Selective Knockdown of Ceramide Synthases Reveals Opposite Roles of Different Ceramide Species in Cardiac Homeostasis

<b>Background/Objectives:</b> Sphingolipids are a class of lipids that play important structural and functional roles in the cell. Specific ceramide species are distinguishable through the fatty acid that is acylated to the sphingosine backbone, leading to distinct biological activities. Generally, long-chain (LC) ceramides (16:0 and 18:0) drive metabolic dysfunction resulting in the progression of different disease states, while very long-chain (VLC) ceramides (22:0 and 24:0) are thought to be either beneficial against disease progression or benign. In this study, we sought to alter the cellular composition of LC and VLC ceramides in ventricular HCMs to investigate how alterations in these lipids can affect the transcriptome of otherwise healthy HCMs. <b>Methods:</b> Here, we used specific siRNA to knockdown the ceramide synthases responsible for the production of LC and VLC ceramides in ventricular HCMs and investigated the changes in the transcriptome of HCMs with <i>CERS2</i> or <i>CERS5/6</i> silenced compared to control conditions. <b>Results:</b> Knocking down <i>CERS2</i> led to an increase in cell death as well as widespread reductions in cellular VLC sphingolipids. Additionally, we demonstrated that VLC sphingolipid species may play a protective role in maintaining cardiovascular function and that reducing these lipids may contribute to cardiac dysfunction. Similarly, knocking down CERS5 and CERS6 led to reduced LC ceramides and also resulted in profound changes in gene transcription. Interestingly, multiple genes and pathways were affected in the opposite direction when compared to the changes observed with the <i>CERS2</i> knockdown. <b>Conclusions:</b> Taken together, our results suggest pathways through which VLC ceramides may contribute to cardiac protection, and pathways where LC ceramides may promote HCM stress and the development of cardiac disease.