Triggering of cardiac preconditioning through Na+/K+-ATPase

See article by Pierre et al.[6](pages 488–496) in this issue. It has been two decades since the first reports describing the phenomenon of ischemic preconditioning (IP). Data generated during these years describe a wide range of pharmacological triggers of IP, different intracellular signalling pathways, and some end-effectors of the powerful protection that IP exerts against cell death [1]. One of the most recent mechanisms that has been proposed to explain the protection induced by IP involves Na+/K+-ATPase. It is well accepted that the kinetics of Na+ recovery during reperfusion determines the influx of Ca2+ through the reverse mode of the Na+/Ca2+-exchanger and, consequently, the degree of injury [2], and it has been consistently reported that IP accelerates Na+ kinetics during early reperfusion by preserving Na+/K+-ATPase activity [3]. Recently, it has been proposed that the cardioprotective effects of IP are at least in part mediated by attenuated calpain activation, resulting in decreased displacement of Na+/K+-ATPase from the membrane-cytoskeleton complex, preserved Na+ pump activity, and attenuated Ca2+ influx through NCX [4]. By contrast, inhibition of Na+/K+-ATPase with ouabain at the onset of reperfusion completely abolished the protective effects afforded by IP. Recently, several groups have proposed that Na+/K+-ATPase has dual functions. In addition to pumping Na+ and K+ across cell membranes, it also relays an extracellular ouabain signal to intracellular compartments via activation of different protein kinases [5]. In this issue of Cardiovascular Research, Pierre et al. [6] add new evidence to the growing body of studies that support the existence of this signal-transducing function of Na+/K+-ATPase, and demonstrate that activation of this pathway by perfusion of isolated rat hearts with ouabain induces cardioprotection. Although, as the authors recognize, these results could apparently be in contradiction to the classical notion that preservation of Na+/K+-ATPase activity and its control of Na+ homeostasis is protective, an effect of ouabain on intracellular Na+ concentration in their study seems extremely unlikely: ouabain was given as a transient stimulus (for 4 min followed by washout) and the concentration used (10 μM) had negligible positive inotropic effects. The author's group has made important contributions helping to unravel many of the mechanisms that underlie the function of Na+/K+-ATPase as a signal transducer. In earlier studies they demonstrated that tyrosine kinases of the Src family are involved in the ouabain activation of the signal-transducing function of Na+/K+-ATPase [7]. After observing that Na+/K+-ATPase interacts with caveolin-1 and that ouabain regulates this interaction and induces the formation of a Na+/K+-ATPase-Src-caveolin-1 signaling complex in a cell line, they proposed that signaling mediated by Na+/K+-ATPase resides in membrane microdomains such as caveolae [8]. Whether the same localization occurs in myocytes needs further investigation. The question whether Src interacts with Na+/K+-ATPase directly or indirectly has been recently resolved: SH2 and kinase domains of Src interact with CD2 and CD3 domains of alpha subunit of Na+/K+-ATPase. These interactions maintain Src in an inactive state, and binding of ouabain to the Na+/K+-ATPase-Src complex activates associated Src, which subsequently provokes downstream protein tyrosine phosphorylation [9]. The epidermal growth factor (EGF) receptor is one of the proteins identified as substrates of Src [10]. Its phosphorylation activates the signaling cascade Ras/Raf/ERK1/2 leading to stimulation of mitochondrial reactive oxygen species (ROS) generation and the activation of NF-κB. This pathway seems to be essential for the observed hypertrophic effects of ouabain on cardiomyocytes. Another substrate of Src is phospholipase C-γ1 (PLC-γ1), which activates the protein kinase C (PKC) pathway [11]. In their paper, Pierre et al. [6] demonstrate, by using pharmacological inhibitors, the involvement of activated Src and PKCε in the cardioprotective effects of transient perfusion with ouabain. Whether the EGF pathway also contributes to this preconditioning-like protection and what are the cardioprotective mediators and final effectors linked to the proposed signaling pathway were not addressed by this study. However, the involvement of PKCε connects this study with the conclusions of different groups that propose this enzyme as a critical mediator of IP. Cardiac-specific overexpression of PKCε has been shown to confer protection against reperfusion-induced injury [12], while IP fails to protect PKCε knockout mice [13]. The role of PKCε in IP has been recently reviewed by Inagaki et al. [14]. In their paper two proposed mechanisms, both associated with the mitochondria, are analyzed as possible effectors for explaining the cardioprotective effects of PKCε: opening of the KATP channel and inhibition of mitochondrial permeability transition pore opening during reperfusion. In parallel to the study published in this issue, Pierre and co-authors have provided more evidence supporting the possibility that ouabain may be cardioprotective in the therapeutic dose range [15]. Continuous perfusion of isolated hearts with ouabain at concentrations without inotropic response before ischemia initiated a cardioprotective signaling pathway that induced ROS production and required mitochondrial KATP channel opening. The observations by Pierre et al. open new perspectives in the study of the signal-transduction function of Na+/K+-ATPase by demonstrating that its transient activation has strong physiologically relevant consequences, such as induction of cardiac tolerance to ischemic stress, and point to the interest of developing drugs that stimulate the signaling function of the Na+/K+-ATPase without affecting its ion pumping activity. In summary, Pierre et al. [6] propose that the activation of the signaling function of Na+/K+-ATPase as a new cardioprotective stimulus should be added to the long list of described preconditioning triggers. The focus of research efforts should probably move from triggers to final effectors if we want to translate this powerful cardioprotective effect of IP into a clinically relevant setting. Partially supported by grants CICYT SAF 2005-01758 and FIS-RECAVA.