let-7e replacement yields potent anti-arrhythmic efficacy via targeting beta 1-adrenergic receptor in rat heart

Beta-adrenoceptor (β-AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let-7e in the up-regulation of β1-AR and arrhythmogenesis in acute myocardial infarction (AMI) in rats. β1-AR expression was significantly up-regulated and let-7a, c, d, e and i were markedly down-regulated in the infarcted heart after 6 and 24 hrs myocardial infarction. Forced expression of let-7e suppressed β1-AR expression at the protein level, without affecting β1-AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let-7e by let-7e antisense inhibitor (AMO-let-7e) enhanced β1-AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let-7e (len-pre-let-7e) significantly inhibited β1-AR expression in rats, whereas len-AMO-let-7e up-regulated β1-AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of β1-AR by endogenous let-7e. Len-negative control (len-NC) did not produce significant influence on β1-AR expression. Len-pre-let-7e also profoundly reduced the up-regulation of β1-AR induced by AMI and this effect was abolished by len-AMO-let-7e. Importantly, len-pre-let-7e application significantly reduced arrhythmia incidence after AMI in rats and its anti-arrhythmic effect was cancelled by len-AMO-let-7e. Notably, anti-arrhythmic efficacy of len-pre-let-7e was similar to propranolol, a non-selective β-AR blocker and metoprolol, a selective β1-AR blocker. Down-regulation of let-7e contributes to the adverse increase in β1-AR expression in AMI and let-7e supplement may be a new therapeutic approach for preventing adverse β1-AR up-regulation and treating AMI-induced arrhythmia.


Introduction
Acute myocardial infarction (AMI) or heart attack as a result of blockage of a coronary artery in the clinical setting is a common cause of mortality and morbidity worldwide. It occurs after a prolonged period of myocardial ischemia, which can result in cardiac electrophysiological disturbance, haemodynamic disorder, metabolic disorders, necrosis and apoptosis of cardiomyocytes [1,2], accompanied by abnormal alterations of gene expression such as aberrant regulation of a number of ion channels [3] and badrenoceptors (b-AR) [4,5], and reduction in connexin43 [6,7], which can often lead to arrhythmias and acute and chronic heart failure.
b-adrenoceptor plays a pivotal role in regulating cardiac function and heart rate (HR). And b-AR blocker is commonly used to treat patients with arrhythmia and heart failure [8,9]. To date, three subtypes of b-AR have been identified pharmacologically in the heart: b 1 -AR, b 2 -AR and b 3 -AR. b 1 -AR and b 2 -AR are the major subtypes that modulate cardiac contractility and HR by stimulating the G-protein/ adenylate cyclase/protein kinase A pathway [10,11]; however, b 3 -AR produces negative inotropic effects by coupling to inhibitory G proteins [12]. In physiological conditions, b 1 -AR and b 2 -AR subtypes are expressed at a ratio of 70:30 [13,14]. However, b-AR expression can be affected by many pathological processes of the heart such as AMI and chronic heart failure. For example, b-AR expression is increased in the acute phase of myocardial infarction [4,5]; in contrast, it is decreased in chronic heart failure [15].
Dynamic expression regulation of b-AR and functional desensitization are considered adaptive or protective mechanisms in the heart. Regulation of b-AR expression involves a variety of factors and pathways in cardiac cells. Ihl-Vahl et al. reported that subtype-selective increase in b 1 -AR induced by AMI is associated with a transcriptional up-regulation, and occupation or activation of b 1 -AR is not involved in this subtype-specific up-regulation of b 1 -AR [4]. Bengtsson et al. reported that a protein synthesis inhibitor cycloheximide regulates b 1 -AR gene expression by acting on repressive elements in the promoter region of the gene in brown adipocytes [16]. Calcium mobilization and protein kinase C activation participate in the down-regulation of b-AR in activated B cells by allogenic stimulation and/or soluble factors [17].
In this study, we, for the first time, displayed the involvement of let-7, a conserved and abundant miRNA in the heart [30], in the upregulation of b 1 -AR in AMI in rats, which provides new insight into the mechanisms for regulation of b 1 -AR expression and overexpression of miRNA let-7e potentially inhibited AMI-induced arrhythmia in rat. This indicates that targeting miRNA let-7e may be a promising therapeutic strategy for modulating b 1 -AR.

Animals
Healthy male Wistar rats (200 AE 20 g; Vitalriver, Beijing, China) used in this study were kept under standard animal room conditions (temperature 21 AE 1°C; humidity 55-60%) with food and water ad libitum for 1 week before experimental interventions. All experimental procedures were in accordance with, and approved by the Institutional Animal Care and Use Committee of the Harbin Medical University.

Rat model of AMI
Rats were anaesthetized with ketamine (60 mg/kg) and xylazine (6 mg/kg). Tracheal cannula was performed with a polyethylene tube and ventilated with the TOPO small animal Ventilato (Kent, OH, USA), and then the chest was opened through the fourth intercostal space and propped ribs by a rib spreader. The pericardium was opened carefully to expose the heart. The left anterior descending coronary artery (LAD) was ligated using a 5/0 silk thread to create infarction of the LV free wall. Cardiac infarction was confirmed by apparent S-T segment elevation in ECG and cyanosis of the myocardium. The sham procedure consisted of a superficial suture in the epicardium of the LV.

MiRNA microarray and data analysis
We performed miRNA expression profiling with the heart samples from with or without AMI (6 hrs) rats. RNA samples 5 lg were labelled with the Exiqon miRCURY Hy3/Hy5 power labelling kit and hybridized on the miRCURY LNA Array (version 11.0) station. Scanning was performed with the Axon GenePix 4000B microarray scanner. GenePix pro version 6.0 was used to read image and analyse raw intensity. The threshold value for significance used to define up-regulation or down-regulation of miRNAs was a fold change >1.5 or <0.5.

Western blot
Total protein was extracted with RIPA Lysis Buffer (Beyotime, Shanghai, China) mixed with 1% proteinase inhibitors, and degenerated by admixing with 59 loading buffer (Beyotime) at 100°C for 5 min. Extracted protein samples (120 lg from NRVCs and 60 lg from tissues) were separated in 10% SDS-PAGE and blotted to nitrocellulose membrane. The blots were blocked with 5% non-fat milk overnight at 4°C, probed with a primary antibody to b 1 -AR (1:20 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), or to b 2 -AR (1:1000 dilution; Abcam, Cambridge, MA, UK), or to GAPDH (1:1000 dilution; Jinshan, Shanghai, China), or to b-actin (1:500 dilution; Jinshan) in 5% non-fat milk, and incubated at 4°C overnight. The membranes were washed with PBS-T and PBS, and then incubated with secondary antibody (LI-COR Bioscience, Lincoln, NE, USA) for 1 hr at room temperature. Finally, western blot bands were collected by Imaging System (LI-COR Biosciences) and quantified with odyssey v1.2 software by measuring intensity in each group with GAPDH as an internal control, but for the ischaemia tissues using b-actin as an internal control [31]. The results were expressed as fold changes by normalizing the data to the values from the control group.

Quantitative reverse transcription-PCR (qRT-PCR)
After experimental treatment, total RNA samples were isolated from cultured NRVCs and cardiac tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocol. RNA (0.5 lg) was then reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) to obtain first-strand cDNA. Levels of let-7a/c/d/e/i, miR-1 and b 1 -AR mRNA were determined using SYBR Green I incorporation methods on ABI 7500 fast Real Time PCR system (Applied Biosystems), with U6 as an internal control of miRNA or GAPDH as an internal control of b 1 -AR mRNA. The sequences of primers used in the qRT-PCR experiments (Invitrogen, Shanghai, China) were listed in Table S1.  Construction of plasmid carrying the 3 0 UTR of b 1 -adrenergic receptor (ADRB1) and luciferase assay Targetscan predicts the presence of a putative binding site for let-7 in the 3 0 UTR of ADRB1 mRNA, the gene encoding b 1 -AR, which is highly conserved among mammals. A segment containing the let-7 miRNA binding sites flanked by the Hand lll and Sac I restriction sites and a scramble sequence as a negative control (NC) were synthesized by Invitrogen. The sequences were inserted separately into the pMD18T-simple vector (Invitrogen), and then transferred into the pMIR-REPORT TM Luciferase miRNA Expression Reporter Vector (Ambion, Austin, TX, USA). pRL Renilla Luciferase Reporter vector (pRL-TK, Promega, Madison, WI, USA) was used as an internal control. The plasmids and miRNAs were transfected grouply with X-tremeGENE siRNA transfection reagent (Roche, Mannheim, Germany) into HEK293 cells. The dual luciferase reporter assay kit (Promega) and the GlouMax biological fluorescent tester were used to detect the activity of the luciferase 36 hrs after transfection. The results were expressed as fold changes by normalizing the scaled data. The sequence of human and rat ADRB1 3 0 UTR inserted into the vectors were as follows: The human

Cardiomyocytes isolation and culture from neonatal rats
The neonatal Wistar rats of 1-3 days old were disinfected by 75% ethanol. The chest was opened to expose the heart. The heart was isolated and sheared into 1-to 3-mm pieces. The cardiac tissues were digested into single cardiomyocytes by 0.25% trypsin. Dispersed cells were placed in high glucose DMEM containing 10% fetal bovine serum (FBS) and 1% benzylpenicillin-streptomycin to terminate the digestion procedure. Pooled cell suspensions were centrifuged and resuspended in DMEM supplemented with 10% fetal bovine serum, and 100 U/ml penicillin and 100 lg/ml streptomycin. The resuspension was transferred onto a 100-mm culture dish for 30 min., then changed to a new dish, and repeated for three times. This procedure allowed for preferential attachment of fibroblasts to the bottom of the culture dish and left nonadherent cardiomyocytes in cell suspension. The cells were plated into a 6-well plate at a density of 1 9 10 6 per well, and incubated at 37°C in a humidified atmosphere of 5% CO 2 and 95% air.

Construction of HIV-1-based lentivirus carrying pre-let-7e
Lentivirus vectors expressing mature let-7e, anti-miRNA-oligo of let-7e (AMO-let-7e) or NC sequence were constructed by Invitrogen (Invitrogen). Briefly, the precursor sequence of let-7e and its antisense fragment were synthesized by Invitrogen. The synthesized fragments were annealed and inserted into the pcDNA TM 6.2-GW/EmGFP-miR vector (Invitrogen). Then, lentivirus plasmid was constructed by BP and LR recombination into pLenti6.3/TO/V5-DEST vector (Invitrogen) through the Gateway recombination technology. The constructed pLenti6.3/TO/ V5-DEST plasmid and an optimized mix of the three packaging plasmid (pLP1, pLP2 and pLP/VSVG; Invitrogen, China) were cotransfected into 293FT procedure cell line by liposome reagent to package lentivirus. The virus liquid was collected and concentrated 48 hrs after cotransfection, and the titre of the lentivirus liquid was determined.

In vivo lentivirus infection and beta-blocker administration
Thoracotomy was performed in the fourth left intercostal space to expose the heart of rats. Virus-containing solution (20 ll, 1 9 10 8 TU) including len-NC, precursor let-7e (len-pre-let-7e), AMO-let-7e (len-AMO-let-7e) or len-pre-let-7e and len-AMO-let-7e was injected using an insulin syringe into LV wall of rat heart. Rats in the sham and MI groups underwent the same procedures but received the same volume of saline (20 ll) with the constructs. Rats in b-blocker groups received non-specific b-AR blocker propranolol (10 mg/kg/day, Sigma-Aldrich, St. Louis, MO, USA) and specific b 1 -AR blocker metoprolol (80 mg/kg/ day; Sigma-Aldrich) by intragastric administration for 7 days. Ligation of LAD was performed at day 7 after injection and administration. All surgery equipments were sterilized to ensure the minimum level of infection. Rats were given penicillin (1 9 10 5 Units/day, im) for 7 days. After surgery, the rats were given food and water ad libitum.

Ultrasound imaging and haemodynamics parameter measurements
Rats were anaesthetized and pre-thoracic fur was removed by a Nair TM depila-tory cream (Church & Dwight Co., Inc., Princeton, NJ, USA). Haemodynamic measurements were performed by the Vevo â 2100 High-Resolution Imaging system (Visual Sonics, Toronto, ON, Canada) in rats 6 hrs after ligation or 7 days after treatments with len-pre-let-7e or len-AMO-let-7e. Rats were positioned on a RatPad (part of the VisualSonics Vevo Integrated Rail System II) equipped with integrated heater. Body temperature was maintained at 37°C. Pre-warmed Aquasonic Clear â Ultrasound Gel (Parker Laboratories, Inc., Fairfield, NJ, USA) was used as a coupling agent between the ultrasound scan-head and the skin. Two-dimensional targeted M-mode traces were obtained with the transducer (MicroScan MS 250-0206) held immobilized. LV end-diastolic and end-systolic wall thickness and LV internal diameter were measured from at least three consecutive cardiac cycles. Ejection fraction (EF) and fractional shortening (FS) were calculated based on the Vevo â 2100 High-Resolution Imaging system (Visual Somics).

Electrocardiogram (ECG) monitoring and arrhythmia scoring
As described above, rats were infected with len-NC, precursor let-7e (len-pre-let-7e), AMO-let-7e (len-AMO-let-7e) or len-pre-let-7e + len-AMOlet-7e, or administrated with either b-AR blocker propranolol or metoprolol. Ligation of LAD was performed on day 7 after infection and administration. Then, standard lead II digital ECG tracings were recorded for evaluation of arrhythmias using the BL-420F bio-function experiments system (Taimeng, Chengdu, China). Arrhythmia score was evaluated from an ECG recorded during 30 min. period after the LAD ligation using Score F as described by Curtis and Walker [32,33], based on the analysis of premature ventricular contractions (PVCs), ventricular tachycardia (VT), ventricular fibrillation (VF), ventricular fibrillation terminated spontaneously (SVF), and VF termination was not spontaneous (NVF). PVCs were identified by the presence of a premature QRS complex; VT was classified as three or more consecutive PVCs. The arrhythmia scoring system F is as follows: 0 = <50 PVCs, 1 = ≥50 PVCs, 2 = 1-5 episodes of VT, 3 = ≥6 episodes of VT, 4 = 1 SVF or 1 episode of NVF or both, 5 = 2-5 episode of NVF, 6 = ≥5 episode of NVF.

Data analysis
All data were statistically analysed by using one-way ANOVA followed by Turkey's multiple comparison test. Data of arrhythmia score were statis-tically analysed by using Wilcoxon signed-rank test. Differences were considered as statistically significant when P < 0.05. Data are presented as mean AE SD or mean AE SEM.

Results
Up-regulation of b 1 -AR and down-regulation of let-7 in infarcted hearts We first compared the expression levels of b 1 -AR between the infarcted and non-infarcted LV tissues in a rat model of AMI. b 1 -AR expression was up-regulated by 3.0 AE 0.8-fold (P < 0.01) at the protein level 6 hrs after AMI (Fig. 1A), compared to non-ischaemic area. Similar results were observed in the infarcted tissue 24 hrs after AMI (Fig. 1A). In contrast to the up-regulation of b 1 -AR, b 2 -AR expression was markedly down-regulated at the protein level 6 hrs (P < 0.01) and 24 hrs (P < 0.01) after AMI (Fig. 1B), compared to non-ischemic area. These data are consistent with previous reports on selective upregulation of b 1 -AR in acute ischemic heart [2,3].

Regulation of b 1 -AR by let-7 in cardiomyocytes in vitro
The reciprocal alterations of b 1 -AR and let-7 in terms of their expression in AMI suggest a targeting relationship between them. To exploit this notion, we performed miRNA gene target prediction using Target-Scan 6.0 database, and we indeed identified a binding site in the 3 0 UTR of b 1 -AR mRNA for all members of the let-7 family, which is highly conserved among human, rat and mouse ( Fig. 2A). We then experimentally verified the regulation of human and rat b 1 -AR by let-7c, d and e with luciferase activity assay in HEK-293 cells ( Fig. 2B and C). Results showed that let-7c, d, and e significantly inhibited luciferase activity elicited by the pMIR-REPORT TM luciferase vector containing their target sequence, but cotransfection with let-7c, d, e and mutant Luc-ADRB1 3 0 UTR were unable to inhibit the luciferase activity and mutant let-7c, d or e alone did not influence the luciferase activity. All these data indicate the specificity of let-7 action on ADRB1 3 0 UTR ( Fig. 2B and C).
Efficient transfection of let-7e was verified by 6.9 AE 0.9-fold elevation of this miRNA in NRVCs. On the other hand, AMO-let-7e, a specific inhibitor for let-7e, reduced the let-7e level to below the baseline control level, indicating a knockdown of both exogenous and endogenous let-7e. The NC had no effect on let-7e expression (Fig. 3A).
Overexpression of let-7e significantly inhibited b 1 -AR expression. AMO-let-7e resulted in a higher b 1 -AR protein level than the control group, indicating a relief of tonic inhibition of b 1 -AR by endogenous let-7e in NRVCs. Negative control of let-7e did not show any effect on b 1 -AR expression (Fig. 3B). The expression of b 1 -AR at mRNA level was unaffected by let-7e and AMO-let-7e (Fig. 3C).

Regulation of b 1 -AR by let-7e in vivo
let-7e was increased by 1.5 AE 0.3-fold in the rat hearts administered with len-pre-let-7e compared with the control animals. And as expected, it was decreased in the len-AMO-let-7e group. Len-NC had no effect on let-7e expression (Fig. 4A). We also measured miR-1 expression in the tissue with the same treatments and found no difference in miR-1 expression among the groups, indicating that the observed changes of let-7e expression were specifically elicited by the lentivirus vector carrying the pre-let-7e or AMO-let-7e (Fig. 4A). Furthermore, we evaluated the effects of let-7e on b 1 -AR expression in the heart. As shown in Figure 4B, b 1 -AR expression was significantly down-regulated in the injected area of the heart in the len-pre-let-7e group, compared with control group. While the len-AMO-let-7e application caused conspicuous increase in b 1 -AR beyond the control level, presumably as a result of inhibition of the receptor by endogenous let-7e. Len-NC did not exert any effects on b 1 -AR expression and len-pre-let-7e had no effect on b 1 -AR mRNA level (Fig. 4C). These results established let-7e as a regulator of b 1 -AR expression in rat heart.

let-7e inhibits up-regulation of b 1 -AR in ischaemic heart
To investigate the regulation of b 1 -AR by let-7e in the infarcted rats, lentivirus vectors containing pre-let-7e, AMO-let-7e or a scrambled sequence were injected into the LV wall at five points within the expected infarct area 7 days prior to AMI. Of note, len-let-7e profoundly mitigated AMI (6 hrs)-induced b 1 -AR overexpression, and which was abolished by co-treated with len-AMO-let-7e (Fig. 5). Len-AMO-let-7e alone did not significantly affect the ischemic b 1 -AR overexpression. b 1 -AR expression was not changed in the len-NC group (Fig. 5). Indicating that miRNA let-7e decreased the up-regulation of b 1 -AR in AMI rats.

Anti-arrhythmic effects of let-7e in AMI rats
Acute myocardial infarction was characterized by induction of cardiac arrhythmias. Electrocardiography recording was performed after ligation of left coronary artery of rat subjected to the pre-treatments with len-pre-let-7e, len-AMO-let-7e or b 1 -AR blocker 7 days ago. Arrhythmia score was calculated from 30 min. period of ECG recording after LAD ligation based on the occurrence of PVCs and episodes of VT and NVF. Notably, len-pre-let-7e infection showed a significant reduction in arrhythmia score from 3.3 AE 0.4 for AMI to 1.7 AE 0.4 (P < 0.05). By comparison, rats with treatment of propranolol (10 mg/kg/day), a non-selective b-AR blocker had an arrhythmia score of 2.1 AE 0.3 (P < 0.05), and those treated with metoprolol (80 mg/kg/day), a selective b 1 -AR blocker had a score of 2.4 AE 0.2 (P < 0.05). All three groups showed significant anti-arrhythmic effects in AMI rats. In contrast, len-AMO-let-7e increased arrhythmia score from 3.3 AE 0.4 to 5.8 AE 0.5 (P < 0.05). Len-NC had no effect on the score (Fig. 6B). These data indicate that let-7e has high antiarrhythmic efficacy, which is similar to classic b-blockers. Consistent with arrhythmia score results, len-pre-let-7e significantly reduced

Effects of let-7e on cardiac function and heat rate
Echocardiogram was performed on rats 7 days after treatments with len-pre-let-7e, len-AMO-let-7e or len-NC. Results showed that there were no significant changes in the cardiac function and HR in normal rats among groups (Table 1). These data indicate that local application of let-7e appears not sufficient to make significant effects on the cardiac function and HR.
To valuate cardiac function in AMI rats with different treatments, echocardiogram was performed 6 hrs after LAD ligation. Results showed that len-pre-let-7e, metoprolol and propranolol significantly caused deterioration of cardiac function, reflected by decreased EF and FS in AMI rats (Table 2). Len-AMO-let-7e enhanced EF from 67.9 AE 1.4 to 81.4 AE 2.3 (P < 0.05) and FS from 46.8 AE 5.5 to 56.0 AE 5.2 (P < 0.05). Both metoprolol and propranolol significantly lowered HR from 355 AE 16 to 280 AE 24 (P < 0.05) and from 355 AE 16 to 264 AE 27 (P < 0.05) in AMI rats respectively. However, len-pre-let-7e had no significant effect on HR of AMI rats (Table 2).

Discussion
The aim of this study was to investigate the miRNA mechanisms for the abnormal up-regulation of b 1 -AR and its effect on arrhythmogenesis in the setting of AMI. There are several new findings in this study. First, our data revealed that let-7 was significantly down-regulated, along with selectively increase in b 1 -AR expression, in the infarcted area of LV tissue. Second, we experimentally established b 1 -AR as a target gene for the members of the let-7 family. Third, we demonstrated that let-7e replacement could ameliorate the abnormal up-regulation of b 1 -AR expression in AMI. And finally, let-7e application markedly inhibited arrhythmia incidence in AMI rats. Taken together,  it is plausible that deregulation or, specifically, down-regulation of let-7e contributes to the adverse increase in b 1 -AR expression and let-7e supplement shows a potential anti-arrhythmic effect in ischemic heart, which may be a new therapeutic approach for preventing adverse b 1 -AR up-regulation and treating ischemic arrhythmia. b 1 -adrenoceptor, a predominant subtype of b-AR, exerts a positive inotropic and chronotropic effects in the heart. In certain pathophysiological conditions, b-AR expression is subtype-selective in the heart, such as that b 2 -AR is up-regulated in the transplanted human heart [34], b 1 -AR up-regulated in AMI [4,5] and b 1 -AR down-regulated in chronic heart failure [15]. Acute myocardial infarction is a type of acute coronary syndrome and can lead to decreased cardiac output as a result of impaired cardiac pump function. To compensate for the decreased cardiac output, one of reactions in the infarcted heart is up-regulation of b 1 -AR expression [4,5], but not other type of b-AR. In this study, term b 1 -AR up-regulation refers to increase in the total number of the receptor not including receptor internalization or externalization. Several factors or pathways have been reported to be involved in the regulation of b 1 -AR expression in the heart. IhI-VahI et al. reported that AMI induces increase in b 1 -AR subtype as a result of a transcriptional regulation [4], possibly by cAMP pathway [35,36]. Above studies show that the increased b 1 -AR by cardiac ischemia is regulated by the transcription of ADRB1 gene (encoding b 1 -AR) possibly via cAMP/PKA/CREB pathway [4,36]. However, other mechanisms for regulation of b 1 -AR expression cannot be ruled out, such as post-transcription regulation of the receptor.
A variety of studies have demonstrated that let-7, an abundant and conserved miRNA, participates in various pathophysiological processes, such as cancer growth and formation [37] and axon regeneration [38]. In silico prediction with Targetscan and miRanda showed that ADRB1 is a target of let-7; the seed sequence is conserved among species, such as rat, mouse and human beings. We also demonstrated that b 1 -AR is a target gene for let-7 revealed by luciferase reporter assay and Western blot analysis. We further verified that let-7a, c, d, e, and i were down-regulated in the acute ischemic tissue and forced overexpression of let-7e inhibited b 1 -AR expression and knockdown of this miRNA by AMO-let-7e increased b 1 -AR expression in neonatal rat cardiomyocytes. However, let-7e did not inhibit the mRNA level of ADRB1 gene, indicating that let-7e regulates b 1 -AR expression by disrupting mRNA translation, not by degrading the mRNA. Importantly in vivo study, we demonstrated that b 1 -AR expression was regulated by let-7e via locally applying this miRNA to rat heart. Notably, len-AMOlet-7e caused an overshoot of b 1 -AR expression relative to the baseline control level, strongly suggesting that this cardiac-enriched miRNA exerts important tonic inhibition of b 1 -AR in the heart. let-7 family shares the same seed sequence (5 0 GAGGUAG 3 0 ) and are highly conserved across species in both their sequences and functions [39]. Study also demonstrated that the let-7 family shares the same downstream targets in human embryonic stem cells [40]. In this study, we freely chose let-7c, d and e from five decreased members of let-7 family for luciferase activity assay. As expected, all three miRNAs significantly inhibited luciferase activity, indicating that ADRB1 3 0 UTR is direct target of let-7 family. The results from let-7e are most likely applicable to other members of the let-7 family based on their same mechanism of action conferred by their same seed site. The most important finding is that len-pre-let-7e significantly lowered the incidence of arrhythmia induced by AMI in rats; as expected, len-AMO-let-7e increased the incidence of AMI-induced arrhythmia.
Our results also showed that efficiency of its antiarrhythmic effects was very similar with that of classic b-AR blocker propranolol, a nonselective b-blocker, and metoprolol, a selective b 1 -AR blocker, and that are commonly used to treat a variety of cardiac arrhythmia, including ischemic arrhythmia. Cardiac function and HR were not influenced by either len-pre-let-7e or len-AMO-let-7e in normal rats. However, in AMI rats, len-pre-let-7e reduced cardiac function but not affected HR. However, both propranolol and metoprolol inhibited cardiac function and slowed HR. These may suggest that local intramuscular injection of let-7e to LV wall is unable to affect b 1 -AR level in the sinoatrial node that controls HR. The present data indicate that like b-AR blocking agent, let-7e has pronounced antiarrhythmic effect in the setting of AMI.
Previous study demonstrated that only b 1 -AR expression is upregulated in AMI. However, the mechanisms involved in subtypeselective regulation of b 1 -AR are not fully understood. Several studies showed that adenylyl cyclase and cAMP participate in up-regulation of b 1 -AR in AMI by enhancing transcription of the receptors [35,36,41,42]. Recently, Wang et al. [43] reported that let-7f regulates the expression of b 2 -AR, a predominant subtype [44], in lung epithelial H292 cells. On the basis of these data, one would expect that downregulation of let-7 should also influence expression of b 2 -AR in addition to up-regulation of b 1 -AR. However, it is known that AMI only causes b 1 -AR up-regulation, without altering b 2 -AR. Clearly, mechanisms other than let-7 may also participate in the regulation of b 2 -AR expression in the heart. This study does not provide explanation of this issue. More importantly, our study also showed that let-7e presented a potential antiarrhythmic efficacy in AMI rats as b-AR blocker did. let-7e exerted its beneficial effect mainly through inhibiting the up-regulated b 1 -AR induced by ischemia. However, we definitely could not rule out other molecular targets of let-7, which are involved in its antiarrhythmic effect in the rats with AMI. Our study reveals a new mechanism of b 1 -AR regulated by let-7e and find that let-7e exerts a potential antiarrhythmic effect by targeting b 1 -AR in AMI rats. let-7e might be a promising target for intervention of b 1 -AR in the pathological condition, and let-7e supplement may be a new therapeutic approach for preventing and treating ischaemia-induced arrhythmia.