Targeting phospholipid metabolism in cardiac ischemia-reperfusion injury protection mediated by PITPNC1

Background Cardiac ischemia-reperfusion injury (IRI) remains a challenge in the management of coronary artery diseases. The pathophysiology of IRI involves alterations in energy metabolism, which is predominantly dependent on the oxidative phosphorylation of fatty acids and other lipids. Any disruption of this process during IRI exacerbates microvascular disorder and cardiac dysfunction. Specifically, phospholipid metabolism plays a crucial role in maintaining the stability of organelle lipid membranes, including the endoplasmic reticulum (ER), endosome membranes, etc., the disorder of which may promote the progression of cardiovascular diseases, particularly under hypoxic/ischemic conditions. Yet, the underlying mechanisms remain to be elucidated. Purpose Our study aims to investigate the role of PITPNC1-mediated phosphatidylinositol (PI) metabolic reprogramming in protection against IRI. Methods We performed mRNA sequencing and lipidomic mass spectrometry (MS) analysis in the ischemic area of the mouse I/R model and the human cardiac microvascular endothelial cell oxygen-glucose deprivation (OGD/R) model. In vitro, transmission electron microscopy (TEM) was applied to observe changes in membrane structures. Immunoprecipitation (IP) followed by LC/MS was employed to identify PITPNC1-interacting proteins. Immunofluorescence (IF) was used to verify the localization and co-localization of PI metabolism-related molecules, and several phenotypic changes were validated. Cardiac dysfunction and chronic remodeling post IRI was assessed through echocardiography and Masson’s trichrome staining. Results Our multi-omics data identified significant alterations in the metabolism of various lipids, notably, several glycerophospholipid species, particularly in the PI family, along with changes in their phosphorylation regulation, which were more pronounced after IRI. Furthermore, PI transfer protein family, especially PITPNC1, was identified as a key regulator of PI metabolism. PITPNC1 expression was significantly decreased in both I/R and OGD/R models. In vitro, PITPNC1-OE was found to promote cell proliferation, protect against late-stage apoptosis, facilitate cell cycle progression, and improve cell viability after OGD/R injury (Fig.2 I-L). TEM revealed that PITPNC1-OE partially restored ER morphology and maintained the continuity of other organelle membranes (e.g., endosomal membrane) (Fig.2 M). IF and IP-LC/MS showed that PITPNC1 regulates PI phosphorylation/dephosphorylation by anchoring the PI family on membrane structures and recruiting downstream metabolic enzymes and thus affects the morphology and function of the ER (Fig.2 N). In vivo, PITPNC1-OE significantly alleviated cardiac dysfunction as evidenced by improved ejection fraction and anatomical structure (Fig.2 O-P). Conclusions Targeting PITPNC1-mediated phosphatidylinositol metabolism protects against cardiac IRI by promoting ER integrity.