered significantly after the infusion of Ucn-2 at 150 µg/ Kg (59.4% ± 3.1; Fig.5A and B) and at 300 µg/Kg and (62.3% ± 1.2); but not when Ucn-2 was administrated at Gene ID Fold Change P value miR-139-3p −3.09 0.003 miR-10a-5p −2.28 0.110 miR-324-3p 1.96 0.016 miR-145 1.88 0.144 miR-125a-3p 1.75 0.050 miR-494 1.66 0.256 miR-30c-2 1.64 0.035 miR-322 1.62 0.013 miR-181b 1.58 0.036 miR-320 1.51 0.014 miR-221 1.47 0.085 miR-126 −1.47 0.224 miR-500 1.44 0.027 miR-140 1.42 0.146 miR-15b −1.41 0.092 miR-99b 1.40 0.127 miR-466b 1.40 0.167 miR-99a 1.39 0.042 miR-128 1.38 0.226 miR-200c 1.36 0.023 miR-872 1.36 0.138 miR-450a 1.36 0.092 miR-24-2 1.35 0.076 miR-30e 1.35 0.053 miR-196c 1.35 0.067 miR-152 -1.35 0.253 miR-7a 1.34 0.010 miR-29a-3p 1.34 0.010 miR-324-5p 1.33 0.097 miR-598-3p 1.33 0.079 miR-361 1.32 0.011 miR-214 −1.31 0.015 let-7d 1.31 0.338 miR-25 1.31 0.093 miR-199a-5p −1.30 0.378 miR-342-3p −1.30 0.097 miR-137 −1.30 0.034 miR-340-5p 1.30 0.030 Table 2. miRNAs identified to be dysregulated. List of expressed miRNAs defined as dysregulated in miRNA microarray analysis in the Ucn-1 group as compared with the I/R group. miRNAs are classified by higher rate of change in Ucn-1 treatment versus untreated I/R. Expressed miRNAs are filtered based on “Fold change >1.3 or <−1.3”. Bold letters is for miRNAs with significant changes at p < 0.05. I/R, ischemia/reperfusion; Ucn-1 (10 nM) was applied 5 minutes before reperfusion. www.nature.com/scientificreports/ 5 SCIentIFIC RepoRts | 7: 8898 | DOI:10.1038/s41598-017-09198-x 50 µg/Kg (51.8% ± 3.5). Similarly, as illustrated in Fig.5C the shortening fraction (SF) was reduced in I/R group (51.1% ± 8.1) comparing with Sham; whereas the infusion of Ucn-2 at 150 µg/Kg (68.1% ± 8.9) and at 300 µg/ Kg (68.1% ± 1.1) recovered significantly the SF; but not when it was administrated at 50 µg/Kg (49.7% ± 5.4). Therefore, Ucn-2 was used at 150 µg/Kg to examine the expression of miR-125a-3p, miR-324-3p, and miR-139-3p in hearts’ samples taken 24 hours after surgery. Figure5D and E shows that the expression of miR-125a-3p and miR-324-3p was downregulated in I/R group as compared to Sham. Ucn-2 infusion partially recovered the levels of miR-125a-3p (Fig.5D); meanwhile, it reverted significantly the expression of miR-324-3p (Fig.5E). In contrast, the expression of miR-139-3p was enhanced under I/R, but following the administration of Ucn-2 miR-139-3p was efficiently downregulated as illustrated in Fig.5F. Altogether, these data confirm in I/R animal model that infusion of Ucn-2 right before reperfusion regulates the expression of, at least, significantly miR-324-3p and miR-139-3p. The transfection of cardiac myocytes with lentivirus overexpressing miRNAs alters the expression of several genes. Using miRNA targets prediction, a group of candidate target genes were identified and selected for measurement of mRNA expression in neonatal rat ventricular cardiac myocytes (NRVMs) transfected with lentivirus overexpressing miRNAs. Table1 shows a list of selected genes known to regulate different cardiac pathophysiological processes related to cardioprotection, apoptosis and cell survival (e.g. BRCA1, IGF1, MTFR1, NRG1, STAT2, …); to adverse remodeling and fibrosis (e.g. NFAT5, CAMTA2, CREBBP, MAP3K12, COL5A2, DNAJB6, …); or to cell metabolism and stress (XBP1, MTFR1, FoxO1, TAZ, …), among other actions. First, we examined whether Ucn-1, under I/R, changes the expression of miRNAs in NRVMs in the same way as in adult cardiac myocytes. Figure2 confirms that the addition of Ucn-1 in reperfusion significantly upregulated the expression of miR-125-3p and miR-324-3p, meanwhile it downregulated miR-139-3p. Second, Fig.6A shows that the transfection of NRVMs with lentivirus over-expressing miR-125a-3p (LV-miR-125a-3p) significantly downregulated the expression of BRCA1, CPT2, MAP3K12, MTFR1, TAZ, and XBP1. Similarly, Fig.6B shows that cardiac myocyte transfection with LV-miR-139-3p significantly attenuated the expression of PDE4a and FoxO1. However, as shown in Fig.6C, cell transfection with LV-miR-324-3p unusually upregulated the expression of STAT2 and BIM genes, meanwhile it downregulated NFAT5 and CASQ1. Furthermore, we examined whether Ucn-1 regulates the expression of these genes in adult cardiac myocytes under I/R. Figure7 shows that the expression of BRCA1, STAT2, BIM, FoxO1 and MAP3K12 decreased under I/R, meanwhile cells treatment with Ucn-1 in reperfusion significantly reversed the expression of these genes. Moreover, Ucn-1 evoked changes in others genes regulated by miR-125a-3p, miR-324-3p and miR-139-3p were Figure 2. miRNAs detected by gene chip and qRT-PCR in perfused rat hearts. (A) List of up- and downregulated miRNAs detected by microarray in samples from Langendorff-perfused heart treated with Ucn-1 (10 nM) versus I/R (n = 4). (B) qRT-PCR conducted with samples from Langendorff-perfused heart treated with Ucn-1 vs I/R showing significant changes in the expression of miR-125a-3p, miR-139-3p and miR-324-3p but not of miR-30c-2, miR-29a-3p, or miR-320. Values are shown in logarithmic scale and are means ± S.E.M, (n = 6). “**” indicates significance at p < 0.01 as compared with I/R. www.nature.com/scientificreports/ 6 SCIentIFIC RepoRts | 7: 8898 | DOI:10.1038/s41598-017-09198-x not significantly different (data not shown). These data reveal that Ucn-1, through miR-125a-3p, miR-324-3p and miR-139-3p, might target multiple genes involved in different signaling pathway implicated in cardioprotection, which will induce synergetic beneficial effects. Figure 3. Urocortin-1 regulates miR-125a-3p, miR-324-3p and miR-139-3p expression in isolated cardiac myocytes. Bar graphs showing fold changes in the expression of miR-125a-3p (A), miR-324-3p (B) and miR- 139-3p (C) in isolated cardiac myocytes treated 10 minutes with increasing concentrations of Ucn-1 (2, 10 and 50 nM). Values are means ± S.E.M, (n = 4). “*” indicates significance at p < 0.05. www.nature.com/scientificreports/ 7 SCIentIFIC RepoRts | 7: 8898 | DOI:10.1038/s41598-017-09198-x Figure 4. Urocortin-1 regulates the expression of miRNAs in isolated cardiac myocytes through ERK1/2 and Epac2 activation. (A–C) Bar graphs show changes in the expression of miR-125a-3p (A), miR-324-3p (B) and miR-139-3p (C) in isolated cardiac myocytes treated with Ucn-1 (10 nM) in reperfusion. (D) Western blot and summary data showing ERK 1/2 phosphorylation in cells exposed to I/R (30 minutes/10 minutes). (E) Western blot and summary data showing Epac2 examined in cells exposed to I/R (30 minutes/18 hours each). “I/R” is for cells undergoing I/R protocols. “+Ast” is for cells subjected to I/R pre-treated 10 minutes with 0.5 µM astressin to inhibit CRF-R2 before the addition of Ucn-1. “+PD” is for cells pre-treated 10 minutes with 5 µM PD 098059 to inhibit ERK1/2, before the addition of Ucn-1. “+ESI-05” is for cells pretreated 10 minutes with ESI-05 (10 µM) to inhibit Epac2, before the addition of Ucn-1. “+8CPT” is for cells treated with 10 µM 8CPT at the onset of reperfusion. Values are shown in logarithmic scale and are means ± S.E.M, (n = 6–9 independent cells preparation). “*” and “**” indicate significance at p < 0.05 and p < 0.01. www.nature.com/scientificreports/ 8 SCIentIFIC RepoRts | 7: 8898 | DOI:10.1038/s41598-017-09198-x Figure 5. Infusion of urocortin-2 improves cardiac function in I/R and regulates the expression of miRNAs. (A) Cardiac function analyzed by echocardiography in M-mode 24 hours after surgery in three experimental models. Images are from “Sham” (n = 5); from “I/R” group corresponding to rats undergoing occlusion of the left coronary artery (LCA) for 40 minutes followed by reperfusion (n = 6); and for I/R rats infused with Ucn-2 (150 µg/Kg) 5 minutes before reperfusion (n = 5). (B,C) Bar graphs summarize ejection fraction (EF, %) and shortening fraction (SF, %) measured in rats infused with 50, 150 or 300 µg/Kg of Ucn-2. (D–F) Bar graphs showing the expression of miR-125a-3p, miR-324-3p and miR-139-3p, respectively in hearts from Sham, I/R animal model, and from I/R rats infused with Ucn-2 (150 µg/Kg; +Ucn-2). Values are shown in logarithmic scale. Data show means ± S.E.M. (n = 5–7). “*” and “**” indicate significance at p < 0.05 and p < 0.01, respectively. www.nature.com/scientificreports/ 9 SCIentIFIC RepoRts | 7: 8898 | DOI:10.1038/s41598-017-09198-x Figure 6. Quantification of mRNA expression by qRT-PCR of microRNAs targets genes. Bar graphs show rate changes of genes mRNA expression assessed by qRT-PCR in neonatal rat cardiomyocytes infected with lentivirus overexpressing miR-125a-3p (A), 139-3p-3p (B) and miR-324-3p (C). Values are shown in logarithmic scale compared to cells infected with the GFP vector. Values are means ± S.E.M (n = 8). “*” and “**” indicate significance at p < 0.05 and p < 0.01, respectively. Figure 7. Urocortin-1 regulates the expression of BRCA1, STAT2, BIM, FoxO1 and MAP3K12 in isolated cardiac myocytes under I/R. Bar graphs show rate changes of genes’ mRNA expression assessed by qRT-PCR in adult rat cardiac myocytes (control); in untreated cells undergoing I/R protocols myocytes (I/R); and in cells treated with Ucn-1 in I/R (10 nM, +Ucn1). Values are in logarithmic scale normalized to control and are means ± S.E.M (n = 8). “*” and “**” indicate significance at p < 0.05 and p < 0.01, respectively. “&” indicates significance between I/R and control at p < 0.05.