Generation of fibroblast-specific NAT10 knockout mice

Floxed NAT10 (exon 4) C57BL/6 genetic background mice and fibroblast-specific tamoxifen-inducible Col1a2−Cre−ER(T) C57BL/6 genetic background mice were sourced from Shanghai Model Organisms in Shanghai, China. To create fibroblast-specific NAT10 knockout (KO) mice with tamoxifen-inducible expression, we crossed homozygous NAT10fl/fl mice with Col1a2−Cre−ER NAT10fl/fl mice. At 4 weeks of age, to activate CreER(T) and enable Cre/loxP-mediated deletion in fibroblasts expressing Cre, a tamoxifen dose of 100 mg/kg/d was administered intraperitoneally for 5 consecutive days in corn oil to both control (NAT10fl/fl) and Col1a2−Cre−ER NAT10fl/fl mice. The deletion efficiency was validated by real-time qPCR and Western Blot (Supplementary Fig. 1).

Myocardial infarction-induced cardiac fibrosis model

Approval for all animal experimental protocols was granted by Nanchang University's Laboratory Animal Research Committee. Male BALB/c mice, aged between 8 and 10 weeks, were sourced from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences, located in Shanghai, China. The animal experiments were subsequently conducted at the Center for Experimental Animal Science, Nanchang University, situated in Nanchang, Jiangxi Province, China. Myocardial infarction (MI) was induced in the mice under anesthesia with pentobarbital, achieved by ligating the mid-left anterior descending artery (LAD) using a 7/0 silk thread. A sham operation was also performed, which involved no ligation of the LAD. Following an eight-week period post-MI, the mice were euthanized through CO2 inhalation, and their heart tissues were collected. These tissues were promptly pre-cooled in liquid nitrogen and stored at −80 °C for subsequent investigations.

Hematoxylin and Eosin (H&E) Staining and Masson staining

For the H&E staining, heart tissues were fixed in 4% paraformaldehyde (PFA, Sangon Biotech) for 24 h at 4 °C, then dehydrated through graded ethanol series (70%−100%), cleared in xylene, and embedded in paraffin. Serial Sects. (5 μm thickness) were cut using a Leica RM2235 microtome. Slides were imaged using Nikon Eclipse Ci-L microscope (200 × magnification). Infarct size was quantified as percentage of total left ventricular area using ImageJ (NIH), with necrosis identified by loss of striations, eosinophilic cytoplasm, and nuclear pyknosis. To evaluate the morphological alterations and assess the degree of cardiac fibrosis, heart specimens were immersed in 4% Paraformaldehyde (PFA) sourced from Sangon Biotech in Shanghai, China, and subsequently embedded in paraffin. For the assessment of cardiac fibrosis, Masson's Trichrome staining was employed utilizing a kit procured from Solarbio in Beijing, China. Cross-sections of the paraffin-embedded hearts, each measuring 5 µm in thickness, underwent staining according to the Masson's Trichrome staining protocol. These stained sections were then imaged under a light microscope at a magnification of 200 × for detailed analysis. The fibrotic fraction was quantified by utilizing Image J software from the National Institutes of Health, Bethesda, MD, USA, which involved calculating the ratio of the blue-stained (fibrotic) area to the entire myocardial area.

Immunohistochemistry (IHC)

An immunohistochemical (IHC) assay was performed to quantify global RNA ac4C acetylation levels in heart tissues, adhering to a previously established protocol. Here is a concise outline of the procedure: The paraffin-embedded heart slides were first dried at 90 °C for a duration of four hours. Subsequently, the slides underwent dewaxing in xylene and were then rehydrated through a graded series of ethanol solutions. After allowing the tissue sections to cool, they were treated with 0.3% hydrogen peroxide for 15 min to effectively block endogenous peroxidase activity. The sections were then rinsed with PBS for five minutes and blocked with 3% BSA at 25 °C for a period of 30 min. Following the blocking step, the sections were washed with PBS and incubated overnight at 4 °C with primary antibody against ac4C (Cat No. 68498–1-Ig, Proteintech, Wuhan, China) and used at a 1:200 dilution. The next day, after washing the sections with PBS, they were incubated with a horseradish peroxidase (HRP)-labeled secondary antibody for 30 min at 37 °C. Following this incubation, the sections were washed with PBS, dehydrated, cleared, and mounted. Hematoxylin was used as the nuclear counterstain, while diaminobenzene served as the chromogen. This IHC assay enabled the visualization and assessment of relative ac4C RNA acetylation levels within the heart tissue samples.

RNA isolation and qPCR

RNA was extracted from murine left ventricle tissue and fibroblast samples utilizing TriZol reagent sourced from Thermo Fisher Scientific, located in Waltham, MA, USA. The extraction process involved the sequential use of chloroform, isopropanol, and ethanol. For the purpose of real-time reverse transcription quantitative polymerase chain reaction (qPCR), an input of 1 µg of RNA was utilized. The qPCR analysis was conducted using the SYBR Green system, also provided by Thermo Fisher Scientific. To quantify each mRNA species, specific primers were employed, with their sequences detailed in Supplemental Table 1. The mRNA quantification was normalized against the expression level of the housekeeping gene GAPDH. The results were expressed as fold change values (calculated as 2–ΔΔCt), comparing the levels of the target mRNA to those of the reference gene.

Western blotting

To prepare lysates from murine left ventricle tissues or cells, the procedure involved several key steps: Initially, the tissues or cells were digested in 1X lysis buffer containing protease and phosphatase inhibitors, along with PMSF sourced from Sangon Biotech. This digestion was facilitated using a Qiagen Tissue Lyser at 4 °C for a duration of 8 min at a speed of 50 rpm. Post-digestion, the lysates were gently agitated for 1 h at 4 °C and subsequently sonicated to ensure thorough cell disruption and protein release. The protein concentration in the lysates was determined using the PierceTM BCA protein assay kit provided by Thermo Fisher. For SDS-PAGE electrophoresis, a quantity of 20 µg of protein from each lysate sample was loaded onto a 10% polyacrylamide gel. Following electrophoresis, the SDS-PAGE gels were transferred onto PVDF membranes (sourced from Millipore, Billerica, MA, USA). These membranes were then blocked with 5% BSA (obtained from Sigma-Aldrich, CA, USA) for 1 h at room temperature (25 °C). The primary antibody (NAT10, a-SMA, Collagen I, Collagen III, Fibronectin 1 and β-actin) were all obtained from Proteintech (Wuhan, China), used at a concentration of 1:1000, was incubated with the membrane at 4 °C with gentle agitation for 1 h. Subsequently, secondary antibodies conjugated with horseradish peroxidase (HRP) were applied to the membrane and incubated for 1 h at room temperature with agitation. The blots were developed using the Tanon High-signature ECL Western Blotting Substrate sourced from Shanghai, China. This comprehensive procedure enabled the preparation of lysates, accurate quantification of protein concentration, effective separation of proteins via SDS-PAGE, successful transfer of proteins onto PVDF membranes, efficient antibody incubation, and the ultimate visualization of Western blot signals through the use of an ECL substrate.

Global RNA ac4C acetylation quantification

To quantify global ac4C acetylation in total RNA samples, the procedure outlined below was executed using the Total RNA ac4C acetylation Quantification Kit (Colorimetric) supplied by Genelily Biotech, Shanghai, China, adhering strictly to the manufacturer's guidelines. The process involved utilizing the total RNA extracted from each sample for acetylation quantification. Specifically, the colorimetric detection-based kit, tailored for this purpose, was utilized. The assay was conducted meticulously, adhering to the comprehensive instructions provided by Genelily Biotech. This kit facilitated the precise measurement of global ac4C acetylation levels within the total RNA samples, offering crucial insights into the overall acetylation profile of the RNA molecules.

ac4C Dot Blot

The total RNA samples underwent a heating process at 75 °C for 5 min, followed by a 1-min cooling period. Subsequently, the cooled samples were transferred onto Amersham Hybond-N + membranes (0.45 mm, sourced from Solarbio, Beijing, China). Post-crosslinking, blocking, and an overnight incubation at 4 °C, the membranes were subjected to incubation with an anti-N4-acetylcytidin antibody (diluted 1:1000, ab252215, abcam, MA, USA). This was followed by a secondary wash and probing with an anti-rabbit IgG antibody, conjugated with HRP, at 25 °C for an hour. After undergoing three washes, the membranes were visualized using a Chemiluminescent HRP substrate provided by Millipore (Billerica, MA, USA). To ascertain the quantity of input RNA, the membrane was exposed and stained with methylene blue staining buffer under gentle agitation for 30 min. Ultimately, the membrane was rinsed thoroughly with ribonuclease-free water.

Adult mouse cardiac fibroblast isolation and immunofluorescence staining

The hearts of euthanized adult mice were excised, finely chopped, and rinsed with water. A digestive solution comprising 0.25% trypsin and 5 mg/mL of Liberase TL was introduced to the chopped tissue, which underwent further digestion at 37 °C for 30 min while being agitated. The resultant mixture was centrifuged at 1500 rpm for 5 min, yielding a cell pellet that was resuspended in fibroblast growth medium. These cells were plated onto gelatin-coated plates and incubated at 37 °C. After 24 h, non-adherent cells were discarded, leaving the adherent cells to be cultivated in fibroblast growth medium. Fibroblasts from passages 1 or 2 were utilized for subsequent experimental procedures. To stimulate myofibroblast differentiation, 10 ng/mL of TGFβ1 was incorporated into the culture medium for 24 h prior to harvesting. Immunofluorescence staining was carried out using specific primary antibodies, including anti-ac4C (1:400, Cat No. 68498–1-Ig, Proteintech, Wuhan, China) or anti-α-SMA (1:400, Cat No. 67735–1-Ig, Proteintech, Wuhan, China), which were incubated overnight at 4 °C. Following washing, the cells were incubated with the corresponding secondary antibody for 2 h at room temperature. Ultimately, the stained cells were mounted using a DAPI-containing mounting medium and visualized using a Zeiss LSM800 confocal microscope. The fluorescent intensity was quantified by utilizing Image J software.

ac4C-MeRIP-qPCR

The ac4C-MeRIP procedure was executed utilizing an RNA immunoprecipitation kit, adhering strictly to the manufacturer's guidelines provided by Genelily Biotech (Shanghai, China). In summary, cells were lysed with 1 mL of RIP lysis buffer for a duration of 10 min, with a 100 μL aliquot of the lysis buffer being preserved at −80 °C for future use. Protein A/G beads were mixed with either an anti-ac4C antibody (diluted 1:50, ab252215, abcam) or normal rabbit IgG (diluted 1:50, catalog No. 2729S; CST, Beverly, MA, USA) and incubated for a period of 2 h. Following this, 450 μL of the lysate was incubated at 4 °C for another 2 h. RNA was then extracted from the beads, and subsequent quantitative Real-time qPCR analysis was conducted as previously outlined.

RNA Stability assay

Cardiac fibroblasts were subjected to treatment with Actinomycin D (at a concentration of 5 μg/mL, catalog number HY-17559, sourced from MedChemExpress, Princeton, NJ, USA) for varying durations of 0, 1, 3, and 6 h in 6-well plates. Total RNA was extracted following a protocol designed for quantitative Real-time qPCR analysis. A linear regression analysis was then conducted to ascertain the half-life of TGFBR1 mRNA.

Dual-Luciferase Reporter Assay

The CDS of TGFBR1 (NM_009370.6) containing predicted ac4C sites was cloned into pmirGLO dual-luciferase vector (Promega) downstream of firefly luciferase. Site-directed mutagenesis (Q5® Kit, NEB) generated cytidine-to-uridine mutations at ac4C sites. Cardiac fibroblasts were seeded in 24-well plates (5 × 104 cells/well) and co-transfected with 500 ng pmirGLO-TGFBR1 (WT or mutant) and 50 ng pRL-TK Renilla vector (Promega) using Lipofectamine 3000 (Thermo Fisher). After 24 h, cells were treated with 10 ng/mL TGF-β1 (PeproTech) for 48 h. Finally, Firefly luciferase assay was detected with 20 μL lysate and 100 μL Luciferase Assay Reagent II. Readings were taken on GloMax® Navigator (Promega). Firefly/Renilla ratios were normalized to empty vector controls.

AAV9-TGFBR1 Vector Production and In Vivo Delivery

The TGFBR1 overexpression construct was generated by cloning full-length mouse TGFBR1 cDNA (NM_009370.6) into the pAAV-CAG vector (Genelily Biotech) using In-Fusion® HD Cloning (Takara Bio). The CAG promoter drives constitutive expression of TGFBR1. A control vector only was generated in parallel. AAV9 serotype vectors were produced by triple transfection of HEK293T cells (ATCC CRL-3216) using polyethylenimine (PEI, Polysciences). Cells were co-transfected with pAAV-CAG-TGFBR1 or pAAV-CAG, pAAV2/9 rep-cap plasmid, and pAdDeltaF6 helper plasmid (Genelily Biotech). Viral particles were harvested 72 h post-transfection, purified by iodixanol gradient centrifugation, and concentrated using Amicon® Ultra-15 centrifugal filters (Millipore). For the in vivo delivery, mice received 1 × 1011 vg of AAV9-TGFBR1 or AAV9-null control via tail vein injection 1 weeks post-MI. Injection volume was 100 μL in PBS. Expression was confirmed by qPCR and western blotting of heart lysates at endpoint.

Statistical analysis

All experiments were carried out with either three or five independent replicates, and statistical evaluations were conducted utilizing GraphPad Prism version 9.0. The data presented in all figures are depicted as the mean, accompanied by the standard deviation. For comparisons between two groups, a t-test was used. For comparisons among multiple groups, One-way or Two-way ANOVA was used followed by a Tukey's post hoc test. Statistical significance was established at a p-value of less than 0.05 (two-tailed).