Lactate-induced liquid-liquid phase separation modulates alternative splicing to promote cardiac regeneration

Background The poor prognosis and high mortality rates associated with heart failure remain critical global challenges. Current therapeutic strategies are limited by their inability to replace lost cardiomyocytes, as the regenerative capacity of the adult mammalian heart is negligible. Compelling evidence suggests that inducing a fetal-like metabolic state in cardiomyocytes can unlock endogenous regenerative potential. Consequently, elucidating the role of metabolites and metabolic reprogramming as key regulatory mechanisms in cardiac proliferation and regeneration is a pressing question in the field. Methods and Results Our preliminary data demonstrate that continuous administration of sodium lactate to increase myocardial lactate concentration in postnatal and adult mice significantly enhances cardiac repair. This treatment induced cardiac regeneration following apical resection in neonatal mice and promoted cardiomyocyte proliferation in the border zone, thereby improving cardiac function after myocardial infarction in adult mice. Recognizing the pleiotropic systemic effects of lactate, we employed a targeted screening approach based on sequencing and experimental validation. This led to the identification of a regenerative strategy centered on inhibiting the lactate exporter MCT4. Specific interference with MCT4 function caused lactate accumulation within cardiomyocytes and effectively promoted proliferation and repair. SnRNA-seq revealed significant enrichment of alternative splicing pathways in the regenerative cardiomyocyte subpopulation. Mechanistically, through integrated quantitative lactylome and proteomic analyses, we identified lactate acting as a signaling molecule. Elevated intracellular lactate promoted lactylation of DDX17, an RNA-binding protein. This post-translational modification inhibited DDX17's phase separation, consequently reducing its alternative splicing activity. This, in turn, prevented the excessive splicing of NOTCH2 into its short isoform, favoring the production of the long isoform, which promoted cardiomyocyte dedifferentiation and proliferation. Conclusion Our study identifies a novel mechanism wherein lactate, retained through MCT4 inhibition, acts as a signaling molecule to modulate alternative splicing via DDX17 lactylation-dependent phase separation. This metabolic-epigenetic crosstalk represents a critical regulatory axis for cardiac regeneration. Investigating how RNA-binding proteins (RBPs) influence alternative splicing through phase separation, as initiated by metabolic cues like lactate, opens a new avenue for understanding and enhancing heart repair. These findings provide a compelling scientific rationale for targeting lactate metabolism as a potential therapeutic strategy for the prevention and treatment of heart failure.