Macrophage MST4 exacerbates cardiac microvascular ischaemia/reperfusion injury by phosphorylating ACLY

Abstract Background Cardiac microvascular dysfunction is a contributing factor in the development of cardiac ischaemia/reperfusion (I/R) injury. The STE20-type kinases play important role in cardiovascular diseases. However, the impact of mammalian Ste20-like kinase 4 (MST4) on cardiac microvascular I/R injury remains unknown. Purpose To decipher the role of MST4 in cardiac microvascular I/R injury and the underlying mechanisms. Methods Single-cell RNA sequencing of I/R mouse heart was performed to explore the expression changes of MST4 in different cell types. MST4 flox (MST4 f/Y) mice was crossed with myeloid-specific LysM-Cre mice to generate myeloid-specific MST4 knockout mice (MST4 CKO). Bone marrow transplantation was used to testify the role of myeloid-derived MST4 in cardiac microvascular I/R injury. Bone-marrow-derived macrophage (BMDM) and cardiac microvascular endothelial cell (CMEC) were used to dissect molecular mechanisms. Adeno-associated virus and adenovirus were constructed to overexpress ATP Citrate Lyase (ACLY) with mutation of serine 455 (S455A) in vivo and in vitro, respectively. Phosphoproteomics, coimmunoprecipitation coupled with liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis and metabolomics were performed to decipher the downstream of MST4. Results MST4 is dominantly upregulated in CCR2+ monocyte-derived macrophages. Myeloid-specific deficiency of MST4 improves cardiac performance in I/R mouse model. Wild type (WT) mice transplanted with BMDM from MST4 CKO mice also exhibited improved cardiac function after I/R, however, MST4 CKO mice transplanted with BMDM from WT mice showed deteriorated cardiac performance. Metabolomics showed that primary bile acid biosynthesis pathway was upregulated in BMDM overexpressing MST4. The level of 7α-hydroxyl3-oxo-4-cholestenoic acid (7-HOCA), an intermediate of bile acid, was dramatically increased in I/R mice or BMDM overexpressing MST4, which can be reversed by knocking down MST4. Phosphoproteomics and LC-MS/MS analysis discovered that ACLY was one of the most important substrates of MST4 in the process of I/R injury. ACLY was directly phosphorylated by MST4 at serine 455, which increased ACLY enzyme activity. Mechanistically, ACLY promoted cholesterol catabolism through the bile acid synthesis pathway, leading to the accumulation of 7-HOCA in the macrophage-endothelial cell microenvironment. High level of 7-HOCA was able to promote endothelial cell mtDNA release, leading to endothelial cell pyroptosis through inflammasome pathway. However, mutation of serine 455 of ACLY blocked the role of MST4. Conclusions Our data defined a previously unrecognized role of MST4/ACLY pathway in cardiac microvascular I/R injury. MST4 led to the accumulation of 7-HOCA by phosphorylating ACLY, furthermore, 7-HOCA promoted endothelial cell injury. Targeting MST4/ACLY pathway ameliorated microvascular I/R injury, which may provide a novel therapeutic strategy for I/R injury.