Landscape of protein l-lactylation in murine heart development: insights from proteomic analysis
Y Li, J Du, L Zhang, H Yang, B Xiong, X Zhou, Q SheAbstract
Background
During embryonic heart development, cardiomyocytes undergo a significant metabolic shift where glycolysis predominates as the energy source, leading to lactate production. Although lactate is a well-known metabolic substrate and an important protein post-translational regulator through lactylation, its roles in the heart development remain largely undefined.
Purpose
This study aimed to profile the dynamic changes of protein lactylation during murine heart development and to identify specific protein targets of this modification using mass spectrometry-based proteomics.
Methods
Hearts were harvested from C57BL/6 mice at embryonic days (E) 10.5, E13.5, E16.5 and postnatal day (P) 0. Publicly available single-cell RNA sequencing (scRNA-seq) data was analysed to assess the expression patterns of lactylation-related genes in different developmental stages. Global lactylation levels were examined via immunoblotting. To identify specific modification sites, quantitative proteomic analysis using pan-anti-l-lactyl-lysine antibody-based enrichment followed by liquid chromatography-tandem mass spectrometry was utilized. Differentially modified sites (|Fold Change| ≥1.5) were identified and subjected to fuzzy c-means clustering and functional enrichment analysis.
Results
Analysis of scRNA-seq data revealed that glycolysis-related genes, Ldha and Ldhb, are predominantly expressed in cardiomyocytes. Ldha (catalysing lactate production) expression peaked in early development and declined thereafter, whereas Ldhb showed an inverse pattern, suggesting high intracellular lactate levels in the early embryonic heart. Consistent with this, western blotting demonstrated that global cardiac lactylation levels were highest at E10.5 and gradually decreased by P0. The quantitative proteomic analysis identified a widespread downregulation of lactylation sites at P0 compared to E10.5 and E13.5. Clustering analysis revealed that six out of seven clusters exhibited an early-stage upregulation pattern. Functional enrichment indicated that hyper-lactylated proteins in early development were significantly enriched in cytoskeleton organization and motor protein pathways. Notably, extensive lactylation modifications were identified on critical sarcomeric components, including proteins associated with the thick filament (Titin [120 sites], Myh7 [43 sites], Myh6, Mybpc3), Z-disk (CapZ complex, Actn2), thin filament (Troponin, Tropomyosin), and M-band (Myomesin, Obscurin). Furthermore, we identified significant lactylation sites on key enzymes driving glycolysis/gluconeogenesis and critical regulators of the cell cycle, indicating a broader regulatory scope.
Conclusions
Extensive protein L-lactylation occurs during early murine heart development, correlating with the glycolytic metabolic state. The preferential modification of cytoskeletal and sarcomeric proteins suggests a potential link between metabolism and the structural proteome of the developing heart.Dynamic cardiac protein lactylomeSpatial landscape of lactylation