Endothelial HSPA1A preserves microvascular integrity in HFpEF via HIF-1α-dependent glycolysis and NNT K1079 lactylation.

BACKGROUND: HFpEF is characterized by microvascular rarefaction and endothelial metabolic dysfunction, but the mechanisms linking these abnormalities remain unclear. METHODS: HFpEF was induced in mice by a high-fat diet plus l-NAME. Whole-heart lactylome profiling identified altered lactylation sites. Mechanistic studies included endothelial-targeted AAV9 manipulation and NNT-WT/NNT-K1079R rescue in vivo, complemented by experiments in primary cardiac microvascular endothelial cells. RESULTS: Endothelial HSPA1A was markedly reduced in HFpEF. Endothelial-targeted HSPA1A knockdown aggravated diastolic dysfunction, microvascular rarefaction and exercise capacity, whereas HSPA1A overexpression alleviated these features. Mechanistically, HSPA1A interacted with BAG2 to restrain CHIP-dependent HIF-1α ubiquitination, thereby stabilizing HIF-1α and sustaining endothelial glycolysis and lactate availability. Endothelial HIF-1α knockdown attenuated HSPA1A-mediated protection in vivo. Consistent with impaired lactate availability, lactate levels and protein lactylation were reduced in HFpEF hearts, and NNT-K1079 was identified as a prominently decreased lactylation site. Lactate increased NNT lactylation, whereas p300 inhibition or knockdown attenuated this response. Compared with NNT-WT, the NNT-K1079R mutant showed reduced lactate responsiveness, impaired redox defense and angiogenic function. To test whether NNT-K1079 mediates HSPA1A protection, NNT epistasis and rescue experiments were performed. Endothelial NNT knockdown weakened HSPA1A-mediated protection, whereas NNT-WT, but not NNT-K1079R, restored microvascular density and diastolic function. In turn, loss of NNT-K1079 lactylation disrupted mitochondrial ROS/ATP homeostasis, thereby suppressing HSF1-dependent HSPA1A transcription. CONCLUSIONS: HSPA1A sustains NNT-K1079 lactylation and mitochondrial redox homeostasis through the BAG2-CHIP-HIF-1α-glycolysis/lactate pathway. In turn, NNT-K1079 lactylation sustains HSPA1A expression, forming a protective feedback loop whose disruption contributes to microvascular rarefaction and diastolic dysfunction in HFpEF.