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.