Neutralization of hexokinase 2-targeting miRNA attenuates the oxidative stress-induced cardiomyocyte apoptosis

2.1 Culture of rat cardiomyocytes and H₂O₂ treatment

The embryonic cardiomyocyte cell line H9c2 is commonly used to study heart disease including I/R injury [32]. H9c2 cell line (American Type Culture Collection) was cultured in high glucose-DMEM (GIBCO, Waltham, MA, USA) containing 10% FBS (Atlas Biologicals, Fort Collins, CO, USA) and 1% antibiotics (GIBCO). The cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂. For H₂O₂ treatment, 9M of stock H₂O₂ was added to the culture media at a final concentration of 100 μM to 600 μM.

2.2 Cell viability assay

To measure cell viability, CCK reagent (cell counting kit-8, Dojindo) was added to each well for a final concentration of 0.5 mg/mL and the cells were incubated at 37°C for 2 hours. The absorbance of the medium was measured at 450 nm using a microplate reader.

2.3 PI/Annexin V double staining for apoptosis

To determine the apoptosis of cells, the annexin V-FITC/propidium iodide (PI) apoptosis detection kit (BD Biosciences) was used. Briefly, cells were collected and re-suspended in 200 ml of buffered medium. Annexin V solution (10 ml) was added to the cell suspension, which was then incubated for 15 min in the dark at RT. Then, 5 μl of PI was added, and the cells were analyzed by flow cytometry (BD ACCURI C6 cytometer, BD Biosciences). Annexin V/PI double negative cells represented viable cells, annexin V positive/PI negative cells represented early apoptotic cells, and annexin V/PI double positive cells represented late apoptotic cells.

2.4 Tetra methyl rhodamine methyl ester (TMRM) staining

Mitochondrial membrane potential was determined by using a tetramethylrhodamine methyl esters (TMRM) fluorescent dye (Invitrogen) [33]. In healthy cells with functioning mitochondria, TMRM accumulates in active mitochondria with intact membrane potentials. Loss of the mitochondrial membrane potential decreases TMRM signal. To evaluate mitochondrial membrane permeability, the cells were loaded with 200 nM of TMRM for 30 minutes at 37°C in medium. TMRM signal was detected by a laser confocal microscopy with absorbance peak at 548 nm and emission peak at 574 nm.

2.5 Immunocytochemistry

The cells were cultured in 4-well slide chambers, washed twice with PBS, and fixed in 1% para-formaldehyde solution for 10 min. The cells were then washed twice with PBS before permeabilization using 0.1% Triton X-100 for 10 min. Next, the cells were blocked for 1 h in blocking solution (2% bovine serum albumin and 10% horse serum in PBS) and incubated with Cytochrome C antibody (Santa Cruz Biotechnology). FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were then used. Immunofluorescence was detected by a laser confocal microscopy (LSM710; Carl Zeiss).

2.6 Transfection of miRNAs and anti-miRNAs

Transfections of miRNA mimics and anti-miRNAs were performed using siLentFect™ Lipid reagent (Life Science Research). Mature specific miRNAs (Genolution Pharmaceuticals, Inc., Korea) were used at a final concentration of 100 nM. Anti-miRNAs was used at a final concentration of 50 nM. After 4 hours of incubation in a CO2 incubator at 37°C, the medium was changed to 10% FBS con taining DMEM.

2.7 Western blot

The cells were washed once in PBS and lysed in lysis buffer (Cell Signaling Technology) with protease and phosphatase inhibitor cocktail. Protein concentrations were determined using the BCA protein assay kit (Thermo Science). Proteins were separated in a SDS–polyacrylamide gel and transferred to the PVDF membrane (Millipore). After blocking the membrane with 0.1% Tris-buffered saline–Tween 20 (TBS-T, 0.1% Tween 20) containing 10% skim milk for 1 hr at room temperature, the membrane was washed twice with TBS and incubated with primary antibodies for overnight at 4 °C. The membrane was washed three times with 0.1% TBS-T for 5 min and then incubated for 1 hr at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. After extensive washing, the bands were detected by enhanced chemiluminescence reagent (ECL, Santa Cruz Biotechnology). The band intensities were quantified using NIH Image J version 1.34e software. The primary antibodies for HKII and β-actin were from Cell Signaling (28675) and Sigma (A1978), respectively.

2.8 Luciferase assay using the 3’UTR of HK2

The 3’UTR sequences of HK2 was amplified using primers with XbaI (forward) and EcoRI (reverse) endonuclease sites. The 3’UTR fragment was then cloned into the pmirGLO vector. HeLa cells were plated at a density of 1×10⁵ cell/well in a 12 well plate, and then transfected with either pmirGLO control vector or pmirGLO vector with HK2 3’UTR using Lipofectamine LTX. After 48 hours, relative luciferase activity was measured by using Dual Luciferase assay kit (Promega) according to the manufacturer’s instructions. The Renilla luciferase was used for normalization.

2.9 Rat I/R injury model

All experimental procedures for animal studies were approved by the Committee for the Care and Use of Laboratory Animals, and performed in accordance with the Committee’s Guidelines and Regulations for Animal Care (CKU 01-2019-008). I/R-injury was produced in male Sprague-Dawley rats (200±50 g) by surgical occlusion of the left anterior descending coronary artery. Briefly, after induction of anesthesia with zoletil (0.8 ml/kg) and rompun (0.2 ml/kg), the rats were intubated, for ventilation (62 strokes/min, tidal volume 8–10 ml/kg). After intubation, the third and fourth ribs were cut to open the chest, and the heart was exteriorized through the intercostal space. The left coronary artery was then ligated 2–3 mm from its origin with a 6-0 prolene suture (Ethicon, Somerville, NJ, USA). Reperfusion was conducted after 1 hour of ischemia. For anti-miR181 treatment, microRNA (5 μg/head) and reagent mixture were prepared in 60 μl and injected from the injured region to the border using a Hamilton syringe (Hamilton Co., Reno, NV, USA) with a 30 gauge needle. Throughout the operation, animals were ventilated with 95% O₂ and 5% CO₂ using a Harvard ventilator (Holliston, MA, USA). Animals were sacrificed 2 weeks after the surgery for analysis.

2.10 Statistical analysis

Quantitative data were expressed as the means±S.E.M (standard error of measurement) of at least 3 independent experiments. For statistical analysis, one-way ANOVA with Bonferroni correction was performed using the OriginPro 8 SR4 software (ver. 8.0951, OriginLab Corporation, Northampton, MA, USA). A p value of less than 0.05 was considered to be statistically significant.

3.1 H₂O₂ increased apoptosis by disrupting mitochondrial membrane potential

It has been reported that H₂O₂ induces apoptosis of cardiomyocytes [34]. In the present study, H₂O₂ was used to simulate the oxidative stress. According to the data, H₂O₂ decreased cell viability of cardiomyocytes in a concentration-dependent manner. To be specific, 300 μM or higher concentra-tions of H₂O₂ significantly decreased cell viability of cardiomyocytes after 6 hours of treatment (Figure 1A). Since 6 hours of treatment with 500 μM of H₂O₂ induced more than 20% of cell death of cardiomyocytes, this particular condition was use for further experiments.

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Fig. 1

H₂O₂ increased apoptosis by disrupting mitochondrial membrane potential. (A) Cell viability of cardiomyocytes exposed to increasing concentrations of H₂O₂ for 6 hours. ^*p < 0.01, ^**p < 0.001 compared to untreated control. The quantitative data were expressed as the means±SEM of at least 3 independent experiments. Cell viability was determined by using CCK. (B) Upper panel: Morphological examination of H₂O₂ treated cardiomyocytes. The cells were exposed to 500 μM of H₂O₂ for up to 6 hours. Scale bar = 200 μm. Bottom panel: The percentage of cells undergoing apoptosis were determined by flow cytometry using PI/Annexin-V double-staining. (C) Mitochondrial membrane potential was determined by tetramethylrhodamine methyl esters (TMRM) staining. The cells cultured in a chamber slide were treated with 500 μM of H₂O₂ for up to 6 hours and then loaded with 200 nM of TMRM for 30 minutes at 37°C in medium. The fluorescent signal of TMRM was detected using a confocal microscopy. DAPI was used to stain nuclei. Scale bar = 50 μm. (D) Time dependent changes of mitochondrial and cytosolic expressions of HK2 in H₂O₂-treated cardiomyocytes. VDAC: voltage dependent anion channel, VDAC and β-actin are used as loading controls for mitochondrial and cytosolic fraction, respectively.

When the cardiomyocytes were treated with 500 μM of H₂O₂, cell detachment was obvious from 6 hours (Figure 1B, upper panel) and the percentage of the cells positively stained for both PI and anne-xin V, which indicates late apoptosis [35], increased suggesting the type of cell death inflicted by H₂O₂ includes apoptosis (Figure 1B, bottom panel). Intact mitochondrial membrane effectively accumulates TMRM [36]. TMRM staining indicated that H₂O₂ disrupted mitochondrial membrane potential in a time-dependent manner (Figure 1C), and the amount of HK2 in mitochondrial fraction time-dependently decreased, suggesting H₂O₂ increased detachment of HK2 from mitochondrial outer membrane (Figure 1D).

To identify miRNAs targeting HK2, 3 different miRNA-target protein prediction databases (Target-Scan, miRWalk, and miRNA.org) were used. According to these databases, 4 miRNAs were simultaneously predicted to target rat HK2 (miR-9, miR-139, miR-425, and miR-181) (Figure 2A). When cardiomyocytes were transfected with those 4 miRNAs, only miR-181 significantly decreased the expression of HK2 (Figure 2B). Therefore, miR-181 was selected as a primary candidate miRNA that may facilitate the expression of HK2 under oxidative stress. To further verify whether miR-181 directly regulates the expression of HK2, a luciferase assay using pmirGLO vector containing the 3’UTR of rat HK2 was conducted (Figure 2C). The luciferase assay are used to evaluate the effect of miRNA-dependent post-transcriptional regulation of target genes [37]. Transfection of 100 nM of miR-181 to cardiomyocytes significantly decreased the expression of luciferase gene linked to the 3’ untranslated region (3’UTR) of HK2 (pmirGLO-HK2) compared to the control (Figure 2D). This indicated that the expression of HK2 is directly regulated by miR-181.

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Fig. 2

Identification of miR181 as miRNA targeting HK2. (A) Screening of candidate miRNAs targeting HK2. To identify miRNAs directly targeting HK2, in silico screening using 3 different miRNA-target gene prediction databases (miRWalk, TargetScan, and microRNA.org) were utilized. A total of 4 different miRNAs were predicted to target HK2. (B) Effect of candidate miRNAs on the expression of HK2. Cardiomyocytes were transfected with each candidate miRNAs (100 nM, each) and the expression of HK2 in total protein was evaluated by Western blot. (C) Construction of luciferase vector containing the 3’UTR sequences of HK2. The miR-181 binding sequence within the 3’UTR of HK2 was amplified using primers with XbaI (forward) and EcoRI (reverse) endonuclease sites. The 3’UTR fragment was then cloned into the pmirGLO vector. (D) MicroRNA-181 targets HK2. HeLa cells were plated at a density of 1×10⁵ cell/well in a 12 well plate, and then transfected with either pmirGLO control vector or pmirGLO vector with HK2 3’UTR. To verify the effect of miR-181 on the expression of HK2, the cells were further treated with either scrambled miRNA or miR-181 (100 nM, each) for 48 hours. Relative luciferase activity was measured by using Dual Luciferase assay kit (Promega) according to the manufacturer’s instructions. ^*p < 0.05 compared to untreated control.

Since it was hypothesized that miR-181 contributed to the down-regulation of mitochondrial HK2 expression, endogenous miR-181 was neutralized using anti-miR-181 and its effect on mitochondrial HK2 was examined. When cardiomyocytes were exposed to H₂O₂ with or without miR-181 neutralization using anti-miR-181, mitochondrial HK2 expression was attenuated compared to H₂O₂ only treated group (Figure 3A). Upon morphological examination, anti-miR-181 apparently prevented cardiomyocytes from H₂O₂-induced detachment (Figure 3B) and H₂O₂-induced decrease of cardiomyocyte viability was significantly recovered by anti-miR-181 (Figure 3C), indicating suppression of miR-181 may be an effective way to enhance cell survival. Furthemore, neutralization of miR-181 using anti-miR-181 decreased the percentage of the cells positively stained for both PI and annexin V (Figure 3D), suggesting that the neutralization of miR-181 was effective in preventing H₂O₂-induced cardio-myocyte apoptosis.

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Fig.3

Effect of miR181 neutralization on H₂O₂-induced cardiomyocyte apoptosis. (A) Effect of anti-miR-181 on the expression of mitochondrial HK2. Cardiomyocytes were transfected with anti-miR-181 (100 nM) and exposed to 500 μM of H₂O₂ for 6 hours. The expression of HK2 in mitochondrial fraction was evaluated by Western blot. (B) Morphological examination of anti-miR-181 treated cardiomyocytes under oxidative stress. The cells with or without neutralization of miR-181 using anti-miR-181 (100 nM) were exposed to 500 μM of H₂O₂ for 6 hours. Scale bar = 200 μm. Rea: transfection reagent only. (C) Cell viability of cardiomyocytes exposed to 500 μM of H₂O₂ for 6 hours with or without miR-181 neutralization using anti-miR-181. ^*p < 0.05 compared to untreated control, ^**p < 0.05 compared to H₂O₂ treated group. The quantitative data were expressed as the means±SEM of at least 3 independent experiments. Cell viability was determined by using CCK. Rea: transfection reagent only. (D) The cells with or without miR-181 neutralization were treated with 500 μM of H₂O₂ for 6 hours and the percentage of cells undergoing apoptosis were determined by flow cytometry using PI/Annexin-V double-staining.

In a mechanistic point of view, cleaved form of caspases indicates their activation [38]. H₂O₂ induced cleavage of caspase-3 and -9 in cardiomyocytes but it was attenuated by anti-miR-181 (Figure 4A). Neutralization of miR-181 also attenuated H₂O₂-induced mitochondrial membrane potential disruption (Figure 4B) and ctyochrome C release (Figure 4D). Additionally, to investigate the effect of miR-181 neutralization in vivo, anti-miR-181 (5 μg/head) was directly delivered to the heart via a local injection immediately after I/R-injury. Masson’s trichrome staining is a common staining method for collagen [39]. Masson’s trichrome staining of 2 weeks post-injured heart indicated that anti-miR-181 attenuated cardiac fibrosis which commonly follows a massive death of cardiomyocytes (Figure 4D). Taken altogether, it was speculated that anti-miR-181 suppressed cardiac fibrosis by neutralizing endogenous miR-181 which mediates cardiomyocyte apoptosis following I/R-injury.

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Fig. 4

Anti-apoptotic effect of miR-181 neutralization. (A) Effect of anti-miR-181 on the activation of caspases. Cardiomyocytes were transfected with anti-miR-181 (100 nM) and exposed to 500 μM of H₂O₂ for 6 hours. The expression of cleaved caspase 9 and 3 was evaluated by Western blot. Rea: transfection reagent only. (B) Effect of miR-181 neutralization on the H₂O₂-induced mitochondrial membrane potential change. Mitochondrial membrane potential was determined by TMRM staining. The cells were treated with 500 μM of H₂O₂ for 6 hours with or without anti-miR-181 pretreatment. The fluorescent signal of TMRM was detected using a confocal microscopy. DAPI was used to stain nuclei. Scale bar = 50 μm. (C) Immunocytochemical detection of cytochrome C following miR-181 neutralization. The cells cultured in a chamber slide were treated with 500 μM of H₂O₂ for up to 6 hours with or without anti-miR-181 pretreatment. The fluorescent signal of cytochrome C was detected using a confocal microscopy. DAPI was used to stain nuclei. Scale bar = 50 μm. (D) Effect of anti-miR-181 on cardiac fibrosis. Representative images of Masson’s trichrome-stained sections of each group demonstrating fibrosis. The heart samples were harvested at 14 days after I/R-injury. Scale bar = 2 mm.