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

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) mouse hearts subjected to IRI ex vivo, and IGF1-TG and Akt-Tg mouse hearts in order to identify 20 differentially regulated candidates as potential modifiers of IRI, and began testing their functional roles in an in vitro model. OBJECTIVE: 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 abundance 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 abundance 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 abundance, 10 phosphopeptides were upregulated, and 330 phosphopeptides were downregulated. In the IGF1-Tg and Akt-Tg hearts, 6700 proteins and 23000 phosphopeptides were quantified. Out of the significantly regulated proteins, 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 24 out of 45 subunits of Complex I of the electron transport chain (p<0.05). CONCLUSION: 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.