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MiR-135a Protects against Myocardial Injury by Targeting TLR4
2021 Volume 69 Issue 6 Pages 529-536
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2021 Volume 69 Issue 6 Pages 529-536
Emerging evidence highlights the importance of microRNAs (miRNAs) as functional regulators in cardiovascular disease. This study aimed to investigate the functional significance of miR-135a in the regulation of cardiac injury after isoprenaline (ISO) stimulation and the underlying mechanisms of its effects. Murine models with cardiac-specific overexpression of miR-135a were constructed with an adeno-associated virus expression system. The cardiac injury model was induced by ISO injection (60 mg/kg per day for 14 d). In vitro, we used H9c2 cells to establish a cell injury model by ISO stimulation (10 µM). The results indicated that miR-135a was increased during days 0–6 of ISO injection and was then downregulated during days 8–14 of ISO injection. The expression of miR-135a was consistent with the in vivo findings. Moreover, mice with cardiac overexpression of miR-135a exhibited reduced cardiac fibrosis, lactate dehydrogenase levels, Troponin I, inflammatory response and apoptosis. Overexpression of miR-135a also ameliorated cardiac dysfunction induced by ISO. MiR-135 overexpression in H9c2 cells increased cell viability and decreased cell apoptosis and inflammation in response to ISO. Conversely, miR-135 silencing in H9c2 cells decreased cell viability and increased cell apoptosis and inflammation in response to ISO. Mechanistically, we found that miR-135a negatively regulated toll-like receptor 4 (TLR4), which was confirmed by luciferase assay. Furthermore, the TLR4 inhibitor eritoran abolished the adverse effect of miR-135 silencing. Overall, miR-135a promotes ISO-induced cardiac injury by inhibiting the TLR4 pathway. MiR-135a may be a therapeutic agent for cardiac injury.
Cardiovascular disease is one of the leading causes of death worldwide.1) During the pathology of various cardiovascular diseases (including hypertension, coronary heart disease, and myocardial ischaemia), sympathetic overactivity promotes the occurrence and development of the disease and leads to the transformation to heart failure.2) Studies have shown that isoproterenol (ISO), a β-adrenergic agonist, is significantly elevated in the early stages of myocardial ischaemia and early onset of hypertension.3) ISO promotes the production of a large number of oxygen free radicals in the heart,4) aggravates myocardial ischaemia, and can induce non-ischaemic myocardial injury, which leads to cardiac dysfunction.5) Indeed, during ISO insult, myocardiocytes undergo excessive cell apoptosis and necrosis, which subsequently attracts large amounts of inflammatory cells and promotes the inflammatory cascade.6) Cardiac fibrosis is also a main feature of ISO-induced cardiac injury with massive extracellular matrix deposition, leading to cardiac dysfunction.7) For decades, the usage of beta-adrenergic blockers has not decreased the morbidity and mortality of heart failure worldwide. Thus, a new therapeutic method for ISO-induced cardiac injury is urgently needed.
MicroRNAs (miRNAs) are a new class of non-coding RNAs with a molecular weight of approximately 19 to 25 nucleotides. They negatively regulate genes by promoting degradation or suppressing gene expression.8) Recent studies have found that miR-135a is involved in cardiovascular diseases. MiR-135a has been reported to inhibit endothelial cell proliferation and migration, which negatively regulates pathophysiological angiogenesis.9) MiR-135a has also been shown to protect against cardiac ischaemia–reperfusion injury.10,11) However, Zheng found that miR-135a accelerated myocardial depression in sepsis.12) Thus, the results on the effect of miR-135a on cardiovascular disease have not been consistent. In this study, we are the first to use an ISO-induced cardiac injury model to evaluate the role of miR-135a in cardiovascular disease.
Enzyme-linked immunosorbent assay (ELISA) kits for tumour necrosis factor α (TNFα, #ab208348), interleukin (IL)-1 (ab197742), and IL-6 (ab203360) were purchased from Abcam (Cambridge, U.K.). The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) kit was purchased from Millipore (#ATP110, MA, U.S.A.). The caspase-3 activity detection assay came from Beyotime (#C1115, Shanghai, China). Isoproterenol was purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). CD45 (ab10558), CD68 (ab955), and toll-like receptor 4 (TLR4) (ab13867) antibodies were purchased from Abcam. The antibody glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Cell Signaling Technology Inc. (Danvers, MA, U.S.A.).
AnimalsMale C57BL/6J mice aged 8–10 weeks (weighing 23.5–27.5 g) were purchased from the Chinese Academy of Medical Sciences & Peking Union Medical College. The animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Animal Care and Use Committee of our hospital. Mice were subjected to ISO injection (dissolved in sterile saline, filled in osmotic minipumps (Durect, Cupertino, CA, U.S.A.), 60 mg/kg per day for 14 d). Two weeks before the injection of ISO, mice were subjected to retroorbital venous plexus injection of Ad-miR-135a to overexpress miR-135a. After 14 d of ISO injection, the mice were sacrificed, and the hearts were removed.
Echocardiographic and Haemodynamic MeasurementsEchocardiography was performed after the final injection of ISO. A Mylab30CV (ESAOTE) ultrasound system was used. M-mode tracings and pulse Doppler were recorded by a 15 MHz probe. The left ventricular (LV) ejection fraction, LV fractional shortening, and E/A ratio were calculated.
A pressure–volume loop was constructed with a microtip catheter transducer (Millar Instruments, Houston, TX, U.S.A.). We inserted this catheter though the right carotid artery of the mice and finally reached the LV. When the recording was stable, we used a Millar Pressure–Volume System to record all the data and then used PVAN software to analyse the data.
Mice Overexpression of MiR-135aThe adenovirus vector that overexpressed miR-135a (Ad-miR-135a) and the negative control (Ad-NC) were constructed by Vigene Biosciences, Inc. (Shanghai, China). Two weeks before ISO injection, mice were subjected to retroorbital venous plexus injection of miR-135a (each with 60 µL, 5.0–6.5 × 1013 GC/mL) as previously described.13)
Histological AnalysisHearts were removed and fixed in 10% formalin, embedded in paraffin and cut into 25 µm sections. Haematoxylin–eosin (H&E) staining and picrosirius red (PSR) staining were performed to detect gross heart and LV collagen deposition. We used a digital image analysis system (Image-Pro Plus, version 6.0) to analyse PSR-stained sections. We used anti-CD45 and anti-CD68 antibodies to label inflammatory cells in heart tissue by immunohistochemical staining.
ELISACells were lysed, and proinflammatory cytokine levels were detected by utilizing commercially available ELISA kits for the quantification of TNF-α, IL-1 and IL-6 according to the manufacturer’s protocol. Optical density values were measured at 450 nm on an ELISA plate reader (Synergy HT, BioTek, Vermont, VT, U.S.A.).
Lactate Dehydrogenase (LDH), TNI Level, and Caspase-3 Activity(LDH and caspase-3 activities were measured according to the manufacturer’s protocol (Beyotime, Shanghai, China). The TroponinI (TNI) level was measured according to the manufacturer’s protocol (Life Diagnostics, Inc., West Chester, PA, U.S.A.).
H9c2 Cell Line CultureH9c2 cardiomyocytes were obtained from the Bank of the Chinese Academy of Science (Shanghai, China). We used Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) to culture cells. Cells were seeded in a 10 cm dish to passage. Cells were transfected with Ad-miR-135a to overexpress miR-135a (MOI = 75) for 6 h and then were cultured for 24 h to detect the expression of miR-135a. For the in vitro model, cells were transfected with Ad-miR-135a for 6 h and then were stimulated with ISO (10 µM) for 24 h. Cells were transfected with miR-135a inhibitor (sequence: 5′-UCA CAU AGG AAU AAA AAG CCA UA-3′) and inhibitor control (anti-miRcon) sequence: 5′-CAG UAC UUU UGU GUA GUA CAA-3′) with Lipo2000™ Transfection Reagent (Beyotime, Shanghai, China). Cells were treated with resatorvid (100 nM, MedChemExpress Company) to inhibit TLR4. A CCK-8 assay (Beyotime, Shanghai, China) was used to detect cell proliferation.
TUNEL StainingTUNEL staining was performed according to the manufacturer’s protocol. Briefly, cells were fixed with 4% paraformaldehyde for 5 min and then were permeabilized with 0.1% Triton™ X-100. Heart samples were fixed with 10% formalin and embedded in paraffin. Samples were first incubated with a TUNEL reagent containing terminal deoxynucleotidyl transferase and fluorescent isothiocyanate-deoxyuridine triphosphate (dUTP) according to the protocol of the ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit (#ATP110, Millipore). H9c2 cell nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI). Heart tissue samples were stained with haematoxylin. A microscope (OLYMPUS DX51) was used to capture images. The percentage of apoptotic cells in vitro was calculated (at least 10 fields on each slide, percentages of apoptotic cells = TUNEL-positive/DAPI-positive nuclei); the number of apoptotic cells in vivo was calculated (at least 10 fields on each slide) by at least three independent individuals in a blinded manner.
RT-PCR and Western Blot AnalysisWe used TRIzol reagent to isolate total RNA. We used a SmartSpec Plus Spectrophotometer (Bio-Rad, Hercules, CA, U.S.A.) to detect mRNA purity with the OD260/OD280 ratio. A total of 2 µg of mRNA was reverse transcribed into cDNA with a cDNA Synthesis Kit (Roche Diagnostics, Basel, Switzerland). We used a LightCycler 480 SYBR Green I Master kit (Roche Diagnostics) for amplification. We used GAPDH as reference. The primers used are listed below:
For Western blotting, we used radioimmunoprecipitation (RIPA) lysis buffer to lyse cells. The total protein concentration was detected by the bicinchoninic acid (BCA) method and then loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. When proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), they were incubated with primary antibodies at 4 °C overnight. Blots were developed with enhanced chemiluminescence (ECL) reagents (Bio-Rad) and captured by a ChemiDoc MP Imaging System (Bio-Rad). We used GAPDH as a reference.
Luciferase AssayH9c2 cells were transfected with TLR4-luc in combination with Ad-miR-135a and miR-135 inhibitor. After treatment, cells were collected and washed and then lysed with passive buffer (Promega). Then, luciferase activity was detected by using a GloMax® 20/20 Luminometer (Promega).
Data AnalysisAll data were expressed as the mean ± standard deviation (S.D.). SPSS 23.3 was used to analysis data. When comparing data between two groups, Student’s t-test was used. When comparing data among four groups, one-way ANOVA followed by Tukey’s post hoc test was used. We defined significant differences with a p-value less than 0.05.
We first detected the expression pattern of miR-135a in ISO-stimulated mouse hearts. As shown in Fig. 1A, the miR-135a level was increased from day 2 to day 6 of ISO injection but dropped sharply from day 8 to day 14 of ISO injection. We then detected a change in expression of miR-135a in H9c2 cardiomyocytes treated with ISO. Consistent with the in vivo results, the miR-135a level was increased after 0–6 h of ISO stimulation then dropped after 12–48 h of ISO stimulation (Fig. 1B). These results indicate that miR-135a participates in the pathology of cardiac injury. We then established a cardiac injury model by ISO injection. MiR-135a was overexpressed in mouse hearts with Ad-miR-135a (Fig. 1C). As a result, ISO injection induced significant cardiac injury, as evidenced by H&E staining (Fig. 1D), and increased LDH and TNI levels in heart tissue in the Ad-NC/ISO group. However, the levels of LDH and TNI were sharply suppressed in the Ad-miR-135a/ISO group (Fig. 1E). The hearts in the Ad-miR-135a/ISO group showed ameliorated damage, as seen in the H&E image (Fig. 1D). We also detected cardiac fibrosis levels by PSR staining and found that miR-135a overexpression inhibited ISO-induced LV collagen volume (Fig. 1F) and restrained the transcription of fibrotic molecules, such as collagen I, collagen III, α-SMA, and TGFβ (Fig. 1G). These data suggest that miR-135a protects against cardiac injury induced by ISO stimulation.
A. MiR-135a expression was assessed in mouse hearts after ISO infusion (n = 6, * p < 0.05 vs. 0 d). B. MiR-135a expression was determined in cardiomyocytes after ISO treatment (n = 6, * p < 0.05 vs. 0 h). C. MiR-135a expression was analysed in mouse hearts after Ad-miR-135a injection (n = 6, * p < 0.05 vs. Ad-NC). D. H&E staining was performed on mouse heart tissue sections after ISO infusion (n = 5). E. LDH and TNI levels were determined in hearts (n = 6). F. PSR staining was performed and quantified results (n = 5). G. mRNA levels were determined of the indicated markers (n = 6). * p < 0.05, ** p < 0.01.
As cardiac inflammation and apoptosis are other features of cardiac injury, we evaluated these characteristics after ISO insult. As shown in Figs. 2A and B, the numbers of CD45-labelled leukocytes and CD68-labelled macrophages were increased in ISO-insulted mouse hearts but were reduced by miR-135a overexpression. The proinflammatory factor levels (TNF-α, IL-1, and IL-6) were remarkably decreased by miR-135a (Fig. 2C). The TUNEL-positive cell number, as detected by TUNEL staining, was increased in the Ad-NC/ISO group but reduced in the Ad-miR-135a/ISO group (Fig. 2D). The activity of caspase-3, which promotes apoptosis, was also elevated in the Ad-NC/ISO group but dropped in the Ad-miR-135a/ISO group (Fig. 2E). Taken together, miR-135a may be a protective factor in ISO-induced cardiac injury.
A and B. CD45 and CD68 staining and quantitative results are shown of mouse hearts after ISO infusion (n = 5). C. Proinflammatory factors were assessed in heart tissue (n = 6). D. TUNEL staining and quantitative results are shown of mouse hearts (n = 5, arrow indicates TUNEL-positive cells). E. Caspase-3 activity was assessed in mouse hearts (n = 6). ** p < 0.01.
Cardiac dysfunction is the decompensatory effect of ISO-induced cardiac injury. We evaluated cardiac function and heart haemodynamic parameters. As shown in the echocardiography data, the heart rate (HR) was increased in ISO-stimulated mouse hearts, but miR-135a did not effectively decrease HR (Fig. 3A). Nevertheless, other parameters, such as LV end diastolic dimension (LVIDd), LVEF, and LVFS, were significantly improved by miR-135a during ISO insult. During the final ISO injection, mice were subjected to haemodynamic measurements. As shown in Fig. 3B, the end diastolic pressure (EDP) and maximum rate of LV pressure rise and decay (dp/dtmax, dp/dtmin) were all improved by miR-135a.
A. Echocardiographs are shown from mouse hearts after ISO infusion (n = 10). B. Haemodynamic measurements are shown of mouse hearts after ISO infusion (n = 10). * p < 0.05, ** p < 0.01.
It is unknown whether miR-135a directly affects cardiomyocytes. We then performed in vitro experiments, stimulating H9c2 cardiomyocytes with ISO. Cells were transfected with Ad-miR-135a to overexpress miR-135a (Fig. 4A). Cell viability was detected by CCK-8 assay. ISO induced an abrupt drop in cell viability; however, overexpression of miR-135a preserved cell viability in response to ISO insult. ISO also increased cell apoptosis, as evaluated by an increased number of TUNEL-positive cells and elevated caspase-3 activity. These apoptosis-associated indexes were blurred by miR-135 overexpression (Figs. 4C, D). We also detected the level of proinflammatory factors in cells under ISO stimulation. The increased inflammation was suppressed by miR-135 overexpression in cells (Fig. 4E).
H9c2 cardiomyocytes were transfected with Ad-miR-135a and treated with ISO. A. The miR-135a expression level was determined. B. Cell viability results are shown for each group. C. TUNEL staining images and quantification results are shown (arrow indicates TUNEL-positive cells). D. Caspase-3 activity was assessed in cells. E. The level of proinflammatory factors was determined in cells. ** p < 0.01. All in vitro studies were performed 3 times.
We then determined whether anti-miR-135a would exert adverse effects. Cells were transfected with miR-135a inhibitor (Fig. 5A). With ISO stimulation, cell viability was reduced in the miR-135a inhibitor group compared with cells transfected with the control sequence (Fig. 5B). Cell apoptosis and caspase-3 activities were also elevated in the miR-135a inhibitor group compared with the control group (Figs. 5C, D). Pro-inflammatory factors were also elevated in cells transfected with miR-135a inhibitor (the levels were significantly elevated when compared with those in control cells, Fig. 5E).
H9c2 cardiomyocytes were transfected with miR-135a inhibitor and treated with ISO. A. The miR-135a expression level was assessed. B. Cell viability was determined in each group. C. TUNEL staining images and quantification results are shown (arrow indicates TUNEL-positive cells). D. Caspase-3 activity was determined in cells. E. The level of proinflammatory factors was determined in cells. ** p < 0.01. All in vitro studies were performed 3 times.
We tried to identify the specific target of miR-135a in cardiomyocytes. The luciferase results showed that miR-135a targeted TLR4 in cardiomyocytes, as the miR-135a inhibitor increased the promoter activity of TLR4, while miR-135a overexpression decreased the promoter activity of TLR4 (Fig. 6A). We then detected the TLR4 protein level. Similarly, the miR-135a inhibitor elevated the protein expression of TLR4, while miR-135a overexpression reduced the protein expression of TLR4 (Fig. 6B). To further confirm this result, we used a TLR4 inhibitor (resatorvid). Cells were transfected with miR-135a inhibitor, treated with TLR4 inhibitor and then stimulated with ISO. A recovery of cell viability was observed in cells treated with both TLR4 inhibitor and miR-135a inhibitor when compared to cells treated with miR-135a inhibitor only (Fig. 6C). The same trends were observed in apoptosis and the inflammatory response. Cells treated with both TLR4 inhibitor and miR-135a inhibitor exhibited less apoptosis and fewer proinflammatory factor levels than cells treated with miR-135a inhibitor only (Figs. 6D–F).
A. A luciferase assay for TLR4 was performed in cells transfected with Ad-miR-135a or miR-135a inhibitor. B. The TLR4 level was determined in H9c2 cells transfected with Ad-miR-135a or miR-135a inhibitor. C–F. Cells were transfected with miR-135a inhibitor and TLR4 inhibitor, resatorvid. C. Cell viability was determined for each group. D. TUNEL staining images and quantification results are shown (arrow indicates TUNEL-positive cells). E. Caspase-3 activity was assessed in cells. F. The level of proinflammatory factors was determined in cells. ** p < 0.01. All in vitro studies were performed 3 times.
Sympathetic hyperactivity is a main feature of many cardiac diseases, including hypertension, myocardial infarction, and ischaemia/reperfusion injury. Increased catecholamine is the most harmful factor that leads to heart failure.2) ISO-induced cardiac injury involves cardiac damage, cardiac fibrosis, myocardial inflammation, and cardiomyocyte apoptosis. These changes would lead to cardiac systolic and diastolic dysfunction and would eventually lead to heart failure.4) MiRNAs are small molecular RNAs that target the 3′-UTRs of many genes with negative regulation. Previous studies have reported that miR-135a participates in cardiovascular diseases, including cardiac ischaemia/reperfusion injury and cardiac fibrosis. However, the results of these studies are controversial. Whether miR-135a affects ISO-induced cardiac injury remains unclear. In this study, we first revealed that during heart ISO insult, heart tissue and cardiomyocytes undergo an elevated level of miR-135a at the very beginning and then drop to the basal level later in the pathological process. Elevated miR-135a exerts protective effects in hearts under ISO insult, including anti-myocardial damage, anti-cardiac fibrosis, anti-inflammation, and anti-apoptosis effects. We also observed that miR-135a preserved cardiac function during heart ISO insult.
MiRNAs participate in many pathophysiological processes, such as cell differentiation, tumorigenesis, cell survival, death and fate change.14) In recent years, many miRNAs have been found to manipulate the pathological processes of cardiovascular disease. Nuclear miR-320 was found to prompt diabetes-induced cardiac remodelling by targeting metabolism-associated genes.15) MiR-199a accelerates cardiac hypertrophy by regulating autophagy.16) MiR-208a participates in the process of ISO-induced cardiac injury.6) Previous studies have reported that miR-135a is involved in cardiovascular diseases. By targeting β-Catenin/GSK-3β signalling, inhibition of miR-135a was reported to alleviate pulmonary arterial hypertension.17) Lu et al. reported that miR-135a downregulated FOXO1, which promoted vascular smooth muscle cell inflammatory damage.18) Icli et al. found that miR-135a-3p suppressed angiogenesis in endothelial cells.9) These results indicate that miR-135a exerts negative effects in vascular disease. In cardiac disease, both Wang et al. and Zhu et al. reported that miR-135a protected against myocardial ischaemia/reperfusion injury.10,11) Wu et al. also found that miR-135a suppressed cardiac fibrosis by targeting fibroblasts.19) These controversial results have led to confusion about the effect of miR-135a on cardiovascular disease. In this study, we used an ISO-induced cardiac injury model. We found that overexpressing miR-135a inhibited myocardial damage, cardiac fibrosis, and cardiomyocyte apoptosis and improved cardiac function. We also confirmed these effects in vitro, as miR-135a overexpression exerted protective effects in H9c2 cardiomyocytes, while the miR-135a inhibitor exerted the opposite effects. Taken together, our study provides valid evidence that during heart insult, miR-135a is beneficial.
During mechanism exploration experiments, we observed reduced TLR4 expression when cardiomyocytes overexpressed miR-135a. TLR4 is a member of the Toll-like receptor family that acts as a damage-related pattern recognition receptor.20) During cardiac injury, many damage-related molecules are released in damaged cardiomyocytes, which activate TLR4 in the cell membrane of other cardiomyocytes and immune cells.21) Once activated, TLR4 signals to the downstream target Myd88, which subsequently activates mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signalling. Both MAPKs and NF-κB signalling are strong proinflammatory and proapoptotic pathways that directly target the transcription of those genes.22) A previous study found that miR-135a targets TLR4 in RAW264.7 cells, which suppresses atherogenesis.23) In this study, we found that the expression of TLR4 was changed with miR-135a. Our luciferase data confirmed that in cardiomyocytes, miR-135a inhibits TLR4 promoter activity. We also confirmed this mechanism by using a TLR4 inhibitor and found that the negative effects of the miR-135a inhibitor on ISO-induced cell injury were ameliorated by the TLR4 inhibitor. These data suggest that miR-135a targets TLR4 in cardiomyocytes.
In summary, our data revealed for the first time that miR-135a acts as a beneficial molecule that ameliorates ISO-induced cardiac injury by directly targeting cardiomyocytes. These protective effects are mediated by targeting the TLR4 signalling protein, which negatively regulates TLR4 transcriptional activity. Thus, increasing the level of miR-135a may become a new method for treating many cardiac diseases.
This work was supported by Grants from the National Natural Science Foundation of China (81873486, 81770327), Natural Scientific Fund of Jiangsu Province (BK20161226), Jiangsu Province’s Key Provincial Talents Program (ZDRCA2016043), and Jiangsu Province’s 333 High-Level Talents Project (BRA2017539).
The authors declare no conflict of interest.
