Inhibition of ceramide synthesis improves the outcome in ischemia/reperfusion injury using human-induced pluripotent stem cell derived cardiomyocyte

Introduction and aim Ceramides are proven to be biologically active in apoptosis, inflammation, mitochondrial dysfunction, and as a second messenger in various signaling pathways1. However, the data linking the role of ceramides in ischemia/reperfusion injury (I/R injury) are lacking. We aimed to establish an I/R injury model using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) and to evaluate ceramide levels, ceramide synthesis pathway, and outcome of CM with inhibition of ceramide synthesis during I/R injury. Methods HiPSC technology has been used to generate functional human CMs to elucidate the underlying mechanisms of the pathophysiology of the human heart. Results In our model, we observed an increase of mRNA levels of genes regulating ceramide synthesis after 6 h of ischemia followed by 16 h reperfusion, such as SPTLC1 (1.1±0.08 vs 1.0, p=0.2), CerS2 (1.6±0.3 vs 1.0, p<0.001), CerS4 (1.3±0.1 vs 1.0, p=0.02), CerS5 (1.3±0.1 vs 1.0, p=0.03), and SMPD (1.6±0.1 vs 1.0, p=0.008) compared to control. Also, both long- and very long-chain ceramide species levels measured with mass spectrometry were increased significantly after 6 h ischemia followed by 16 h reperfusion compared to control (C14:0: 1,1±0.3 pmol/million cells vs 0,3±0,2 pmol/ million cells, p=0.02 and C24:1: 26,3±7,1 pmol/ million cells vs 9,6±3,4 pmol/ million cells, p=0.02). Inhibition of ceramide synthesis with Fumonisin B1 (FB1) significantly increased the viability after 6h of ischemia followed by 16 h of reperfusion compared to CMs incubated without inhibitors (32.2%±1.5% vs 26.9%±2.6%, p=0.04). Interestingly, we identified two mechanisms with which the viability improves after incubation with ceramide inhibitor. The first mechanism observed could be the restoration of both intracellular calcium baseline (control 29±1.2, I/R 55±5.7 and I/R with FB1 35.6±2.5, p<0,001) and peak (control 45.1±5.6, I/R 94.3±5.7 and I/R with FB1 56.5±7.5, p<0,001) levels to nearly the same levels as observed in control samples. A possible cause of increased calcium oscillations after 6 h of ischemia followed by 3 h of reperfusion in the first place could be an upregulation of the RyR2 levels detected by qPCR (2.5±0.4 vs control 1.0, p=0.008). The second mechanism of improving viability in I/R injury could be a decrease of generation of reactive oxygen species (ROS) detected by MitoSOX dye after incubation with FB1 inhibitor to nearly the same levels as observed in control (control 22±5.1, I/R 33.8±5.8 and I/R with FB1 30.7±5.9, p=0,06). Conclusion We conclude that ceramides have important implications in either mediating or causing injury and their inhibition improves the outcome of I/R injury by decreasing ROS generation and improving calcium oscillations. FUNDunding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Jena University Hospital, Clinic for Internal Medicine 1Interdisciplinary Center for Clinical Research Jena