ZNFX1 suppresses apoptosis-associated mRNA stability in cardiomyocyte to protect against myocardial infarction
Aims: Cardiomyocytes apoptosis is the predominant pathological feature following myocardial infarction (MI) and significantly impacts disease progression and outcomes. Zinc finger NFX1-type containing 1 (ZNFX1), an RNA helicase family member, remains relatively understudied in molecular biology and its role in cardiovascular diseases remains unclear. This study aims to explore the involvement of ZNFX1 in MI and uncover its mechanisms underlying cardiomyocyte apoptosis. Methods and results: Male C57BL/6 mice were administered AAV9-ZNFX1 or AAV9-sh-ZNFX1 with the cTNT promoter via tail vein injection for the purpose of cardiomyocyte-specific ZNFX1 overexpression or knockdown. Concurrently, left anterior descending coronary artery ligation (LAD) was performed to induce MI. Echocardiography was utilized to evaluate the cardiac function. Triphenyl tetrazolium chloride (TTC), Hematoxylin and eosin (HE) and Masson's trichrome staining were used to evaluate the cardiac infarction and cardiac remodeling. TUNEL assay, flow cytometry, western blot and quantitative reverse transcription PCR (qRT-PCR) were employed to evaluate apoptosis. The cardiomyocyte viability was quantified using the CCK-8 assay. RNA sequencing (RNA-seq) and RNA immunoprecipitation assays (RIP) were employed to investigate the interaction between ZNFX1 and apoptosis-associated genes. The expression of ZNFX1 was decreased in MI myocardium and hypoxia-treated cardiomyocytes. Overexpression of ZNFX1 significantly attenuated cardiac dysfunction, reduced infarct size, inhibited collagen deposition and alleviated cardiac hypertrophy which was ascribed to MI in mice, whereas knockdown of ZNFX1 produced the opposite effects. RNA-seq identified apoptosis as a possible regulated pathway of ZNFX1, overexpression of ZNFX1 represses the cardiomyocyte apoptosis that gives rise to MI while knockdown of ZNFX1 deteriorates it. Mechanically, ZNFX1 binds with mRNA of apoptosis genes that contain highly structured 3'UTR and prompts their decay.   Conclusion: ZNFX1 plays a protective role in MI by degrading mRNA of apoptosis-related genes, which often possess highly structured 3'UTRs. This highlights ZNFX1 as a potential novel molecular target for treating cardiac dysfunction associated with MI. Keywords: ZNFX1, apoptosis, mRNA stability, helicase activity, UPF1, myocardial infarction Overall design: The mouse hearts were separately harvested from MI and MI+ZNFX1 groups and the primary cardiomyocytes were harvested from hypoxia and hypoxia+ZNFX1 groups. The RNA libraries were sequenced using the Illumina NovaseqTM 6000 platform, conducted by LC Bio-Technology CO., Ltd. (Hangzhou, China). Briefly, the heart RNA samples were isolated and purified by TRIzol (ThermoFisher Scientific). The amount and purity of RNA samples were controlled by NanoDrop ND-1000 (NanoDrop, DE). Also, RNA integrity was tested by Bioanalyzer 2100 (Agilent). mRNA containing PolyA (polyadenylate) was captured by Oligo (dT) magnetic beads (Dynabeads Oligo (dT), ThermoFisher Scientific) through two rounds of purification. Finally, Illumina NovaseqTM 6000 was used to sequence in PE150 mode according to standard procedures. FPKM values for each gene were determined based on gene length and the number of reads mapped. Differential expression analysis was carried out using the DESeq2 R package (version 1.20.0). Genes with a P value less than 0.05 and a fold change greater than 2 were identified as differentially expressed. Gene Set Enrichment Analysis (GSEA) was conducted using tools available on OmicStudio (https://www.omicstudio.cn/tool). The heatmap was generated using the R (https://www.r-project.org/) on the OmicStudio platform (https://www.omicstudio.cn/tool).