Proteomic Discovery of Functionally Important Pathways in Myocardial Ischemia-Reperfusion Injury

Date

2014-02-04

Authors

Ahmed, Kamran

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Abstract

BACKGROUND: Coronary heart disease, a source of myocardial ischemia-reperfusion injury (IRI), is the world's leading cause of death and disability. Insulin-like growth factor 1 (IGF1) transgenic (Tg) mouse hearts are protected from IRI, whereas Akt-Tg mouse hearts recover poorly from IRI. Surprisingly, Akt is a downstream component of IGF1 signaling. The Akt-Tg phenotype can be rescued by cardiac gene transfer of activated PI3-kinase (PI3K), another component of the IGF1 pathway, suggesting that PI3K-dependent but Akt-independent pathways are key determinants of IRI. To discern such pathways, we analyzed the proteomic and phosphoproteomic changes in wild-type (WT), IGF1-Tg, and Akt-Tg mouse hearts, identified 20 differentially regulated candidates as potential modifiers of IRI, and began testing their functional roles in an in vitro model. We hypothesize that the cardioprotection observed in IGF1 overexpression is a result of PI3K-dependent but Akt-independent signaling pathways. METHODS: WT hearts were collected at 4 time points of ex-vivo Langendorff IRI and analyzed with liquid chromatography-tandem mass spectrometry to determine protein expression and phosphorylation changes. IGF1-Tg and Akt-Tg hearts were analyzed at baseline. Protein network analysis was performed using Cytoscape software. The functional effects of candidates with expression or phosphorylation differences ≥2-fold were assessed in rat neonatal ventricular myocytes using in vitro redox-based viability assays and cell proliferation studies. RESULTS: In the WT IRI studies, 6403 proteins and 22833 phosphopeptides were quantified. During IRI, no proteins changed in expression, 45 phosphopeptides were upregulated, and 975 phosphopeptides were downregulated. In the IGF1-Tg and Akt-Tg hearts, 6700 proteins and 23000 phosphopeptides were quantified. In vitro knockdown of rho-associated protein kinase 2 (ROCK2) increased the viability signal by 17% in normoxia and 33% in simulated IRI (p<0.05) and increased EdU incorporation from 28.9% to 40.15% (p<0.00001). Network analysis of Akt-Tg hearts revealed significant downregulation of 23 out of 45 subunits of Complex I (p<0.05). CONCLUSIONS: Dephosphorylation of the cardiac phosphoproteome is the dominant pattern in IRI, which may reflect phosphatase activation or reduced ATP levels inhibiting kinase activity. ROCK2 knockdown increased the viability signal by stimulating proliferation in vitro. Whether ROCK2 is involved in cardiomyogenesis in the adult heart will be addressed in future studies. Akt-Tg hearts may be susceptible to IRI due to a reduced ATP reserve caused by Complex I downregulation

General Notes

The 52nd Annual Medical Student Research Forum at UT Southwestern Medical Center (Tuesday, February 4, 2014, 3-6 p.m., D1.502)

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Citation

Ahmed, K., Luo, Y., Taneja, S., Keshishian, H., Carr, S. & Rosenzweig, A. (2014, February 4). Proteomic discovery of functionally important pathways in myocardial ischemia-reperfusion injury. Poster session presented at the 52nd Annual Medical Student Research Forum, Dallas, TX. Retrieved from https://hdl.handle.net/2152.5/1641

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