Myocardial ischemic postconditioning against ischemia-reperfusion is impaired in ob/ob mice

Abstract

Ischemic postconditioning (IPCD) significantly reduces infarct size in healthy animals and protects the human heart. Because obesity is a major risk factor of cardiovascular diseases, the effects of IPCD were investigated in 8- to 10-wk-old leptin-deficient obese (ob/ob) mice and compared with wild-type C57BL/6J (WT) mice. All animals underwent 30 min of coronary artery occlusion followed by 24 h of reperfusion associated or not with IPCD (6 cycles of 10-s occlusion, 10-s reperfusion). Additional mice were killed at 10 min of reperfusion for Western blotting. IPCD reduced infarct size by 58% in WT mice (33+/-1% vs. 14+/-3% for control and IPCD, respectively, P<0.05) but failed to induce cardioprotection in ob/ob mice (53+/-4% vs. 56+/-5% for control and IPCD, respectively). In WT mice, IPCD significantly increased the phosphorylation of Akt (+77%), ERK1/2 (+41%), and their common target p70S6K1 (+153% at Thr389 and +57% at Thr421/Ser424). In addition, the phosphorylated AMP-activated protein kinase (AMPK)-to-total AMPK ratio was also increased by IPCD in WT mice (+64%, P<0.05). This was accompanied by decreases in phosphatase and tensin homolog deleted on chromosome 10 (PTEN), MAP kinase phosphatase (MKP)-3, and protein phosphatase (PP)2C levels. In contrast, IPCD failed to increase the phosphorylation state of all these kinases in ob/ob mice, and the level of the three phosphatases was significantly increased. Thus, although IPCD reduces myocardial infarct size in healthy animals, its cardioprotective effect vanishes with obesity. The lack of enhanced phosphorylation by IPCD of Akt, ERK1/2, p70S6K1, and AMPK might partly explain the loss of cardioprotection in this experimental model of obese mice.

Figure 1

Experimental protocols used for investigating the effects of ischemic postconditioning (IPCD) in wild-type and ob/ob mice (CAO, coronary artery occlusion; CAR, coronary artery reperfusion).

Figure 2

Infarct size (expressed as percentage of the area at risk) measured in wild-type (WT) and ob/ob mice subjected to 30 min of coronary artery occlusion and 24 h of reperfusion in the absence or presence of postconditioning (Control and IPCD, respectively). Open circles represent individual values and closed circles indicate the average. *p<0.05 vs Control; †p<0.05 vs respective WT.

Figure 3

Western blot analysis of Akt and its phosphorylated form at Ser 473 (panel A), ERK 1/2 and its phosphorylated forms at Thr 202 and Tyr 204 (panel B), p70S6K1 and its phosphorylated form at Thr 389 due to the effect of Akt (panel C), p70S6K1 and its phosphorylated form at Thr 421 and Ser 424 due to the effects of ERK1/2 (panel D), AMPK and its phosphorylated form (panel E), PTEN (panel F), MKP-3 (panel G) and PP2C (panel H) in wild-type (WT) and ob/ob mice performed at baseline (i.e., in the absence of ischemia-reperfusion). Values are expressed as mean ± SEM (n=5 per group for WT mice and n=5 per group for ob/ob mice). *p<0.05 vs WT.

Figure 4

Western blot analysis of Akt and its phosphorylated form at Ser 473 (panel A) as well as ERK 1/2 and its phosphorylated forms at Thr 202 and Tyr 204 (panel B) in wild-type (WT) and ob/ob mice submitted to 30 min of coronary artery occlusion and 10 min of reperfusion in the absence (Control) or presence of postconditioning (IPCD). Values are expressed as mean ± SEM (n=3 per group for WT mice and n=4 per group for ob/ob mice). *p<0.05 vs respective Control; †p<0.05 vs respective WT.

Figure 5

Western blot analysis of p70S6K1 and its phosphorylated forms. Panel A: Thr 389 due to the effect of Akt; panel B: Thr 421 and Ser 424 due to the effects of ERK 1/2 in wild-type (WT) and ob/ob mice submitted to 30 min of coronary artery occlusion and 10 min of reperfusion in the absence (Control) or presence of postconditioning (IPCD). Values are expressed as mean ± SEM (n=3 per group for WT mice and n=4 per group for ob/ob mice). *p<0.05 vs respective Control; †p<0.05 vs respective WT.

Figure 6

Western blot analysis of AMPK and its phosphorylated form (Thr 172) in wild-type (WT) and ob/ob mice submitted to 30 min of coronary artery occlusion and 10 min of reperfusion in the absence (Control) or presence of postconditioning (IPCD). Values are expressed as mean ± SEM (n=3 per group for WT mice and n=4 per group for ob/ob mice). *p<0.05 vs respective Control; †p<0.05 vs respective WT.

Figure 7

Western blot analysis of PTEN (panel A), MKP-3 (panel B) and PP2C (panel C) in wild-type (WT) and ob/ob mice submitted to 30 min of coronary artery occlusion and 10 min of reperfusion in the absence (Control) or presence of postconditioning (IPCD). Values are expressed as mean ± SEM (n=3 per group for WT mice and n=4 per group for ob/ob mice). *p<0.05 vs respective Control; †p<0.05 vs respective WT.