Effect of knocking down CERS2 or a double knockdown (KD) CERS5/6 in ventricular cardiomyocytes on gene expression under conditions of hypertrophy induced by phorbol 12-myristate 13-acetate (PMA) or controls.

Sphingolipids are an important class of lipids that play imperative structural and functional roles in the body. Ceramides constitute the central essential structural backbone of more complex sphingolipids, thus alterations in the genes that regulate their expression could dysregulate the entire cellular sphingolipid metabolic scheme. The fatty acid acylated to the sphingosine backbones distinguishes ceramide species both structurally and functionally. Higher plasma concentrations of 16:0 ceramide are associated with an increased risk of heart failure, while higher concentrations of 22:0 plus 24:0 ceramide are associated with lower risk. Here, we silenced the ceramide synthase genes (CERS) responsible for the production of long-chain (LC) 16:0 ceramide (CERS5/6) or very long-chain (VLC) 22:0 and 24:0 ceramide (CERS2) in immortalized human ventricular cardiomyocytes and examined how these silenced genes altered the cardiomyocyte’s ability to maintain homeostasis in control conditions and under cardiac hypertrophy in response to PMA treatment. Taken together, our data provides support for the importance of the different ceramide species in maintaining cardiovascular homeostasis and in the development and progression of cardiac hypertrophy. To investigate the role of LC and VLC ceramides in maintaining cardiomyocyte homeostasis and during the development and progression of cardiac hypertrophy, we silenced CERS2 or CERS5/6 in a human ventricular cardiomyocyte cell line using siRNA. We then treated the KD or control cells with the hypertrophy inducing compound PMA or controls and analyzed how reductions in the respective ceramide species altered the hypertrophic response. We performed gene expression analysis using data obtained from RNA-seq of the 8 different treatment groups (n=3).