Cardioprotection of Immature Heart by Simultaneous Activation of PKA and Epac: A Role for the Mitochondrial Permeability Transition Pore

Abstract

Metabolic and ionic changes during ischaemia predispose the heart to the damaging effects of reperfusion. Such changes and the resulting injury differ between immature and adult hearts. Therefore, cardioprotective strategies for adults must be tested in immature hearts. We have recently shown that the simultaneous activation of protein kinase A (PKA) and exchange protein activated by cAMP (Epac) confers marked cardioprotection in adult hearts. The aim of this study is to investigate the efficacy of this intervention in immature hearts and determine whether the mitochondrial permeability transition pore (MPTP) is involved. Isolated perfused Langendorff hearts from both adult and immature rats were exposed to global ischaemia and reperfusion injury (I/R) following control perfusion or perfusion after an equilibration period with activators of PKA and/or Epac. Functional outcome and reperfusion injury were measured and in parallel, mitochondria were isolated following 5 min of reperfusion to determine whether cardioprotective interventions involved changes in MPTP opening behaviour. Perfusion for 5 min preceding ischaemia of injury-matched adult and immature hearts with 5 µM 8-Br (8-Br-cAMP-AM), an activator of both PKA and Epac, led to significant reduction in post-reperfusion CK release and infarct size. Perfusion with this agent also led to a reduction in MPTP opening propensity in both adult and immature hearts. These data show that immature hearts are innately more resistant to I/R injury than adults, and that this is due to a reduced tendency of MPTP opening following reperfusion. Furthermore, simultaneous stimulation of PKA and Epac causes cardioprotection, which is additive to the innate resistance.

Keywords: development; immature heart; ischaemia/reperfusion injury; mitochondria.

Figure 1

Perfusion protocols for experimentation. Graphical representations of the experimental protocols used for ex vivo heart perfusion in experiments measuring (A) CK activity and area of infarct size in experiments and (B) experimental protocols for mitochondrial isolation experiments Data labels are the durations, in minutes, of each phase of the protocol. Colour blocks indicate the phases of the experiment; blue = standard perfusion with Krebs Henseleit (KH) buffer; orange = perfusion with KH containing a drug; grey = drug washout period. C = control group; I = intervention group; EI = extended ischaemic time; I/R = ischaemia/reperfusion.

Figure 2

Activity of creatine kinase in coronary effluent during perfusion experiments. (A) Adult heart; (B) P14 hearts 30 min global ischaemia; (C) P14 hearts 50 min global ischaemia. Normalised to coronary flow rate. Error bars represent mean ± SE. n = 6 per group.

Figure 3

Total activity for CK in each age and treatment group for the cAMP analogue experiments. Total activity taken as estimated area under activity–time curves summed by trapezoidal addition of mean values for each time point. Bars represent mean area under curve of activity/time curves ±SE; * = p < 0.05 vs. same age group control. Significance tested by Student’s t-test. n = 6 per group.

Figure 4

Cross-sectional proportionate infarct size vs. age group and intervention. Mean ± SE; * = p < 0.05 vs. same age group control. Two separate P14 groups were used with different ischaemic durations (see text). Statistical testing by Student’s t-test compared against same age group control. n = 6 per group.

Figure 5

Isoprenaline-induced MPTP inhibition in hearts not exposed to I/R injury. Change in absorbance of (A) adult or (B) P14 mitochondrial extract following addition of calcium to mitochondrial suspension with or without perfusion with isoprenaline. Baseline—peak change in OD at baseline; Ca²⁺ Added—change in OD after calcium addition. * = p < 0.05 vs. baseline swelling in same group; data expressed as mean value ± standard error of the mean. Significance testing by ANOVA. n = 6 per group.

Figure 6

Summary of effects of interventions on MPTP activity. Error bars = 1 SEM. * = p < 0.05, vs. IR group in corresponding age group. # = p < 0.05 vs. adult age group in corresponding intervention. $ = p < 0.05 vs. isoprenaline in corresponding age group. Significance testing by 2-way ANOVA. n = 6 per age group per drug treatment.