Reperfusion of ischaemic cells causes intracellular Ca2+ oscillations as the sarcoplasmic reticulum (SR) takes up and releases Ca2+, leading to hypercontracture and cell death. In other systems, nicotinic acid adenine nucleotide phosphate (NAADP) acts as a second messenger to stimulate Ca2+ release from acidic intracellular Ca2+ stores, which in turn triggers Ca2+ release from the SR. We hypothesised that NAADP signalling is involved in the Ca2+ fluctuations that occur at reperfusion.
We examined the effects of a novel NAADP inhibitor, Ned-19, on ischaemia-reperfusion injury in isolated adult rat ventricular cardiomyocytes (ARVC). The sensitivity of mitochondrial permeability transition pore (mPTP) was measured in ARVC using a laser-induced oxidative stress model. SR Ca2+ release was measured by treating cells loaded with fluorescent dye, fluo-4-AM, with caffeine. Cardioprotection was tested by exposing ARVC to metabolic ischaemia-reperfusion. Ned-19 was found to significantly delay the time to mPTP opening by 76%±16%, 55%±20%, 47%±19% and 44%±17% (all p<0.05) at concentrations of 100 μmol/l, 10 μmol/l, 1 μmol/l and 0.1 μmol/l, respectively, compared with the control group. Concentrations of Ned-19 at 100 μmol/l, 10 μmol/l and 1 μmol/l, but not 0.1 μmol/l, significantly inhibited caffeine-stimulated SR Ca2+ release (71.6%±2.0%, 34.2%±1.9%, 55.6%±5.5% and −14%±21%, respectively) indicating non-specific effects at higher concentrations. A low dose of 0.1 μmol/l Ned-19 increased the survival of cells following metabolic ischaemia-reperfusion to 46%±19% from 29% (control).
In conclusion, we have shown the involvement of NAADP in SR Ca2+ release and mPTP opening, and that by inhibiting NAADP signalling at reperfusion with Ned-19, cardiomyocytes may be protected against ischaemia-reperfusion injury.
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