Role of cardiac proteoform alterations in the pathogenesis of phospholamban R14DEL cardiomyopathy

The National Heart, Lung, and Blood Institute awarded $108,656 to the University of Wisconsin–Madison on March 3, 2026, under the Cardiovascular Diseases Research program (CFDA 93.837) to characterize the molecular mechanisms of phospholamban R14DEL cardiomyopathy.

The recipient will use high-resolution mass spectrometry–based proteomics, human clinical samples, and patient-specific human induced pluripotent stem cell–derived cardiomyocytes to investigate how cardiac proteoform alterations drive disease pathogenesis in the PLN-R14DEL mutation. The work employs top-down and bottom-up proteomics approaches to identify the molecular mechanisms, pathways, and proteins involved in pathological remodeling. This research addresses a gap in understanding why certain carriers of the mutation remain asymptomatic despite the mutation's association with dilated cardiomyopathy, ventricular dysfunction, and arrhythmia. Current treatments for affected patients focus on symptom management rather than disease prevention, and the study aims to enable mechanistic insights that could inform future therapeutic strategies.

Place of performance is Madison, Wisconsin. The award period of performance runs through July 1, 2026. The assistance type is a Project Grant, awarded to support basic and translational cardiovascular research under NHLBI's competitive grant mechanism.

Abstract

Dilated cardiomyopathy (DCM) is the most common form of heart disease and is characterized by patients developing ventricular dilation, contractile dysfunction and ventricular arrhythmias, which can ultimately lead to heart failure. A pathogenic mutation in the phospholamban (PLN) gene, resulting in the deletion of amino acid arginine 14 (PLN-R14DEL), has been associated with the onset of DCM. PLN is a transmembrane protein in the sarcoplasmic reticulum (SR) that is dynamically regulated by its post-translational modifications (ptms) and plays a crucial role in calcium (CA2+)-handling and heart contractility. The dysregulation of PLN's PTM state when mutated impacts its structure and ability to regulate SR CA2+ atpase (SERCA2A) and the translocation of CA2+ ions. However, the molecular mechanism of pathogenesis remains unclear as a notable subset of carriers remain asymptomatic in later age, contributing to the high phenotypic variability observed with this mutation. Current treatments for PLN-R14DEL patients focus on symptom management rather than preventing disease progression, emphasizing the urgent need for a deeper understanding of PLN-R14DEL and its downstream consequences. Herein, i propose to leverage the power of high-resolution mass spectrometry (MS)-based proteomics, human clinical samples and patient-specific human induced pluripotent stem cell derived cardiomyocytes (hipsc-CMS) to enable a deeper characterization of disease pathogenesis. I will use top-down proteomics to characterize proteoforms - protein products from a single gene resulting from genetic mutations, alternative splicing, or ptms - which will be integrated and bioinformatically analyzed with bottom-up proteomics data sets to identify the molecular mechanisms, pathways and proteins involved in pathological remodeling. My novel top-down proteomics methods enable the characterization of proteoforms from diverse subcellular regions (membrane- bound, sarcomere, SR, mitochondria, etc.) with minimal sample requirements. These technological advancements will help bridge the GAP between the genotype and phenotype of PLN-R14DEL patients. Aim 1 will comprehensively examine the molecular composition of human cardiac tissue from late-stage DCM patients with the PLN-R14DEL mutation compared to DCM patients with no PLN mutation and healthy donors with no cardiac history. To address the discrepancies between symptomatic and asymptomatic carriers, aim 2 will utilize patient- specific hipsc-CMS and proteomics to characterize the molecular and physiological differences between patients that carry the same mutation, yet express high phenotypic variability. The success of this work will provide a transformative understanding of the underlying biological mechanisms altered within PLN-R14DEL DCM and can aid in the pursuit of novel therapeutic strategies or targets for treating and perhaps preventing this devastating disease.