ABSTRACT: Background: Hypertrophic cardiomyopathy (HCM) has traditionally been regarded as a disease of the sarcomere, however it is in the midst of a paradigm shift with growing recognition of contributions beyond the sarcomere to the heterogeneity of HCM phenotypes. Innovative approaches are essential to uncover novel determinants and mechanisms underlying this heterogeneity. Top-down proteomics has emerged as a powerful method for analysis of proteoforms the myriad protein products arising from genetic variants, post-translational modifications (PTMs), and splicing isoforms from a single gene offering a more precise lens to understand the disease heterogeneity in HCM. Yet, how proteoforms are altered on the global scale in HCM has not been investigated.
Methods: Global top-down proteomics was performed on myocardial samples from patients with advanced obstructive HCM and nonfailing controls. Specifically, serial protein extraction enabled by the photocleavable surfactant, Azo was employed to solubilize diverse categories of proteins from minimal tissue, including membrane proteins. Subsequently, high-sensitivity top-down mass spectrometry was utilized to detect and quantify proteoforms across various cellular compartments.
Results: Using this global top-down approach, we detected ~2000 proteoforms across disparate cellular compartments, including the sarcoplasmic reticulum (SR), cytoskeleton, mitochondria, and nucleus, in advanced obstructive HCM tissues compared to non-failing controls. Quantitative analysis uncovered significant alterations not only in sarcomeric but also cytoskeletal, mitochondrial, nucleosome, and SR proteoforms. Notably, we discovered a significant proteoform crosstalk among the sarcomere, SR, and cytoskeleton. Moreover, we identified a previously unrecognized decrease in succinylated mitochondrial proteoforms as a critical feature of advanced obstructive HCM proteoform landscape, alongside a marked reduction in acetylation of nucleosome proteins.
Conclusions: This study represents the most comprehensive analysis of proteoform landscape in HCM to date. We have identified broad proteoform alterations spanning multiple cellular compartments in advanced obstructive HCM, indicating that a complex network of perturbations underlie HCM. These findings highlight pathways beyond the sarcomere that may contribute to HCM pathophysiology, offering potential targets for development of therapeutic interventions.
Ying Ge 