Preconditioning Human Ventricular Cardiomyocytes with Brief Periods of Simulated Ischaemia

Objective: The aim was to test for "ischaemic" preconditioning in monolayer cultures of quiescent human ventricular cardiomyocytes.

Methods: Stabilised cardiomyocytes (n = 8 plates per group) were preconditioned with varying periods of simulated ischaemia and reperfusion, followed in all groups by 90 min of sustained "ischaemia" with or without 30 min of reperfusion. Cellular injury was assessed by trypan blue exclusion and survival was assessed by culturing the cells for 24 h postintervention. In addition, separate groups of cell plates (n = 8 per group) which had first been preconditioned with 20 min ischaemia and 20 min reperfusion were exposed to either 30, 60, or 90 min sustained ischaemia or 90 min sustained ischaemia with 30 min reperfusion. The supernatants and/or cell homogenates were analysed for hydrogen ion, lactate, lactate dehydrogenase (LDH), and adenine nucleotides and degradation products.

Results: Preconditioning (PC) decreased trypan blue uptake following subsequent sustained ischaemia, with the 20 min ischaemia/20 min reperfusion (20/20) regimen having the most profound effect [control ischaemia: 37.0(SEM 2.1); 10/10: 23.9(1.5); 20/20: 15.4(1.4); 30/30: 25.8(2.1) percent blue stained cells, p < 0.05 by ANOVA/Duncan]. The 20/20 preconditioning regimen resulted in less hydrogen ion [control: 2.1(0.4); PC: 1.4(0.1) mmol.g-1 protein, p < 0.05] and less LDH release [control: 20.7(3.1); PC: 11.9(4.2) units.g-1 protein, p < 0.05]. At 90 min of sustained ischaemia, the control group had produced significantly greater lactate [intracellular: control 1.55(0.62); PC 0.54(0.23) mol.g-1 DNA, p < 0.05; extracellular: control 0.47(0.09); PC 0.33(0.07) mol.g-1 DNA, p < 0.05]. There were no differences in ATP depletion in the two groups.

Conclusions: Ischaemic preconditioning can be induced in human cardiomyocytes independent of other cell types. The effect can be established in human cell cultures.