Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry-based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction (n = 16) compared to nonfailing donor hearts (n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease.

Keywords: hypertrophic cardiomyopathy; phosphorylation; posttranslational modifications; proteoform; top-down proteomics.

Fig. 1.

A complex sarcomeric proteoform landscape. (A) Control tissue from left ventricle (LV) of nonfailing donor hearts (Ctrl, n = 16) and HCM tissues procurred via surgical septal myectomy procedure (HCM, n = 16). (B) Schematic representation of cardiac sarcomere, consisting of thin (green) and thick filaments (pink) flanked by Z-disk. (C) Sarcomeric protein interactome showing a complex network of interactions between myofilament and Z-disk proteins. (D) Representative base peak chromatograms showing separation and detection of major sarcomeric proteins by LC-MS for Ctrl and HCM tissues. (E and F) Representative deconvoluted mass spectra showing the proteoforms of (E) myofilament and (F) Z-disk proteins.

Fig. 2.

Coordinated decrease in cTnI and ENH2 phosphorylation in HCM tissues. Representative deconvoluted mass spectra for (A) cTnI and (B) ENH2 from donor hearts (black) and HCM tissues (red). Mono- and bis-phosphorylation are denoted by red p and pp, respectively. (C) Total protein phosphorylation (P_tot) calculated by mol Pi/mol protein for cTnI and ENH2 in Ctrl (n = 16) and HCM (n = 16). Horizontal bars represent the mean of the group and error bars represent SEM in gray for Ctrl and black for HCM. Groups were considered significantly different at P < 0.05. (D) Localization of cTnI phosphorylation to Ser22/23 and (E) localization of ENH2 phosphorylation to Ser118 by ECD. (F) Linear correlation between cTnI phosphorylation and ENH2 phosphorylation (R² = 0.9276).

Fig. 3.

Altered phosphorylation of cTnT, Tpm1.1, and MLC-2v. Representative deconvoluted mass spectra from donor hearts (black) and HCM tissues (red) for (A) cTnT, (B) Tpm1.1, and (C) MLC-2v. Monophosphorylation is denoted with red p. Total phosphorylation (P_tot) calculated by mol Pi/mol protein for (D) cTnT, (E) Tpm1.1, and (F) MLC-2v for Ctrl (n = 16) and HCM (n = 16). Horizontal bars represent the mean of the group and error bars represent SEM (gray, Ctrl; black, HCM). Groups were considered statistically different at P < 0.05.

Fig. 4.

Differential tropomyosin isoform expression in HCM. (A) Representative deconvoluted spectra showing Tpm proteoforms Tpm1.2, Tpm1.1, and pTpm1.1 proteoforms. (B) Zoom-in to baseline of deconvoluted mass spectra to show lower abundance Tpm proteoforms, Tpm2.2, Tpm3.12, and pTpm3.12. Monophosphorylation denoted by red p.

Fig. 5.

Top-down MS characterization of cypher proteoforms in HCM. Representative deconvoluted mass spectra for (A) cypher-5 and (B) cypher-6 from donor hearts (black) and HCM tissues (red). Sequence tables showing MS/MS cleavages from CAD and ECD for (C) cypher-5 and (D) cypher-6. Mono, bis-, tris, tetra-phosphorylation indicated by red p, pp, 3p, “4p”, respectively. Schematic showing protein backbone cleavages is shown, with fragments produced by CAD and ECD shown in red and blue, respectively.