Post-conditioning restores pre-ischaemic receptor coupling in rat isolated hearts

Abstract

Background and purpose: Ischaemic preconditioning (IPC) and ischaemic post-conditioning (IPoC) activate signal transduction pathways that are also involved in receptor de- and re-sensitization such as phosphatidylinositol (PI) 3-kinase. Therefore, IPC and IPoC may affect post-infarct receptor coupling.

Experimental approach: Rat isolated hearts (Langendorff mode, constant flow) were exposed to 45 min flow arrest followed by 120 min reperfusion, including IPC or IPoC. Control hearts were perfused without a 45 min flow arrest. Left ventricular developed pressure (LVdevP) was determined. Thirty min after reperfusion, hearts were exposed to parathyroid hormone-related peptide (PTHrP) or isoprenaline for 10 min to monitor receptor responsiveness. Reperfusion injury was quantified by enzyme release.

Key results: IPC and IPoC significantly reduced enzyme release compared with ischaemia and reperfusion alone by 75% and 62% respectively. Wortmannin or chelerythrine inhibiting either PI 3-kinase or protein kinase C, respectively, attenuated protection. Application of PTHrP 30 min after reperfusion did not change LVdevP in hearts exposed to ischaemia (+1 +/- 11%), but IPoC restored the normal and non-ischaemic response to PTHrP characterized by a negative inotropism (-8.3 +/- 3.9% and -12.9 +/- 6.1%). IPC restored a small negative inotropic effect (-4.4 +/- 4.7%). Application of a PTHrP receptor antagonist during the 45 min flow arrest attenuated receptor desensitization (DeltaLVdevP: -6.1 +/- 1.7%). Wortmannin but not chelerythrine attenuated the re-sensitizing effect of IPoC on post-ischaemic receptor coupling (DeltaLVdevP: +6.2 +/- 10.5 and -15.0 +/- 7.7%). As observed with PTHrP receptors, IPoC restored beta-adrenoceptors (DeltaLVdevP: +9.3 +/- 11.8% vs. 62.3 +/- 15.8%).

Conclusions and implications: IPoC restores PTHrP receptor coupling in a PI 3-kinase-dependent way. A similar mechanism may allow beta-adrenoceptor re-sensitization.

Figure 1

Schematic overview of the experimental design. The study was performed in three steps (A–C). Flow was stopped during the ischaemic periods, which are indicated in black. Functional data were recorded before any intervention (basal, B), 30 min after the onset of reperfusion (R1), 10 min after the addition of PTHrP (100 nmol·L⁻¹) or isoprenaline (ISO, 100 nmol·L⁻¹) (Response, Re) or at the end of the experiments (R2). Where indicated wortmannin (W, 100 nmol·L⁻¹) or chelerythrine (Che, 1 µmol·L⁻¹) were added. When isoprenaline was given during ischaemia, its concentration was 10 nmol·L⁻¹. When the activity of PTHrP released during ischaemia was inhibited, a PTHrP antagonist [PTHrP-AG = PTHrP(7–34), 100 nmol·L⁻¹] was given. I/R, ischaemia and reperfusion; IPC, ischaemic preconditioning; IPoC, ischaemic post-conditioning; PTHrP, parathyroid hormone-related peptide.

Figure 3

Effect of ischaemic post-conditioning (IPoC) and its inhibition by wortmannin (W) and chelerythrine (Che) on the activation of the reperfusion injury salvage kinase pathway as monitored by p70rsk activity, measured by phosphorylation of a p70rsk substrate peptide and quantified on a microplate reader. Data are expressed as absorbance at 450 nm and are means ± SEM (n = 5). *P < 0.05 vs. ischaemia and reperfusion (I/R).

Figure 2

Effects on reperfusion injury, of ischaemia and reperfusion (I/R), ischaemic preconditioning (IPC), ischaemic post-conditioning (IPoC), IPoC with wortmannin (IPoC + W) and IPoC with chelerythrine (IPoC + Che). Reperfusion injury was determined by lactate dehydrogenase (LDH) release into the perfusate. Samples were collected after 5, 10 and 15 min and the area under curve (AUC) was calculated. Data are shown in comparison with normoxic time controls (C). Data are expressed as means ± SEM, n = 8 in each group. *P < 0.05 vs. C.

Figure 5

Acute effects of parathyroid hormone-related peptide (PTHrP) (100 nmol·L⁻¹) on functional parameters for the ischaemia and reperfusion (I/R), ischaemic preconditioning (IPC), ischaemic post-conditioning (IPoC), normoxic control (C) group, IPoC with wortmannin (W) and IPoC with chelerythrine (Che). PTHrP was perfused, 30 min after the onset of reperfusion, for 10 min. Data shown in this figure are the changes, 10 min after drug application. Data are given for left ventricular developed pressure (LVdevP) and represent means ± SEM; n = 8 in each group. *P < 0.05 vs. C; #P < 0.05 vs. baseline before application. For the absolute values before application of PTHrP or vehicle see Figure 4. AG, antagonist.

Figure 4

Functional recovery during reperfusion in groups with ischaemia and reperfusion (I/R), ischaemic preconditioning (IPC), ischaemic post-conditioning (IPoC), IPoC and wortmannin (W), IPoC and chelerythrine (Che) and normoxic controls (C). Left ventricular developed pressure (LVdevP) was measured during the first 30 min of reperfusion. Data are means ± SEM; n = 8–16 in each group. P < 0.05 n.s. vs. C. AG, antagonist.

Figure 6

Effect of cardiac protection and parathyroid hormone-related peptide (PTHrP) receptor stimulation on recovery over 120 min. Left ventricular developed pressure (LVdevP) was measured for the ischaemia and reperfusion (I/R), ischaemic preconditioning (IPC), ischaemic post-conditioning (IPoC), normoxic control (C) group and expressed as percentage of initial values (mean: 145 ± 8 mmHg). In the PTHrP groups, the peptide was perfused at 30 min of reperfusion and maintained for 10 min, before the peptide was washed out again. Data are means ± SEM; n = 8 in each group, *P < 0.05 vs. C or C + PTHrP.

Figure 7

Effect of ischaemic post-conditioning (IPoC) on β-adrenoceptor coupling in hearts with desensitized β-adrenoceptors. Normoxic control hearts were compared with hearts undergoing 45 min flow arrest in the presence of isoprenaline (10 nmol·L⁻¹) subsequently followed by reperfusion (I/R) or IPoC and reperfusion. Isoprenaline (100 nmol·L⁻¹) was perfused 30 min after the onset of reperfusion, and changes in heart function were determined. Data shown in this figure are the changes 5 min after drug application. Data are given for left ventricular developed pressure (LVdevP) and represent means ± SEM; n = 8 in each group; *P < 0.05 vs. C; #P < 0.05 vs. I/R.