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Description
Phospholamban (PLN) is a transmembrane protein that regulates cardiomyocyte calcium handling and contraction. PLN function is dynamically regulated by post-translational modifications (PTMs), most notably through phosphorylation. A pathogenic deletion of arginine-14 (PLN-R14del), is associated with dilated cardiomyopathy (DCM), and defined by a high mortality rate with minimal treatment options. Moreover, the molecular mechanism of pathogenesis remains unclear, as some R14del carriers are asymptomatic. We use mass spectrometry (MS)-based top-down proteomics to investigate proteoform alterations in two systems: 1) human cardiac tissue from healthy donors and late-stage DCM patients (R14del carriers and noncarriers); 2) human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from symptomatic and asymptomatic patients. Proteins were extracted from cryopulverized tissue or hiPSC-CMs using a two-stage extraction. Cytosolic proteins are depleted, while PLN and additional cardiac proteins were extracted with Azo (MS-compatible surfactant). Proteins were buffer exchanged and analyzed with reverse phase liquid chromatography coupled to tandem MS. Characterization of the proteoform landscape in R14del patients revealed multiple PLN proteoforms. Notably, we observed a significant decrease in the total phosphorylation levels of R14del samples compared to both donor samples and DCM patients without the mutation, providing a novel insight into the endogenous phosphorylation potential of PLN-R14del. Additionally, we observed dysregulation of phosphorylation in key sarcomere/Z-disk proteins, further associating contractile dysfunction with proteoform alterations. In the hiPSC-CM model, preliminary analysis revealed that the asymptomatic patient line contained PLN phosphorylation levels comparable to the isogenic control line; importantly, there was a significant increase in phosphorylation in both when compared to the symptomatic carrier. These results suggest that proteoforms may factor into variable disease expressivity and that our method is capable of delineating distinct phenotypes between patients that share the same mutation, which can aid in the pursuit of therapeutic strategies for precision medicine and bridge the gap between genotype and phenotype.
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