Abstract 4143634: Macrophage MST4 Exacerbates Cardiac Microvascular Ischemia/reperfusion Injury by Phosphorylating ACLY

Background: Cardiac microvascular dysfunction is a contributing factor to cardiac ischemia/reperfusion (I/R) injury. The impact of mammalian Ste20-like kinase 4 (MST4) on cardiac microvascular I/R injury remains unknown. Aims: To decipher the role of MST4 in cardiac microvascular I/R injury and the underlying mechanisms. Methods: Single-cell RNA sequencing was performed to explore the expression changes of MST4 in different cardiac cell types. Myeloid-specific MST4 knockout mice (MST4 CKO) and bone marrow transplantation were used to testify to the role of myeloid-derived MST4 in cardiac microvascular I/R injury. Bone-marrow-derived macrophage (BMDM) and cardiac microvascular endothelial cells (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. Results: MST4 was dominantly upregulated in CCR2+ monocyte-derived macrophages. Myeloid-specific deficiency of MST4 or wild type mice that were transplanted with BMDM from MST4 CKO mice improves cardiac performance in the I/R mouse model. Metabolomics showed that the 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. A high level of 7-HOCA was able to promote endothelial cell mtDNA release, leading to endothelial cell pyroptosis through the inflammasome pathway. However, mutation of serine 455 of ACLY blocked the role of MST4. Conclusions: Our data defined a previously unrecognized role of the 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 the MST4/ACLY pathway ameliorated cardiac microvascular I/R injury, which may provide a novel therapeutic strategy for I/R injury.