Inhibition of ceramide synthesis improves the outcome of ischemia/reperfusion injury in cardiomyocytes derived from human induced pluripotent stem cell.

Abstract

Background: Ceramides are bioactive sphingolipids that have physiological effects on inflammation, apoptosis, and mitochondrial dysfunction. They may play a critical role in the harm of ischemia/reperfusion (IR). Ceramides and IR injury are not well-studied, and there is a lack of human data.

Methods and results: Current studies aimed to investigate the role of ceramide buildup in cardiomyocytes (CMs) death using CMs derived from human induced pluripotent stem cell (hiPSC) as a model for simulating IR injury in vitro. In our model, serum- and glucose-free media was used to expose hiPSC-derived CMs to hypoxia (3% O₂) for 6 h (hrs), followed by reoxygenation (20% O₂) for 16 h. In contrast to normoxia (control) or hypoxia (ischemia), our data showed that following IR, there was an increase in the formation of mitochondrial superoxide and the mRNA levels of genes regulating ceramide synthesis, such as CerS2 and CerS4 in CMs. Further, there was a considerable rise in the levels of total ceramide, long-chain (C16:0, C18:0, and C18:1), and very long-chain (C22:0 and C24:1) ceramide species in CMs following reperfusion in comparison to control or ischemic CMs. Interestingly, compared to CMs exposed to IR without inhibitor, our data showed that inhibition of ceramide formation with fumonisin B1 (FB1) significantly lowered ceramide levels, reduced apoptosis, improved mitochondrial function, and enhanced survival of CMs exposed to IR. Furthermore, we used a transgenic mouse model, in which the CerS2 gene was overexpressed in the CMs of α-MHC-CerS2 mice, to validate the basic idea that ceramide contributes to heart disease in vivo. Our results showed that the heart tissues of α-MHC-CerS2 mice had significant levels of long-chain and very long-chain ceramides, which causes increased apoptosis, proinflammatory cytokines, interstitial inflammatory cell infiltration, and collagen deposition.

Conclusions: Results from both in vitro and in vivo experiments show that ceramides have a significant role in either mediating or inducing damage to CMs. Additionally, in vitro findings show that ceramide reduction improves the outcome of IR injury by lowering intracellular Ca²⁺ [Ca²⁺]_i concentration and improves mitochondrial function changes during IR.