Downregulated ALKBH5 contributes to myocardial ischemia/reperfusion injury by increasing m6A modification of Trio mRNA

Jiaxin Li; Jieshan Chen; Mingyi Zhao; Zhetao Li; Nanbo Liu; Heng Fang; Miaoxian Fang; Ping Zhu; Liming Lei; Chunbo Chen

doi:10.21037/atm-22-1289

Downregulated ALKBH5 contributes to myocardial ischemia/reperfusion injury by increasing m⁶A modification of Trio mRNA

Ann Transl Med. 2022 Apr;10(7):417. doi: 10.21037/atm-22-1289.

Authors

Jiaxin Li^#^{1

2}, Jieshan Chen^#³, Mingyi Zhao^#⁴, Zhetao Li^{1

5}, Nanbo Liu⁵, Heng Fang^{1

6}, Miaoxian Fang², Ping Zhu^{1

4}, Liming Lei^#², Chunbo Chen^#^{1

2

6

7}

Affiliations

¹ The Second School of Clinical Medicine, Southern Medical University, Guangzhou, China.
² Department of Intensive Care Unit of Cardiac Surgery, Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China.
³ Department of Emergency, Maoming People's Hospital, Maoming, China.
⁴ Department of Cardiac Surgery, Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China.
⁵ Department of Medical Sciences, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China.
⁶ Department of Critical Care Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China.
⁷ Department of Critical Care Medicine, Maoming People's Hospital, Maoming, China.

^# Contributed equally.

Abstract

Background: The modification of N⁶-methyladenosine (m⁶A) is a dynamic and reversible course that might play a role in cardiovascular disease. However, the mechanisms of m⁶A modification in myocardial ischemia/reperfusion injury (MIRI) remain unclear.

Methods: A mouse model of MIRI and a cell model of oxygen-glucose deprivation/reperfusion (OGD/R) HL-1 cells were employed. In an in vivo study, the total RNA m⁶A modification levels were determined by dot blot, and the key genes related to m⁶A modification were screened by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In an in vitro study, the effects of AlkB homolog 5 (ALKBH5), an RNA demethylase, on cell proliferation, cell injury, and apoptosis were detected by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, lactate dehydrogenase (LDH) and cardiac troponin-I (cTnI) levels, and flow cytometry. Besides, the m⁶A modification-changed and differentially expressed messenger RNA (mRNA) were determined by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) in ALKBH5-overexpressed HL-1 cells. Finally, the mRNA levels of the promising targeted gene were examined by RT-qPCR and its m⁶A modification levels were examined by MeRIP-qPCR.

Results: Our results showed that RNA m⁶A modification was involved in MIRI, in which ALKBH5 was downregulated. Functionally, by overexpressing or silencing ALKBH5 in experimental cells, we verified its protective properties on cell proliferation, cell injury, and apoptosis in the process of MIRI. Besides, we provided a mass of latent different mRNAs with m⁶A modification variation in ALKBH5-overexpressed HL-1 cells. Mechanistically, we further screened the most potential targeted mRNAs and suggested that triple functional domain (Trio) mRNA could be upregulated by ALKBH5 by reducing m⁶A level of Trio.

Conclusions: This study demonstrated that the downregulated ALKBH5 might contribute to MIRI process by increasing the m⁶A modification of Trio mRNA and downregulating Trio.

Keywords: AlkB homolog 5 (ALKBH5); N6-methyladenosine modification (m6A modification); myocardial ischemia/reperfusion injury (MIRI).