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Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry
Published on: July 21, 2017
Age-related changes in lysosomal abundance in mouse hearts assessed by Lysotracker fluorescence imaging and autophagy
Jawaher Albulushi1, Hannah Coghlan1, Mohesh Moothanchery2
1Institute of Systems, Molecular and Integrative Biology, Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, UK.
Insights
Whole-organ imaging of mouse hearts using In Vivo Imaging System (IVIS) and Lysotracker Red revealed preserved lysosomal abundance with aging. This rapid method aids in studying cardiac aging and lysosome function.
Area of Science:
- Cardiovascular Biology
- Cellular Biology
- Aging Research
Background:
- Lysosomal function is critical for maintaining cardiac proteostasis and cellular health.
- The regulation of lysosomal function during cardiac aging is not well understood.
- Novel methods are needed for rapid, scalable quantification of lysosomal abundance in intact organs.
Purpose of the Study:
- To evaluate whole-organ fluorescence imaging using In Vivo Imaging System (IVIS) as a novel approach.
- To quantify lysosomal abundance in intact ex vivo mouse hearts.
- To assess the utility of IVIS for rapid and scalable analysis prior to molecular investigation.
Main Methods:
- Ex vivo hearts from young and aged mice were labeled with Lysotracker™ Red.
- Whole-heart acidic-vesicle-associated fluorescence signals were quantified using IVIS.
- Gene expression of lysosomal and autophagy-related markers (Lamp2, Atp6v1a, Sqstm1, Cd63, Atg12, Nfe2l2, M6pr) was assessed via RT-qPCR.
Main Results:
- Whole-heart Lysotracker fluorescence did not significantly differ between young and aged mice, indicating preserved overall lysosomal pools.
- Gene expression analysis showed maintained lysosomal acidification (Atp6v1a) and structure (Lamp2), with a minor upregulation of Sqstm1 suggesting altered autophagy.
- Atria exhibited higher Lysotracker signals than ventricles, consistent with known regional differences in acidic vesicular stores.
Conclusions:
- IVIS-based Lysotracker imaging offers a rapid, whole-organ method for assessing acidic vesicle distribution in intact hearts.
- This approach enables scalable screening of lysosome-associated physiology and complements molecular analyses.
- The method supports integrated investigations into lysosomal and autophagy pathways during cardiac aging, despite limitations in optical depth penetration and organelle specificity.
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.
