Introduction: Toxic lipid metabolites such as ceramide accumulate in diabetic cardiomyocytes (CMs) leading to structural and functional impairment. However, deleterious effects and mechanisms of lipotoxicity and the underlying mechanism of mitochondrial function have yet to be elucidated in human CMs.

Methods and results: To investigate the role of ceramides in diabetic cardiotoxicity, CMs were transfected with SPTLC1-overexpression plasmids, a subunit of the SPT complex critical for ceramide de novo synthesis. Overexpression of SPTLC1 in CMs resulted in increased total ceramide levels (p<0.02). Among all ceramide species analyzed, it was found that the levels of long-chain (C16:00 and C18:00) and very long-chain (C24:00) ceramide species were much in SPTLC1-overexpressed-CMs compared with CTRL–CMs. Furthermore, higher levels of apoptosis and cellular oxidative stress were confirmed by increased levels of cleaved caspase-3 (1.72±1.13, p<0.008) and MitoSOX staining (1.9±0.2, p<0.001), respectively. Overexpression of SPTLC1 in hiPSC-derived CMs significantly increased levels of the dynamin-related protein 1 (DRP1) and mitochondrial fission factor (MFF). Furthermore, the auto/mitophagic protein levels of LC3B (2.6±0.4, p<0.001), as determined by the ratio of lipidated LC3-II (autophagic) to non-lipidated LC3-I (non-autophagic), and PINK-1 (2.5±0.7, p<0.002) were elevated. Moreover, these cells showed decreased mRNA levels of the mitochondrial fusion MFN2 and OPA1 compared with CTRL-CMs. Furthermore, decreased mitochondrial basal respiration (18.8±2.18 versus 8.0±1.26, p<0.01), respiratory capacity (31.87±2.2 versus 10.32±1.3, p<0.01), and ATP production (15.41±2.37 versus 6.84±0.77, p<0.01) were observed, indicating an impaired mitochondrial function in the SPTLC1-overexpressed-CMs. Incubation of SPTLC1-overexpressed-CMs with myriocin improved FA ß-oxidation (and glucose oxidation as well as ATP production (6.84±0.77 versus 10.58±1.51, p<0.02) compared to non-treated SPTLC1-overexpressed-CMs. In addition, protein levels of PGC-1α (0.47±0.1, p<0.009) and AMP-activated protein kinase (p-AMPKα) (0.42±0.12, p<0.006) were decreased in SPTLC1-overexpressed-CMs. Interestingly, myriocin abolished the adverse effect of SPTLC1 overexpression in the CMs. This alteration in PGC-1α, a regulator of mitochondrial biogenesis, and p-AMPKα, a regulator of cellular energy and auto/mitophagy, confirmed our assumption that ceramide accumulation is involved in mitochondrial dysfunction.

Conclusion: Our results suggest that ceramide accumulation impaired mitochondrial function, increased auto/mitophagy and cell apoptosis in CMs. Moreover, inactivation of the de novo ceramide synthesis signaling pathway improves the mitochondrial morphology and metabolism, revealing a potential strategy to treat metabolic cardiomyopathy.