Peroxynitrite - matrix metalloproteinase and erythropoietin receptor signaling pathways in ischemic heart disease

Abstract

In the industrialized world, cardiovascular diseases (CVD) are responsible for almost 50% of the total cause of death, and among CVD, ischemic heart disease (IHD) represents the highest subpopulation. In spite of huge number of many preclinical and clinical studies, there is still no cardioprotective drug on the market to reduce infarct size in IHD. In the pathomechanism of myocardial ischemia/reperfusion injury, peroxynitrite (ONOO-) – matrix metalloproteinase (MMP) signaling as well as erythropoietin (EPO)-induced cytoprotective pathways have well-established roles. ONOO- – MMP pathway is influenced by hyperlipidemia, which is one of the most common comorbidit of IHD. Moreover, in spite of the promising preclinical findings with EPO against acute myocardial infarction (AMI), EPO failed to reduce infarct size in clinical trials and showed significant safety problems. Therefore, our aims were (i) to obtain novel information on the role of ONOO- – MMP and (ii) EPO receptor- mediated signaling pathways in IHD. Therefore, we investigated i) the correlation of markers of peroxynitrite–MMP signaling with hyperlipidemia and with cardiac function in patients with significant coronary stenosis and ii) the cardioprotective effects of EPO, the prolonged half-life EPO analogue, darbepoetin alpha (Dpa), and two novel selective non-erythropoietic EPO receptor ligand dimeric peptides (AF41676 and AF43136) in a rat model of AMI. We found in coronary artery disease patients, that serum nitrotyrosine (NTyr), a widely accepted marker of ONOO- generation, positively correlated with MMP-9 activity, but not with MMP-2 activity. NTyr positively correlated with total and LDL cholesterol, serum triglyceride, and creatinine, while it negatively correlated with HDL cholesterol and with left ventricular ejection fraction (LVEF). MMP-2 activity correlated positively with total and LDL cholesterol. In statin-treated patients, a significantly reduced serum NTyr level was found compared to statin-naives; however, MMP activities were not different in statin-treated and in statin-naive patients. MMP-2 activity was significantly increased in smoker patients compared to the non-smokers, while NTyr or MMP-9 activity did not show any difference related to smoking. MMP-9 activity correlated positively with urea nitrogen and negatively with LVEF, respectively. Serum creatinine correlated negatively with LVEF. In a pilot cell culture study, EPO at 100 U/mL significantly decreased cell death compared to vehicle in rat neonatal cardiomyocytes subjected to simulated ischemia/reperfusion. In further studies (studies 1–4), in vivo AMI was induced by 30 min coronary occlusion and 120 min reperfusion in male Wistar rats. Test compounds and positive controls (B-type natriuretic peptide, BNP or cyclosporine A, CsA) were administered iv. before the onset of reperfusion. Infarct size (IS) was measured by standard TTC staining. In study 1, 5000 U/kg EPO reduced infarct size significantly compared to vehicle. In study 2, Dpa showed maximal infarct size-reducing effect at 5 μg/kg compared to the vehicle. In study 3, in the dose-response curve of AF41676, 3 mg/kg was the most effective dose compared to the vehicle. The positive control BNP significantly decreased infarct size in studies 1–3 by approximately 35%. In study 4, AF43136 at 10 mg/kg decreased infarct size, similarly to the positive control CsA compared to the appropriate vehicle. We demonstrated for the first time in the literature that (i) serum NTyr correlates with MMP-9 activity, (ii) lipid parameters correlate with NTyr and MMP-2 activity, (iii) myocardial function correlates with creatinine, NTyr and MMP-9 activity, and (iv) creatinine correlates with NTyr and urea nitrogen with MMP-9 activity in patients with CAD. Therefore, studying the biomarkers of peroxynitrite–MMP pathway in large prospective trials may reveal their predictive or diagnostic avails. Moreover, we proved first time in the literature, that selective, non-erythropoietic EPO receptor ligand dimeric peptides AF41676 and AF43136 administered before reperfusion are able to reduce infarct size in a rat model of AMI. Non-erythropoietic EPO receptor peptide ligands may be promising cardioprotective agents in IHD.