Purpose of Study Cardiac acetylcholine (ACh) signaling is protective, but the role of ACh in ischemic preconditioning (IPC) remains largely unknown. We studied the effect of selective alpha-7 nicotinic ACh receptor (a7nAChR) antagonism by methyllycaconitine (MLA) on the functional benefits of IPC and the effects of this on mitochondrial complexity and inner mitochondrial membrane potential (ψM).
Methods Used Male Sprague Dawley rats (n=17, 322±17 g) were heparinized and anesthetized with 80 mg/kg pentobarbital IP, and their hearts excised and perfused at constant pressure with a non-circulating Langendorff apparatus. Left ventricular (LV) pressure (LVDP) and heart rate (HR) were continually measured with a fluid filled latex balloon attached to a pressure transducer. Treatment groups were: ischemia-reperfusion (IR)(n=6): 20 min. perfusion, 30 min. of global ischemia, 45 min. of reperfusion; IPC (n=5): 10 min. perfusion, 3 min. ischemia with 2 min. reperfusion repeated 3 times prior to IR protocol, IPC+MLA (n=6): 6 min. perfusion, 4 min. of infusion of MLA at 233 nM, IPC with MLA during reperfusion periods, then IR. Mitochondria were isolated from the LV free wall, stained for ψM and for size, and examined by Flow Cytometry with a BD LSRFortessa. Controls (C) (n=4) were freshly excised hearts from similar animals with identical anesthesia.
Summary of Results IPC increased LV work product (LVDP times HR) as a percent of pre-ischemia (%P) during reperfusion compared to IR control, and this effect was attenuated by MLA pretreatment (IR=24.1±4.5%P, IPC=49.8±2.8%P, IPC+MLA=33.8±3.5%P, p<0.01). IPC reduced end diastolic pressure from IR levels, and this was partially prevented by MLA treatment (IR=78.8±7.7 mm Hg, IPC=18.8±6.6 mm Hg, IPC+MLA=46.3±8.6 mm Hg, p<0.05). IPC maintained mitochondrial structural complexity compared to IR (C=65±6% of total mitochondria, IPC=61±5%, IR=32±4%, p<0.01). MLA reduced the effect of IPC on ψM in intact mitochondria to IR levels (IR=67±10% of intact population, IPC=88±3%, IPC+MLA=71±4%, p<0.01).
Conclusions Signaling through the a7nAChR is necessary for the effect of IPC on maintaining ψM and cardiac contractile function after IR injury.
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