A targeted knockdown of Agps in H9c2 cells lowered the level of plasmalogen lipids that disturbed mitochondrial function

Abstract

Plasmalogens belong to a subclass of phospholipids called ether lipids. The peroxisome synthesize these lipids and involve the limiting AGPS enzyme. In addition, mutations in the Agps gene are associated with cardiac manifestations. We hypothesized that decreased plasmalogens in cells negatively affect mitochondrial function and cell survival. We aimed to evaluate the metabolic and functional consequences of plasmalogens deficiency in a cardiomyoblastic cell line (H9c2 cells) modified using a CRISPR-Cas9 strategy targeting the Agps gene. We characterized this cell line using qPCR and immunoblotting to explore the influence on markers related to mitochondrial function [biogenesis, fatty acid metabolism (FA)], endoplasmic reticulum (ER) stress and apoptosis. AGPS reduction was validated at the gene (−58%) and protein (−78%) level, and is associated with decreased plasmalogens (−76%) that we analysed using mass spectrometry. This deficiency leaded to mitochondrial perturbations as revealed by changes on the expression of genes involved in biogenesis: Pgc-1α and ß (−27%; −61%), dynamics: Opa1 and Mff (−15%) as well as in the use of FA: Cd36 and Cpt1 (−52%; −51%) and in the oxidation of FA: Vlcad and Lcad (−22%; −13%). Consistently, using mass spectrometry, we observed decreased acylcarnitines (̴75%) used as markers of mitochondrial FA metabolism. This is accompanied with signs of increased ER stress and apoptosis as suggested by the increase in Chop gene expression (+31%) and the decreased uncleaved form of the Caspase-3 protein (−32%), respectively. Finally, we were able to normalize the levels of plasmalogen in our cells using alkylglycerols. Collectively, these results demonstrate that lowering plasmalogens affects mitochondrial function and cell stress in H9c2 cells. The normalisation of plasmalogens using alkylglycerols is encouraging to further characterize their metabolic and functional benefits.