Downregulation of heat shock protein 90 alpha family class A member 1 inhibits necroptosis in cardiac microvascular endothelial cells and alleviates sepsis-induced cardiomyopathy.

Background: Sepsis-induced cardiomyopathy (SIC) is a disease of cardiac dysfunction caused by sepsis and represents one of the most serious complications of sepsis. Cardiac microvascular endothelial cells (CMECs) play an important role in supporting and nourishing cardiomyocytes (CMs) and blood vessels. SIC may cause injury to CMECs, thereby accelerating the disease progression. In addition, heat shock protein 90 alpha (HSP90α) is encoded by heat shock protein 90 alpha family class A member 1 (HSP90AA1). As a stress-responsive molecule, HSP90α exhibits markedly elevated expression under pathological conditions such as inflammation, ischemia, and hypoxia. Furthermore, HSP90α plays a crucial role in disease progression.

Objectives: Using mice as models, this study aims to investigate the effects of Hsp90AA1 (the mouse ortholog of human HSP90AA1) on the function and necroptosis of mouse cardiac microvascular endothelial cells (MCMECs), and to clarify whether Hsp90AA1 is involved in the pathological process of SIC through the nuclear factor-kappa B (NF-κB) signaling pathway.

Methods: Heterogeneity of capillary endothelial cells (Cap ECs) in SIC was analyzed via single-cell RNA sequencing (scRNA-seq), followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA). Sepsis models were established both in vitro and in vivo by inducing MCMECs with lipopolysaccharide (LPS) and performing cecal ligation and puncture (CLP) surgery on mice, respectively. Cell viability was detected by cell counting kit-8 (CCK-8). Hsp90aa1 expression was detected by Western blot. Phosphorylated receptor-interacting protein kinase 3 (p-RIPK3), phosphorylated mixed lineage kinase domain-like pseudokinase (p-MLKL), and phosphorylated nuclear factor-kappa B p65 subunit (p-NF-κB p65) expression was detected by immunofluorescence. MCMEC tube formation and migration were separately detected by the tube formation assay and the Transwell migration assay. Serum creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), intercellular adhesion molecule-1 (ICAM-1), and vascular cellular adhesion molecule-1 (VCAM-1) levels in mice were determined by enzyme-linked immunosorbent assay (ELISA). Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured by transthoracic echocardiography (TTE).

Results: scRNA-seq techniques showed a significant increase in the number of heat shock protein 90 alpha family class A member 1-positive Cap ECs (Hsp90aa1⁺Cap ECs) in SIC. Additionally, these cells exhibited upregulation of necroptosis and NF-κB signaling pathways. After LPS treatment, MCMEC viability was decreased, and p-RIPK3, p-MLKL, and Hsp90aa1 expression was increased. Whereas, Hsp90aa1 knockdown restored cell viability of LPS-treated cells, decreased p-RIPK3 and p-MLKL expression, and improved tube formation and cell migration. Down-regulation of Hsp90aa1 reduced LPS-induced p-NF-κB p65 expression. In CLP model mice, LVEF and LVFS were decreased; CK-MB, cTnI, ICAM-1, and VCAM-1 levels were elevated; and p-RIPK3 and p-MLKL expression was increased. Knockdown of Hsp90aa1 alleviated cardiac injury in CLP mice.

Conclusion: Knockdown of Hsp90aa1 can alleviate SIC by inhibiting necroptosis in CMECs via downregulation of the NF-κB signaling pathway.