Analysis of the murine cardiac proteome and phosphoproteome using Tandem-Mass-Tag Mass Spectrometry (TMT-MS)

by Jannis Jelten
Doctoral thesis
Date of Examination:2025-10-13 // postponed to 2025-11-20
Date of issue:2025-09-22
Advisor:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Stephan E. Lehnart
Persistent Address: http://dx.doi.org/10.53846/goediss-11498

 

 

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Abstract

English

The heterozygous deletion of arginine 14 in phospholamban (PLN Arg14del) is a cause of arrhythmogenic cardiomyopathy. Patients present with a high rate of ventricular tachycardias and life-threatening malignant ventricular arrhythmias, with excess mortality starting from as early as 25 years old. In the present study, a mass spectrometry based workflow was established to detect early changes in the cardiac proteome and phosphoproteome of PLN Arg14del mice. Wild type and diseased mice were sacrificed at 12 weeks of age, at which time the PLN Arg14del mice are known to show a distinct phenotype with electrophysiological changes but no significant cardiac remodeling yet. The resulting data set provides the most in-depth proteome and phosphoproteome dataset of PLN Arg14del cardiomyocytes reported to date. PLN Arg14del mutant mice show reduced phosphorylation of phospholamban at serine 16 as well as threonine 17, supporting the idea of reduced affinity of the mutant isoform to PKA and CaMKII. Furthermore, phosphorylation of the muscle specific ankyrin isoform sAnk1 at serine 55 is decreased in PLN Arg14del mice, but the biological effect of this remains unclear. PLN Arg14del mutant mice also show reduced expression of the E3 ubiquitin-protein ligase Itch and the Sodium-Calcium exchanger NCX1. Itch regulates important signaling proteins from the Wnt/β-Catenin pathway as well as mTOR and NF-κB pathway and its involvement in the development of heart failure has already been described. The PLN Arg14del heterozygous cardiomyocytes also showed a reduction in mitochondrial, myofibrillary and dystrophin complex proteins compared to wild type cardiomyocytes, hinting at a reduced cardiac output at an early disease stage.
Keywords: Tandem-Mass-Tag; Mass spectrometry; Phospholamban Arg14del; Proteome; Phosphoproteome; Murine cardiomyocytes; Heart failure; Arrythmogenic cardiomyopathy