Dataset Information

Mass spectrometry-based redox and protein profiling reveals distinct protein expression and oxidation patterns in failing human hearts

ABSTRACT: Oxidative stress plays a key role in development and progression of cardiovascular diseases and it is correlated with left ventricular dysfunction and heart failure (HF). Oxidative environments lead to the formation of intra- and intermolecular disulfide bonds, as well as to plethora of other reversible and irreversible oxidative amino acid modifications, affecting the functionality of the proteins. Here we report that heart failure due to ischemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM) is correlated with increase in oxidative stress compared to non-failing control hearts, manifested through decreased GSH/GSSG ratio in failing heart tissue samples and adaptations of cardiac redox proteome which occur in correlation with two different heart pathologies.

INSTRUMENT(S): maXis

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Heart

SUBMITTER: Tamara Tomin

LAB HEAD: Ruth Birner-Gruenberger

PROVIDER: PXD021261 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

Mass Spectrometry-Based Redox and Protein Profiling of Failing Human Hearts.

Tomin Tamara T Schittmayer Matthias M Sedej Simon S Bugger Heiko H Gollmer Johannes J Honeder Sophie S Darnhofer Barbara B Liesinger Laura L Zuckermann Andreas A Rainer Peter P PP Birner-Gruenberger Ruth R

International journal of molecular sciences 20210211 4

Oxidative stress contributes to detrimental functional decline of the myocardium, leading to the impairment of the antioxidative defense, dysregulation of redox signaling, and protein damage. In order to precisely dissect the changes of the myocardial redox state correlated with oxidative stress and heart failure, we subjected left-ventricular tissue specimens collected from control or failing human hearts to comprehensive mass spectrometry-based redox and quantitative proteomics, as well as glu ...[more]

PMID: 33670142

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Project description:Background: The phospholamban (PLN) p.Arg14del mutation belongs to the established causes of dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, CRISPR/Cas9 gene editing and patient heart samples to investigate the molecular pathomechanisms of PLN p.Arg14del cardiomyopathy. Methods and results: Patient dermal fibroblasts carrying the PLN p.Arg14del mutation were reprogrammed into hiPSC, isogenic controls were established by CRISPR/Cas9. Cells were differentiated into cardiomyocytes and characterized in 2D and 3D-engineered heart tissue (EHT) format. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+-load dependent irregular beating pattern and lower peak force compared to isogenic controls. While this data support the reported super-inhibitory effect of PLN p.Arg14del on the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, an unchanged SR Ca2+ content argued against disturbed SR Ca2+ cycling as the sole cause of contractile dysfunction. Label-free proteomic analysis of p.Arg14del EHTs revealed less endoplasmic reticulum (ER), ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the rough ER, large lipid droplets in close association with mitochondria and reduced mitochondrial number. Follow-up experiments confirmed impairment of the rough ER/mitochondria compartment and enhanced oxidative stress in PLN p.Arg14del. Relevance for human disease was demonstrated by immunohistochemistry of human heart samples. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison to ischemic heart failure - and non-failing donor heart samples. Surprisingly, transduction of PLN p.Arg14del EHTs with the Ca2+ binding protein GCaMP6f reversed the contractile, molecular and morphological disease phenotype. Conclusion: This study identified impairment of the rough ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+-scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.

Project description:Heart failure is associated with high morbidity and mortality and its incidence increases worldwide. MicroRNAs (miRNAs) are potential markers and targets for diagnostic and therapeutic applications, respectively. We determined myocardial and circulating miRNA abundance and its changes in patients with stable and end-stage heart failure before and at different time points after mechanical unloading by a left ventricular assist device (LVAD) by small-RNA-sequencing. MiRNA changes in failing heart tissues partially resembled that of fetal myocardium. Consistent with prototypical miRNAM-bM-^@M-^Starget-mRNA interactions, target mRNA levels were negatively correlated to changes in abundance for highly expressed miRNAs in heart failure and fetal hearts. The circulating small RNA profile was dominated by miRNAs, and fragments of tRNAs and small cytoplasmic RNAs. Heart- and muscle-specific circulating miRNAs (myomirs) increased up to 140-fold in advanced heart failure, which coincided with a similar increase in cardiac troponin I protein, the established marker for heart injury. These extracellular changes nearly completely reversed 3 months following initiation of LVAD support. In stable heart failure, circulating miRNAs showed less than 5-fold differences compared to normal, and myomir and cardiac troponin I levels were only captured near the detection limit. These findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers. Total RNA isolated from human left ventricular myocardium of failing hearts due to dilated or ischemic cardiomyopathy before and after mechanical unloading by a left ventricular assist device, and fetal myocardium compared to non-failing postnatal myocardium was subjected to multiplexed small RNA-sequencing on the Illumina platform. mRNA gene expression data using Illumina HumanHT-12v4 beadarrays for a subset of the myocardial samples is available (GSE52601).

Dataset Information

Mass spectrometry-based redox and protein profiling reveals distinct protein expression and oxidation patterns in failing human hearts

Publications

Mass Spectrometry-Based Redox and Protein Profiling of Failing Human Hearts.

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