A role for the RISK pathway and K(ATP) channels in pre- and post-conditioning induced by levosimendan in the isolated guinea pig heart

Abstract

Background and purpose: Myocardial reperfusion injury prevents optimal salvage of the ischaemic myocardium, and adjunct therapy that would significantly reduce reperfusion injury is still lacking. We investigated whether (1) the heart could be pre- and/or post-conditioned using levosimendan (levosimendan pre-conditioning (LPC) and levosimendan post-conditioning (LPostC)) and (2) the prosurvival kinases and/or the sarcolemmal or mitochondrial K(ATP) channels are involved.

Experimental approach: Isolated guinea pig hearts were treated with two 5 min cycles of levosimendan (0.1 microM) interspersed with vehicle perfusion, or two 5 min cycles of ischaemia/reperfusion, before coronary artery ligation (CAL) for 40 min at 36.5 degrees C. Hearts were treated with mitochondrial or sarcolemmal K(ATP) channel blockers before LPC or LPostC. For post-conditioning, hearts received three 30 s cycles of ischaemia/reperfusion or levosimendan/vehicle. Hearts were pretreated with levosimendan immediately before CAL (without washout). Cardiac function, infarct size and reperfusion injury salvage kinase activity was assessed.

Key results: LPC and LPostC halved the infarct size compared with controls (P<0.05). Treatment with K(ATP) channel blockers before LPC or LPostC reversed this decrease. Pretreating hearts with levosimendan increased activity of extracellular signal-regulated kinase (ERK) 42/44 on reperfusion and had the most marked infarct-lowering effect (P<0.05).

Conclusions and implications: (1) Hearts could be pharmacologically pre- and post-conditioned with levosimendan; (2) levosimendan pretreatment is the most effective way to reduce infarct size, possibly by increasing ERK 42/44 activity; (3) benefits of LPC and LPostC were abolished by both K(ATP) channel blockers and (4) LPC may be useful before elective cardiac surgery, whereas LPostC may be used after acute coronary artery events.

Figures

Figure 1

Experimental perfusion protocol used to induce ischaemic and levosimendan pre-conditioning and to pretreat the hearts with levosimendan (a). Myocardial infarct size in control, untreated hearts; hearts pre-conditioned using ischaemic pre-conditioning (IPC), or levosimendan pre-conditioning (LPC) or the combination of both; or hearts pretreated with levosimendan (LPT) (b). LD, Langendorff perfusion; WH, working heart perfusion; CAL, coronary artery ligation; n=6–9; *P<0.05 vs control.

Figure 2

Experimental perfusion protocol used to induce levosimendan pre-conditioning in the absence or presence of KATP channel blockers (a). Effects of KATP channel blockers (5HD or GBD) on the myocardial infarct size in hearts pre-conditioned with levosimendan (b). n=6–8; LD, Langendorff perfusion; WH, working heart perfusion; CAL, coronary artery ligation; Levo, levosimendan; 5HD, 5-hydroxydecanoic acid; GBD, glibenclamide; 5HD Control, 5HD without intermittent levosimendan pre-conditioning; LPC+5HD, 5HD with intermittent levosimendan pre-conditioning; GBD Control, GBD without intermittent levosimendan pre-conditioning; LPC+GBD, GBD with intermittent levosimendan pre-conditioning.

Figure 3

Experimental perfusion protocol used to induce ischaemic (IPostC) or levosimendan post-conditioning (LPostC) in the presence and absence of KATP channels blockers (a). Myocardial infarct size in control hearts, hearts post-conditioned with ischaemia or hearts post-conditioned with levosimendan (b). n=4–5; *P<0.05 vs control; LD, Langendorff perfusion; WH, working heart perfusion; CAL, coronary artery ligation; Levo, levosimendan; 5HD, 5-hydroxydecanoic acid; GBD, glibenclamide.

Figure 4

Aortic output recovery (%) for control hearts and hearts pretreated with levosimendan (LPT), or pre-conditioned using ischaemia (IPC) or levosimendan (LPC) (a). Aortic output recovery (%) for control hearts and hearts post-conditioned with ischaemia (IPostC), or levosimendan (LPostC) or in combination with KATP channel blockers (b). n=6–9; *P<0.05 vs control.

Figure 5

Representative blots for total ERK and phospho-ERK 1/2 and densitometry showing the effect of levosimendan pre-conditioning (LPC) or levosimendan pretreatment (LPT) on ERK 44 (a) and ERK 42 (b) phosphorylation of the ischaemic tissue at 5 and 10 min reperfusion. n=6; *P<0.05 vs basal; #P<0.05.