P425Cytoprotection by the NO-donor SNAP and BNP against ischemia/reperfusion injury in rat engineered heart tissue

Abstract

Purpose: In vitro drug screening and disease modeling is a rapidly growing research field and may help to replace animal experiments. The aim of this study was to test the response of engineered heart tissue (EHT) to ischemia/reperfusion (I/R) and the effect of known cardioprotective molecules. Method: Fibrin-based mini-EHT were generated in a 24-well format from neonatal rat heart cells and cultured at 37°C, 7% CO2 and 40% O2 for 15-22 days (DMEM, 10% horse serum). 24 h after the last medium change, EHTs were subjected to 180 min ischemia (93% N2 and 7 % CO2 gas flow) followed by 120 min reperfusion (40% O2) or remained at 40% O2 as time-matched controls. The following treatments were applied during I/R (n=6 each): fresh EHT medium (positive control), the NO-donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP, 10-7, 10-6, 10-5 M) or the particulate guanylate cyclase activator brain type natriuretic peptide (BNP, 10-9, 10-8, 10-7 M). Beating rate and force of contraction were monitored during the entire experiment; pH, Troponin I and LDH release and glucose consumption were measured in EHT medium after I/R. Result: During the 180 min ischemia, EHTs stopped to beat or beat at significantly lower rate. Most EHTs started to beat during reoxygenation and survived until the end of a 2-day follow up period. The cardioprotective interventions did not significantly affect contractile activity during the ischemic phase. In contrast, fresh medium as well as SNAP and BNP increased rate and/or rate x force product during reoxygenation. Troponin I (ranged between 0,2-0,8 ng/ml) and LDH release (0,07-0,08 U/mL) as well as glucose consumption (3-3,5 mmol/L; fresh medium 4,8 mmol/L) and medium pH (7,5) was not significantly affected by SNAP or BNP. Conclusion: The 24-well EHT test system is applicable to test cardioprotective compounds in vitro and monitor several functional and biochemical endpoints, which otherwise could be only measured in vivo or ex vivo heart preparations. Grants: German Research Foundation (DFG Es/88-12), the European Commission (FP7 Angioscaff, FP7 Biodesign), NKFP 07 1-ES2HEART-HU (OM-00202/2007), National Development Agency - New Hungary Development Plan (TAMOP-4.2.2-08/1/2008-0013, TAMOP-4.2.1/B-09/1); OTKA PD 106001.