Post-conditioning with gradually increased reperfusion provides better cardioprotection in rats

Abstract

Background: Rapid and complete reperfusion has been widely adopted in the treatment of patients with acute myocardial infarction (AMI), but this process sometimes can cause severe reperfusion injury. This study aimed to investigate different patterns of post-conditioning in acute myocardial ischemia-reperfusion injury, and to detect the role of mitogen activated protein kinase (MAPK) during the injury.

Methods: RATS WERE RANDOMLY DIVIDED INTO FIVE GROUPS: sham group, reperfusion injury (R/ I) group, gradually decreased reperfusion group (GDR group, 30/10-25/15-15/25-10/30 seconds of reperfusion/ischemia), equal reperfusion group (ER group, 20/20 seconds reperfusion/ischemia, 4 cycles), and gradually increased reperfusion group (GIR group, 10/30-15/25-25/15-30/10 seconds of reperfusion/ischemia). Acute myocardial infarction and ischemic post-conditioning models were established in the rats. Six hours after reperfusion, 3 rats from each group were sacrificed and myocardial tissues were taken to measure the expressions of phosphorylation of extracellular signal-regulated protein kinase (P-ERK), phosphorylated c-Jun N-terminal kinase (P-JNK), mitogen-activated protein kinase p38 (p38 MAPK), tumor necrosis factor-α (TNF-α), caspases-8 in the myocardial tissue, and cytochrome c in the cytosol using Western blot. Hemodynamics was measured at 24 hours after reperfusion, the blood was drawn for the determination of cardiac enzymes, and the heart tissue was collected for the measurement of apoptosis using TUNEL. One-way analysis of variance and the Q test were employed to determine differences in individual variables between the 5 groups.

Results: Three post-conditioning patterns were found to provide cardioprotection (P<0.05) compared with R/I without postconditioning. GIR provided the best cardioprotection effect, followed by ER and then GDR. Apoptotic index and serum marker levels were reduced more significantly in GIR than in ER (P<0.05). The enhanced cardioprotection provided by GIR was accompanied with significantly increased levels of P-ERK 1/2 (1.82±0.22 vs. 1.54±0.32, P<0.05), and lower levels of p-JNK, p38 MAPK, TNF-α, caspase-8, caspase-9 and cytochrome in the cytoplasm (P<0.05), compared with ER. The infarct size was smaller in the GIR group than in the ER group, but this difference was not significant (16.30%±5.22% vs. 20.57%±6.32%, P<0.05). All the measured variables were improved more significantly in the GIR group than in the GDR group (P<0.05).

Conclusion: Gradually increased reperfusion in post-conditioning could attenuate reperfusion injury more significantly than routine method, thereby the MAPK pathway plays an important role in this process.

Keywords: Apoptosis; Ischemia-reperfusion injury; Postconditioning.

Figure 1

Experimental protocols demonstrating that GIR was more similar to than either GDR or ER in gradual reperfusion.

Figure 2

Detection of apoptotic cells using TUNEL staining (A), with TUNEL-positive cells dyed green. Apoptotic indexes (B) were lower in the GIR group than those in the R/I, GDR and ER groups. Compared with the R/I group, *P<0.05; compared with the ER group, ^#P<0.05; compared with the GDR group, ^ΔP<0.05.

Figure 3

Expression of phosphorylated ERK, p38 and JNK MAPK in all groups. The GIR group had a higher expression of P-ERK and a lower level of P-p38/JNK compared with the R/I, GDR and ER groups. Compared with the R/I group, *P<0.05; compared with the ER group, ^#P<0.05; compared with the GDR group, ^ΔP<0.05.

Figure 4

Expression of TNF-α and caspase-8 in all groups, with a lower expression of TNF-α and caspase-8 in the GIR group than in the R/I, ER and GDR groups (P<0.05); compared the with R/I group, *P<0.05; compared with the ER group, ^#P<0.05; compared with the GDR group, ^ΔP<0.05.

Figure 5

Cytosolic cytochrome c expression in all groups. There was a significantly lower expression in the GIR group than in the R/I, GDR and ER groups (P<0.05). Compared with the R/I group, *P<0.05; compared with the ER group, ^#P<0.05; compared with the GDR group, ^ΔP<0.05.