MiRNAs regulate the cardiomyocyte (CM) cell cycle at the post-transcriptional level, affect cell proliferation, and intervene in harmed CM repair post-injury. The present study was undertaken to characterize the role of let-7i-5p in the processes of CM cell cycle and proliferation and to reveal the mechanisms thereof. In the present study, we used real-time qPCR (RT-qPCR) to determine the up-regulated let-7i-5p in CMs during the postnatal switch from proliferation to terminal differentiation and further validated the role of let-7i-5p by loss- and gain-of-function of let-7i-5p in CMs in vitro and in vivo. We found that the overexpression of let-7i-5p inhibited CM proliferation, whereas the suppression of let-7i-5p significantly facilitated CM proliferation. E2F2 and CCND2 were identified as the targets of let-7i-5p, mediating its effect in regulating the cell cycle of CMs. Supperession of let-7i-5p promoted the recovery of heart function post-myocardial infarction by enhancing E2F2 and CCND2. Collectively, our results revealed that let-7i-5p is involved in the regulation of the CM cell cycle and further impacts proliferation, which may offer a new potential therapeutic strategy for cardiac repair after ischemic injury.
The loss of cardiomyocytes (CMs) and their insufficient replacement is a major contributor to the pathogenesis of many cardiovascular diseases, such as myocardial infarction (MI), cardiovascular fibrosis, and heart failure . Thus, CM proliferation and regeneration are keys to repair the myocardium following injury. However, adult CMs are viewed as terminally differentiated cells and have very limited potential for self-renewal and cell cycle re-entry in mammals . Promoting the re-entry of CMs into the cell cycle is a potential path to functional recovery from injury . Recently, research on the cell cycle has increasingly focussed on genes’ networks, especially on non-coding RNAs and their signature effects in controlling gene expression at the epigenetic, transcriptional, and post-transcriptional levels .
MiRNAs are abundant in higher eukaryotes  and a vital class of small (19–28 nts) endogenous nonprotein-coding RNAs that negatively regulate target mRNAs at the post-transcriptional level. Evidence that miRNAs are implicated in a wide range of cell cycle molecular networks has largely come from their effect on the synthesis of cyclins, cyclin-dependent protein kinases, cyclin-dependent kinase inhibitors, and transcription factors [6–8]. The let-7 family of miRNAs, which is highly conserved and the largest of all miRNA family, are ubiquitously expressed in most somatic cells and controls multiple targets to repress cell cycle regulators and block cell cycle progression required for proper development and tumor suppression . In CMs, the let-7 family reduces EdU incorporation (a uridine analog that marks newly synthesized DNA) and ki-67 positivity (a marker of proliferation antigen) in vitro, and let-7i-5p was proposed to be a strong suppressor of CM proliferation in vitro . Let-7i-5p functions to maintain cancer stem cell growth and aggravate carcinogenesis, and it is significantly down-regulated in the plasma of young ST-segment-elevation MI (STEMI) sufferers , indicating a high chance of participation of let-7i-5p during CMs dysfunction. However, it still remains largely unknown whether let-7i-5p operates CM proliferation in vivo.
Integrated bioinformatics analyses have revealed that both E2F2 and CCND2 are potential targets of let-7i-5p. CCND2, a kind of D-type cyclin, interacts with cyclin-dependent kinase 4 or 6 to affect G1 progression of cell cycle. E2F2, more highly expressed in proliferating cells, is described as an opposing molecule that controls the G1-to-S-phase transition. Aberrant expression of E2F2 and CCND2 can lead to abnormal cellular proliferation . E2F2 is a downstream target of let-7b in regulating glioma cell proliferation . E2F2 and CCND2 interact with let-7a to affect the development of prostate cancer by regulating the cell cycle of prostate cells . Given that the let-7 family members exert similar functions because they share a common seed region , let-7i-5p appears to function by combining with CCND2 and E2F2.
Therefore, we propose the hypothesis that let-7i-5p could regulate the cell cycle and promote CM proliferation via E2F2 and CCND2. To test our hypothesis, the loss- and gain-of-function effects of let-7i-5p were assessed in CMs in vitro and in vivo and in a MI model to further explore the underlying mechanism. The specific characteristics of let-7i-5p might be well used to potently regulate CM proliferation in the treatment of ischemia injury.
All protocols were approved by the Institutional Animal Care and Use Committee of the University of Southern Medical University. The present study conformed to the rules of the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH 8th edition, 2011).
Ventricular CM isolation and culture
Ventricular tissue isolation
Mouse ventricular CMs were isolated by enzymatic disassociation of 1-day-old (P1), 7-day-old (P7), and 10-day-old (P10) C57BL/6J mouse hearts using the following methods. C57BL/6J mice were purchased from the Laboratory Animal Center of Southern Medical University (Guangzhou, Guangdong, China). Neonatal mice were killed via 2% isoflurane inhalation and cervical dislocation. Then, the chest was opened along the sternum to allow access to the chest cavity and the heart. Left ventricles from neonatal mice were harvested from perfused and digested hearts and dissected into small pieces to dissociate them in transfer buffer (digested in 0.25% trypsin (Gibco, CA, U.S.A.) at 4°C overnight).
Enzymatic tissue digestion
A second digestion of the tissue was performed in collagenase type II (Gibco, CA, U.S.A.) with BSA (Sigma, Darmstadt, Germany) at 37°C for 15 min, two times under constant stirring. The supernatant was collected in FBS (Gibco, CA, U.S.A.) after each step. To separate the cells, the supernatant was centrifuged, and the pellet was resuspended in Dulbecco’s modified Eagle’s medium/nutrient F-12 Ham (DMEM/F12) 1:1 medium (HyClone, Utah, U.S.A.) supplemented with 10% FBS, 100 U/ml of penicillin, and 100 mg/ml of streptomycin (Sigma, Darmstadt, Germany). The collected cells were seeded on to 100-mm plastic dishes for 3 h at 37°C with 5% CO2 in a humidified atmosphere. The supernatant, composed mostly of CMs, was then collected and pelleted.
Ventricular CMs culture
Injection of adenovirus or adeno-associated virus 9 vectors in neonatal or adult mice
Based on the time to reach the peak of expression and the time course of expression of different types of virus, we used adenovirus (AdV) to deliver let-7i-5p to neonatal mice and adeno-associated virus 9 (AAV9) to adult mice [17–20]. The AdV and AAV9-containing green fluorescent protein (GFP) vectors overexpressing or depleting let-7i-5p was synthesized by Vigene (Shandong, China). The neonatal P1 C57BL/6J mice were anesthetized by cooling on an ice bed for 5 min. After the fourth intercostal was dissected, mice were intramyocardially injected with Ad-negative control (Ad-NC) or Ad-let-7i-5p, at a dose of 2 × 1010 viral genome particles per animal, using an insulin syringe with an incorporated a 30-gauge needle (BD, NJ, U.S.A.). The virus was injected into the neonatal heart at three sites randomly, and the total volume injected into each heart was 20 µl. Then, mice were placed under a heat lamp and warmed for several minutes until recovery [19,20]. The hearts of the injected mice were collected 8 days after AdV injection. Bright field and fluorescence images of the tissues (heart, kidney, spleen, liver, and lung) were captured to examine GFP fluorescence with a Bruker In-Vivo FX Pro system (Bruker, MA, U.S.A.). Adult mice were intramyocardially injected with AAV9 vectors (AAV9-NC or AAV9-anti-let-7i-5p) at a concentration of 1 × 1011 viral genome particles per animal. GFP fluorescence was used to determine the distribution of AdV-mediated let-7i-5p expression in the myocardium. To monitor AAV9 vectors transfer to the CMs over time in vivo, mice were injected with AAV9 vectors as described above, sedated using isoflurane, and imaged on a Bruker In-Vivo FX Pro systems as previously described .
Plasmids, miRNA mimic and inhibitor transfection
Plasmids overexpressing E2F2 or CCND2 and depleting E2F2 or CCND2 were synthesized by Vigene (Shandong, China). Let-7i-5p mimics and inhibitors were synthesized by Ribobio (Guangzhou, China). Isolated P1 mouse CMs were seeded at 70% confluence, and after a 48-h culture, 5 μl Lipofectamine 2000 (Invitrogen, CA, U.S.A.) and 50 nM plasmids or mimics or inhibitors were added to Opti-MEM (Gibco BRL, Paisley, U.K.). The mixture was added to the cells after incubation at room temperature for 20 min. This medium was replaced after a 6-h incubation at 37°C with the same volume of DMEM/F12 medium. RNA or protein was isolated or immunofluorescence analysis of the cells was conducted on cells after 48 h.
RNA isolation and real-time qPCR
Real-time qPCR (RT-qPCR) experiments were performed according to the MIQE guileline of RT-qPCR . Total RNA was isolated from dissected ventricular heart tissue samples or isolated CMs using the E.Z.N.A. Total RNA Kit II (Norcross, GA, U.S.A.) according to the manufacturer’s instructions. The RNA concentration and quality were measured using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher, U.S.A.) which measured the absorbance at 260 and 280 nm. Samples with an A260:A280 ratio ≥ 2.0 were selected for further analysis. For the quantitation of mRNA expression, the PrimeScript™ RT Master Mix (TaKaRa, Dalian, China) was used according to the manufacturer’s instructions. Briefly, 1 μg total RNA, 2 μl of 5× gDNA Eraser Buffer, 1 μl gDNA Eraser, and RNase-free dH2O, were combined in a total reaction volume of 10 μl and incubated at 42°C for 2 min to eliminate the genomic DNA. A total of 10 μl of the reverse thanscription reaction mixture (consisting of 4 μl 5× PrimeScript Buffer 2, 1 μl PrimeScript RT Enzyme Mix 1, 1 μl RT Primer Mix, and 4 μl RNase-free dH2O) was then added, and mixture was incubated at 37°C for 15 min, followed by 85°C for 5 s to generate the cDNA. RT-qPCR was performed with the SYBR Premix ExTaq™ Kit (TaKaRa, Dalian, China) on Light cycler 480 (Roche, Basel, Switzerland). Briefly, the 20 μl reaction mixtures were incubated at 95°C for 30 s for the initial denaturation, followed by 40 cycles at 95°C for 5 s and 60°C for 34 s. To normalize gene expression, β-actin was used as a reference gene and the 2−ΔΔCt method was used [22,23]. For the quantitation of miRNA expression, the miRNA First Strand cDNA Synthesis (Sangon, Shanghai, China) and MicroRNAs Quantitation PCR Kit (Sangon, Shanghai, China) were used according to the manufacturer’s instructions. U6 was used as a reference gene to normalize miRNA expression using the 2−ΔΔCt method [23–26]. All primers were used to amplify cDNA with different concentration gradients. The average CT value and ΔCT value of these primers were calculated. The ΔCT value was mapped by the log value of the cDNA concentration gradient. When the absolute slope of the obtained straight line was close to zero, the amplification efficiencies of these primers were equal. Then, the relative quantitation was carried out by the 2−ΔΔCt method. We took the difference between the CT values of the target gene and internal reference gene in each group as the ΔCT of each group and subtracted the average value of ΔCT of each group from that of control group to obtain the ΔΔCT of each group; next, 2−ΔΔCt was used to calculate the relative expression level of each group [22,23]. The unpaired, two-tailed Student’s t test was used for the statistical comparison of the two groups, and one-way ANOVA followed by the least significant difference (LSD) post hoc test was used for the statistical comparison of more than two groups. All primers were designed by Sangon Biotech (Shanghai, China). The sequences of the primers are shown in Supplementary Table S1.
All animal procedures were approved by the Institutional Animal Care and Use Committee at the Southern Medical University and followed the NIH Guidelines for the Care and Use of Laboratory Animals. MI surgeries were performed on 8-week-old male C57BL/6J mice. These mice were singly housed in a temperature-controlled (approximately 22°C) environment with a 12-h light/dark cycles with food and water available ad libitum. The animals were habituated to the animal facilities for at least 1 week before use. Surgeries were performed on sedated mice (2% isoflurane) and an ALC-V8S rodent ventilator (ALCBIO, Shanghai, China) was used to supply oxygen during the surgical procedure. Lateral thoracotomy at the fourth intercostal space was performed by blunt dissection of the intercostal muscles after skin incision, and the pericardium was then removed. The left anterior descending (LAD) coronary artery was permanently ligated with a 5-0 silk suture (Ningbo Medical Needle Co., Ningbo, China) and successful infarction was confirmed using an electrocardiogram (Supplementary Figure S3B). The thoracic wall and skin were closed with 5-0 silk suture after surgery. After ligation, AAV9-NC, AAV9-anti-let-7i-5p, the mixture of AAV9-anti-let-7i-5p and AAV9-si-E2F2, or the mixture of AAV9-anti-let-7i-5p and AAV9-si-CCND2 was immediately injected into the myocardium bordering the infarct zone using an insulin syringe with an incorporated 30-gauge needle. After surgery, the skin was disinfected, and the animals were revived while being maintained on a thermal insulation blanket. Buprenorphine (0.03–0.06 mg/kg) was injected subcutaneously for pain relief. EdU was administered intraperitoneally (500 μg per animal) on alternate days up to day 12 post-MI. Hearts were collected 28 days after infarction as described below.
To evaluate the cardiac dimensions in MI mice, heart function was evaluated by transthoracic 2D echocardiography performance on sedated mice with isoflurane at 0, 7, 14, 21, and 28 days post-surgery using a Vevo 2100 Imaging System (Visual Sonics, ON, Canada) equipped with a 40-MHz probe. Two-dimensionally guided M-mode images of the short axis at the papillary muscle level were recorded. The average of at least three measurements was used for every data point from each mouse.
Animals were anesthetized with 2% isoflurane and then killed by injecting with 10% KCl. Hearts were excised, briefly washed in 0.9% NaCl, and then weighed. For histologic analysis, hearts were fixed in 10% formalin at room temperature and embedded in paraffin. For immunofluorescence analysis, hearts were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, CA, U.S.A.), and frozen sections of heart tissue were assessed by immunofluorescence.
Masson’s trichrome staining
Mouse hearts were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Masson’s trichrome staining was performed according to standard procedures. Briefly, the heart tissue slides were deparaffinized with xylene and rehydrated and incubated in Weigert Hematoxylin iron for 5 min, differentiated in hydrochloric acid (HCl)–ethanol, and then sequentially incubated in Ponceau Acid Fuchsin for 5 min, phosphomolybdic acid for 5 min, and Aniline Blue (Leagene, Beijing, China) for 5 min. Masson’s trichrome staining was used for the detection of fibrosis. The fibrotic area was measured and quantitated with ImageJ software.
Cells were fixed in 4% paraformaldehyde (PFA) (Leagene, Beijing, China), permeabilized with 0.2% Triton X-100 in PBS and blocked with PBS containing 1% BSA. Cells were then stained for 2 h at room temperature with the following primary antibodies diluted in blocking solution: cardiac troponin T (Abcam, ab8295, 1:200, Cambridge, U.K.), vimentin (Abcam, ab24525, 1:200, Cambridge, U.K.), histone H3 phosphorylated at Ser10 (pH3) (Abcam, ab47297, 1:200, Cambridge, U.K.), and Aurora B (Abcam, ab2254, 1:200, Cambridge, U.K.). Cells were washed with PBS and incubated for 1 h at room temperature with the respective secondary antibodies: goat anti-mouse IgG/Alexa Fluor 488 (bs-0296G-AF488, 1:200), goat anti-rabbit IgG/Alexa Fluor 594 (bs-0295G-AF594, 1:200), or goat anti-rat IgG/Alexa Fluor 647 secondary antibodies (bs-0310G-AF647, 1:200) (Biosynthesis, Beijing, China). To reveal EdU incorporation, cells stained with EdU were processed using a Click-iT EdU Alexa Fluor 555 Imaging Kit (Invitrogen, CA, U.S.A.) according to the manufacturer’s instructions. When indicated, the cells were further stained with Hoechst 33342 (Bioworld, MN, U.S.A.). Frozen sections (5 μm) were fixed with acetone. Then, the slides were permeabilized with 0.2% Triton X-100 PBS and were processed for immunofluorescence as described for cultured CMs. For wheat germ agglutinin (WGA) staining, heart sections were deparaffinized, rehydrated, and then incubated for 1 h at room temperature with WGA conjugated to Alexa Fluor 555 (Invitrogen, CA, U.S.A.) in PBS. The slides were then washed with PBS. Using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining (Roche, Basel, Switzerland), apoptotic cell death was determined according to the manufacturer’s instructions. To analyze the mono- and binuclear CM numbers, CMs were labeled with cardiac-specific Troponin T (cTnT) primary antibody as well as Hoechst 33342 (Bioworld, MN, U.S.A.). cTnT-positive CMs and their respective nuclei were counted under a fluorescence microscope. Image acquisition was performed using an LSM 880 confocal microscope (Zeiss, Oberkochen, Germany).
Isolated mouse CMs were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Dingguo Changsheng, Beijing, China) with protease inhibitors and phosphatase inhibitors, and the protein concentrations were determined using the BCA Protein Quantitative Analysis kit (Fudebio-tech, Hangzhou, China). Protein samples were mixed with Laemmli buffer containing 5% β-mercaptoethanol and were equally loaded on to 8–12% SDS/PAGE. Proteins were transferred to PVDF membranes (Millipore, U.S.A.), which were incubated at room temperature for 2 h in blocking buffer (5% BSA in TBS and Tween 20 (TBST) buffer). The membranes were then incubated with the following primary antibodies overnight at 4°C: E2F2 (Santa Cruz, Biotechnology sc-633, 1:200, Dallas, Texas, U.S.A.), CCND2 (Santa Cruz Biotechnology, sc-593, 1:200, Dallas, Texas, U.S.A.), and β-actin (Biosynthesis, bs-0061R, 1:5000, Beijing, China). The membranes were washed three times with TBST and incubated for 1 h at room temperature with a donkey anti-rabbit IgG H&L antibody (Abcam, ab205722, 1:10000, Cambridge, U.K.). The protein bands were visualized using ECL reagents (Millipore, U.S.A.) and the chemiluminescence imaging GeneGnome XRQ system (Syngene, MD, U.S.A.). The relative density was calculated using ImageJ software and the intensity of each protein band was normalized to that of β-actin.
miRNA target prediction
Putative miRNA targets were identified using the target prediction tools TargetScan (http://targetscan.org/index.html)  and RNA22 v2 microRNA Target Detection (https://cm.jefferson.edu/rna22/Interactive/) . Briefly, the sensitivity and specificity values were kept at 63 and 61%, respectively. The minimum number of paired-up bases was maintained at 12 while the maximum folding energy was maintained at −14 kcal/mol .
Luciferase reporter assay
Wild-type E2F2 3′-UTR (E2F2-WT), E2F2 3′-UTR mutant derivatives devoid of the let-7i-5p binding site (E2F2-MU1, E2F2-MU2, and E2F2-MU1+2), wild-type CCND2 3′-UTR (CCND2 -WT) and CCND2 3′-UTR mutant derivatives devoid of the let-7i-5p binding site (CCND2-MU1, CCND2-MU2, CCND2-MU3, and CCND2-MU1+2+3) were inserted into the psiCHECK™-2 vector (SaichengBio Co. Ltd., Guangzhou, China). To detect the transfection efficiency of E2F2 3′-UTR and CCND2 3′-UTR, the vectors were transfected into isolated CMs. At 24, 48, or 72 h after transfection, RT-qPCR was performed to detect the relative expression of the E2F2 3′-UTR and CCND2 3′-UTR. β-actin was used as a reference gene with the 2−ΔΔCt method [22,23]. Let-7i-5p mimics were cotransfected with the E2F2-WT or E2F2-Mus, CCND2-WT or CCND2-MUs psiCHECK™-2 vectors into isolated P1 mouse CMs using Lipofectamine 2000. Cell were harvested 48 h after transfection, and the luciferase activity was then determined using a luciferase reporter system according to the provided protocol (Promega, Madison, U.S.A.).
The data are presented in graphs compared with the control-treated groups. SPSS 20.0 software was adopted for the statistical analysis. The samples, enumerated CMs, means, S.D., and standard errors in each figure and Supplementary figures are shown in Supplementary Tables S2 and S3, respectively. An unpaired two-tailed Student’s t test was used for the statistical comparison of two groups, and one-way ANOVA followed by the LSD post hoc test was used for the statistical comparison of more than two groups. A value of P<0.05 was considered significant.
Let-7i-5p expression increases during heart development and affects neonatal mouse CM proliferation
To validate our assumption that let-7i-5p participates in CM proliferation, we initially evaluated mouse CM proliferation activity using pH3 immunostaining at embryonic day 16.5 (E16.5), P1 and P56. We found that the number of pH3-positive CMs decreased (Figure 1A,B). The efficiency of primer amplification of the target gene and internal reference gene were equal (Supplementary Figure S1A), so we used the 2−ΔΔCT method for the following RT-qPCR relative quantitations. Next, we measured let-7i-5p expression using RT-qPCR in mouse hearts at E16.5, E18.5, P1, P3, P7, and P56, as well as CMs isolated from mice at P1, P7, and P10, and found that the expression of let-7i-5p gradually increased with age (Figure 1C,D). Those results suggest that let-7i-5p may play a potential role in regulating CM proliferation. To assess the functional consequences of let-7i-5p on CM proliferation, neonatal mouse ventricular myocytes (NMVMs) (P1) were transfected with let-7i-5p mimic (mi-let-7i-5p) or the mimic negative control (mi-NC) for 48 h (Figure 1E). The let-7i-5p mimic significantly decreased NMVM number and led to a decreased number of EdU- and pH3-positive CMs (Figure 1F–K). Taken together, the above data suggested that let-7i-5p might negatively regulate NMVMs proliferation.
Let-7i-5p overexpression decreases the proliferation of NMVMs
Let-7i-5p overexpression inhibits the proliferation of neonatal mouse CM proliferation
To further explore whether overexpression of let-7i-5p could inhibit CM proliferation in neonatal mice, we delivered an AdV via intramyocardial injection to increase let-7i-5p levels in vivo for 8 days and performed GFP disruption to determine the specificity of Ad-let-7i-5p. We found that Ad-let-7i-5p was restricted to heart tissue (Figure 2A,B and Supplementary Figure S1B). We found that there were no significant differences in body weight, heart morphology, heart/body weight, or CM size between the two groups (Figure 2C–E and Supplementary Figure S1C,D). Fewer EdU-positive (1.24 ± 0.16 to 0.54 ± 0.23%) (Figure 2F,G) and pH3-positive CMs (0.31 ± 0.09 to 0.12 ± 0.10%) resulted from the overexpression of let-7i-5p (Figure 2H,I). Importantly, the population of mononucleated CMs was decreased in CMs isolated from Ad-let-7i-5p compared with Ad-NC hearts (Figure 2J). The numbers of EdU-labeled both mononuclear and binuclear CMs decreased with let-7i-5p overexpression (Figure 2K). Thus, we demonstrated that let-7i-5p inhibited the proliferation of neonatal mouse CM proliferation.
Let-7i-5p overexpression decreases the proliferation of NMVMs
Inhibition of let-7i-5p promotes CM proliferation
To further investigate the function of let-7i-5p in CMs, we reduced let-7i-5p in CMs by transfection with a let-7i-5p inhibitor (Figure 3A). Transfection of isolated NMVMs with let-7i-5p inhibitor resulted in a 1.23 ± 0.05-fold increase in the number of CMs (Figure 3B,C). Approximately 12.62 ± 1.35% of CMs were positive for EdU, while control transfection (in-NC) resulted in 6.19 ± 1.10% of CMs positive for EdU, indicating a significant increase in DNA synthesis (Figure 3D,E). In addition, 3.10 ± 0.31% of CMs transfected with let-7i-5p inhibitor were positive for mitosis as shown by pH3 staining (Figure 3F,G), and the 2.01 ± 0.26% of Aurora-B-positive adult CMs indicated cytokinesis (Figure 3H,I). Collectively, these data demonstrated that the reduction in let-7i-5p promotes CM proliferation in the neonatal mouse heart in vitro.
Let-7i-5p suppression increases the proliferation of NMVMs
Let-7i-5p suppression promotes the proliferation of adult mouse CM proliferation
To investigate whether suppression of let-7i-5p promotes CM proliferation in adult mice, the 8-week-old mice’s hearts were injected with AAV9-NC or AAV9-anti-let-7i-5p. AAV9 vectors were exclusively restricted to the heart tissue (Figure 4A), and AAV9-anti- let-7i-5p led to the suppression of let-7i-5p (Figure 4B). We measured the weights of AAV9-NC and AAV9-anti-let-7i-5p mice and found no significant difference between the two groups (Figure 4C,D). Additionally, the HW:BW ratio and CM size were not significantly different between the AAV9-NC and AAV9-anti-let-7i-5p mice (Figure 4E and Supplementary Figure S2A,B). In contrast, the inhibition of let-7i-5p promoted proliferation as evidenced by increased numbers of both EdU-positive (0.47 ± 0.30 to 1.17 ± 0.25%) and pH3-positive CMs (0 ± 0 to 0.13 ± 0.08%) (Figure 4F–I). Moreover, the percentage of mononucleated CMs was increased in adult CMs isolated from AAV9-anti-let-7i-5p compared with AAV9-NC hearts (Figure 4J). Mono- and binucleated CMs labeled with EdU also increased compared with the controls, with an especiallly significant increase in mononuclear CMs (Figure 4K). Collectively, the above data show that let-7i-5p suppression stimulates CM proliferation.
Let-7i-5p suppression increases the proliferation of adult mouse ventricular myocytes
E2F2 and CCND2 are the direct targets of let-7i-5p
Next, we sought to identify downstream targets of let-7i-5p. TargetScan-based miRNA-UTR binding predictions suggested that E2F2 and CCND2 were potential target genes of let-7i-5p, for which there were two potential binding sites in E2F2 and three in CCND2 (Figure 5A). We then used RNA22 to calculate the folding energy of these potential binding sites and selected cut-offs according to a previous study maintaining the maximum folding energy at −14 kcal/mol . The results showed that the folding energy of E2F2 binding site 1 was −16.3 kcal/mol and of CCND2 binding site 1 was −15.6 kcal/mol (Figure 5A). Thus, we next verified whether E2F2 or CCND2 was the direct target of let-7i-5p. Luciferase reporter assays confirmed the let-7i-5p binding predictions for the E2F2 and CCND2 3′-UTRs. The efficiencies of primer amplification of β-actin and E2F2 or CCND2 were equal (Supplementary Figure S3A,B). The E2F2 3′-UTR and CCND2 3′-UTR increased gradually during 24–48 h of transfection, but decreased after 72 h of transfection compared with 48 h of transfection (Supplementary Figure S3C,D). Cotransfection of the E2F2 and CCND2 3′-UTR reporters with the let-7i-5p mimic conferred lower luciferase activity compared with the cells cotransfected with scrambled RNA. Single mutations of both E2F2 and CCND2 3′-UTR binding site 1 can abolish this repression, suggesting that let-7i-5p directly targetted both E2F2 binding site 1 and CCND2 3′-UTR binding site 1 (Figure 5B,C). Moreover, the binding sequence was highly consistent between rats, mice, and humans, according to the NCBI database (Supplementary Figure S3E). To further illuminate the relationship between them, we found that overexpression of let-7i-5p inhibited both E2F2 and CCND2 proteins, whereas the suppression of let-7i-5p led to higher protein expression of E2F2 and CCND2 in vitro by Western blotting (Figure 5D–F). Thus, we discovered that let-7i-5p directly binds to both E2F2 and CCND2 mRNAs.
E2F2 and CCND2 are targets of let-7i-5p
E2F2 and CCND2 are involved in let-7i-5p-regulated CM proliferation
To investigate whether CCND2 and E2F2 are involved in let-7i-5p-induced CM proliferation, the E2F2 and CCND2 proteins were expressed from the E2F2 and CCND2 plasmids, as verified by Western blotting (Supplementary Figure S4A–D). Overexpression of let-7i-5p inhibited the level of E2F2 protein, while cotransfection of E2F2 and let-7i-5p mimic reversed E2F2 protein levels (Figure 6A,B). The same phenomenon was observed for CCND2 and the delivery of let-7i-5p mimic to CMs (Figure 6C,D). Moreover, cotransfection with either E2F2 or CCND2 and let-7i-5p mimic reversed the suppressive influence of let-7i-5p mimic, as observed by the EdU incorporation rate of CMs (Figure 6E,F). These results reveal that let-7i inhibits CM proliferation by targetting E2F2 and CCND2.
E2F2 and CCND2 are involved in the inhibitory effect of let-7i-5p on the proliferation of CMs
Inhibition of let-7i-5p promotes cardiac function in response to MI
To further test the regenerative potential of let-7i-5p in an adult mouse model of MI, we developed an MI model by LAD ligation and injected AAV9-anti-let-7i-5p, AAV9-NC, AAV9-si-CCND2, or AAV9-si-E2F2 into mouse hearts following MI establishment (Supplementary Figure S5A,B). Noninvasive transthoracic echocardiography was used to measure the cardiac function at 0, 7, 14, 21, and 28 days after MI (Supplementary Figure S5A), including left ventricular end-diastolic dimension (LVEDd), left ventricular end-systolic dimension (LVESd), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS). The results showed that inhibition of let-7i-5p significantly improved functional parameters compared with AAV9-NC groups, and cotransfection with either si-E2F2 or si-CCND2 and anti-let-7i-5p reversed the cardiac function (Figure 7A–E and Supplementary Figure S5C). Additionally, a clear reduction in infarct size (Figure 7F,G) was observed, along with reduced TUNEL staining (Figure 7H,I), which is a measure of apoptosis, at 28 days after MI, whereas MI size and apoptosis increased after inhibiting both let-7i and E2F2 or CCND2 (Figure 7F–I). To determine whether proliferation was occurring, we performed EdU staining at the border zone 28 days after MI. Suppression of let-7i-5p increased the positive rate of EdU staining of CMs in the infarct border zone, while the number of EdU-positive CMs decreased after inhibiting let-7i-5p and E2F2 or CCND2 at the same time. (Figure 7J,K). Collectively, our data show that suppression of let-7i-5p has a positive influence on CM proliferation and cardiac repair in adult hearts post-MI by simultaneously regulating E2F2 and CCND2. Thus, let-7i-5p leads to decreased E2F2 and CCND2 expression, causing the CMs to exit the cell cycle, thereby weakening CM proliferation (Figure 8).
Suppression of let-7i-5p promotes heart function post-MI in adult mice
Diagram of the biomechanism diagram of let-7i-5p influencing CM proliferation
Here, we have demonstrated that the progressively increased expression of let-7i-5p during heart growth is negatively correlated to CM proliferation, and knockdown of let-7i-5p enhances cardiac proliferation both in vitro and in vivo. Mechanistically, let-7i-5p negatively regulated the cell cycle of CMs induced by CCND2 and E2F2. CCND2 and E2F2 activation rescued the CM proliferation phenotype induced by let-7i-5p reduction in adult CMs. Moreover, down-regulation of let-7i-5p promoted cardiac repair and improved heart function post-MI. Thus, our findings illuminate an important function of the miRNA let-7i-5p in CM cell cycle progression.
It is now increasingly recognized that the postnatal mammalian heart retains a regenerative capacity that is lost within 7 days, coinciding with the time when CMs withdraw from the cell cycle after birth . During this period, the dynamic expression of miRNAs plays an important role in modulating CM proliferation . By quantitating the expression levels of the miRNAs in CMs from mouse heart tissues at different ages including P1, P7, and P10, we found that let-7i-5p levels in P10 CMs was increased by more than two-fold compared with CMs in P1 neonatal heart tissue, which was in accordance with the trend observed in heart tissue from embryonic to adult mice. This phenomenon was accompanied by the dynamic physiological processes that occur with age and the gradual decline in proliferative ability of CMs, which is consistent with prior studies of CMs. Intriguingly, when let-7i-5p was knocked down in adult mouse CMs, we observed an increase in CM proliferation, as evidenced by a significant increase in DNA synthesis, mitosis, and cytokinesis of CMs. In contrast, the up-regulation of let-7i-5p led to a decline in the proliferation of neonatal CMs. Accordingly, our results suggest that the up-regulation of let-7i-5p is closely associated with the loss of proliferative ability.
To further assess the mechanism by which let-7i-5p regulates CM proliferation and cardiac regeneration, we used bioinformatics analysis to find that CCND2 and E2F2 were the targets of let-7i-5p with high possibility. Both of them are positive regulators of the cell cycle. CCND2, belonging to the highly conserved cyclin family, functions as a regulatory subunit of CDK4 or CDK6. The activity of CCND2 is required for the G1/S transition . Increased CCND2 activates cell cycle progression in human induced pluripotent stem cell-derived CMs (hiPSC-CMs) . Moreover, the overexpression of CCND2 is sufficient to stimulate CM cell cycle activity in adult hearts . E2F2 is part of the E2F family that regulates the cell cycle and apoptosis in the heart and is involved in the tumorigenesis of various cancers . Consistent with prior studies, the knockdown of E2F2 or CCND2 reduced the augmentation of CM proliferation modulated by let-7i-5p suppression, and even worsened cardiac function post-MI, while up-regulation of E2F2 or CCND2 rescued the inhibition caused by let-7i-5p overexpression. Thus, let-7i-5p interacts with E2F2/CCND2 to modulate the cell cycle of CMs.
Of note, the current study suggests that let-7i-5p functions differently under distinct pathological condition of the heart. On the one hand, as a suppressor in multiple tumor types, let-7i-5p negatively regulates mouse CM proliferation both in vitro and in vivo. Adult mice that suffer from MI display improved cardiac function upon let-7i-5p suppression. On the other hand, let-7i-5p is beneficial for angiotensin (Ang) II-mediated cardiac remodeling by negatively regulating cardiac inflammation and fibrosis via suppressing the expression of interleukin-6 and collagens . This disparity in function may derive from the differences in pathophysiology between myocardial ischemia and Ang-II-mediated cardiac remodeling. When coronary arteries are occluded, an enormous number of myocardial cells necrose and apoptose, triggering an instant and intense inflammatory cascade that releases myriad components of the innate immune system, thus affecting both CMs and other cells [37,38]. In contrast, myocardial hypertrophy occurs as a response to deteriorated blood pressure condition stimulated by high levels of Ang-II. Thus, let-7i-5p may inhibit CM proliferation in the ischemic model and fibrosis in the Ang-II model [36,39], and suppression of let-7i-5p induces an overall effect of improved heart function after MI without exaggerated fibrosis.
The present study has two limitations. First, we verified that the suppression of let-7i-5p promoted CM re-entry into the cell cycle and improved post-MI repair; however, the high levels of let-7i-5p in adult mouse heart remain unclear and must be clarified in future analyses. Second, the targets of let-7i-5p are many, and we only reported two targets. Given the pleiotropic effects of miRNAs, potentially effective therapeutic strategies based on the inhibition of let-7i-5p require careful consideration.
In conclusion, our findings have demonstrated that let-7i-5p down-regulates the expression of E2F2 and CCND2, and further represses CM proliferation, which is induced by CCND2 and E2F2 activation. This novel observation implies the therapeutic potential of let-7i-5p in CM proliferation, cardiac repair, cardiac regeneration, and related disorders.
Let-7i-5p was proposed to be a strong suppressor of CM proliferation in vitro. Let-7i-5p functions to maintain cancer stem cell growth and aggravate carcinogenesis, and it is significantly down-regulated in the plasma of young STEMI sufferers. However, the role of let-7i-5p in the processes of CM cell cycle and proliferation in vivo is unknown.
Overexpression of let-7i-5p inhibited CM proliferation, whereas the suppression of let-7i-5p significantly facilitated CM proliferation. Suppression of let-7i-5p promoted the recovery of heart function post-MI by enhancing E2F2 and CCND2.
Inhibition of let-7i-5p might be a novel strategy for cardiac repair after ischemic injury.
J.B., Y.W., W.L., and Y.L. conceived the project and designed experiments. Y.H., G.J., B.L., Y.C., and L.Z. performed the experiments. G.C., X.C., and J.Z. analyzed the data. Y.H., G.J., and J.B. wrote the manuscript.
The authors declare that there are no competing interests associated with the manuscript.
This work was supported by the National Natural Science Foundation of China [grant numbers 81771857, 81571698, 81271640 (to J.B.)].
adeno-associated virus 9
cardiac-specific Troponin T
Dulbecco’s modified Eagle’s medium/nutrient F-12 Ham
left anterior descending
least significant difference
neonatal mouse ventricular myocyte
PBS and Tween 20
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
wheat germ agglutinin
These authors contributed equally to this work.