Age-related changes in lysosomal abundance in mouse hearts assessed by Lysotracker fluorescence imaging and autophagy gene expression analysis

Abstract Purpose Lysosomal function is essential for cardiac proteostasis and cellular health, yet its regulation during ageing remains poorly defined. We aimed to determine whether whole-organ, fluorescence imaging using an In Vivo Imaging System (IVIS) provides a novel, rapid and scalable approach for quantifying lysosomal abundance in intact ex vivo hearts prior to deeper molecular analysis. Methods Ex vivo hearts from young (2–4 months) and aged (18 months) mice were labelled with Lysotracker™ Red and imaged using IVIS, to quantify whole-heart acidic-vesicle-associated fluorescence signals. Expression of lysosomal and autophagy-related genes ( Lamp2 , Atp6v1a , Sqstm1 , Cd63 , Atg12 , Nfe2l2 , M6pr ) was assessed by RT-qPCR. Results Whole-heart Lysotracker fluorescence did not differ significantly between age groups, indicating preservation of overall acidic-vesicle pool. Expression of Atp6v1a and Lamp2 was unchanged, suggesting maintained acidification capacity and lysosomal structure, whereas minor, upregulation of Sqstm1 might indicate increased autophagic demand and altered vesicle trafficking, which warrants further investigation. No statistically significant changes in M6pr , Atg12 , or Nfe2l2 were detected, suggesting transcriptional stability in enzyme trafficking, core autophagy, and oxidative stress pathways. Regionally, atria showed higher Lysotracker signal than ventricles, consistent with known enrichment of acidic vesicular stores in atrial physiology. Conclusion IVIS-based Lysotracker imaging provides a rapid whole-organ approach for assessing acidic vesicle distribution in intact hearts, enabling scalable screening of lysosome-associated physiology. While limited by depth-dependent optical attenuation and lack of organelle specificity, this approach complements molecular analysis and supports integrated investigation of lysosomal and autophagy pathways during cardiac ageing. Graphical Abstract