Activation of cardiac muscarinic M3 receptors induces delayed cardioprotection by preserving phosphorylated connexin43 and up-regulating cyclooxygenase-2 expression

Abstract

Background and purpose: Activation of muscarinic M(3) mucarinic acetylcholine receptors (M(3)-mAChRs) has been previously shown to confer short-term cardioprotection against ischaemic injuries. However, it is not known whether activation of these receptors can provide delayed cardioprotection. Consequently, the present study was undertaken to investigate whether stimulation of M(3)-mAChRs can induce delayed preconditioning in rats, and to characterize the potential mechanism.

Experimental approach: Rats were pretreated (24 h), respectively, with M(3)-mAChRs agonist choline, M(3)-mAChRs antagonist 4-DAMP or M(2)-mAChRs antagonist methoctramine followed by the administration of choline. This was followed by 30 min of ischaemia and then 3 h of reperfusion. Ischaemia-induced arrhythmias and ischaemia-reperfusion (I/R)-induced infarction were determined. The phosphorylation status of connexin43 (Cx43) after 30 min ischaemia, and the expression level of Hsp70, cyclooxygenase-2 (COX-2) and iNOS effected by administration of choline were also measured.

Key results: Compared to the control group, pretreatment with choline significantly decreased ischaemia-induced arrhythmias, reduced the total number of ventricular premature beats, the duration of ventricular tachycardia episodes and markedly reduced I/R-induced infarct size. Furthermore, choline attenuated ischaemia-induced dephosphorylation of Cx43, and up-regulated the expression of Hsp70 and COX-2. Administration of 4-DAMP abolished these changes, while methoctramine had no effect.

Conclusions and implications: Our results suggest that stimulation of M(3)-mAChRs with choline elicits delayed preconditioning, which we propose is the result of up-regulation of the expression of COX-2 and inhibition of the ischaemia-induced dephosphorylation of Cx43. Therefore, M(3)-mAChRs represent a promising target for rendering cardiomyocytes tolerant to ischaemic injury.

Figure 1

Ischaemia-induced myocardial infarction. IS was expressed as a percentage of the AR. Values are mean ± SEM, obtained from seven to eight rats. **P < 0.01 versus MI; #P < 0.05 versus Choline.

Figure 3

Distribution of VPBs. A 30 min period of myocardial ischaemia induced frequent arrhythmias, which presented a peak at around 15 min (A); administration of choline markedly decreased the total number of VPBs, especially the peak at around 15 min (B), and this beneficial effect was abolished by pretreatment with 4-DAMP (D), but not with methoctramine (C). Values are mean ± SEM, n= 12.

Figure 2

Ventricular arrhythmias occurred during 30 min of occlusion of the LAD in all groups. (A) Examples of normal rhythm, VPBs, VT and VF as labelled. Compared to the MI group, in both the Choline and Meth groups, the arrhythmic scores (B), VT duration (C) and incidence of VF (D) were markedly decreased, while in the 4-DAMP group these measurements increased again. Values are mean ± SEM, n= 12. *P < 0.05, **P < 0.01 versus the MI group; ##P < 0.01, #P < 0.05 versus the Choline group.

Figure 4

Changes in Cx43 determined after the 30 min occlusion of the LAD. (A) Top panel, representative Western blots for sarcolemmal Cx43 and GAPDH. Bottom panel, a histogram summarizing the Western blots expressed as a percentage of the total sarcolemmal Cx43 content. (B) Top panel, representative Western blots for total Cx43; bottom panel, histogram depicting the analysis of the total Cx43 content. Values are mean ± SEM, obtained from n= 4–5 samples in each group. ##P < 0.01 versus control group, *P < 0.05 versus MI, #P < 0.05 versus Choline.

Figure 5

Effect of choline treatment on Hsp70 (A), COX-2 (B) and iNOS (C) expression. Top panels, representative Western blot showing expression of Hsp70, COX-2 and iNOS 24 h following treatment in each group; bottom panels, histogram showing relative level of expression of each protein. Values are mean ± SEM, obtained from four samples per group. #P < 0.05 versus control, ##P < 0.01 versus Choline. *P < 0.05 versus Choline.