Reperfusion injury and ischemic preconditioning: two sides of a coin?

See article by Xiao and Allen [1] (pages 244–253) in this issue. A substantial body of presently available evidence supports the contention that cytosolic accumulation of sodium during ischemia is instrumental to the elevation of intracellular calcium during ischemia and reperfusion through prolonged reversed mode operation of the Na+/Ca2+-exchanger [1–10], which eventually leads to irreversible injury. The study by Xiao and Allen [1] published in this issue of Cardiovascular Research further elaborates on the original hypothesis by Lazdunski et al. [2] and previous work of the authors [11] that the Na+/H+-exchanger is pivotal in causing myocardial reperfusion injury following prolonged ischemia and adds to the ongoing debate on the subject. The study provides experimental evidence that the Na+/H+-exchanger is relatively inactive during ischemia, but rapidly re-activates early during reperfusion causing an increase in cytosolic Na+ which secondarily results in cytosolic Ca2+ overload by the Na+/Ca2+-exchanger operating in reversed mode. Inhibition of Na+/H+-exchange during reperfusion greatly improved functional recovery, which corroborates results obtained previously by others [2–8,10]. Additionally, they demonstrate that re-activation of the Na+/H+-exchanger is attenuated or prevented during reperfusion of hearts preconditioned by short preceding ischemia/reperfusion episodes. Finally, they report the observation that the preconditioning ischemic episodes per se do not affect the activity of the Na+/H+-exchanger. From this study the authors find support for their hypothesis that the mechanism of cardio-protection by preconditioning is linked to the mechanism by which inhibition of the Na+/H+-exchanger prevents reperfusion injury. Three aspects of the process of ischemic preconditioning have been distinguished and critically reviewed [12]: the initiating trigger delivered during the preconditioning ischemic episodes, the activation of signalling pathways and the end-points. There is considerable consensus that preconditioning merely delays the onset of irreversible damage but certainly does not prevent it. This holds for all end-point indices of preconditioning, contracture development, electrical uncoupling, increase of cytosolic [Ca2+], contractile dysfunction, onset of necrosis and infarct size reduction (see for review Ref. [12]). Thus, any proposed mechanism underlying preconditioning should basically explain the retardation of deteriorating metabolism and ion homeostasis. The qualitative and quantitative outcome of interventions such as reperfusion during the course of ischemia critically depends on the timing of the intervention. The observation that the preconditioning episodes did not affect the activity of the Na+/H+-exchanger before the onset of prolonged ischemia [1] suggests that inactivation of the Na+/H+-exchanger does not represent a preconditioning trigger. In addition, the authors demonstrate that the Na+/H+-exchanger is rather inactive during ischemia. At least, they do not observe obvious differences between ventricular performance and cytosolic sodium accumulation in the presence or absence of a specific inhibitor of the Na+/H+-exchanger, neither in control hearts nor in preconditioned hearts [1]. Therefore, it is not immediately obvious in what manner the Na+/H+-exchanger itself could possibly have contributed to the retardation of metabolic and ionic deterioration in the preconditioned hearts. At this stage, it can not be excluded that re-activation of the Na+/H+-exchanger upon reperfusion in the preconditioned hearts did not become apparent, because at the time of reperfusion the bio-energetic and ionic conditions were better preserved. Regardless of the exact mechanism by which a critical calcium overload condition causes ischemia/reperfusion damage, it is quite undisputed that [Na+]i dynamics underly it by activating reversed mode Na+/Ca2+ exchange of sufficiently large magnitude and duration. Indeed, from a 23Na–NMR study in the isolated rat heart Imahashi et al. recently concluded that intracellular sodium accumulation during ischemia is the substrate for reperfusion injury and that its kinetics coupled to Ca2+ influx during reperfusion determines the degree of injury [3]. An Editorial commentary dedicated to this study reviewed potential mechanisms of sodium influx during ischemia versus re-flow and the controversies regarding the involvement of the Na+/H+-exchanger, but also stressed the important implications of the issue with respect to potential beneficial clinical interventions [4]. Another not explicitly discussed potential contributor to sodium accumulation is the Na+/HCO3− symporter. Recently, it was demonstrated that inhibition of the Na+/HCO3− symporter attenuated the development of hypercontracture and reperfusion injury, additive to inhibition of the Na+/H+-exchanger [10]. Contribution by non-inactivating Na-channels to sodium accumulation was discussed [4]. However, because Na-channel blockade reduces hypoxic sodium loading and sodium-dependent calcium overload [13], action potential dependent Na+ entry must be considered, because at physiological heart rates it could contribute substantially to sodium accumulation during reperfusion. Na-channel dependent Na+ entry during the action potential may be estimated to be about 2 to 4 μmol/l per beat. The requirements for a Na+/Ca2+-exchanger dependent calcium overload condition to occur during ischemia and during early reperfusion are complex. The process involves the interplay between thermodynamic driving forces and kinetic properties not only of the sarcolemmal Na+/Ca2+-exchanger (see for review Ref. [14]), but also of the Na+/H+-exchanger [10,15], the Na+–HCO3− symporter [10], the Na+/K+–ATPase [14,15], Na-channel activation (see above), the SR Ca2+–ATPase and ryanodine receptor [14], the mitochondrial Na+/Ca2+-exchanger and Ca2+-uniporter [16,17] and last but not least the free energy of ATP hydrolysis, ΔGATP, from which all driving forces are ultimately derived [14]. ΔGATP progressively decreases during the course of ischemia in a biphasic manner [18]. During ischemia the first phase of moderate decrease of ΔGATP coincides with ongoing anaerobic glycolytic activity, development of acidosis and lactate production, the plateau phase of extracellular K+ accumulation [18,20], extrusion of calcium [20], maintenance of low [Ca]i and [Na]i[21] (see for review Ref. [14]). During this first phase the free energy of the Na+/Ca2+-exchanger gradually decreases from about 6.5 kJ/mol to near thermodynamic equilibrium [22]. After depletion of glycolytic substrate stores a substantial secondary decrease of ΔGATP follows which is associated with further K+ loss and membrane depolarization and a gradual increase of [Ca]i and [Na]i[18,21,22]. During this phase the free energy of the Na+/Ca2+-exchanger remains about zero [22]. It is during this phase that reperfusion may cause injury. Preconditioning retards the onset of the second phase. Near thermodynamic equilibrium of the Na+/Ca2+-exchanger may be formulated in terms of trans-sarcolemmal ion gradients by in which V is membrane potential, F the Faraday constant and R and T gas constant and temperature, respectively. This formulation states that any process increasing [Na]i (see above) or decreasing membrane potential (K+ loss and/or action potentials) forces the Na+/Ca2+-exhanger into reversed mode until equilibrium is re-established by an increase of [Ca]i. The opposite occurs with reduction of [Na]i by the Na+/K+–ATPase or by washout of extracellular K+ and membrane repolarization. Detrimental effects by reperfusion of tissue suffering from substantial potential loss, which is particularly obvious in prolonged hypoxia or ischemia [18,19], may thus at least in part be explained by limited repolarization during washout of extracellular K+. It also follows from the near equilibrium formulation that the maximal contribution that the Na+/Ca2+-exchanger can make to changes of [Na]i equals three times the change of [Ca]i. Taking into consideration calcium buffering of myocardial tissue [23] it can be estimated that even at [Ca]i as high as a few μmol/l this contribution is a few mmol/l at most. It is not immediately clear how to distinguish between specific reperfusion dependent injury and aggrevation by reperfusion of pre-existing irreversible damage inflicted during the course of ischemia. Nevertheless, the specific reperfusion dependent effects, as far as calcium overload plays a role, may be appreciated from the time derivative of the above equilibrium formulation during the course of reperfusion. The time derivative is a function of [Na]i and membrane potential as well. A negative sign of the time derivative would preclude additional reperfusion dependent injury. A positive sign sets the stage for potential injury, the extent of which depends on its magnitude and duration. All mechanisms contributing to intracellular sodium dynamics discussed above except one contribute to a positive time derivative. Only the Na+/K+–ATPase can possibly add a negative component. Therefore, from a thermodynamic perspective, the rate at which ΔGATP is restored and the consequent kinetic activation of the electrogenic Na+/K+–ATPase relative to the rate of Na+ entry ultimately determines whether or not and to what extent reperfusion may cause additional irreversible injury.