ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection

Clara Bredow; Fabien Thery; Eva Katrin Wirth; Sarah Ochs; Meike Kespohl; Gunnar Kleinau; Nicolas Kelm; Niclas Gimber; Jan Schmoranzer; Martin Voss; Karin Klingel; Joachim Spranger; Kostja Renko; Markus Ralser; Michael Mülleder; Arnd Heuser; Klaus-Peter Knobeloch; Patrick Scheerer; Jennifer Kirwan; Ulrike Brüning; Nikolaus Berndt; Francis Impens; Antje Beling

doi:10.1093/cvr/cvae026

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection

Cardiovasc Res. 2024 Feb 3:cvae026. doi: 10.1093/cvr/cvae026. Online ahead of print.

Authors

Clara Bredow¹, Fabien Thery^{2

3}, Eva Katrin Wirth^{4

5}, Sarah Ochs¹, Meike Kespohl^{1

4}, Gunnar Kleinau⁶, Nicolas Kelm¹, Niclas Gimber⁷, Jan Schmoranzer⁷, Martin Voss¹, Karin Klingel⁸, Joachim Spranger^{4

5}, Kostja Renko⁹, Markus Ralser¹⁰, Michael Mülleder¹⁰, Arnd Heuser¹¹, Klaus-Peter Knobeloch^{12

13}, Patrick Scheerer^{4

6}, Jennifer Kirwan^{14

15

16}, Ulrike Brüning^{14

15

16}, Nikolaus Berndt¹⁷, Francis Impens^{2

3

18}, Antje Beling^{1

4}

Affiliations

¹ Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Biochemistry, Berlin, Germany.
² Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
³ VIB-UGent Center for Medical Biotechnology, Ghent, Belgium.
⁴ Deutsches Zentrum für Herz-Kreislauf-Forschung, partner site Berlin, Berlin, Germany.
⁵ Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Endocrinology, Diabetes and Nutrition, Berlin, Germany.
⁶ Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Medical Physics and Biophysics, Group Protein X-ray Crystallography and Signal Transduction, Charitéplatz 1, Berlin, Germany.
⁷ Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Advanced Medical Bioimaging Core Facility, Berlin, Germany.
⁸ University of Tübingen, Cardiopathology, Institute for Pathology and Neuropathology, Tübingen, Germany.
⁹ German Federal Institute for Risk Assessment (BfR), German Centre for the Protection of Laboratory Animals (Bf3R), Berlin, Germany.
¹⁰ Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Core Facility - High-Throughput Mass Spectrometry, Berlin, Germany.
¹¹ Animal Phenotyping Platform, Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.
¹² University of Freiburg, Institute of Neuropathology, Freiburg, Germany.
¹³ CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
¹⁴ Berlin Institute of Health (BIH) @ Charité, Metabolomics Platform, Berlin, Germany.
¹⁵ Max-Delbrück-Center (MDC) for Molecular Medicine, Berlin, Germany.
¹⁶ Berlin Institute of Health (BIH), Berlin, Germany.
¹⁷ Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute for Computational and Imaging Science in Cardiovascular Medicine, Berlin, Germany.
¹⁸ VIB Proteomics Core, Ghent, Belgium.

PMID: 38309955
DOI: 10.1093/cvr/cvae026

Abstract

Aims: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signaling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown.

Methods and results: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modeling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced upregulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modeling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system.

Conclusions: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

Keywords: ISG15; ISGylation; glycolysis; metabolism; mitochondrial function; virus infection.