Methods for detection of cardiac glycogen-autophagy

Area of Science:

• Cell Biology
• Metabolism
• Autophagy

Background:

• Glycogen-autophagy (glycophagy) is a selective autophagy pathway for glycogen degradation.
• STBD1 and GABARAPL1 are key proteins in glycophagy.
• Assessing glycophagy activity is crucial for understanding its role in metabolism and disease.

Purpose of the Study:

• To develop and validate tools for quantifying cardiac glycophagy activity.
• To investigate basal glycophagy flux in cardiac tissue.

Main Methods:

• Developed antibodies against GABARAPL1 for immunoblotting.
• Utilized a GFP-GABARAPL1 cardiac cell line to measure lysosomal flux.
• Quantified endogenous glycophagy flux in rat ventricular myocytes and mouse hearts using lysosomal inhibitors (bafilomycin, chloroquine) and measuring glycogen accumulation.

Main Results:

• Antibodies detected endogenous GABARAPL1.
• Lysosomal flux was quantifiable using GFP puncta in the cell line.
• Combined bafilomycin and chloroquine treatment significantly increased glycogen content in myocytes and hearts, indicating basal glycophagy flux.

Conclusions:

• Established methodologies for assessing cardiac glycophagy.
• These tools enable further research into glycophagy's role in cardiac health and disease.
• Provides a comprehensive toolbox for tracking cardiac glycophagy activity.