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Proteomic analysis of the cardiac myocyte secretome reveals extracellular protective functions for the ER stress response.

ABSTRACT: The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG but not TM. Proteomic analyses of TG- and TM-conditioned media showed that the secretion of most proteins was inhibited by TG and TM; however, secretion of several ER-resident proteins, including GRP78 was increased by TG but not TM. Simulated ischemia, which decreases ER/SR calcium also increased secretion of these proteins. Mechanistically, secreted GRP78 was shown to enhance survival of NRVMs by collaborating with a cell-surface protein, CRIPTO, to activate protective AKT signaling and to inhibit death-promoting SMAD2 signaling. Thus, proteins secreted during ER stress mediated by ER calcium depletion can enhance cardiac myocyte viability.

SUBMITTER: Blackwood EA

PROVIDER: S-EPMC8597053 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Proteomic analysis of the cardiac myocyte secretome reveals extracellular protective functions for the ER stress response.

Blackwood Erik A EA Thuerauf Donna J DJ Stastna Miroslava M Stephens Haley H Sand Zoe Z Pentoney Amber A Azizi Khalid K Jakobi Tobias T Van Eyk Jennifer E JE Katus Hugo A HA Glembotski Christopher C CC Doroudgar Shirin S

Journal of molecular and cellular cardiology 20200425

The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG bu ...[more]

PMID: 32339566

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Project description:Cardiac mesenchymal stem cells (C-MSCs) are a novel mesenchymal stem cell (MSC) subpopulation derived from cardiac tissue, which are reported to be responsible for cardiac regeneration. Notch signaling is believed to aid in cardiac repair following myocardial injury. In this study, we have investigated the role of extracellular vesicles (EVs) from Notch1 engineered C-MSCs on angiogenesis and cardiomyocyte (CM) proliferation in ischemic myocardium. C-MSCs were isolated from Notch1flox mice (C-MSCNotch1 FF). Notch1 gene deletion was accomplished by adenoviral vector-mediated Cre recombination, and Notch1 overexpression was achieved by overexpression of Notch1 intracellular domain (N1ICD). EVs were isolated by using the size exclusion column method. Proteomic composition of EV was carried out by mass spectrometry. A mouse myocardial infarction (MI) model was generated by permanent left anterior descending (LAD) coronary artery ligation. Intramyocardial transplantation of Notch1 knockout C-MSCs (C-MSCsNotch1 KO) did not have any effect on cardiac function and scar size. On the other hand, transplantation of N1ICD-overexpressing C-MSCs (C-MSCsN1ICD) showed significant improvement in cardiac function and attenuation of fibrosis as compared to the control (PBS) group and non-modified C-MSC groups. C-MSCsN1ICD differentiated into smooth muscle cells and formed new vessels. Proteomics profiling identified several proteins, such as lysyl oxidase homolog-2 and biglycan, as highly enriched proteins in EV-C-MSCsN1ICD. Go term analysis indicated that EV-C-MSCsN1ICD were enriched with bioactive factors, potent pro-repair proteins responsible for cell migration and proliferation. EV-C-MSCs Notch1FF and EV-C-MSCsN1ICD were strongly proangiogenic under both in vitro and in vivo conditions. EV-C-MSCsN1ICD caused dense tube formation in vitro and increased neovasculogenesis in the peri-infarct area in vivo. Furthermore, EV-C-MSCsN1ICD attenuated endothelial cell (EC) and CM apoptosis under oxidative stress and ischemic injury. Similarly, EV-C-MSCNotch1 FF and EV-C-MSCN1ICD treatment improved cardiac function and decreased fibrosis in mice post-MI. EV-C-MSCsN1ICD were very effective in improving cardiac function and decreasing fibrosis. Notch1 signaling is a strong stimulus for cardiac regeneration by C-MSCs. EVs secreted by Notch1-overexpressing C-MSCs were highly effective in preventing cell death, promoting angiogenesis and CM proliferation, and restoring cardiac function post-MI. Overall, these results suggest that Notch1 overexpression may further enhance the effectiveness of EVs secreted by C-MSCs in cell-free therapy.

Dataset Information

Proteomic analysis of the cardiac myocyte secretome reveals extracellular protective functions for the ER stress response.

Publications

Proteomic analysis of the cardiac myocyte secretome reveals extracellular protective functions for the ER stress response.

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