Spatial Metabolomics Reveals GLA ‐Mediated Imbalance of Gal 2 Cer / GalCer as a Therapeutic Target in Myocardial Ischemia–Reperfusion Injury

ABSTRACT Myocardial ischemia–reperfusion (I/R) injury, a major complication of reperfusion therapies for acute myocardial infarction that exacerbates myocardial necrosis, impairs cardiac function, and worsens patient prognosis. Profound metabolic disturbances drive myocardial I/R injury; however, their spatial heterogeneity remains unclear. Using MALDI mass spectrometry imaging with spatial segmentation, we mapped the spatiotemporal metabolite dynamics in mouse heart tissue across multiple time points (30 min ischemia to 7 days reperfusion), aiming to identify critical metabolic pathways and regulatory targets underlying I/R injury. Glycerophospholipids and sphingolipids exhibited pronounced heterogeneity. Glycerophospholipid metabolism remained dysregulated throughout the I/R process, with long‐chain phospholipids (PA, PE, PC, and PS) exhibiting a distinct distribution gradient. They were enriched in the remote zone, downregulated in the border zone, and markedly reduced in the injured zone 6 h post‐reperfusion. Sphingolipid metabolism was dynamically reprogrammed, with galactosylceramide (GalCer) accumulating and digalactosylceramide (Gal 2 Cer) depleting in injured and border zones. Mechanistically, α‐galactosidase (GLA), which hydrolyzes Gal2Cer to GalCer, was upregulated in the border zone post‐I/R. Functional validation further demonstrated that both cardiomyocyte‐specific GLA knockdown (via AAV‐shGla) and pharmacological inhibition (using the GLA inhibitor GR181413A) effectively reduced myocardial infarct size, alleviated pathological remodeling, and improved cardiac function in I/R‐injured mice. Collectively, our spatial metabolomics revealed that GLA‐mediated Gal2Cer/GalCer imbalance is a critical regulator of myocardial I/R injury, and targeting GLA represents a promising therapeutic strategy.