HNEAP Regulates Necroptosis of Cardiomyocytes by Suppressing the m5C Methylation of Atf7 mRNA

Abstract PIWI‐interacting RNAs (piRNAs) are highly expressed in various cardiovascular diseases. However, their role in cardiomyocyte death caused by ischemia/reperfusion (I/R) injury, especially necroptosis, remains elusive. In this study, a heart necroptosis‐associated piRNA (HNEAP) is found that regulates cardiomyocyte necroptosis by targeting DNA methyltransferase 1 (DNMT1)‐mediated 5‐methylcytosine (m5C) methylation of the activating transcription factor 7 (Atf7) mRNA transcript. HNEAP expression level is significantly elevated in hypoxia/reoxygenation (H/R)‐exposed cardiomyocytes and I/R‐injured mouse hearts. Loss of HNEAP inhibited cardiomyocyte necroptosis and ameliorated cardiac function in mice. Mechanistically, HNEAP directly interacts with DNMT1 and attenuates m5C methylation of the Atf7 mRNA transcript, which increases Atf7 expression level. ATF7 can further downregulate the transcription of Chmp2a, an inhibitor of necroptosis, resulting in the reduction of Chmp2a level and the progression of cardiomyocyte necroptosis. The findings reveal that piRNA‐mediated m5C methylation is involved in the regulation of cardiomyocyte necroptosis. Thus, the HNEAP‐DNMT1‐ATF7‐CHMP2A axis may be a potential target for attenuating cardiac injury caused by necroptosis in ischemic heart disease.


SUPPLEMENTARY MATERIAL
Myocardial infarct size measurement.After reperfusion or sham treatment, 1.0%Evans blue dye (EBD) (Sigma, St. Louis, E2129, Missouri, USA) was injected into the coronary circulation of the mice to delineate the myocardial perfusion region.
Evans Blue Dye Assays.Mice were injected with 1% EBD at a dose of 100mg/kg through tail vein injection.After 24h, the mice were operated on with I/R or Sham operation, and the hearts were taken out and placed in the Tissue-TekOCT compound for rapid freezing.Using Leica cryostat for frozen sections (7nm), the frozen sections were anti-stained with wheat germ agglutinin (WGA) conjugate (AAT Bioquest, USA) in the ratio of 1:200, and the staining results were observed by confocal fluorescence microscope (Leica, TCS SP8) to quantify EBD positive cells.
Propidium iodide (PI) staining.After being treated with H/R, the cells were washed with PBS and stained with 30μg/ml PI (Solarbio, Beijing, China) at 37℃ for 30 min under dark conditions.After washing 3 times, 4% paraformaldehyde was fixed at room temperature for 15-25min.Washing again 3 times with PBS, then the cells were anti-stained with 4-diamino-2-phenylindole (DAPI, Solarbio, Beijing, China) solution (1μg/ml) and observed under the fluorescence microscope (Nikon ECLIPSE Ti-S).
The number of PI-positive nuclei was counted and quantified.
Lactate dehydrogenase (LDH) activity assay.LDH assay kit (Solarbio, Beijing, China) was used to determine the activity of LDH released from cell culture medium according to the instructions.After adding all the reagents according to the steps, mix them well, let them stand at room temperature for 3-5min, and use the Microplate reader (Synergy Neo2, BioTek, USA) to measure the absorbance value at the wavelength of 450nm.LDH activity was calculated according to beer's law.
Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay.TUNEL assay kit (Yeasen, Shanghai, China) was used to determine the apoptosis according to the instructions.Cell slides or tissue sections were fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100.After cleaning the slides with PBS, they were incubated with 1×Equilibration buffer for 30min.The cells were incubated with Terminal deoxynucleotidyl transferase (TdT) hybridization solution for 1h at 37℃ in the dark.After the slides were cleaned, nuclei were labeled with DAPI.
Transfection of HNEAP agomir and antagomir.All HNEAP agomir or antagomir and their negative controls were obtained from GenePharma (Shanghai, China).

RNA pull down.
Cardiomyocytes transfected with biotinylated HNEAP and NC (GenePharma, Shanghai, China) were incubated in a lysis buffer containing protease inhibitor and RNA inhibitor.Then 10% volume of the lysis mixture was used as input, and 90% volume of the mixture was incubated with streptavidin magnetic beads (Biomake, Shanghai, China).Add 1/3 volume of 4×loading buffer to the collected sample and boil it for 10min.After SDS-PAGE gel electrophoresis, the pull-down materials of HNEAP and NC were collected and analyzed by LC-MS/MS.2'-O-methylation at the 3'end.RTL-P is used to detect 2'-O-methylation at the 3' end, and the small RNA samples were extracted using mirVanamicroRNA isolation kit (Ambion, AM1556, USA).T4-RNA ligase (Takara, 2050A, Beijing, China) can connect 3' RNA adapter with the small RNA.In the presence of anchored or unanchored RT primers, the ligated products were reverse-transcribed using dNTPs at a high (50μM) or low (0.5μM) concentration, respectively.The product was amplified by PCR with specific primers and detected in agarose gel.
or its negative control (sh-NC) adenovirus and transfected with anta, and then were treated with H/R.(c) The protein levels of Atf7 were detected by western blot assay.Supplementary

m 5 C
-IP-qPCR.To quantify the specific gene level of m 5 C-modification, total RNA was extracted from cardiomyocytes or mouse hearts.Then the total RNA was fragmented, the RNA sample (10% of total volume) was used as the input control and the rest of the RNA sample was incubated with m 5 C-antibody-conjugated beads in immunoprecipitation buffer containing RNase inhibitor at 4℃ for 2h.The m 5 C-containing RNA sample was eluted from the beads.qRT-PCR was performed with gene-specific primers for both the input control and m 5 C-immunoprecipitated samples.Nuclear and cytoplasmic RNA extraction.Collect cardiomyocytes and add CEB-Amixed solution containing DTT and protease inhibitor.The cells were completely resuspended and incubated on ice for 10 minutes.Add precooled CEB-B to the test tube.16000g, centrifuged for 5 minutes.Take the supernatant as the cytoplasmic extract.After the precipitation was suspended with NEB, the supernatant was taken by vortex centrifugation as the nuclear extract.The whole process was operated in a low temperature environment, and the obtained samples were extracted by TRNpure Trizol Reagent or stored at -80℃.Fluorescence in situ hybridization (FISH).HNEAP in cardiomyocytes can be labeled by specific fluorescent probes, and all steps are carried out following the instructions of the FISH Kit (gene Pharma, Shanghai, China).After 4% paraformaldehyde fixation and 0.1% Triton X-100 permeabilization, cardiomyocytes were dehydrated and dried in different concentrations of ethanol solution.Then add the hybridization buffer containing the fluorescent probe preheated at 37℃ in advance, denature it at 73℃, and incubate it at 37℃ in dark conditions overnight.After DAPI labeling, the distribution of HNEAP was observed under a confocal microscope.

( d )
Quantitative analysis of the percentage of necroptotic cardiomyocytes (n=5 independent experiments).(e) The activity of LDH in cardiomyocytes (n=6 independent experiments).Data are presented as Mean ± SD.One-way ANOVA test (d, e).Supplementary Figure 7. Knockdown of Chmp2a induced cardiomyocyte necroptosis.(a-c) Cardiomyocytes transfected with Chmp2a-siRNA (si-Chmp2a) or its negative control (si-NC).(a) The protein levels of Chmp2a were detected by western blot assay.(b) Quantitative analysis of the percentage of necroptotic cardiomyocytes (n=5 independent experiments).(c) The activity of LDH in cardiomyocytes (n=3 independent experiments).(d) The representative western blot showing the expression of RIPK1 and RIPK3 in cardiomyocytes after infected with sh-Atf7 or sh-NC and transfected with HNEAP.(e) The activity of LDH in cardiomyocytes after transfected with sh-Atf7 or sh-NC and transfected with HNEAP (n=3 independent experiments).(f) Cardiomyocytes were transfected with si-Chmp2a or si-NC after HNEAP knockdown, and treated with H/R.Quantitative analysis of the percentage of necroptotic cardiomyocytes (n=5 independent experiments).Data are presented as Mean ± SD.One-way ANOVA test (b, c, e, f).Supplementary Figure 8. Model of HNEAP function in cardiomyocytes necroptotic signaling.HNEAP participates in the regulation of cardiac necroptosis by targeting DNMT1/ATF7/CHMP2A pathway.In our model, HNEAP inhibits DNMT1-mediated m 5 C methylation of Atf7 mRNA transcript, which leads to an increase of Atf7 expression, reduced Atf7-antagonistic Chmp2a expression and thus leads to cardiomyocytes necroptosis.

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