In-depth phosphoproteomic profiling of the insulin signaling response in heart tissue and cardiomyocytes unveils canonical and specialized regulation

• Published in: Cardiovascular diabetology 2024-07, Vol.23 (1), p.258-16, Article 258
• Main Authors: Achter, Jonathan Samuel, Vega, Estefania Torres, Sorrentino, Andrea, Kahnert, Konstantin, Galsgaard, Katrine Douglas, Hernandez-Varas, Pablo, Wierer, Michael, Holst, Jens Juul, Wojtaszewski, Jørgen Frank Pind, Mills, Robert William, Kjøbsted, Rasmus, Lundby, Alicia
• Format: Article
• Language: English
• Subjects: Adapter proteins; Adipocytes; AKT1 protein; AKT2 protein; Animals; Cardiac muscle; Cardiometabolic; Cardiomyocytes; Congestive heart failure; Coronary artery disease; Datasets; Diabetes; Diabetes mellitus; Fatty acids; Glucose; GTPase-Activating Proteins - genetics; GTPase-Activating Proteins - metabolism; Heart diseases; Heart failure; Insulin; Insulin - metabolism; Insulin receptors; Insulin resistance; Insulin signaling; Kinases; Liquid chromatography; Male; Mass spectrometry; Mass spectroscopy; Metabolism; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Cardiac - metabolism; Oxidation; Peptides; Phosphoproteins - metabolism; Phosphoproteomics; Phosphorylation; Physiology; Protein deficiency; Protein transport; Proteomics; Proto-Oncogene Proteins c-akt - metabolism; Receptor, Insulin - metabolism; Scientific imaging; Signal Transduction; Skeletal muscle; TOR protein; Ventricle
• ISSN: 1475-2840; 1475-2840
• DOI: 10.1186/s12933-024-02338-4

Insulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent. Insulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements. We quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (Insr ) itself as well as the Insulin receptor substrate protein 1 (Irs1 ). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized