Clinically-relevant consecutive treatment with isoproterenol and adenosine protects the failing heart against ischaemia and reperfusion

Abstract

Background: Consecutive treatment of normal heart with a high dose of isoproterenol and adenosine (Iso/Ade treatment), confers strong protection against ischaemia/reperfusion injury. In preparation for translation of this cardioprotective strategy into clinical practice during heart surgery, we further optimised conditions for this intervention using a clinically-relevant dose of Iso and determined its cardioprotective efficacy in hearts isolated from a model of surgically-induced heart failure.

Methods: Isolated Langendorff-perfused rat hearts were treated sequentially with 5 nM Iso and 30 μM Ade followed by different durations of washout prior to 30 min global ischaemia and 2 hrs reperfusion. Reperfusion injury was assessed by measuring haemodynamic function, lactate dehydrogenase (LDH) release and infarct size. Protein kinase C (PKC) activity and glycogen content were measured in hearts after the treatment. In a separate group of hearts, Cyclosporine A (CsA), a mitochondria permeability transition pore (MPTP) inhibitor, was added with Iso/Ade. Failing hearts extracted after 16 weeks of ligation of left coronary artery in 2 months old rats were also subjected to Iso/Ade treatment followed by ischaemia/reperfusion.

Results: Recovery of the rate pressure product (RPP) in Iso/Ade-treated hearts was significantly higher than in controls. Thus in Iso/Ade treated hearts with 5 nM Iso and no washout period, RPP recovery was 76.3±6.9% of initial value vs. 28.5±5.2% in controls. This was associated with a 3 fold reduction in LDH release irrespective to the duration of the washout period. Hearts with no washout of the drugs (Ade) had least infarct size, highest PKC activity and also showed reduced glycogen content. Cardioprotection with CsA was not additive to the effect of Iso/Ade treatment. Iso/Ade treatment conferred significant protection to failing hearts. Thus, RPP recovery in failing hearts subjected to the treatment was 69.0±16.3% while in Control hearts 19.7±4.0%. LDH release in these hearts was also 3 fold lower compared to Control.

Conclusions: Consecutive Iso/Ade treatment of normal heart can be effective at clinically-relevant doses and this effect appears to be mediated by glycogen depletion and inhibition of MPTP. This intervention protects clinically relevant failing heart model making it a promising candidate for clinical use.

Figure 1

Outline of the experimental protocols of series 1 and 2. Panel A – experimental protocol of the Series 1. Iso/Ade-0, Iso/Ade-5 and Iso/Ade-10 – consecutive perfusion with isoproterenol and adenosine with 0, 5, and 10 min washout period respectively; Iso/Ade Mix – perfusion with the mixture of isoproterenol and adenosine. Panel B – experimental protocol of the Series 2. Control; Iso/Ade – consecutive isoproterenol/adenosine treatment with no washout period (similar to that of Iso/Ade-0 in the Series 1); CsA – perfusion with cyclosporine A; and Iso/Ade + CsA – consecutive isoproterenol/adenosine treatment combined with perfusion with Cyclosporine A.

Figure 2

Outline of the experimental protocol of series 3. Panel A – model of surgically-induced heart failure. CAL – left anterior descending coronary artery ligation; SO – sham-operated heart. Panel B – experimental protocol of series 3. The CAL and SO hearts were divided to two groups each: Control CAL, Iso/Ade CAL, Control SO and Iso/Ade SO. The Iso/Ade protocol was similar to those of Iso/Ade-0 group in the Series 1 (Figure 1A) and Iso/Ade group in Series 2 (Figure 1B).

Figure 3

Lactate dehydrogenase (LDH) activity and infarct size in hearts of the Series 1. The experiments were carried out on Control (n = 7) and Iso/Ade groups with 0 (n = 9), 5 (n = 9), and 10 (n = 9) min washout periods. Panel A - LDH activity in the effluent perfusate during 30 min reperfusion. Differences between Control and all three Iso/Ade groups (measured for the corresponding data points) were statistically significant (P < 0.05) at all data points. There was no significant difference in LDH activity between the Iso/Ade groups. Panel B – Infarct size. Mean ± SEM (columns) and representative images of the corresponding heart slices stained with 2,3,5-triphenyltetrazolium chloride (TTC). ** P < 0.01, *** P < 0.001 vs. Control; # P < 0.05 vs. Iso/Ade group with zero min washout period.

Figure 4

Protein Kinase C (PKC) activity in hearts of Experimental Series 1. PKC activity was measured using non-radioactive PepTag® assay and is expressed as Mean ± SEM of a ratio of fluorescence intensity of phosphorylated and non-phosphorylated PepTag® C1 peptide (Panel B) which is proportional to the PKC activity. PKC activity was measured in Control (C, n = 10), Iso/Ade-0 (n = 9) and Iso/Ade-10 (n = 9) groups. Panel A – Representative image of the phosphorylated and non-phosphorylated PepTag® C1 peptide under UV light. ** P < 0.01 vs. Control.

Figure 5

Glycogen content in hearts of Experimental Series 1. Glycogen content in myocardium of Control hearts (n = 8), hearts treated consecutively with Iso and Ade (Iso/Ade group, n = 8) and hearts treated with the mixture of Iso and Ade (Iso/Ade Mix, n=7) was measured prior to ischaemia. * P < 0.05 vs. control.

Figure 6

LDH activity and infarct size in hearts in Experimental Series 2. The experiments were carried out on Control (n = 9), Iso/Ade (n = 9), cyclosporine A (CsA, n = 6) and Iso/Ade + CsA (n = 8) groups of hearts. The perfusion protocol of Iso/Ade group in this series of experiments was similar to that of Iso/Ade-0 group in Experimental Series 1. Panel A – LDH activity in the effluent perfusate during 30 min reperfusion. Differences between control and three other groups (measured for the corresponding data points) were statistically significant (P < 0.05) at all data points. There was no significant difference in LDH activity between the three intervention groups. Panel B – Infarct size. Mean ± SEM (columns) and representative images of the corresponding heart slices stained with TTC. ** P < 0.01, *** P < 0.001 vs. Control.

Figure 7

LDH activity in hearts of Experimental Series 3. LDH activity was measured in the effluent perfusate during 30 min reperfusion. The perfusion protocol of Iso/Ade groups in this series of experiments was similar to that of Iso/Ade-0 group in the Experimental Series 1. LDH activity is presented for Control and Iso/Ade groups in sham-operated hearts (SO, n = 6 in each group) and failing hearts after the left descending coronary artery ligation (CAL, n = 5 in control and n = 6 in Iso/Ade group). * P < 0.05 vs. corresponding Control (either SO or CAL); # P < 0.05 vs. Control CAL.