Uhrf1 regulates H3K9me2 modification of mTOR to inhibit the effect of autophagy in myocardial ischemia-reperfusion injury

Changes in Uhrf1 expression in the in vivo MIRI mouse model

The mice were divided into three groups, which were subjected to myocardial ischemia for 30 minutes and then reperfusion for 2, 6, or 12 hours. The expression of Uhrf1 in the infarcted areas (IA) and non-infarcted areas (NIA) were detected by qRT-PCR (Figure 1). The Uhrf1 level in the non-infarcted areas was significantly lower than the infarcted areas at 2, 6 and 12 hours after myocardial ischemia-reperfusion in vivo.

Figure 1. Detection of the changes of Uhrf1 in myocardial ischemia-reperfusion injury model of mice in vivo. The relative mRNA expressions of Uhrf1 in the infarcted and non-infarcted regions of the mice hearts were detected by qRT-PCR at 2, 6 and 12 h after myocardial ischemia-reperfusion injury. Data shown are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. N=3 per group.

Protective effect of Uhrf1 in MIRI

The protective effect of Uhrf1 on MIRI was investigated by comparative analysis of overexpression and knockdown of Uhrf1 in an in vitro MIRI model. To establish stable cardiomyocytes that exhibit overexpression and knockdown of Uhrf1, we analyzed the expression of Uhrf1 plasmid transfection and screened the knockdown efficiency of different siRNAs on Uhrf1. The sequences for siRNA were in Table 1. The efficacy of overexpression and knockdown of Uhrfl was verified by qRT-PCR (Figure 2A, 2B) and western blot (Figure 2C, 2D). We found the protein level of Uhrf1 increased 1.4 fold in the overexpression cells and siRNA-002 (Si2) significantly knocked down the expression of Uhrf1 in cardiomyocytes. In subsequent in vitro experiments, Si2 was used to knock down the Uhrf1 (Si group).

Figure 2. Detection of transfection efficiency of overexpression and knockdown of Uhrf1 plasmid. (A) The relative mRNA expressions of Uhrf1 in Myocardial ischemia-reperfusion model in vitro with Uhrf1 plasmid were determined by qRT-PCR. NC, negative control of plasmid transfection; Uhrf1, Uhrf1 overexpression. (B) The relative mRNA expressions of Uhrf1 in Myocardial ischemia-reperfusion model in vitro with Uhrf1 siRNA plasmid were determined by qRT-PCR. NC, negative control of RNAi; si1-3, siRNA targeting different mRNA regions. (C) Western blot was used to detect the expression level of Uhrf1 protein in each group. GAPDH serves as a loading control. Model, in vitro oxidative stress model; NC, negative control of RNAi; si, RNAi knockdown of Uhrf1; Uhrf1, Uhrf1 overexpression. (D) Expression of Uhrf1 protein relative to GAPDH data from 3 biological repeats is shown. Data shown are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N=3 per group.

To confirm the effect of Uhrf1 in cardiomyocyte apoptosis, the mRNA and protein levels of Bcl-2, procaspase-3, cleaved caspase-3 and Bax (Figure 3A) were determined by qRT-PCR, western blot (Figure 3B, 3C) and immunocytochemistry analyses (Figure 3D). Our results show that overexpression of Uhrf1 reduces cardiomyocyte apoptosis after MIRI, decreases the expression of the apoptosis factors Bax, pro- and cleaved caspase-3 at both the mRNA and protein levels, and increases the expression of apoptosis inhibitory factor Bcl-2. Knockdown of Uhrf1in cardiomyocyte showed the opposite trend after MIRI.

Figure 3. The effect of Uhrf1 on cardiomyocyte apoptosis during myocardial ischemia-reperfusion injury. (A) The relative mRNA expressions of Bax, Bcl-2 and Caspase-3 in myocardial ischemia-reperfusion model in vitro were determined by qRT-PCR. (B) Western blot was used to detect the expression level of Bax, Bcl-2 and Caspase-3 protein in each group. GAPDH serves as a loading control. (C) Expression of Bax, Bcl-2 and Caspase-3 protein relative to GAPDH data from 3 biological repeats is shown. (D) Detection of Bax, Bcl-2 and Caspase-3 protein expression and semi quantitative analysis by immunofluorescence microscopy (x200). Scale bar, 100 μm. Data shown are mean ± SD. *P < 0.05, **P <0.01, ***P <0.001, ****P < 0.0001. N=3 per group. Model, in vitro oxidative stress model; NC, negative control of RNAi; si, RNAi knockdown of Uhrf1; Uhrf1, Uhrf1 overexpression.

To explore the regulatory effect of Uhrf1 on the cell cycle and the activity of cardiomyocytes, we measured the expression of Ki67 (Figure 4A–4D), the activity of cardiomyocytes (Figure 4E), and the beating frequency of cardiomyocytes in each group (Figure 4F). Our results show that overexpression of Uhrf1 in MIRI increases Ki67 expression at both the mRNA and protein levels. Knockdown of Uhrf1 showed the opposite trend, suggesting that Uhrf1 can increase cardiomyocytes proliferation after MIRI. Using CCK8 to detect cell viability, we found that cardiomyocytes overexpression of Uhrf1 significantly increased the cell viability after MIRI, and there was no significant change in the viability of cardiomyocytes with Uhrf1 knockdown. Moreover, we observed that cardiomyocytes over-expressing Uhrf1 had higher beating frequency than the blank plasmid control after MIRI, while knockdown of Uhrf1 in cardiomyocytes resulted in lower beating frequency than the blank plasmid control.

Figure 4. The effect of Uhrf1 on cell cycle and viability in myocardial ischemia-reperfusion injury. (A) The relative mRNA expressions of Ki67 in Myocardial ischemia-reperfusion model in vitro were determined by qRT-PCR. (B) Western blot was used to detect the expression level of Ki67 protein in each group. GAPDH serves as a loading control. (C) Expression of Ki67 protein relative to GAPDH data from 3 biological repeats is shown. (D) Detection of Ki67 protein expression and semi quantitative analysis by immunofluorescence microscopy (x200). Scale bar, 100μm. (E) The cardiomyocytes viability in knockdown and overexpression Uhrf1 groups was detected by CCK8. (F) The beating times per minute of cardiomyocytes in overexpression and knockdown group. Data shown are mean ± SD. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. N=3 per group. Model, in vitro oxidative stress model; NC, negative control of RNAi; si, RNAi knockdown of Uhrf1; Uhrf1, Uhrf1 overexpression.

Effect of Uhrf1 on the expression of mTOR during MIRI

To investigate the regulatory effect of Uhrf1 on mTOR in MIRI, we used qRT-PCR to detect changes in the mRNA levels of mTOR after overexpression and knockdown of Uhrf1 (Figure 5A) and confirmed the results by western blot (Figure 5B, 5C) and immunofluorescence (Figure 5D) analysis. We observed that overexpression of Uhrf1 increases mTOR expression at both the mRNA and protein levels. In contrast, knockdown of Uhrf1 only reduced mTOR expression in the mRNA level, and there was no significant change in the protein levels.

Figure 5. Uhrf1 promotes the expression of mTOR in MIRI. (A) The relative mRNA expressions of mTOR in Myocardial ischemia-reperfusion model in vitro were determined by qRT-PCR. (B) Western blot was used to detect the expression level of mTOR protein in each group. GAPDH serves as a loading control. (C) Expression of mTOR protein relative to GAPDH data from 3 biological repeats is shown. (D) Detection of mTOR protein expression and semi quantitative analysis by immunofluorescence microscopy (x200). Scale bar, 100μm. Data shown are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. N=3 per group. Model, in vitro oxidative stress model; NC, negative control of RNAi; si, RNAi knockdown of Uhrf1; Uhrf1, Uhrf1 overexpression.

Uhrf1 regulates mTOR expression through H3K9me2

Uhrf1 can specifically recognize H3K9me2, which suppresses the expression of specific genes in target cells and tissues. Therefore, Uhrf1 can regulate the expression of specific genes by regulating H3K9me2. As previously reported, there are four peaks for H3K9me2 expression on the mTOR gene [4]. Thus, changes in the expression of peaks 1-4 when overexpressing and knocking-down Uhrf1 were investigated by qRT-PCR (Figure 6A). We found overexpression of Uhrf1 inhibits the four expression peaks of H3K9me2 on the mTOR gene and knocking down Uhrf1 promotes expression of these four peaks.

Figure 6. Uhrf1 regulates mTOR expression through H3K9me2. (A) The relative mRNA expressions of Peak1-4 in Myocardial ischemia-reperfusion model in vitro were determined by qRT-PCR. (B) Western blot was used to detect the inhibitory effect of G9a inhibitor on H3K9me2 in cardiomyocytes by different concentrations (0.1 µM, 0.5 μM, 1.0 μM and 2.0 μM). GAPDH and the total histone H3 were used as loading controls. (C) The relative mRNA expressions of mTOR in myocardial ischemia-reperfusion model in vitro were determined by qRT-PCR after adding 1.0 μM G9a inhibitor to Si group. (D) Western blot was used to detect the expression level of mTOR protein after adding 1.0 μM G9a inhibitor to Si group. GAPDH serves as a loading control. (E) Expression of mTOR protein relative to GAPDH data from 3 biological repeats is shown. (F) The relative mRNA expressions of Peak1-4 in Myocardial ischemia-reperfusion model in vitro were determined by qRT-PCR after adding 1.0 μM G9a inhibitor to Si group. Data shown are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N=3 per group. Model, in vitro oxidative stress model; NC, negative control of RNAi; si, RNAi knockdown of Uhrf1; Uhrf1, Uhrf1 overexpression.

In order to further clarify the regulation of mTOR by H3K9me2, the H3K9me2 methyltransferase (G9a) inhibitor BIX01294 was added to the cardiomyocytes. The inhibitory effects of BIX01294 in various concentrations (0.1, 0.5, 1.0, and 2.0 μM) on cardiomyocyte H3K9me2 were evaluated, and the optimal inhibitory concentration was determined (Figure 6B). We observed that when the concentration of BIX01294 was 1.0 μM, the expression of H3K9me2 in cardiomyocytes was effectively inhibited. Changes in the mRNA and protein levels of mTOR were detected after adding G9a inhibitor (BIX01294) at 1.0 μM to the Si group (Figure 6C–6E). We observed that the expression of mTOR is increased in Uhrf1-knockdown cardiomyocytes after G9a inhibitor was added. The changes in the four expression peaks for H3K9me2 on mTOR after G9a inhibitor were further evaluated (Figure 6F). After G9a inhibitor was added, peak 1 and peak 2 decreased, while peak 3 and peak 4 did not change significantly. Thus, these results indicated that the regulation of mTOR expression by Uhrf1 was mediated by regulating H3K9me2.

Effect of Uhrf1 on autophagy through mTOR in MIRI

To detect changes in autophagy after MIRI, the expression of autophagy-related proteins was evaluated (Figure 7A, 7B). After overexpression of Uhrf1, the ratio of LC3-II/LC3-I to Beclin-1 expression was significantly decreased, while the expression of P62 and p-mTOR was markedly increased. This indicated that Uhrf1 promotes the expression of mTOR during MIRI, thereby inhibits the occurrence of autophagy.

Figure 7. The regulation of mTOR on autophagy in MIRI. (A) Western blot was used to detect the expression level of Beclin1, LC-3, P62, total m-TOR and p-mTOR protein. GAPDH serves as a loading control. (B) Expression of Beclin1, LC-3, P62 and p-mTOR protein relative to GAPDH data from 3 biological repeats is shown. Data shown are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N=3 per group. Model, in vitro oxidative stress model; NC, negative control of RNAi; si, RNAi knockdown of Uhrf1; Uhrf1, Uhrf1 overexpression.

Animals

In this study, C57BL/6J mice were purchased from Jackson laboratory. The mice were placed under the standard conditions of animals with 21 ± 1° C temperature and 55%-60% humidity, and they ate and drank at will. In the experiment, C57BL/6J male mice (25-30 g) were selected for in vivo experiment and newborn mice (1-3 days old) for in vitro experiment. Experimental procedures were carried out in accordance with the animal care and use system approved by the Committee of Harbin Medical University.

Myocardial ischemia/reperfusion model preparation

According to the "Guidelines for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996), animal experiments were performed. Nine male C57BL/6J mice aged 2-3 months (25-30 g) were divided into three groups: 2, 6, and 12 hours. A 100% concentrated storage solution was prepared by mixture of 10 g avertin and 10 mL tert-amyl alcohol. When used, it was diluted to 1.2% with sterilized distilled water, and the dosage was 0.7 mL/20 g for intraperitoneal injection of anesthetized mice, which was also used as an analgesic during the experiment. The left anterior descending coronary artery was ligated with a loose knot (6-0 wires). After 30 minutes of ischemia, the loose knots were released. After I/R operation, air is discharged from the chest and the surgical wound is sutured. The hearts of the mice were taken at 2, 6, and 12 hours (according to group) after reperfusion, and the ischemic and non-ischemic areas were separated.

Cardiomyocyte culture

Newborn mice were taken, and a primary culture of neonatal mouse ventricular myocytes was prepared by enzymatic hydrolysis of cardiac tissue. Briefly, the hearts were enzymatically digested in 0.25% trypsin and centrifuged at 1500 r/min for 5 minutes. The cardiomyocytes were cultured at 37° C for 90 minutes for cardiomyocyte purification, and the resultant cardiomyocytes were cultured at a concentration of 1 × 10⁵/mL in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/mL of penicillin and 100 mg/mL of streptomycin for 48 hours in an incubator (37° C, 5% CO₂).

Experimental grouping

The isolated cardiomyocytes were sorted into cardiomyocyte, model, blank plasmid, knock-down Uhrf1, over-expressing Uhrf1 and Si with H3K9me2 methyltransferase (G9a) inhibitor (BIX01294: Cat.No. HY-10587, MedChemExpress, China) groups. Reagent addition for each group is given in Table 2.

Plasmid transfection

For amplification and extraction of Uhrf1 DNA plasmid pRP[Exp]-EGFP/Puro-CAG>mUhrf1 [NM_010931.3]* (Vector Builder), 2000 ng of the Uhrf1 plasmid was mixed with 15 μL of X-treme GENE siRNA Transfection Reagent (Roche, Basel, Switzerland) and dissolved in opti-MEM medium. The mixed liquid was added to the cardiomyocytes at 100 μL/mL culture solution. The siRNA sequence of Uhrf1 was labeled CCTTGCAGACCATTCTCAA, and 7 μL of this siRNA plasmid and blank NC plasmid (purchased from RIBBIO) were mixed with 10 μL of x-treme and dissolved in opti-MEM. The mixed liquid was added to the cardiomyocytes at 100 μL/mL culture solution. The cells were incubated for 24 hours.

Induction of in vitro oxidative stress model

After 24 hours of plasmid transfection, 3% H₂O₂ (1 mol/L) was diluted 100-fold to a final concentration of 10 nmol/μL and passed through a 0.22 μm filter. This was then added 100 nmol H₂O₂ per 1 mL of cardiomyocyte culture solution. Oxidative stress was induced by hydrogen peroxide in serum-free medium for 24 hours, and then exposed to normoxic conditions and DMEM containing glucose for the next 24 hours.

Quantitative real-time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA samples were obtained by reverse transcription using TransScript All-in-One reagent (TransGen Biotech, China). The expression levels of specific genes and GAPDH was determined by quantitative real-time PCR (qRT-PCR) in a CFX96 real-time PCR system. The results of qRT-PCR were normalized to GAPDH expression. Use method 2^-∆∆Ct to calculate the relative quantitative expression. All reactions were repeated three times. The sequences of primers are listed in the Table 3.

Protein extraction and western blot

Cardiomyocytes were lysed in RIPA buffer (150 mM NaCl, 10% glycerol, 50 mM Tris/HCl, and 1% NP-40, pH 8.0) containing phosphatase and protease inhibitors for 30 minutes. Cardiomyocyte fragments were removed by centrifugation at 12,000 rpm for 20 minutes at 4° C. Protein concentrations were determined using a Thermo Pierce BCA Protein Assay Kit. A 50 μg protein sample was boiled in the loading buffer, denatured, and fractionated by SDS-PAGE. Then, the gel containing the protein was placed on a PVDF membrane in a membrane transport tank. After sealing with 5% skimmed milk, the PVDF membrane was incubated overnight with antibodies of Uhrf1, Bax, caspase-3, Bcl-2, Ki67 and mTOR from Bioss (Beijing, China), Beclin-1, P62, LC3 from Proteintech (Wuhan, China), p-mTOR and H3K9me2 from Immunoway (Newark, USA) and GAPDH from Goodhere Biotechnology (Hangzhou, China). On the second day, the second-order fluorescent antibody was incubated for 2 hours. The immune response band was detected by Protein Simple system.

Immunocytochemistry

First, 1 mL of 4% paraformaldehyde was fixed for 15 minutes, and 0.3% Triton at 1 mL/well membrane was added. After 10 minutes, the capsules were washed with goat serum for 15-20 minutes. Meanwhile, 50 μL of primary antibody was diluted with 1% BSA and placed in a wet box at 4° C overnight. Then, the wet box was removed and the small slides were placed back into a 12 well plate and each well was rinsed with phosphate-buffered saline (PBS) three times. Then, in a dark room, 50 μL of the secondary antibody diluted with 1% BSA was added and the plate was preheated at 37° C. The incubation of the secondary antibody was completed after 1 hours. The nuclei were stained with DAPI for approximately 5 minutes and glycerin seals were applied. Under the microscope, three different fields of vision were used to randomly select each slide for scoring. The intensity and positive proportion were semi-quantitatively scored. The intensity score was used to calculate protein expression level.

CCK8 cell viability test

The cardiomyocytes were inoculated into 96 well culture plate, with 1 × 10⁴ cells per well. The plates were pre-cultured in an incubator for 24 hours (37° C, 5% CO₂). Cells in each well were treated with 10 μL of CCK8 reagent (Dojindo) with care taken not to generate bubbles in the pore as they affect the OD value. The plates were incubated in an incubator for 2 hours. The absorbance at 450 nm was measured.

Statistical analyses

Statistical calculation and analysis of counting test results and data were performed and evaluated by ANOVA or Student's t-test for two or multiple group comparisons by using GraphPad Prism 8. The difference in the detection indexes was considered statistically significant when P<0.05. The data in the experiment were expressed as mean ± SD.