MicroRNAs Involved in the Mitogen-Activated Protein Kinase Cascades Pathway During Glucose-Induced Cardiomyocyte Hypertrophy

Animal Studies

Experiments were performed using age- and weight-matched C57/BL6 male mice (8 weeks old, 23 to 25 g body weight; Sippr-bk Lab Animal Ltd., Shanghai, China). Before and during treatment, all animals were housed in a climate-controlled room with a 12-hour light/dark cycle. Animals were fed a standard diet and allowed tap water ad libitum. All procedures were performed in accordance with National Institutes of Health (NIH) guidelines (no. 85-23, revised 1996) for the care and use of experimental animals.

Diabetes was induced by a single injection of streptozotocin (STZ, 150 mg/kg i.p) dissolved in 0.1 mol/L citrate buffer, pH 4.3 (Sigma, St. Louis, MO). Nondiabetic control mice received the citrate buffer only. Three day after the injection of STZ or vehicle, blood glucose levels were measured using the Optium Xceed Diabetes Monitoring System (Abbott Diabetes Care, Alameda, CA). Blood glucose content of ≥18.6 mmol/L was chosen as indicating diabetes in the present study.¹⁷

Cell Culture

Primary neonatal rat myocytes were isolated from newborn Sprague-Dawley rat heart ventricles by collagenase digestion and cultured as described previously.¹⁴ In brief, isolated cardiomyocytes were plated onto cell culture dishes at a density of 3.0 × 10⁴ cells/cm² and were maintained for 24 hours in Dulbecco's modified Eagle's medium (DMEM). The cells were serum starved 24 hours before use for the experiments. All experiments were performed after 48 hours of incubation with 5.5 mmol/L (control; NG) or 25 mmol/L D-glucose (high glucose; HG). A 25-mmol/L quantity of L-glucose was used as an osmotic control. The experiments were repeated with at least three different cardiomyocyte cultures.

In the experiments, to inhibit p38 MAPK, ERK1/2, and JNK, cardiomyocytes were pretreated with or without 5 μmol/L SB-203580, PD-98059 or SP-600125 for 30 minutes and then exposed to 5.5 or 25 mmol/L glucose for 48 hours.

Cardiac Hypertrophy and Cardiomyocyte Cell Size Analysis

At 8 weeks after diabetes was induced, the left ventricle was rinsed in PBS buffer and weighted to calculate the ratio of heart weight to body weight (HW/BW). Cardiac hypertrophy was analyzed by histology. Briefly, LV samples were fixed in 4% paraformaldehyde for 12 hours at room temperature and embedded in paraffin. After that, 5-μm-thick sections were stained with hematoxylin and eosin (H&E) and cardiomyocyte size was measured using NIH ImageJ software on micrographs. Approximately 100 randomly chosen cardiomyocytes from each group (n = 5) were analyzed to measure the cross-sectional cardiomyocyte area.^18,19 Cardiomyocyte cell surface area was determined to assess cellular hypertrophy.¹⁶ Cells were visualized with a Nikon (Tokyo, Japan) microscope and images were captured at 40× magnification. At least 100 cells from randomly selected fields in one well were examined.

Real-Time RT-PCR

Total RNA was extracted from mouse LV tissues using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. Only the highly pure RNAs (ratio of 260/280 > 1.8 and 260/230 > 1.8) were used for downstream assay. Real-time RT-PCR for atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed using the primers as follows: ANP (GenBank number: NM_008725.2) forward, 5′- TGACAGGATTGGAGCCCAGAG-3′; reverse, 5′- AGCTGCGTGACACACCACAAG-3′; BNP (GenBank number: NM_008726.4) forward, 5′-ATCGGATCCGTCAGTCGTTTG-3′; reverse, 5′-CCAGGCAGAGTCAGAAACTGGAG-3′; β-MHC (GenBank number: NM_080728.2) forward, 5′-TAACCCGAGGCAAGCTCACA-3′; reverse, 5′-CACAATCATGCCGTGCTGAC-3′; GAPDH (GenBank number: NM_008084.2) forward, 5′- TGTGTCCGTCGTGGATCTGA-3′; reverse, 5′-TTGCTGTTGAAGTCGCAGGAG-3′. The neonatal rat cardiomyocytes were cultured in DMEM. miR-373 levels were detected by real-time quantitative PCR. The sequences of primers, which were designed for miR-373 and U6 (for normalization), to analyze their expression, were as follows: miR-373 stem-loop primer 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACACC-3′; miR-373 forward, 5′-GTGCTTCGATTTTGGGG-3′; miR-373 reverse, 5′-GTGCAGGGTCCGAGGT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′; and U6 reverse transcript and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. Relative gene expression levels were calculated by the 2^−ΔΔCT method.²⁰

Microarray Analysis and Target Prediction

miRNAs were purified from total RNA by using the mirVana miRNA Isolation Kit (Ambion, Austin, TX). Purification of miRNA from three cardiac tissues in each group was labeled and used for miRNA expression analysis by using the CapitalBio Mammalian miRNA Array V4.0 (CapitalBio Corp.) containing 1320 probes including human, mouse, and rat mature miRNAs. Differentially regulated miRNAs were defined as those with either _0.5- or 1.5-fold changes in expression for both arrays from diabetic or nondiabetic mouse heart. The normalized microarray data were validated by using real-time PCR. The commonly used and accurate Sanger miRNAs database (http://microrna.sanger.ac.uk) was used to predict potential miRNA targets. The top 25% of miRNA targets that had been assigned the highest numbers of miRNAs interaction sites were collected.

GO Category and Pathway Analysis

The top 25% of miRNA targets were subjected to GO term analysis, which was applied to organize genes into hierarchical categories on the basis of biological process, molecular function, and subcellular localization.²¹ Generally, a two-sided Fisher's exact test and χ² test were used to classify the GO category, and the false discovery rate (FDR) was calculated to correct the P value. We computed P values for all of the different genes in all GO biological categories; the threshold of significance was defined as P < 0.01 and FDR < 0.05. Enrichment provides a measure of the significance of the function: As the enrichment increases, the corresponding function is more significant. Within the significant category, the enrichment Re was given by the following: Re (n/n)/(N / N), where n_f is the number of differential genes within the particular category, n is the total number of genes within the same category, N_f is the number of differential genes in the entire microarray, and N is the total number of genes in the microarray.

Meanwhile, the top 25% of miRNA targets were collected, and subjected to KEGG pathway annotation. Similarly, pathway analysis was used to determine the significant pathway of the differential genes. Fisher's exact test and χ² test were used to select t significant pathway, and the threshold of significance was defined as P < 0.01 and FDR < 0.05. The enrichment Re was calculated like the equation above. Furthermore, the network of miRNA-mRNA interaction, representing the critical miRNAs and their targets, was established based on the integration of GO function and KEGG analysis findings.

miRNA Mimic Transfection

The primary neonatal rat cardiomyocytes were seeded in antibiotic-free medium for 24 hours before transfection. Lipofectamine 2000 (Invitrogen) was used to transfect cells with miR-373 (20 nmol/L) mimic (Dharmacon RNA Technologies) for 48 hours according to the manufacturer's instructions. miRIDIAN miRNA mimic negative controls (Dharmacon RNA Technologies) were used for control transfections. Cells were collected after 48 hours of miRNA mimic transfections for quantification of miRNA and protein expression.

Western Blot Analysis Significant Pathways

Total proteins isolated from LV tissues or cultured cardiomyocytes were prepared by standard procedures and quantified by the bicinchoninic acid (BCA) protein assay (Pierce, USA). A total of 30 μg protein in each sample was subjected to 10% SDS-PAGE gel by electrotransfer onto polyvinylidene difluoride membranes (Millipore, USA). The expression or phosphorylation of p38, ERK1/2, and JNK were determined by using specific antibodies against total-p38, -ERK1/2, -JNK and phospho-p38, -ERK1/2, and -JNK, respectively. The primary antibody against MEF2C was used to analyze MEF2C protein expression. After scanning, results were quantified by the NIH ImageJ program.

Statistical Analysis

All experimental data are expressed as mean ± SD and were analyzed by Wilcoxon Mann–Whitney method and Kruskal–Wallis test. A P value of 0.05 or less was considered significant.

Hyperglycemia Induces Cardiac Hypertrophy and Neonatal Rat Cardiomyocyte Hypertrophy

At 72 hours after STZ injection, blood glucose was elevated to 24.0 ± 3.0 mmol/L in diabetic mice versus 8.3 ± 0.6 mmol/L in the C57BL6 controls. Throughout the 8-week study period, mice with STZ-induced diabetes displayed severe hyperglycemia. At the end of 8 weeks after diabetes was induced, increased LVHW/BW ratios were observed in diabetic mice (Figure 1A). At the same time, histological analysis showed that the cross-sectional area of cardiomyocytes increased in the diabetic heart (Figure 1, B and C). Furthermore, ANP, BNP, and β-MHC, markers of cardiac hypertrophy, were upregulated in diabetic hearts as assessed by real-time RT-PCR (Figure 1D). Further in vitro investigation showed that high glucose increased the size of cultured cardiomyocytes exposed to 25 mmol/L glucose for 48 hours (Figure 1E).

Altered Expression of miRNAs and Their Target Genes in the Diabetic Heart

Using miRNA microarray analysis, we evaluated miRNA expression profiles of three diabetic heart samples and three nondiabetic heart samples (fold change: _0.5- or 1.5; false discovery rate: ≤0.05). We identified 19 miRNAs that were deregulated greater than twofold, and 16 were further verified by real-time PCR. Overall, miR-195, miR-199a-3p, miR-700, miR-142-3p, miR-24, miR-21, miR-221, miR-499-3p, miR-208a, and miR-705 have often been found to be upregulated, whereas miR-29, miR-1, miR-373, miR-143, miR-20a, and miR-220b have been found to be down-regulated in STZ-induced diabetes when compared with controls. By using the Sanger database, 3212 target genes of the 16 changed miRNAs were collected.

GO Function and KEGG Pathway Enrichments

To gain a better understanding of the functional roles of the predicted miRNAs target genes and related regulatory network, we looked for target enrichment in GO and KEGG. On the basis of the top 25% miRNA targets, 31 GO terms were found to be involved in the diabetic cardiomyopathy (Table 1). The five high-enrichment GOs were insulin-like growth factor receptor signaling, JNK cascade, leukocyte adhesive activation, transmembrane receptor protein serine/threonine kinase signaling, MAPKKK cascade, and collagen fibril organization. They were involved in proliferative, metabolic, extracellular matrix, adhesive, apoptosis, signal transduction, and heart physiology. KEGG pathway analysis of the top 25% of the predicted targets that were assigned the highest numbers of miRNA target sites revealed 26 KEGG pathways enriched in diabetic cardiomyopathy (Figure 2). Many of these signaling pathways, such as MAPK, Wnt, TGF-β, VEGF, JAK/STAT, and mTOR have been shown to participate in the development of cardiac hypertrophy (Table 2). Among the list of high-enrichment KEGG pathways of these miRNA-targeted genes, MAPK signaling pathway was significantly prominent. A similar phenomenon was also observed in GO analysis.

In addition to providing the interactions of miRNAs and target genes, the common targets of GO analysis and KEGG pathway were filtered out, miRNAs–gene network analysis integrated changed miRNAs, and 192 critical mRNAs were further generated (Figure 3). Intriguingly, the most overrepresented miRNA targets belonged to the MAPK signaling pathway (Figure 4), which is known to be involved in the hypertrophic growth in response to overpressure by affecting the activities of the three best-characterized MAPKs including extracellular signal-regulated kinase (ERK1/2), c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38). These pathway analyses illustrate some of the possible roles of the highly expressed miRNAs in cardiac hypertrophy of diabetic mice.

Experimental Verification the MAPK Signaling Pathway

To further investigate whether MAPK signaling pathway mediates cardiomyocyte hypertrophy induced by hyperglycemia, we investigated the role of JNK, ERK1/2, and p38 MAPK in the heart in diabetes and in the cardiomyocytes exposed high glucose. Our results showed that diabetes in the heart causes significant upregulation of JNK, ERK1/2, and p38 (Figure 5A) in association with morphometrically demonstrable cardiomyocyte hypertrophy and ANP, BNP, and β-MHC upregulation (Figure 1). Exposure of cardiomyocytes to 25 mmol/L glucose for 48 hours showed similar upregulation of JNK, ERK1/2, and p38 MAPK protein levels compared with 5.5 mmol/L glucose (NG) and 25 mmol/L L-glucose (Figure 5B).

miR-373 Mediates Glucose-Induced Cardiomyocyte Hypertrophy via Target Gene MEF2C

To gain insight into the possible functional roles of miRNAs in diabetic cardiomyopathy, miR-373 levels in neonatal rat cardiomyocytes were modulated by transfecting the cells with miRIDIAN miR-373 mimic. A negative control miRNAs mimic was used as control. Transfection with miR-373 mimic in glucose-exposed cells significantly increased the expression of miR-373 and decreased the cell size as measured by cell surface area (Figure 6, A and B). We also observed that the protein level of MEF2C was reduced when overexpressing miR-373 (Figure 6C). The results suggested that MEF2C was a target gene of miR-373.

miR-373 Down-Regulation Is Dependent on p38 MAPK Pathway

To understand the relationship between miR-373 and MAPK signaling pathway in cardiac myocytes, activation of p38 MAPK, ERK1/2, and JNK was evaluated by Western blot. As shown in Figure 7, high glucose significantly increased the phosphorylation levels of p38 MAPK, ERK1/2, and JNK, but miR-373 mimic had no effect on phospho-p38 MAPK, phospho-ERK1/2, and phospho-JNK expression. To further explore the functional role of p38 MAPK, ERK1/2, and JNK, we used SB-203580, PD-98059, and SP-600125 to test whether inhibition of p38 MAPK, ERK1/2, and JNK could influence the effect of miR-373 on cardiac hypertrophy and MEF2C expression. Of note, a p38 MAP kinase inhibitor SB203580 significantly decreased miR-373 levels and inhibited high-glucose–induced hypertrophy (Figure 8, A–C), whereas PD98059 or SP600125 had no effect on miR-373 levels and cell size (data not shown). Furthermore, administration of SB-203580 led to a reduction in MEF2C levels compared with those in the HG group (Figure 8D).