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Targeted glycophagy ATG8 therapy reverses diabetic heart disease in mice and in human engineered cardiac tissues

Nature Cardiovascular Research (2025)Cite this article

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Abstract

Diabetic heart disease is highly prevalent and is associated with the early development of impaired diastolic relaxation. The mechanisms of diabetic heart disease are poorly understood, and it is a condition for which there are no targeted therapies. Recently, disrupted glycogen autophagy (glycophagy) and glycogen accumulation have been identified in the diabetic heart. Glycophagy involves glycogen receptor binding and linking with an ATG8 protein to locate and degrade glycogen within an intracellular phagolysosome. Here we show that glycogen receptor protein starch binding domain protein 1 (STBD1) is mobilized early in the cardiac glycogen response to metabolic challenge in vivo, and that deficiency of a specific ATG8 family protein, γ-aminobutyric acid type A receptor-associated protein-like 1 (GABARAPL1), is associated with diastolic dysfunction in diabetes. Gabarapl1 gene delivery treatment remediated cardiomyocyte and cardiac diastolic dysfunction in type 2 diabetic mice and the diastolic performance of ‘diabetic’ human induced pluripotent stem cell-derived cardiac organoids. We identify glycophagy dysregulation as a mechanism and potential treatment target for diabetic heart disease.

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Fig. 1: Glycophagy mediator proteins are mobilized early in cardiac glycogen response to metabolic challenge in vivo.
Fig. 2: Cardiac GABARAPL1 (Atg8) deficiency is associated with diastolic dysfunction in diabetes in vivo and induces glycogen accumulation in vitro.
Fig. 3: Gabarapl1 knockdown induces diastolic dysfunction and cardiac glycogen accumulation in mice.
Fig. 4: AAV Atg8 gene delivery rescues cardiomyocyte and cardiac diastolic dysfunction in diabetic mice.
Fig. 5: AAV Atg8 gene delivery in vivo expedites cytosolic Ca2+ removal during cardiomyocyte relaxation in diabetic mice.
Fig. 6: AAV Atg8 gene delivery improves diastolic performance in ‘diabetic’ human iPSC-derived cardiac organoids.
Fig. 7: Proposed cellular mechanism.

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Acknowledgements

We acknowledge D. Stapleton for providing the glycogen phosphorylase antibodies. This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRCA; 1027865, 628643, 1082215, 1037320, 1067869, 1157320), the Diabetes Australia Research Trust, Stem Cells Australia, the National Heart Foundation of Australia (NHFA), the New Zealand Marsden Fund (14-UOA-160, 19-UOA-268), the Health Research Council of New Zealand (19/190) and the University of Auckland Faculty Research Development Fund, and the National Institutes of Health, USA (R01 HL155346-01 and R01 HL144509-01). Fellowship support is acknowledged from NHMRCA (J.E.H., R.G.P., E.R.P.) and Heart Foundation of Australia (E.R.P., J.E.H., K.L.W.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Author information

Authors and Affiliations

  1. Department of Physiology, University of Auckland, Auckland, New Zealand

    K. M. Mellor, P. Koutsifeli, L. J. Daniels, M. Annandale, X. Li, S. L. James & L. M. D. Delbridge

  2. Department of Anatomy and Physiology, University of Melbourne, Melbourne, Victoria, Australia

    K. M. Mellor, U. Varma, C. L. Curl, J. V. Janssens, G. B. Bernasochi, A. J. A. Raaijmakers, K. L. Weeks, J. R. Bell, E. R. Porrello & L. M. D. Delbridge

  3. Cedars-Sinai Medical Center, Los Angeles, CA, USA

    J. V. Janssens, D. J. Taylor, K. Raedschelders, J. E. Van Eyk & R. A. Gottlieb

  4. Baker Department of Cardiometabolic Health, University of Melbourne, Melbourne, Victoria, Australia

    K. L. Weeks

  5. Murdoch Children’s Research Institute, Melbourne, Victoria, Australia

    R. J. Mills & E. R. Porrello

  6. Centre for Microscopy and Microanalysis and Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia

    R. G. Parton

  7. Institute of Molecular Medicine, Peking University, Beijing, China

    X. Hu & R. P. Xiao

  8. Department of Microbiology, Anatomy, Physiology and Pharmacology, La Trobe University, Melbourne, Victoria, Australia

    J. R. Bell

  9. Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia

    T. J. O’Brien

  10. Department of Neuroscience, Monash University, Melbourne, Victoria, Australia

    T. J. O’Brien

  11. Department of Physiology, HeartOtago, University of Otago, Dunedin, New Zealand

    R. Katare

  12. School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia

    J. E. Hudson

  13. QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia

    J. E. Hudson

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  1. K. M. Mellor
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Contributions

L.M.D.D. and K.M.M. conceived and designed the experiments. K.M.M., U.V., P.K., C.L.C., J.V.J., L.J.D., G.B.B., A.J.A.R., M.A., X.L., S.L.J., D.J.T., K.R., R.J.M. and R.G.P. performed the experiments. K.M.M., U.V., P.K., C.L.C., J.V.J., L.J.D., G.B.B., A.J.A.R., M.A., X.L., S.L.J., D.J.T., K.R., K.L.W., R.J.M., J.R.B., E.R.P. and J.E.H. analyzed the data. L.M.D.D., K.M.M., E.R.P., J.E.H., J.E.V., X.H., R.P.X., R.K., T.J.O. and R.A.G. contributed materials and analysis tools. L.M.D.D. and K.M.M. wrote the paper.

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Correspondence to L. M. D. Delbridge.

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Extended data

Extended Data Fig. 1 Cardiac function and glycogen handling in diabetic hearts.

a, Ratio of phosphorylated glycogen synthase (pGS Ser641) to total glycogen synthase (GS) is unchanged in T2D mouse hearts (n = 8 animals/group, molecular weight indicated in kilodaltons, kDa, analyzed by unpaired 2-sided T-test). b, Ratio of phosphorylated glycogen phosphorylase (pGP Ser14) to total glycogen phosphorylase (GP) is unchanged in in T2D mouse hearts (Ctrl: n = 8, T2D: n = 10 animals/group, analyzed by unpaired 2-sided T-test). c, Exemplar M-mode and flow Doppler echocardiography traces. d, Echocardiography M-mode-derived ejection fraction and fractional shortening are unchanged with diabetes in type 2 diabetic (T2D, 14 week high fat sugar diet) vs control (Ctrl) mice (%EF n = 10 animals/group. %FS Ctrl: n = 10, T2D: n = 9 animals/group, analyzed by unpaired 2-sided T-test). e, Glycogen content normalized to protein in adult rat cardiomyocyte lysate from Ctrl and streptozotocin (STZ)-induced diabetic rats (8 weeks post-STZ, Ctrl: n = 10, STZ: n = 9 animals/group, analyzed by unpaired 2-sided T-test, p = 0.0037). f, Ca2+ time constant of decay (Tau) in isolated adult cardiomyocytes from control and STZ rats (8 weeks post-STZ, Ctrl: n = 10, STZ: n = 9 animals/group, analyzed by unpaired 2-sided T-test, p = 0.0022). g, Correlation of cardiomyocyte glycogen content and Ca2+ time constant of decay (Tau) in control (blue) and STZ (pink) rat cardiomyocytes (unloaded cells; r, Pearson correlation coefficient). h, Gabarapl1 mRNA is lower in atrial appendage samples from patients with T2D (n = 12 patients/group, analyzed by unpaired 2-sided T-test, p = 0.043). i, GABARAPL1 protein content in fractionated atrial appendage samples from patients with T2D (Homog: Ctrl n = 9, T2D n = 4; Memb, Cyt, Memb:Cyt ratio: n = 5 patients/group, analyzed by unpaired 2-sided T-test). j, STBD1 protein content is increased in atrial appendage samples from patients with T2D (Ctrl: n = 9, T2D: n = 4 patients/group, analzyed by unpaired 2-sided T-test, p = 0.0418). Data presented as truncated violin plots with median and upper & lower quartiles indicated, or bar graphs mean +/- sem. *p < 0.05. Related to Fig. 2.

Source Data

Extended Data Fig. 2 Crispr-Cas9 Gabarapl1-KO mouse validation.

a, Gabarapl1 sequence details for the 8 founder Crispr-Cas9 mouse lines confirming excision of exons 2-4 from the Gabarapl1 gene (Next Generation sequencing). Text in red highlights mutations differing from the predicted sequence. b, Validation of Gabarapl1 knockdown using DNA electrophoresis to identify the presence of the Gabarapl1-KO allele in the heterozygote Gabarapl1-KO (tail sample). c, Immunoblot to confirm the absence of the GABARAPL1 band in the homozygote knockout mouse (membrane-enriched fraction of heart homogenate). d, Heterozygote Gabarapl1-KO mice exhibit ~50% knockdown of the Gabarapl1 gene (qPCR) in the heart, and absence of the Gabarapl1 gene is confirmed in the homozygote Gabarapl1-KO mouse heart (male mice, 30 weeks old, WT n = 4, KO/WT n = 5, KO/KO n = 5 animals/group, analyzed by 1-way ANOVA with Bonferroni post-hoc, p < 0.0001). Data presented as mean ± s.e.m. *p < 0.05. Related to Fig. 3.

Source Data

Extended Data Fig. 3 AAV9-Gabarapl1 gene delivery in vitro and in vivo, cardiac stiffness protocol and iPS cardiomyocyte glycogen.

a, Confirmation of Gabarapl1 mRNA overexpression with AAV-Gabarapl1 in NRVMs (n = 3 independent culture wells/group, analyzed by unpaired 2-sided T-test, p < 0.0001). b, Immunoblot of GABARAPL1 protein expression in mouse hearts 4 weeks post-injection (i.v.) of AAV-Null or AAV-Gab (1010, 1011 or 1012 gc/mouse) with homozygote Gabarapl1-KO mouse heart as negative control. c, AAV vector burden in mouse heart 12 weeks post-injection (i.v.) of 1012 gc/mouse AAV9-cTnT-Null (AAV-Null) or AAV9-cTnT-Gabarapl1 (AAV-Gab) as measured by digital-droplet PCR detection of the WPRE viral element (Ctrl-Null n = 9, T2D-Null n = 9, Ctrl-Gab n = 12, T2D-Gab n = 13 animals/group, analyzed by 2-way ANOVA with Bonferroni post-hoc test). d, M-mode and flow Doppler echocardiography exemplar traces in T2D mice treated with AAV-Null or AAV-Gab. e, Ratio of phosphorylated (Thr172) to total AMPK protein expression is decreased with T2D in mice with Gabarapl1 gene delivery (AAV-Gab, Ctrl-Null n = 9, T2D-Null n = 11, Ctrl-Gab n = 11, T2D-Gab n = 13 animals/group, analyzed by 2-way ANOVA with Bonferroni post-hoc, Ctrl-Gab vs T2D-Gab p = 0.0107). f, Myocardial sections from T2D mice with Gabarapl1 gene delivery (T2D-Gab) stained with Oil Red O for visualization of cellular neutral lipids (scale bar, 100 µm). g, Exemplar images of a non-stretched and stretched cardiomyocyte attached between two glass rods. Cardiomyocyte stretch protocol. h, Glycogen is increased in human iPS cardiomyocytes in response to high glucose exposure (n = 6 wells/group, analyzed by unpaired 2-sided T-test, p = 0.0021). Data presented as mean ± s.e.m. *p < 0.05. Related to Figs. 4, 5 & 6.

Source Data

Extended Data Table 1 Proteomic (LC-MS/MS) analysis of glycogen-associated proteins in cardiac and skeletal muscle in control and acute metabolic stress rats
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Extended Data Table 2 Characteristics of T2D mouse model
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Extended Data Table 3 Primer, siRNA & guide RNA sequences
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Extended Data Table 4 Patient characteristics
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Extended Data Table 5 Absolute glycogen values
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Extended Data Table 6 AAV9-Gabarapl1 T2D mice: systemic and cardiac characteristics
Full size table

Supplementary information

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Source Data Figs. 1–6

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Source Data Extended Data Figs. 1–3 and Source Data Extended Data Tables 2, 5 and 6

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Mellor, K.M., Varma, U., Koutsifeli, P. et al. Targeted glycophagy ATG8 therapy reverses diabetic heart disease in mice and in human engineered cardiac tissues. Nat Cardiovasc Res (2025). https://doi.org/10.1038/s44161-025-00726-x

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