Methods for detection of cardiac glycogen-autophagy

Abstract

Glycogen-autophagy ('glycophagy') is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N-terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.

Keywords: Atg8; GABARAPL1; autophagy; glycogen; glycophagy flux.

Figure 1.

High sequence similarity between GABARAP Atg8 proteins compromises GABARAPL1 antibody specificity. (A) Protein sequence alignment of human GABARAP Atg8 subfamily members. (B) Percentage similarity between protein sequences of GABARAP, GABRAPL2 and GABARAPL1. (C) Characteristics of three commercially available GABARAPL1 antibodies (#1 Proteintech (11010-1-AP), #2 Abcam (ab86497), #3 Cell signaling (26632)). (D) Schematic of Crispr/Cas9 genome editing design targeting Gabarapl1 for gene deletion in mice (green: homologous part of gRNA sequences, red: proto-spacer adjacent motifs). (E) Validation of Gabarapl1 knockout using DNA electrophoresis (tail sample). (F) Immunoblot of total cardiac homogenate shows that Ab#2 and Ab#3, but not Ab#1, detect a specific GABARAPL1 band at 18kDa, validated using the GABARAPL1-KO mouse heart as a negative control (note absence of 18kDa GABARAPL1 (GABL1) band in KO samples for Ab#2 and Ab#3, but not Ab#1). (G) Immunoblot of crude membrane (containing autophagosome membrane structures) and cytosol fractions from cardiac homogenate samples comparing Ab#1 and Ab#3. A specific 18kDa GABARAPL1 band is detected in membrane and cytosol fractions using Ab#3 (validated by KO sample). Ab#1 does not detect the 18kDa GABARAPL1 band in either fraction.

Figure 2.

Quantification of cardiac glycophagy flux in vitro and ex vivo. (A) Autophagosome-lysosome fusion schematic illustrating the mechanism of action of lysosomal inhibitors chloroquine (CQ) and bafilomycin (Baf). (B) Glycophagosome membrane-bound GABARAPL1 puncta visualized using GFP-Gabarapl1 in mouse atria-derived HL1 cardiomyocytes. HL1 cells were transduced with GFP-Gabarapl1 and imaged before (control, Ctrl) and after 2 hours lysosomal inhibition with 100 nM Baf. Lysosomal Gabarapl1 flux is determined by the extent of accumulation of GFP-Gabarapl1 puncta in response to lysosomal inhibition with Baf. Quantification of GFP epifluorescent micrographs presented as the number of puncta-containing cells as a percentage of total cells/60x image, puncta are indicated by white arrows (9-13 images/well, n = 3 independent wells). (C) Glycophagy flux is measured in neonatal rat ventricular myocytes (NRVMs) following 2h treatment with 50 µm CQ, 100 nM Baf or 50 µm CQ + 100 nM Baf. Flux is determined by the extent of glycogen accumulation in response to lysosomal inhibition (n = 5-11 independent wells per group). (D) Immunoblot showing accumulation of macro-autophagy marker LC3BII confirms lysosomal inhibition by CQ, Baf and CQ + Baf treated NRVMs (n = 6-9 independent wells per group). (E) Glycophagy flux is measured in ex vivo isolated perfused mouse hearts following 30min treatment with 20 µm CQ, 1 µm Baf or 20 µm CQ + 1 µm Baf. Flux is determined by the extent of glycogen accumulation in response to lysosomal inhibition (n = 5-10 mice per group). (F) Immunoblot showing accumulation of macro-autophagy marker LC3BII confirms lysosomal inhibition by Baf, but not CQ or CQ + Baf in perfused mouse hearts (n = 5-10 mice per group). Data presented as mean ± SEM, *p < 0.05.