Dataset Information

Sarcomeric Proteoform Landscape in Ischemic Cardiomyopathy Defined by Top-down Proteomics

ABSTRACT: In ischemic cardiomyopathy (ICM), left ventricular systolic dysfunction leads to reduced blood flow and oxygen supply to the heart. Alterations in sarcomeric protein function and expression play prominent roles in the onset and progression of cardiomyopathies; however, the molecular mechanisms underlying ICM remain poorly defined. Herein, we have implemented a top-down liquid chromatography (LC)-mass spectrometry (MS)-based proteomics method for the simultaneous quantification of sarcomeric protein expression and modifications in non-failing donor (n = 16) compared to end-stage failing ICM (n = 16) human cardiac tissues. Our top-down proteomics platform provided a “bird’s eye view” of proteoform families with high mass accuracy and reproducibility. In addition, quantification of post-translational modifications (PTMs) and expression reveal significant changes in various sarcomeric proteins extracted from ICM tissues. Changes include altered phosphorylation and expression of cardiac troponin I (cTnI) and enigma homolog 2 (ENH2) as well as a marked increase in muscle LIM protein (MLP) and calsarcin-1 phosphorylation in ICM hearts. Our results imply that the contractile apparatus of the sarcomere is severely dysregulated during ICM. Thus, this study is the first to uncover significant molecular changes to multiple sarcomeric proteins in end-stage ischemic heart failure patients using LC-MS-based top-down proteomics.

INSTRUMENT(S): maXis

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Heart

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Emily Chapman

LAB HEAD: Ying Ge

PROVIDER: PXD038066 | Pride | 2023-02-20

REPOSITORIES: Pride

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Publications

Defining the Sarcomeric Proteoform Landscape in Ischemic Cardiomyopathy by Top-Down Proteomics.

Chapman Emily A EA Aballo Timothy J TJ Melby Jake A JA Zhou Tianhua T Price Scott J SJ Rossler Kalina J KJ Lei Ienglam I Tang Paul C PC Ge Ying Y

Journal of proteome research 20230217 3

Ischemic cardiomyopathy (ICM) is a prominent form of heart failure, but the molecular mechanisms underlying ICM remain relatively understudied due to marked phenotypic heterogeneity. Alterations in post-translational modifications (PTMs) and isoform switches in sarcomeric proteins play important roles in cardiac pathophysiology. Thus, it is essential to define sarcomeric proteoform landscape to better understand ICM. Herein, we have implemented a top-down liquid chromatography (LC)-mass spectrom ...[more]

PMID: 36800490

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Project description:Background: Lamins A/C (encoded by the LMNA gene) can lead to dilated cardiomyopathy (DCM). Objectives: This study sought to undertake proteomic analysis of myocardial tissue to explore the postgenomic phenotype of end-stage lamin heart disease. Methods: Consecutive patients with end-stage lamin heart disease (LMNA-group, n=7) and ischaemic DCM (ICM-group, n=7) undergoing heart transplantation were enrolled. Samples were obtained from left atrium(LA), left ventricle(LV), right atrium(RA), right ventricle(RV) and interventricular septum(IVS). Liquid chromatography combined with mass-spectrometry was used for protein quantification. We compared protein concentrations in cardiac samples between LMNA and ICM groups. Proteins were considered differentially abundant if the quantitative difference was 1.5-fold and corrected p-value <0.05 at a false discovery rate of 0.01. Gene ontology(GO) enrichment analysis explored the related biological processes. Results: 4,247 proteins were identified in LMNA and ICM samples, of which 633 were differentially abundant in LA, 39 in LV, 181 in RA, 52 in RV, and 85 in IVS. Abundance of lamin A/C was reduced but lamin B (LMNB) increased in LMNA LA/RA tissue compared to ICM, but not in LV/RV. Transthyretin was more abundant in the LV/RV of LMNA compared to ICM while sarcomeric proteins such as titin and cardiac myosin heavy chain were generally reduced in RA/LA of LMNA. Protein expression profiling and GO enrichment analysis revealed sarcopenia, extracellular matrix(ECM) remodeling, deficient myocardial energetics, redox imbalances, and abnormal calcium handling in LMNA samples. Conclusion: Lamin heart disease is a biventricular and biatrial disease, characterized by sarcopenia, aberrant metabolism, and ECM remodeling. LMNB and transthyretin were unexpectedly abundant in the atria and ventricles respectively of patients with end-stage lamin heart disease potentially hinting to the possibility of compensatory responses.

Project description:Introduction: Cell-derived extracellular vesicles ([EVs], i.e., exosomes and microparticles) have been reported to mediate the cardioprotective effects of stem cells. However, a head-to-head comparison of their effects with those of the cells from which they derive has not been reported. Hypothesis: The effects of the EV content of human embryonic stem cell (hESC)-derived cardiac progenitors may be equivalent to those of the parent cells. Methods: Two series of immunodeficient (nude) mice underwent permanent coronary artery ligation. Three weeks later, those with an echocardiographically-determined LV ejection fraction â¤ 50% were randomly allocated to receive transcutaneous echo-guided peri-infarct injections of alpha-MEM media (controls), hESC-derived SSEA-1+ cardiac progenitors (500000 cells in 30 Î¼L), or total EV secreted by those 500000 cells in 48 hours in an equivalent volume. The second series also included sham-operated animals for which the needle was inserted into the myocardium in three places without injection. EVs were collected from the cell-secreted medium by ultracentrifugation and characterized by flow cytometry, and nanosight NTS. Outcomes were assessed after 6 weeks by echocardiography, taking LV end-systolic volume (ESV) as the primary end point. Hearts were processed for the assessment of fibrosis and angiogenesis. Total RNA was extracted from carefully selected animals for gene expression analysis by Affymetrix chip. Only animals with similar VTS at baseline were used for this analysis. All data were collected and analyzed blindly. Results: Cardiac progenitors were successfully generated by exposure of the pluripotent ESC to bone morphogenetic protein-2, purified by anti-CD15 immunomagnetic sorting and then cultured on vitronectin for 48 hours, after which cells or EVs derived from the same cell batch were injected. After 6 weeks in the first series, LVESV [mÂ±SEM] in controls did not significantly differ from the pre-injection value (difference: -2.64 Â± 1.54 ÂµL, p=0.12, n= 12). Conversely, in the cell-treated group, LVESV significantly decreased by -4.20 Â± 0.96 ÂµL (p=0.0007, n=16). A similar decrease was seen in the EV-treated group: -5.73 Â± 1.21 ÂµL (p=0.0003, n=15). Similar patterns were seen for LV end-diastolic volumes: controls (-2.46 Â± 1.19 ÂµL, p=NS), cells (-4.48 Â± 1.47 ÂµL, p=0.009), EV (-4.29 Â± 1.31 ÂµL, p=0.005). For the second series, the VTD of cell- and EV-treated animals remained stable (cell: -1.85 Â± 3.27 ÂµL, p=NS, n= 4; EV: +0.05 Â± 1.53 ÂµL, p=NS, n = 4), whereas sham- and control-treated animals deteriorated significantly (sham: +2.20 Â± 0.35 ÂµL, p = 0.003, n = 5; control: +3.27 Â± 0.87 ÂµL, p = 0.03, n = 4). The trend held for VTS, though differences were not significant. Analysis of gene expression data revealed interesting pathways that were more highly expressed in cell- and EV-treated animals than in controls and sham-operated animals. Conclusions: These data support the hypothesis that EVs may be critical mediators of the paracrine effects of cell therapy, and for the sake of streamlining translational processes, deserve to be considered as potential alternatives to stem cell transplantation. Gene expression levels were compared between mouse hearts.

Dataset Information

Sarcomeric Proteoform Landscape in Ischemic Cardiomyopathy Defined by Top-down Proteomics

Publications

Defining the Sarcomeric Proteoform Landscape in Ischemic Cardiomyopathy by Top-Down Proteomics.

Similar Datasets

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