A hierarchical lectin-based multimodal workflow for spatial mapping of extracellular matrix glycans in fibrotic hearts

Cardiac fibrosis with excessive extracellular matrix (ECM) accumulation is a hallmark of hypertensive heart failure. Characterizing glycan alterations within fibrotic ECM requires analytical strategies that integrate molecular detection with spatial and structural information. However, spatial glycomics approaches applicable to ECM-rich tissue regions remain limited. Here, we established an integrated lectin-based spatial glycomics workflow combining lectin microarray-based tissue glycome mapping, lectin histochemical staining, and low-vacuum scanning electron microscopy (LVSEM). This hierarchical analytical design enables lectin signal detection, spatial localization, and structural visualization within the same tissue section. Using this workflow, we mapped N- and O-glycan distribution in fibrotic regions of hypertensive heart failure rat hearts. Among 45 lectins, Maackia amurensis hemagglutinin (MAH)-reactive sialo-O-glycans were markedly increased in WFA-positive fibrotic regions compared with controls. Glycosidase pretreatment selectively reduced WFA and MAH signals, confirming their specificities for N- and O-glycans, respectively. High-resolution single-slide imaging combining lectin fluorescence histochemistry with LVSEM revealed distinct spatial partitioning of WFA and MAH signals within fibrotic ECM regions. Co-staining with ECM glycoproteins, including periostin, collagen VI α6 chain, cartilage intermediate layer protein 1, and thrombospondin 4, showed partial spatial overlap with lectin signals, suggesting that multiple ECM glycoproteins may contribute to the observed glycan signals. These results demonstrate a lectin-based spatial glycomics workflow integrating glycomic signal detection, spatial localization, and ultrastructural validation, establishing a lectin-based multimodal analytical framework for spatially resolved glycan partitioning in fibrotic tissues.