Up-regulation of miR-208a aggravates high-fat -diet-induced cardiomyocytes injury by targeting IRS-2/PI3K/AKT pathway

doi:10.21203/rs.3.rs-2375837/v1

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Up-regulation of miR-208a aggravates high-fat -diet-induced cardiomyocytes injury by targeting IRS-2/PI3K/AKT pathway

https://doi.org/10.21203/rs.3.rs-2375837/v1

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Background: Previous studies found that the myocardial function is damaged in obese mice induced by a high-fat diet, with the increased miRNA-208a and decreased IRS-2.

Method: Herein, miR-208a(-/-) knockout and C57BL/6J wild-type mice were divided into the normal diet and high‑fat diet groups, separately. Expressions of p-IRS-2, PI3K, p-AKT, NPPA, AKT, IRS-2, MYH6, MYH7 and Glut4 were determined using immunohistochemical staining. Furthermore, western blotting was performed to identify proteins.

Cardiomyocytes were isolated from neonatal rats, and the cardiomyocytes injury model was induced by PA. CCK-8 was conducted to assess the viability of cardiomyocytes, and the glucose uptake of cardiomyocytes was measured using glucose uptake test.

Result: H&E staining revealed that the degree of hypertrophy and swelling of cardiomyocytes and cardiac fibrosis of miR208a-/- knockout mice in the high-fat diet group was significantly increased. Moreover, high levels of IRS-2, p-AKT, AKT, NPPA ,GLUT4, MYH7 and MYH6 expression in miR208a-/- knockout mice. Therefore, AKT, MYH6, IRS-2, NPPA in miR208a-/- knockout mice of high-fat diet.

In PA-treated cardiomyocytes, the viability was markedly declined and glucose uptake was significantly reduced, while the lipid accumulation and fibrosis were significantly enhanced. Most importantly, the cardiomyocytes injury induced by PA was aggravated in miR-208a-3p upregulated cardiomyocytes, but alleviated in miR-208a-3pdownregulated cardiomyocytes.

Conclusion: Our results indicate that the high expression of miR-208a promote the suppression of IRS-2/PI3K/AKT signaling pathway. Meanwhile, miR-208a-3p contributes to promoting PA-induced cardiomyocyte injury, which may be a biomarker of cardiomyocyte injury in lipotoxic cardiomyopathy.

miR-208a-3p

miRNAs

palmitic acid

lipotoxic cardiomyopathy

cardiomyocyte injury

high-fat diet

Cardiovascular disease is a serious threat to human health, especially the elderly over 65 years old ¹. Recently, the cardiovascular complications caused by diabetes have attracted more and more attention. Wherein, lipotoxic cardiomyopathy caused by glucose metabolism disorders has become a health problem of public concerns ^2–4, and its pathogenesis may be due to the accumulation of toxic lipids ⁵. Lipid toxicity can result in oxidative damage and apoptosis of cardiomyocytes, aggravate diastolic and systolic dysfunction, and then induce and aggravate diabetic cardiomyopathies ^6,7. Hence, it is necessary to exhaustively understand the mechanism and find biomarkers to retard cardiomyocytes injury for the lipotoxic cardiomyopathy therapy.

To our knowledge, the key factors of lipid metabolism changes are the increase of free fatty acids (FFAs) in blood and the disorder of lipid metabolism regulation ⁸. Palmitic acid (PA), which is the most abundant FFAs in plasma, is also one of the main fatty acids involved in lipotoxic myocardial injury ⁹. Excessive PA is associated with the apoptosis induction in multiple cell types, e.g. cardiomyocytes ¹⁰. Of note, PA induced lipotoxicity can lead to H9c2 cardiomyocyte damage, which can increase the risk of cardiovascular disease. Therefore, PA can be used to establish myocardial lipid toxicity model ¹¹.

MicroRNAs (miRNAs), a class of small noncoding RNA molecules, are widely exists in eukaryotes ¹². It is evolutionarily conservative, which functions by combining with the 3 'untranslated region (3'UTR) of the target mRNA ¹³. miRNAs function in the occurrence and development of cardiovascular diseases, which is also one of the hotspots of current research ¹⁴. Numerous researches have presented that miRNAs regulate cardiomyocyte differentiation, migration, proliferation and apoptosis ^15–17. Therefore, miRNAs may bring new strategies for the diagnosis and treatment of cardiovascular diseases ¹⁸. MiR-208 is specifically expressed exclusively in cardiomyocytes and it is an essential regulator of cardiovascular disease ¹⁹. The miR-208 family including miR-499 and miR-208a/b. MiR-208a was encoded by an intron of the α-cardiac muscle myosin heavy chain gene (MYH6) and expressed only in the heart ²⁰. Meanwhile, miR-208b which was encoded by an intron of the β-cardiac muscle myosin heavy chain gene (MYH7), expressed in cardiac and skeletal muscles ²¹. A previous study has revealed that miR-208 was up-regulated in the obese mice induced by high fat diet, whereas the insulin resistance and glucose tolerance were significantly improved when miR-208 was down-regulated ²². In addition, another recent clinical study identified that myocardial fibrosis and remodeling were statistically improved in miR-208a knockout mice ²³. Interestingly, IRS-2 (insulin receptor substrate 2) is one of the target genes of miR-208. The insulin receptor substrate(IRS)/phosphatidylinositol-3-kinases (PI3K)/protein kinase (AKT) regulatory pathway is the classical insulin signaling pathway and plays a vital role in insulin metabolism. Therefore, the inhibition of IRS-2/PI3K/AKT pathway could lead to insulin resistance (IR) and glucose metabolism disorder ²⁴, and targeting the IRS-2/PI3K/AKT signalling pathways is emerging as an important contributor to the treatment of metabolic diseases ³⁵. However, the role and molecular mechanism of miR-208a on cardiac function in obese mice induced by high-fat diet remain unclear and how miR-208a plays a role in PA-induced cardiomyocyte injury has not yet been elucidated. Moreover, whether the abnormal cardiac function in obese mice induced by high-fat diet is closely related with miRNA-208a via its downstream IRS-2/PI3K/AKT pathway needs to be further proved.

In this study, we will discuss the role and mechanism of the miRNA-208a on cardiac function through the models including obese mice induced by high-fat diet, the miR-208(-/-) mice, and primary myocardial cells. This study truely have very important significances for the further explore of the mechanism of cardiac dysfunction and prevention in patients with obesity from the cadiac diseases.

In Vivo Study Designs

Animal Experiments

All animal experimental procedures were were performed as directed by the ARRIVE Guidelines and approved by the Ethics Committee of the affiliated Shandong Provincial Hospital of Shandong First Medical University and were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals ²⁶.

The C57BL/6J wild-type mice and miR208a-/+ knockout mice weighing 25–35 g were purchased from Cyagen Biosciences (Suzhou, China). The animals were maintained at 22 ± 2℃, 40–70% relative humidity, and a 12-h light/dark cycle. Water and food was freely available to the mice. Body weight, body length, waistline and food/water consumption were monitored weekly. Homozygous MiR208a-/- knockout mice were obtained by intercrosses of heterozygous miR208a-/+ knockout mice. The genotypes of the offspring mice were identified by RT-PCR. Anaesthesia with isoflurane was used before sampling. A solution of lidocaine and adrenaline was applied locally after blood withdrawal and before mice were allowed to wake up. Blood samples were obtained in heparin lithium tubes. The mice were randomly divided into the following five groups: I) Control (The C57BL/6J wild-type mice of normal diet )(n = 9); II) HFD (The C57BL/6J wild-type mice of highfat diet (HFD)) (n = 12); and III) miR208a-/- mice of normal diet(n = 6); IV) miR208a-/- mice of highfat diet group with saline water intraperitoneal injection group (n = 13); V) miR208a-/- mice of HFD group with omagirn intraperitoneal injection (200 mg/kg)(n = 6). All mice except for mice in the I, III group were fed on a HFD for 20 weeks consecutively. The composition of HFD was 59% fat, 24% carbohydrate and 17% protein, while the normal chow diet consisted of 10% fat. The mice were fed for 20 weeks and the initiation of the experimental modeling was done. Then the mice were sacrifificed by cervical dislocation. Heart tissues were isolated and then were flashfrozen in liquid nitrogen and stored at 80˚C for histological, RNA and protein analysis.

Heart Sampling And Histopathology Morphological Analysis

Mice heart tissues were fixed in paraformaldehyde ( G1101, Servicebio, Wuhan, China) for 4 h, then fixed in 1% osmic acid for 1 h, dehydrated in a graded series of ethanol, and embedded in paraffin. The heart sections (70 nm thick) were obtained using a microtome.The remaining heart sections were then stained using haematoxylin-eosin (H&E) staining and viewed under the microscope (IX-53,OLYMPUS, Japan).

Hematoxylin And Eosin (H & E) Staining

The paraffin-embedded sections were routinely dewaxed and hydrated. Then, the sections were dewaxed using xylene I and II (each for 10 min), and rehydrated in gradient ethanol (100%, 100%,95%, 90%, 80%, and 70%, 5 min for each concentration). After hydration, the sections were conventionally stained with HE (MB9898, Meilunbio, Dalian, China). The sections were then baked, dewaxed, and hydrated followed by staining in hematoxylin for 8 min. Afterword, sections were differentiated in 1% hydrochloric acid solution for 10 s, followed by treatment in bluing solution (0.6% ammonia) for 30 s. After that, the sections were stained with eosin for 3 min and processed for microscopic examination of the morphology of cardiomyocyte. Then dehydrated using gradient alcohol, cleared using xylene, and sealed with neutral gum. The cardiomyocyte of mice was observed under an optical microscope.

Immunohistochemical Staining

The paraffin sections were dewaxed in xylene and rehydrated in gradient alcohol. After blocking in hydrogen-peroxide-solution, sections were incubated with primary antibodies against GLUT4 Mouse Monoclonal antibody(66846-1-Ig, Proteintech), PI3K p85 alpha mouse monoclonal antibody (60225-1-Ig, Proteintech), MYH6 Rabbit Polyclonal antibody (22281-1-AP, Proteintech), MYH7-specific Rabbit Polyclonal antibody (22280-1-AP, Proteintech), NPPA Rabbit Polyclonal antibody (27426-1-AP, Proteintech), Phospho-IRS-2 (Ser 1100) Ab (AF-8383, Proteintech), beta Actin Antibody (AB0035), Abways, AKT Rabbit Polyclonal antibody (10176-2-AP, Proteintech), AKT-phospho-S473 Mouse Monoclonal antibody (66444-1-Ig, Proteintech), IRS-2 Rabbit pAb (A7945, Abclonal) for 1 hour at room temperature. p-IRS-2, PI3K, p-AKT, NPPA, AKT, IRS-2, MYH6, MYH7, and Glut4 expressions were visualised using the diaminobenzidine liquid (DAB, brown colour, ZSGB-BIO, dehydration with alcohol and sealed with neutral balsam). Images of representative fields were captured using Caseviewer 2.0 (Danjier, Inc. China). And the optical density was quantified using the ImageJ (NIH, Bethesda, MD) software.

Western Blot Analysis

The total RNA was extracted from the heart tissues using the RNA-easyTM Isolation Reagent(R701, Vazyme). Equal amounts (25 µg protein/lane) of lysates were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked for 1 hr in using 5% non-fat milk in Tris-buffered saline with Tween (TBST) and incubated overnight at 4℃ with the GLUT4 Mouse Monoclonal antibody (66846-1-Ig, Proteintech), PI3K p85 alpha Mouse Monoclonal antibody (60225-1-Ig, Proteintech), MYH6 Rabbit Polyclonal antibody (22281-1-AP, Proteintech), MYH7-specific Rabbit Polyclonal antibody (22280-1-AP, Proteintech), NPPA Rabbit Polyclonal antibody (27426-1-AP, Proteintech), Phospho-IRS-2 (Ser 1100) Ab (AF-8383, Proteintech), beta Actin Antibody (AB0035, Abways), AKT Rabbit Polyclonal antibody (10176-2-AP, Proteintech), AKT-phospho-S473 Mouse Monoclonal antibody (66444-1-Ig, Proteintech), IRS-2 Rabbit pAb (A7945, Abclonal). The membranes were washed thrice by TBST and incubated with secondary antibodies (1:10000) for 1 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence autoradiography by MiniChemi 610 (Saizhi Venture Technology Co., Ltd.,Beijing,China). β-actin was used as the loading control for total protein. Quantification of bands was performed using the ImageJ (NIH, Bethesda, MD) software.

In Vitro Study Designs

Separation And Culture Of Neonatal Rat Cardiomyocytes

Neonatal rats (1–3 days) were obtained from the shanghai SLAC laboratory animal CO. LTD. The rats killed by cervical dislocation and the hearts were taken out by thoracotomy. The apical tissues were cut and washed in PBS to remove the residual blood in the cardiac cavity. Then, the above tissues were cut into pieces and digested with trypsin. The cell suspension was put into the pre-cooled DMEM medium (contains 10% fetal bovine serum, FBS) to terminate digestion, and then the cell suspension was drawn again for trypsin digestion. Totally, repeat these steps 3 times. The cell suspension obtained from each digestion was inoculated into a culture dish and incubated at 37 ℃ with 5% CO₂ incubator for 2 h. The not adherent cell suspension was aspirated and filtered with a 0.22 µm filter. After centrifugation for 5 min, the precipitates were re-suspended in DMEM medium contains 10% FBS. Non-adherent cells were gathered and cultured as primary cardiomyocytes for subsequent experiments. After that, cardiomyocytes was cultured in DMEM medium contains 10% FBS and 200 µM oleic acid for 2h, which then stimulated with 0, 2, 20, 200, 400 µM PA for 24h, respectively. CCK8 assay was used to determine the optimal concentration.

Cardiomyocytes transfection

MiR-208a-3p agomir, antagomir and negative control miRNAs were synthetized by GenePharma (Shanghai, China). Cardiomyocytes were transfected with miR-208a-3p agomir / antagomir to up-regulate/down-regulate miR-208a-3p by Lipofectamine 2000 (Invitrogen, USA) based on the supplier's protocol and previously description ²⁷.

Cell-Counting Kit-8 (CCK-8) assay

Cell viability was assessed by CCK-8 assay as previously described ²⁸. The microplate reader was utilized to determine the optical density (OD) value at 450 nm wavelength, which reflected the cell viability.

2.4 Quantitative reverse transcriptase-PCR (qRT-PCR)

Trizol reagent (Invitrogen, USA) was applied to separate the total RNA from cardiomyocytes as directed by manufacturer. All procedures were conducted based on previously described ²⁹. qRT-PCR was carried out to measure the level of miR-208a-3p using SYBR Premix Ex TaqTM (Takara, Japan). Real-time PCR was performed with quantitative PCR. All data was normalized to U6.

2.5 Oil red O staining

The lipid droplet formation in cardiomyocytes was detected by oil red O staining following a previously reported method ²⁷.

Immunofluorescence Analysis

Neonatal rat cardiomyocytes were fixed with 4% paraformaldehyde for 15 min and then blocked in 5% bovine serum albumin for 30 min. The cells were then incubated overnight with anti-alpha-SMA (α-SMA, Santa Cruz biotechnology, California, USA) at 4°C, followed by washing with PBS. Then DyLight™ 488 conjugated secondary antibodies (Thermo Scientific) were used to culture cardiomyocytes for 1h. The images were taken under the Olympus fluorescence microscope (Zeiss, New York, New York).

2.7 Glucose Uptake Assay

Glucose uptake fluorometric assay kit (MAK084, Sigma-Aldrich) was utilized to measure the glucose uptake by cardiomyocytes. In short, sorted cardiomyocytes were seeded at 1×10⁵ cells/well in a 96 well plate and starved in 100µL of serum-free medium for 4h. After washing with PBS, cells were plated with 100µL of KRPH buffer containing 2% BSA for 40min to make glucose-starve. Then, the cells were treated with or without insulin (1µM) for 20min, followed by culturing for 20min with 10µL of 10mM 2-DG. Following incubation, cells were lysed with 80µL of extraction buffer and freezed in dry ice, which then heated for 40min at 85°C. Cool cell lysate on ice for 5min and then neutralize by adding 10µL of neutralization buffer. Briefly spin down at 13,000×g to remove insoluble material. 50µl supernatant was mixed with 50µL of the Master Reaction Mix to each of the wells and incubated for 40minutes at 37°C. Fluroscence intensity was quantified at λ ex = 535/λ em = 587nm.

Statistical analysis

All assays in this work were repeated 3 times and the data was expressed at mean ± SD. All data were analyzed by SPSS 22.0 and GraphPad Prism software (Ver 8.0) statistical analysis software, and the comparison were carried out by student’s t-test (two groups) or one-way ANOVA variance analysis with post-test of Tukey’s test (three groups and above). The difference was considered to be statistically significance at p < 0.05.

Effect of HFD on pathological changes in the heart tissues of miR208a-/- knockout mice

Morphological changes in the heart are shown in Figure. 1A. In electron microscopy observations, the heart tissue in the wild mice demonstrated organelles with normal morphology, clear structure, and abundant mitochondria. Compared with the Control group, HFD treatment increased the hypertrophy and swelling of cadiomyocytes. In miR208a-/- knockout mice groups, H&E staining demonstrated that the arrangement of myocardial fibers appeared almost regular, but the cadiomyocytes was hypertrophy and swelling. Moreover, compared with the wild mice groups, the arrangement of myocardial fibers in miR208a-/- knockout mice was disordered, the intracellular substance was not clear, and the cells were hypertrophy and swelling, especially in group IV. Moreover, the survival probability was significantly decreased in HFD group(Figure. 1B). Meanwhile, as presented in Figure. 1B, the survival probability of miR208a-/- knockout mice groups was lower than that of the wild mice groups.

Effects of HFD on p-IRS-2, IRS-2, PI3K, p-AKT, AKT, NPPA, MYH7 and MYH6 protein immunohistochemical staining in the heart

In miR208a-/- knockout mice, IRS-2, p-AKT, AKT, NPPA, GLUT4, MYH7 and MYH6 indicated heavy yellow staining of heart tissue was uniformly distributed throughout the cytoplasmic areas of the cardiocytocytes, indicating high levels of IRS-2, p-AKT, AKT, NPPA ,GLUT4, MYH7 and MYH6 expression (all p < 0.05). However, in cardicmyocytes from the miR208a-/- knockout mice, the protein expression levels of p-IRS-2 were significantly lower than those observed in control group, with fewer brown-yellow cells (all p < 0.05, Figure. 2C). In addition, compared with the model normal diet group, the IRS-2, MYH7 ( Figure. 2A) and MYH6 ( Figure. 2B) expression levels were markedly elevated in the HFD treated groups.

Protein Expression Of Irs-2, Pi3k,akt, Nppa, Myh7 And Myh6

The results of western blot showed that compared with the Control group, HFD group presented a significant increase in the protein levels of PI3K, MYH7, NPPA, but a decrease in the protein levels of IRS-2, AKT,and MYH6 (Figure. 2). Compared with control group, miR208a-/- knockout mice group showed a faintly increase in the protein levels of PI3K, AKT, NPPA, but a decrease in the protein levels of IRS-2, MYH7(all p < 0.05). Among the HFD groups, miR208a-/- knockout mice group showed a significant decrease in the protein levels of PI3K, MYH7, but an increase in the protein levels of AKT, MYH6, IRS-2, NPPA (all p < 0.05), and the omagirn intraperitoneal injection group showed a significantly decrease in the protein levels of PI3K, AKT, MYH7, NPPA, and an increase in the IRS-2, compared to the saline water intraperitoneal injection group(all p < 0.05). At the same time, among miR208a-/- knockout mice groups, protein expression levels of PI3K,MYH6, MYH7, NPPA exhibited slight increases in the heart tissues of mice in the HFD group compared with those in the normal diet group, whilst an opposite trend was observed in IRS-2 and AKT expression. Interestingly, after the omagirn intraperitoneal injection, the HFD groups presented a significant decrease in the protein levels of PI3K and MYH6, MYH7, NPPA (all p < 0.05). It suggested that low expression of miR-208a could further inhibit the increase expression of IRS-2 and PI3K, miR-208a expression could increase AKT expression in myocardial cells after HFD.

Screen the optimal concentration of PA for modeling.

Firstly, the cardiomyocytes were successfully isolated from neonatal rat (Figure.5A), which laid a foundation for our subsequent experiments. As presented in Figure.5B, with the increase of PA concentration, the cell viability gradually decreased. At the concentration of 200µM, the cell viability decreased significantly compared with the Vehicle group. The viability was lower at 400µM, but it was not significantly different from the 200µM group. So we choose the PA concentration of 200µM as the best concentration for modeling. These data indicated that PA stimulation has an effect on cardiomyocytes viability and may cause injury to cardiomyocytes.

PA induces miR-208a-3p up-regulation as well as lipogenesis and fibrosis in cardiomyocytes.

To detect the level of miR-208a-3p in PA-stimulated cardiomyocytes, qRT-PCR was performed. The results was displayed in Figure.6A that miR-208a-3p mRNA level was markedly increased in PA (200µM)-stimulated cardiomyocytes than that in control group. Then, we examined the impact of PA on cardiomyocyte phenotype. Oil red O staining revealed that the PA-stimulated cardiomyocytes had a large amount of lipid accumulation compared with the control group (Figure.6B). The results obtained from immunofluorescence staining demonstrated that the α-SMA expression increased significantly in PA-stimulated cardiomyocytes (Figure.6C). Since the accumulation of lipid in the heart may affect the normal function of cardiomyocytes ³⁰, and α-SMA was a marker of myocardial fibrosis ³¹, these findings suggested that the stimulation of PA resulted in cardiomyocyte injury, and up-regulation of miR-208a-3p may be participated in this process.

Up-regulation of miR-208a-3p contributes to aggravating cardiomyocyte injury.

PA treatment markedly restrained the viability of cardiomyocyte compared with vehicle group, while up-regulation of miR-208a-3p aggravated the inhibitory effect (Figure.7A). On the contrary, suppression of miR-208a-3p reversed the reduction of cardiomyocytes viability induced by PA. Also, the glucose uptake assay presented the similar trends (Figure.7B). Compared with the vehicle group, glucose uptake in PA stimulated cardiomyocytes decreased significantly. Importantly, the PA-induced decrease of glucose uptake was further declined in miR-208a-3p up-regulated cells, but reversed in miR-208a-3p down-regulated cells.

miR-208a-3p overexpression by transfecting agomir into PA-stimulated cardiomyocytes can markedly enhance the lipid deposition induced by PA, while inhibition of miR-208a-3p showed an elimination effect on lipogenesis in PA-stimulated cardiomyocytes (Figure.8A). Consistently, α-SMA expression was clearly enhanced after PA-stimulated cardiomyocytes transfected with agomir, but markedly declined after down-regulation of miR-208a-3p (Figure. 8B). All these data in this chapter revealed that miR-208a-3p aggravates PA-induced cardiomyocytes injury.

In this present work, we have demonstrated for the first time that miR-208a alleviates mice cardiac injury induced by high-fat-diet. The mechanism for this protective effect of miR-208a appeared to be related repressed IRS-2/PI3K/AKT signaling pathway. These results suggest that miR-208a plays an important role in modulating myocardiac function in vivo in obesity. H&E staining revealed that the degree of hypertrophy and swelling of cardiomyocytes and cardiac fibrosis of miR208a-/- knockout mice in the HFD group was significantly increased than the control group. To further investigate the function of miR-208a in HFD mice, we performed a loss of function study by injecting miR-208a omagirn into the miR208a-/- knockout mice. Then, we conducted immunohistochemistry staining to evaluate the expressions of several key proteins from each group. We observed that high levels of IRS-2, p-AKT, AKT, NPPA ,GLUT4, MYH7 and MYH6 expression in miR208a-/- knockout mice. Consistent with this finding, western blot results also showed increased protein levels of AKT, MYH6, IRS-2, NPPA in miR208a-/- knockout miceof HFD group. A downstream target gene of IRS-2, was found to be slightly reduced in the heart tissue of mice in the HFD group compared with that in the control group. When insulin binds to insulin receptor (InsR) by membrane, IRS/PI3K/AKT mechanism is activated. Relative substrate protein family of activated InsR is mainly IRS-1/2 ³². IRS can activate PI3K inducing second messengers to stimulate downstream of AKT ³³. Uptake of circulating glucose through GLUT4 translocation from intracellular compartments to cell membrane would be induced by insulin-stimulated AKT ³⁴.

We also successfully constructed cardiomyocyte injury model with PA. In PA-treated cardiomyocytes, cell viability and glucose uptake were significantly decreased, and lipid accumulation and α-SMA expression were significantly increased. Importantly, miR-208a-3p was highly expressed in PA-treated cardiomyocytes. Furthermore, up-regulation of miR-208a-3p aggravated PA-induced cardiomyocyte injury, while, PA-induced cardiomyocyte injury was reversed in miR-208a-3p downregulated cells. Hence, we concluded that the cardiomyocyte injury induced by PA was at least partially achieved by up-regulating the miR-208a-3p expression.

As a specific marker of myofibroblasts, the abnormal expression of α-SMA is also participated in the change of cardiac function in lipotoxic cardiomyopathy ³⁵. Our data showed that after the stimulation of cardiomyocytes with PA, the glucose uptake of cardiomyocytes is significantly reduced, while the accumulation of lipids and the expression of α-SMA are both markedly increase. Combined with literature reports and our results, it is proved that PA can successfully induce cardiomyocytes injury model, which provides theoretical support for our subsequent experiments.

Mir-208a/b is considered to be cardiac specific miRNAs, which is involved in maintaining the function and survival of cardiomyocytes ³⁶. Previously, Oliveira-Carvalho V et al. revealed that the miR-208a expression is lower in the early stage of cardiac development, and mainly exists in the form of miR-208b. Soon after birth, the miR-208b level gradually decreases and the miR-208a expression increases, which plays a dominant role in the regulation of heart ³⁷. Our findings revealed that the mRNA level of miR-208a-3p was increased in PA stimulated cardiomyocytes. Most importantly, we revealed that up-regulation of miR-208a-3p aggravated the decrease of glucose uptake as well as the increase of lipid accumulation and myocardial fibrosis induced by PA, while inhibition of mir-208a-3p reversed PA induced myocardial injury. These findings revealed that miR-208a-3p can be used as a therapeutic target for cardiomyocytes injury in diabetic cardiomyopathy.

In summing, the present study successfully investigated the role of miR-208a in HFD induced heart injury. Our results indicated that the high expression of miR-208a promote the suppression of IRS-2/PI3K/AKT signaling pathway, thereby inducing the autophagy of cardiac myocytes, enhancing cardiomyocyte viability, and reducing apoptosis. We also revealed that PA can induce cardiomyocyte injury, and this effect can be rescued by inhibiting miR-208a-3p, which provides a biomarker and theoretical basis for molecular therapy of cardiomyocyte injury. MiR-208a is expected to be an important target for the treatment of cardiac diseases in obesity population and maybe the combined use of IRS-2/PI3K/AKT pathway activator can deliver better effect. However, the associated mechanism may be related to multiple targets or pathways, necessitating further confirmation.

Free fatty acids (FFAs); High-fat diets (HFD); Palmitic acid (PA); MicroRNAs (miRNAs); α-cardiac muscle myosin heavy chain gene (MYH6); β-cardiac muscle myosin heavy chain gene (MYH7); Insulin receptor substrate(IRS); Phosphatidylinositol-3-kinases (PI3K); Protein kinase (AKT); Insulin resistance (IR); Hematoxylin and Eosin (H & E); Quantitative reverse transcriptase-PCR (qRT-PCR).

Funding

This work was funded by Science and Technology Development Projects of Jinan City (201805062), and Natural Science Foundation of China (81800753).

Authors’ contributions

Ye Bi ,Yao W and Zhibin W performed the experiments. Ye Bi and Yan W analyzed the data. Ye Bi and XiangLan S wrote the manuscript. Ye Bi, Yao W, Yan W Zhibin W and XiangLan S reviewed and edited the manuscript. All authors read and approved the final manuscript.

Data Availability Statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics declarations

All animal experiments were performed as directed by the ARRIVE Guidelines and approved by the Ethics Committee of Shandong First Medical University. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

All authors have read and approved the content, and agree to submit for consideration for publication in the journal.

Competing interests

The author(s) declare no competing interests.

Acknowledgements

Not applicable.

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No competing interests reported.

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Up-regulation of miR-208a aggravates high-fat -diet-induced cardiomyocytes injury by targeting IRS-2/PI3K/AKT pathway

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Abstract

Figures

Background

Methods

Animal Experiments

Heart Sampling And Histopathology Morphological Analysis

Hematoxylin And Eosin (H & E) Staining

Immunohistochemical Staining

Western Blot Analysis

Separation And Culture Of Neonatal Rat Cardiomyocytes

Cardiomyocytes transfection

Cell-Counting Kit-8 (CCK-8) assay

2.4 Quantitative reverse transcriptase-PCR (qRT-PCR)

2.5 Oil red O staining

Immunofluorescence Analysis

2.7 Glucose Uptake Assay

Statistical analysis

Results

Effect of HFD on pathological changes in the heart tissues of miR208a-/- knockout mice

Protein Expression Of Irs-2, Pi3k,akt, Nppa, Myh7 And Myh6

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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