Cardiomyocyte-derived adiponectin is biologically active in protecting against myocardial ischemia-reperfusion injury

Abstract

Adiponectin (APN) has traditionally been viewed as an adipocyte-specific endocrine molecule with cardioprotective effects. Recent studies suggest that APN is also expressed in cardiomyocytes. However, biological significances of this locally produced APN remain completely unknown. The aim of this study was to investigate the pathological and pharmacological significance of cardiac-derived APN in cardiomyocyte pathology. Adult cardiomyocytes from wild-type littermates (WT) or gene-deficient mice were pretreated with vehicle (V) or rosiglitazone (RSG) for 6 h followed by simulated ischemia-reperfusion (SI/R, 3 h/12 h). Compared with WT cardiomyocytes, myocytes from APN knockout (APN-KO) mice sustained greater SI/R injury, evidenced by greater oxidative/nitrative stress, caspase-3 activity, and lactate dehydrogenase (LDH) release (P < 0.05). Myocytes from adiponectin receptor 1 knockdown (AdipoR1-KD) or AdipoR1-KD/AdipoR2-KO mice had slightly increased SI/R injury, but the difference was not statistically significant. RSG significantly (P < 0.01) increased APN mRNA and protein expression, upregulated AdipoR1/AdipoR2 expression, reduced SI/R-induced apoptosis, and decreased LDH release in WT cardiomyocytes. However, the anti-oxidative/anti-nitrative and cell protective effects of RSG were completely lost in APN-KO cardiomyocytes (P > 0.05 vs. vehicle group), although a comparable degree of AdipoR1/AdipoR2 upregulation was observed. The upregulatory effect of RSG on APN mRNA and protein expression was significantly potentiated in AdipoR1-KD/AdipoR2-KO cardiomyocytes. However, the cellular protective effects of RSG were significantly blunted, although not completely lost, in these cells. These results demonstrated that cardiomyocyte APN is biologically active in protecting cells against SI/R injury. Moreover, this locally produced APN achieves its protective effect primarily through paracrine/autocrine activation of APN receptors.

Fig. 1.

Simulated ischemia-reperfusion (SI/R) injury assessed by lactate dehydrogenase (LDH) release (A) and caspase-3 activation (B). Assays were performed at the end of 3 h of SI and 12 h of reperfusion (SI/R) or 15 h of normal culture (Sham). **P < 0.01 vs. sham SI/R; ^#P < 0.05 and ^##P < 0.01 vs. wild-type (WT) cardiomyocytes with SI/R; n = 14–16 wells/group with cardiomyocytes isolated from 6–8 mice.

Fig. 2.

Effect of rosiglitazone (RSG) on adiponectin (APN) mRNA expression (A), APN protein expression (B), and APN secretion (C) in C57BL/6 cardiomyocytes. Assays were performed 6 h after vehicle or RSG treatment. **P < 0.01 vs. vehicle; n = 14–16 wells/group with cardiomyocytes isolated from 6–8 mice.

Fig. 3.

Effect of RSG on APN protein expression in cardiomyocytes isolated from WT or gene-manipulated mice. Assays were performed 6 h after vehicle or RSG treatment. *P < 0.05 and **P < 0.01 vs. vehicle in the same group; ^#P < 0.05 and ^##P < 0.01 vs. WT cardiomyocytes with the same treatment; n = 14–16 wells/group with cardiomyocytes isolated from 6–8 mice.

Fig. 4.

Effect of RSG on adiponectin receptor 1 (AdipoR1) expression in cardiomyocytes isolated from WT or gene-manipulated mice. Assays were performed 6 h after vehicle or RSG treatment. *P < 0.05 vs. vehicle in the same group; n = 14–16 wells/group with cardiomyocytes isolated from 6–8 mice.

Fig. 5.

Effect of RSG on adiponectin receptor 2 (AdipoR2) expression in cardiomyocytes isolated from WT or gene-manipulated mice. Assays were performed 6 h after vehicle or RSG treatment. *P < 0.05 vs. vehicle in the same group; n = 14–16 wells/group with cardiomyocytes isolated from 6–8 mice.

Fig. 6.

Effect of APN, AdipoR1, and AdipoR2 deficiency on RSG cardioprotection against SI/R injury. Cardiomyocytes were pretreated with vehicle or RSG for 6 h followed by 3 h of SI and 12 h of reperfusion. All data were normalized against mean values of their own vehicle group. *P < 0.05 and **P < 0.01 vs. vehicle in the same group; ^##P < 0.01 vs. WT cardiomyocytes with the same treatment; n = 14–16 wells/group with cardiomyocytes isolated from 6–8 mice.

Fig. 7.

Effect of APN, AdipoR1, and AdipoR2 deficiency on RSG-mediated anti-oxidative/anti-nitrative effect in SI/R cardiomyocytes. Cardiomyocytes were pretreated with vehicle or RSG for 6 h followed by 3 h of SI and 12 h of reperfusion. All data were normalized against mean values of their own vehicle group. *P < 0.05 and **P < 0.01 vs. vehicle in the same group; ^#P < 0.05 and ^##P < 0.01 vs. WT cardiomyocytes with the same treatment; n = 14–16 wells/group with cardiomyocytes isolated from 6–8 mice.