Carnitine acetyltransferase (CrAT)-Solute Carrier Family 25 Member 20 (SLC25A20) mediated acetylcarnitine shuttle links lipid oxidation to epigenetic control of cardiac metabolic maturation

Background During postnatal development, cardiomyocytes experience marked upregulation of lipid metabolism genes, which drives the transition from glycolysis to fatty acid β-oxidation (FAO). Fatty acids (FA) insufficiency hinders the maturation of induced pluripotent stem cell-derived cardiomyocytes, yet the underlying mechanism remains elusive. Purpose To elucidate the mechanism by which FA regulate cardiac metabolic maturation. Methods Transcriptome and Oroboros oxygraph-2k (O2k) were used to determine transcriptional and metabolic transition. Adeno-associated virus 9 and small interfering RNA were constructed to silence gene expression. Chromatin immunoprecipitation followed by sequencing (ChIP-seq), stable isotope tracing, subcellular organelle isolation, and immunofluorescence staining were also used. Results Palmitate markedly activated FAO-related gene transcription in cardiomyocytes, whereas no such activation was observed with glucose treatment. Knockdown of CD36 significantly suppressed FAO-related gene upregulation and FAO capacity. Furthermore, inhibition of CPT1b nearly abrogated FAO-related gene upregulation. While octanoic acid supplementation, which was oxidized independently of CPT1b, restored gene upregulation, indicating FAO was a prerequisite for FA-mediated metabolic maturation. We hypothesized that FAO-generated acetyl-CoA might act as a key mediator to modulate histone acetylation and genes transcription. Results confirmed that inhibition of histone acetyltransferase (HAT) significantly attenuated palmitate-induced global histone acetylation elevation and FAO-related gene upregulation. Histone acetylation site screening and ChIP-seq identified H3K18ac as the key modification regulating metabolic maturation, which was significant enriched at the promoter regions of FAO-related genes. Moreover, different from canonical citrate/acetate pathways, 13C-palmitate based stable isotope tracing uncovered that acetylcarnitine mediated the transport of FA-derived acetyl-CoA from mitochondria to nucleus. Specifically, mitochondrial CrAT converted FA-derived acetyl-CoA to acetylcarnitine, which was shuttled into nucleus via acetylcarnitine transporter SLC25A20. Nuclear CrAT ultimately catalyzed acetylcarnitine back to acetyl-CoA to fuel H3K18ac. Results showed that palmitate markedly increased the perinuclear localization of SLC25A20 and the amount of CrAT in both mitochondria and nucleus. Knockdown of SLC25A20 significantly suppressed palmitate-induced H3K18ac elevation and FAO-related gene transcription, and this inhibitory effect was far more pronounced than blocking canonical citrate/acetate pathways. Conclusion FA played a pivotal role in cardiac metabolic maturation: FAO-derived acetyl-CoA was shuttled to nucleus via CrAT-SLC25A20-mediated acetylcarnitine shuttle, which fueled H3K18ac at the promoter regions of FAO-related genes and promoted transcriptional activation, thereby orchestrating metabolic maturation.