Lysotracker-Based IVIS Imaging Combined with qPCR to Study Age-Related Lysosomal Pathway Changes in the Mouse Heart

Abstract

Lysosomal function plays a critical role in cardiac proteostasis and cellular health, but its regulation during ageing remains poorly understood. In this study, we hypothesised that ageing would alter both the abundance of acidic organelles and the acidification machinery supporting them. We used fluorescence-based IVIS imaging with Lysotracker™ Red in young (2-3 months old) and ageing mouse hearts (18 months old), to quantify whole-heart acidic-vesicle signal and performed RT qPCR analyses of lysosomal and autophagy-related markers, including Lamp2, Atp6v1a, Sqstm1, Cd63, Atg12, Nfe212 and M6pr. Global Lysotracker signal did not differ significantly between young and aged hearts, suggesting that the overall pool of acidic organelles is preserved. Stable Atp6v1a expression supports maintenance of lysosomal acidification machinery, while consistent Lamp2 levels indicate preserved structural stability and chaperone-mediated autophagy. In contrast, Sqstm1 was significantly upregulated, consistent with increased autophagy demand, and Cd63 expression was significantly elevated, reflecting potential alterations in extracellular vesicle trafficking rather than expansion of acidic compartments. No significant changes were observed in M6pr, Atg12, or Nfe212, suggesting that lysosomal enzyme trafficking (M6pr), core autophagy pathways (Atg12), and basal oxidative stress responses (Nfe212) remain largely preserved at the transcriptional level in aged hearts. Regional analysis revealed higher Lysotracker signal in atrial regions compared with ventricles, consistent with known enrichment of acidic vesicular stores and lysosomal Ca²⁺-linked signalling in atrial physiology. Batch effects were present but accounted for statistically, ensuring robustness of the conclusions. Collectively, our findings indicate that cardiac ageing preserves the acidic-vesicle pool and acidification machinery but triggers adaptive transcriptional remodelling of autophagy and vesicular trafficking pathways. These results provide new insight into lysosome-associated regulation in the ageing heart and establish a framework for future studies exploring therapeutic strategies targeting lysosomal pathways in cardiac ageing.