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EDITORIAL FOCUS
Product Safety Laboratories, Eurofins Scientific, Dayton, New Jersey
THE FINE STUDY by Jiang et al. (8) in this issue of the American Journal of Physiology Heart and Circulatory Physiology is an important milestone in the ongoing studies on the role of the mitochondrial ATP-sensitive K+ (mitoKATP) channels in myocardial ischemia. It is not particularly surprising that this channel exists in human cardiac myocytes, because most proteins are highly conserved in mitochondria despite the fact that many of these proteins, including KATP, are encoded from nuclear genes. What is important is finding the presence of an inward rectifying K+ channel (Kir)6.2 reactive protein and its regulation by a PKC, both located within the inner mitochondrial membrane. Now, looking on this as a pharmacologist, this is a more satisfying solution than the translocation of a cytoplasmic PKC isoform that is more typically seen for activation of cell surface receptors. Several excellent studies (5, 11) have shown the importance of PKC in preconditioning, although these studies tended to focus on cytoplasmic isoforms.
These studies are, of course, the beginning of our understanding of the signaling pathways, endogenous ligands, and receptors involved in mitoKATP channel-mediated cardioprotection. What I would like to do in this commentary is to put the data from this study in perspective with previous work and potential future work. Hopefully, the readers will be forgiving of a pharmacologist's view of this work, but this seems reasonable because most of the work in the arena of mitoKATP channels, including that from Jiang et al. (8), use pharmacological tools "as instruments that dissociate and analyze the most delicate phenomena of the living machine" (Claude Bernard). With this caveat, it is important to keep in mind the concepts of ischemic preconditioning and pharmacological mitoKATP channel activation as related, but possibly distinct, biochemical events. We know that mitoKATP channels are involved with both pharmacological cardioprotection (3) and ischemic preconditioning (4), but the pathways leading to either mitoKATP channel activation or PKC activation may be very different. The intriguing data from this study are that both pharmacological activators of mitoKATP channel and PKC activation increase open probability of this channel. This makes it difficult to believe that PKC activation at the mitochondrial level is downstream of pharmacological activation, particularly because 5-hydroxydecanoic acid (5-HD) reduced open probability after phorbol ester treatment. Do PKC and pharmacological activation represent parallel pathways? This will need to be addressed in future studies. Perhaps preconditioning activates PKC through generation of an endogenous ligand binding to some, as yet, undiscovered receptor. Pharmacological activators may bypass this system by directly activating mitoKATP channels, although it would be interesting if the activators do not directly open mitoKATP channels but their interaction with their ligand binding domain (LBD) activates this local mitochondrial PKC. These questions and thoughts bring up several key issues that might truly begin to unlock the secrets behind the cardioprotective effects of KATP openers and ischemic preconditioning.
For preconditioning, a perplexing question may be asked: What is the endogenous mitochondrial ligand that causes activation of mitoKATP channels? From a therapeutic point of view, this is a more direct approach to capturing the clinical utility of activation of mitoKATP channels. Little work has been done to uncover this elusive "savior" of distressed cardiomyocytes, although to be fair, this is a daunting task. Because of the highly stereotyped response to preconditioning, such a mediator will most likely act through a specific LBD on a receptor protein. It could be an integral part of the mitoKATP channel complex or in close proximity where it may cause local activation of PKC. The only meager information I came across in my studies always captured my imagination but only remotely suggested potential places to start thinking about endogenous ligands. Sulfhydryl-containing peptide analogs, such as zofenopril, N-acetyl-L-cysteine, and captopril, protected ischemic myocardium in a stereoselective and 5-HD-reversible manner, suggesting a "lock and key" mechanism (10). The presence of thiol groups was a necessary, but not sufficient, condition for this cardioprotection because only peptide or amino acid-type analogs seemed to have this effect, unlike penicillamine. The stereoselective nature suggests that the thiol group needs to be presented in an exquisitely specific manner, and more than a simple redox reaction is required. Zofenopril is a highly lipophilic molecule and penetrates myocytes readily (10). It would be interesting to determine the effect of the redox state or thiol groups presented on various molecular scaffolds on the model system used by Jiang et al. (8). Whereas these data in no way even slightly open the door to knowledge of the endogenous ligand, it might serve as a useful starting place to at least start thinking about future work in the area of preconditioning.
When thinking about the pharmacological activators of mitoKATP channels, a number of questions come to mind that may be of critical importance. The binding site of these highly studied activators is still unknown. It has long been speculated for the sarcolemmal channels that the pharmacological openers bound to a receptor on the regulatory subunit sulfonylurea receptor (SUR) and reduced sensitivity of the channel to ATP (2, 7). This becomes even more of a mystery for mitoKATP channels because the nature of a regulatory protein(s) that may be related to SUR is still largely unknown. It would be hard to believe that the receptors on the sarcolemmal and mitoKATP channels are different because the active enantiomers for opening sarcolemmal channels are the same as seen for cardioprotection. Even the simplest question of the nature of the cardioprotective effects of KATP channel openers, which is whether these agents can penetrate cells and move rapidly to the mitochondria, has not been clearly established. Just because these openers work in lipid bilayer systems does not mean they can get to that target in vivo. The only evidence I can provide is in the form of a very simple study that only suggests cell penetration into mitochondria but not specific interaction with the critical receptor and was only published in abstract form. P-1075 was radiolabeled and put into the solution perfusing isolated rat hearts. The hearts were subjected to autoradiography, and the results showed that 70% of the label was associated with mitochondria (1). These data are crude and are just a beginning because much more elegant means for following the migration of drugs are available.
Even if we understood the endogenous ligand and receptor crucial for preconditioning or the receptor for pharmacological mitoKATP channel activators, we are faced with the daunting task of deciphering how activation of mitoKATP channels can lead to cardioprotection. Several excellent theories have been tested, including alterations of respiratory state, inhibition of calcium influx into mitochondria, modulation of voltage-dependent anion channels (VDAC), and the shuttling of adenine nucleotides (9, 12). VDAC modulation is particularly intriguing because it is the rate-limiting step for ADP transport into the mitochondria. Examination of this channel alone seems eminently worthy of further study.
The study by Jiang et al. (8) is tantalizing in that it may bring us a little closer to developing therapeutics. Efforts at Bristol-Myers Squibb were hampered by a limited knowledge of KATP, and we developed "selective" mitoKATP openers on purely empirical grounds (it is better to be lucky than good). Our only screen was an isolated rat heart model of ischemia, and, therefore, we were only able to test 
500 compounds. With the model system developed by Jiang et al. (8), perhaps a high- throughput screen could be developed. Unfortunately, the kinetic assay they used is not optimal, but if conditions could be developed under which reasonably high-affinity binding assays could be developed, several things become possible. First, it will be possible to test a sufficiently high number of compounds that we may be able to move away from the benzopyran and cyanoguanidine scaffolds (they were not originally designed specifically for mitoKATP activation) and develop truly selective and highly potent compounds. With such high-affinity compounds, it should be relatively easy to "pull out" the receptor and determine its three-dimensional structure.
Overall, it is a pleasure to read such fine work done by the Milwaukee group, and with the publication of these data, it may be a good time for investigators to reevaluate and rethink future studies in this exciting arena. Understanding of this pathway may unlock a generalized pathway used throughout the body for dealing with stress because most cells in the body have mitochondria and most likely have mitoKATP channels. Such a generalized system might give us an incredible and fundamental understanding of numerous diseases (6). As Claude Bernard said, "We can only preserve life to the extent that we can comprehend it."
FOOTNOTES
Address for reprint requests and other correspondence: G. J. Grover, Director of Pharmacology, Product Safety Laboratories, Eurofins Scientific, 2394 Route 130, Dayton, NJ 08810 (e-mail: garygrover{at}productsafetylabs.com)
REFERENCES
is upstream and PKC-
is downstream of mitoKATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium. Am J Physiol Cell Physiol 287: C1418–C1425, 2004.
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