J Mol Cell Cardiol 32, 677–695 (2000) doi:10.1006/jmcc.2000.1111, available online at http://www.idealibrary.com on

ATP-Sensitive Potassium Channels: A Review of their Cardioprotective Pharmacology

Gary J. Grover1 and Keith D. Garlid2 1 Cardiovascular and Metabolic Diseases Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, N.J. 08543-4000, USA; 2 Department of Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, 200000 NW Walker Road, Beaverton, Oregon 97006-8291 (Received 8 September 1999, accepted in revised form 27 January 2000)

G. J. G  K. A. G. ATP-Sensitive Potassium Channels: A Review of their Cardioprotective Phar- macology. Journal of Molecular and Cellular Cardiology (2000) 32, 677–696. ATP-sensitive potassium channels

(KATP) have been thought to be a mediator of cardioprotection for the last ten years. Significant progress has been made in learning the pharmacology of this channel as well as its molecular regulation with regard to

cardioprotection. KATP openers as a class protect ischemic/reperfused myocardium and appear to do so by conservation of energy. The reduced rate of ATP hydrolysis during ischemia exerted by these openers is not due to a cardioplegic effect and is independent of action potential shortening. Compounds have been synthesized

which retain the cardioprotective effects of first generation KATP openers, but are devoid of vasodilator and cardiac sarcolemmal potassium outward currents. These results suggest receptor or channel subtypes. Recent

pharmacologic and molecular biology studies suggest the activation of mitochondrial KATP as the relevant cardioprotective site. Implications of these results for future drug discovery and preconditioning are discussed.  2000 Academic Press K W: ATP-sensitive potassium channels; Mitochondria; Heart; Myocardial Ischemia, Preconditioning.

Introduction created enthusiasm for further studying the thera- peutic potential of KATP for the treatment of acute Recently several lines of investigation concerning myocardial ischemia.

ATP-sensitive potassium channels (KATP) have con- Recent studies have further fueled interest in verged, suggesting that KATP may be an important KATP by suggesting differential importance of KATP therapeutic target for the treatment of acute myo- subtypes. These studies show that cardioprotection cardial ischemia. Pharmacologic studies starting may not be mediated by sarcolemmal KATP currents, in 1989 showed KATP openers to exert profound suggesting an intracellular site of action (mito- cardioprotective effects in numerous mammalian chondria). Therefore, it is possible to develop agents species. The results of this work were made even which can selectively open the KATP subtype of more relevant by later findings showing pre- interest. conditioning to be mediated (at least in part) by With the heightened interest created by the stud-

KATP activation. Therefore, pharmacologic KATP ac- ies described above, it would be useful to review tivation would be expected to mimic an endogenous what is presently known about KATP , particularly cardioprotective mechanism that also seems to be the recent insights into the molecular biology and operative in man. These converging results have regulation of these channels and how these findings

Please address all correspondence to: Gary J. Grover, Ph.D., Metabolic Diseases Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA. Phone: +609 252-5139; Fax: +609 252-6609.

0022–2828/00/040677+19 $35.00/0  2000 Academic Press 678 G. J. Grover and K. D. Garlid can help us understand their cardioprotective phar- hyperpolarize or depolarize pancreatic b-cells re- macology. We will place some emphasis on the spectively.11,12 These channels are important for newly hypothesized role of mitochondrial channels setting membrane potential in pancreatic b-cells in cardioprotection. In this review, our heightened and the closure of a few channels (as seen by an knowledge of the nature of the molecular regulation increase in extracellular glucose) will depolarize of KATP will be integrated with pharmacologic and these cells and therefore stimulate insulin se- 13,14 physiologic studies so that the reader can better cretion. Sulfonylurea KATP blockers such as gli- understand the complex matrix of studies leading benclamide (glyburide) have utility for treating type to our preset understanding of the role of KATP II diabetes and increase insulin secretion by de- b and pharmacologic modulators of this channel in polarizing -cell membranes. Opening of KATP in myocardial ischemia. smooth muscle cells would be expected to hy- perpolarize sarcolemmal membranes and therefore 15 cause relaxation. Most KATP openers are potent vasodilators and smooth muscle relaxants and their Properties and Molecular Biology of earliest proposed clinical uses were asthma, hyper- Plasma Membrane KATP tension, and urinary incontinence. The role of KATP in normal myocardium is presently unclear, al- 1 KATP were first described by Noma in cardiac vent- though a role in ischemic conditions is well known ricular myocytes. These channels are of inter- and will be discussed in full detail in this review. mediate conductance and are inhibited by Opening cardiac KATP will, of course, enhance a physiologic concentrations of ATP. KATP were ori- repolarizing potassium current and therefore cause 16 ginally termed ATP-dependent potassium channels action potential duration (APD) shortening. KATP because ATP was the first modulator studied, al- are thought to be involved with the early re- though other endogenous modulators have since polarization seen in ischemic cardiac tissue.17–19 This been found and therefore they are now referred to early repolarization is associated with the “injury as “ATP-sensitive” channels. KATP are inhibited by current” of ischemia which is the basis for ST- physiologic levels of ATP and, as ATP falls, channel segment shifts and it is thought that KATP contribute, open probability increases (although the degree of at least in part, to this electrophyiologic ab- ATP reduction needed would rarely be seen under normality.17,20 21–23 physiologic conditions; see review by Edwards and KATP is a complex of two different proteins. 2 Weston ). KATP have been found to be modulated One subunit is an inwardly-rectifying potassium by pH, fatty acids, NO, SH-redox state, various channel (Kir) subunit and it is thought that four nucleotides, G-proteins and various ligands (ad- of these combine to form the channel pore. Two enosine, acetylcholine, benzopyrans, cyano- types of Kir (Kir6.1 and Kir6.2) are thought to be 2–6 7 guanidines, etc.). Larsson et al. showed that associated with KATP at this time. The sulfonylurea pancreatic KATP are opened by long-chain acyl CoA receptor (SUR) is the protein which confers a regu- esters, and Paucek and Garlid (unpublished results) latory role as well as sensitivity of the channel to have observed similar opening of cardiac sar- pharmacologic agents and ATP.22 SUR is a member colemmal channels by acyl CoA esters. KATP appear of the ATP-binding cassette protein superfamily, to be linked to the metabolic state of the cell and also called ABC transporters and is related to CFTR have therefore also been labeled as metabolicaly channels (cystic fibrosis transmembrane regulator, 24 regulated channels. KATP are expressed in numerous also glyburide inhibitable). This ATP transporter, tissue types including skeletal muscle, brain, kidney, CFTR, releases ATP, which interacts with a pur- heart, pancreatic b-cells, and smooth muscle.1,2,8–10 inergic receptor subtype which then opens chloride

KATP are thought to serve as a link between meta- channels. SUR is a related ATP permeant protein. bolism and either secretory activity (insulin, brain) SUR1 is highly expressed in pancreatic b-cells, while or electro-mechanical coupling in muscle. KATP are SUR2 is highly expressed in cardiac and skeletal also found in the inner mitochondrial membrane, muscle cells.25 It is unknown how many ways and these will be described in a later section. these different Kirs and SURs can interact, but data In the case of pancreatic b-cells, insulin secretion suggest different combinations in different tissue is controlled by glucose metabolism. KATP opening types. Currently, it is thought that Kir6.2 and SUR2 23 inhibits secretion due to hyperpolarization while form cardiac sarcolemmal KATP . In the study by reduction of channel activity will increase insulin Inagaki et al.,23 diazoxide did not activate the sar- secretion (see review by Edwards and Weston2). colemmal cardiac channel while it did open pan-

Pharmacologic openers and blockers of KATP will creatic KATP , which is thought to be formed by ATP-Sensitive Potassium Channels 679

21 Kir6.2 and SUR1. This finding suggests the ex- gene-related peptide, adenosine (adenosine A2 re- citing possibility of tissue selectivity, which has ceptor subtype), prostacyclin and b-adrenoceptor already been strongly suggested on the basis of agonists,6,30,31 although some of these effects could pharmacologic evidence. The excellent agreement be due to indirect effects of these various agonists.32 of the pharmacology and molecular biologic data Recent data33 show that extracellular ATP can on KATP (particularly with regard to the phar- enhance KATP current via a P2 receptor, through macology of diazoxide) will be discussed later in activation of adenylyl cyclase, although this is in- this review. dependent of PKA. It is presently unknown how Much still needs to be learned about the mo- these data relate to the concept of a secretory role lecular regulation of KATP , altough we will briefly for SUR. While we have listed the potential for PKA review what is currently known. ATP inhibits KATP or PKC to modulate KATP activity, this issue is still and this inhibition does not require phos- speculative and not all investigators can show sim- 34 phorylation. Inhibition of KATP is produced not only ilar actions for these protein kinases. by intracellular ATP, but its non-hydrolyzable ana- KATP openers are compounds of diverse chemo- logs, suggesting phosphorylation is not critical.2 types that can open this channel in various tissues 28 ADP reduces the sensitivity of KATP for ATP and (see review by Hiraoka and Furukawa ) (see struc- therefore it has been proposed that the channel is tures in Fig. 1). The activation of KATP is inhibited modulated by the ratio of these nucleotides. It is by increased ATP suggesting an interaction or com- thought that ADP competes for the ATP binding petition at the ATP binding (inhibitory) site. The site and ADP without the presence of ATP inhibits KATP activating effects of the pharmacologic openers channel activity, although another ADP site is are inhibited by glyburide, although this inhibition thought to mediate weak agonist activity. This does not appear to be through displacement from may be a nucleotide phosphate site which is also a common receptor site. The effects of openers and stimulated by GDP. blockers of KATP appear to be modulated by the

It has been proposed that KATP openers reduce metabolic state of the cell, with openers being more the affinity of ATP at the regulatory or inhibitory active under ischemic conditions and blockers such 19,35 site. In addition to an inhibitory action of ATP, KATP as glyburide being less active during ischemia. will “run-down” in the absence of ATP and it is Recent studies show tissue differences in the activity 26 thought that ATP can prime KATP for opening. of KATP openers and blockers, suggesting KATP sub- Only MgATP (but not free ATP) can reactivate the types which is consistent with the recent findings channel and non-hydrolyzable forms of ATP are on the molecular biology of these channels.36,37 ineffective. This is thought to require phos- Therefore, the signaling systems involved with phorylation and therefore, channel “run-down” KATP modulation appear complex and as yet, a clear may be mediated by channel dephosphorylation. picture has not been elucidated. We have included It has been proposed that a complex balance of a schematic diagram for proposed modulatory path- phosphorylation and dephosphorylation of tyrosine ways for KATP (Fig. 2). This diagram is to be con- and serine/threonine residues can modulate KATP sidered speculative, as not all investigators agree activity, although much needs to be learned about on some of the proposed pathways. this regulatory mechanism.27 It is presently unclear whether dephosphorylation and phosphorylation are involved with the mechanism of run-down and reactivation by MgATP (see review by Hiraoka and Effects of Pharmacologic Modulators of 28 Furukawa ). KATP on Myocardial Ischemia Occupation of the appropriate receptors as- sociated with G-proteins releases GDP and enhances Because of the metabolic regulation of KATP and its GTP binding. A number of ligands known to interact possible contribution to ischemic injury currents, with Gi increase KATP open probability and include it was natural to presume a role for this channel meditors such as acetylcholine and adenosine. Lig- in the pathogenesis of myocardial ischemia. A good ands such as adenosine (through the A1 receptor means for determination of the role of KATP in subtype) may also activate protein kinase C (PKC) ischemia was to use selective openers and blockers and it is thought that PKC activates KATP in vent- of this channel in various models of ischemia. ricular myocytes by reducing channel sensitivity to Before detailed studies in models of ischemia were ATP at the inhibitory site.29 Protein kinase A (PKA) completed, two schools of thought existed. In one may modulate KATP activity and data suggest it is school, KATP blockers would be protective, not only involved with KATP opening induced by calcitonin in terms of cardioprotection, but also in terms of 680 G. J. Grover and K. D. Garlid

Figure 1 Chemical structures of the major classes of KATP openers.

Figure 2 Scheme of proposed control mechanisms for plasma membrane KATP. Some of these proposed pathways are speculative and for some there are conflicting data (see text).

inhibition of arrhythmogenesis. Another school of KATP openers would exert a cardioplegic action and thought was that KATP openers would be protective protect by inhibiting energy utilization and con- because of their ability to shorten APD (and also tractile function in a manner similar to L-type inhibit ischemic depolarization) and therefore re- calcium channel blockers. This hypothesis led many duce calcium entry. This hypothesis presumed that scientists to label KATP openers as “indirect calcium ATP-Sensitive Potassium Channels 681

effect of cromakalim on the time to ischemic con- tracture in isolated rat hearts. The improved re- perfusion flow observed for some openers was not necessary for protection and was most likely sec- ondary to increased metabolic demand in the health- ier tissue. In this study, we also found that ischemic

depolarization was inhibited by KATP openers, al- though it was difficult to know whether this was the mechanism of protection or a result of protection occuring before ischemic depolarization. Since the publication of these results, we have found lev- cromakalim (showing cardioprotection to be stereo- selective), bimakalim, aprikalim, and P-1075 to protect isolated rat hearts with a similar profile of action.40–42 Interestingly, these agents were pro- tecting hearts at concentrations that did not exert significant negative inotropic effects, therefore dis- tinguishing these agents from calcium channel blockers. Figure 3 Effect of increasing concentrations of cro- Cole et al.35 published a paper using a perfused makalim or diazoxide on the time to onset of contracture during global ischemia in isolated rat hearts. Time to guinea-pig ventricle preparation. They found that contracture is defined as the time (min) during ischemia a high concentration of pinacidil (10 l) exerted in which a 5 mmHg increase in end diastolic pressure is negative inotropic effects and shortened APD, while observed. Figure taken from Garlid et al. (1997) with Β Χ it enhanced post-ischemic recovery of contractile permission. Diazoxide; Cromakalim. function. This study was important in further show-

ing cardioprotective effects for a KATP opener, but also suggested an association between APD short- channel blockers”. As we will see, this hypothesis ening and cardioprotection. These data, however, was inadequate to explain the cardioprotective ef- could not definitively prove a connection between fects of KATP openers. cardioprotection and APD shortening. At this con- l The idea that KATP might be involved in ischemia centration (10 ), pinacidil was certainly opening was first suggested by studies on nicorandil although sarcolemmal KATP . Also of interest in this study was the role of KATP in the protective effects of nicorandil their finding that pinacidil was more effective in was not fully appreciated at the time (this compound shortening APD during ischemia in myocardium, 38 is also an NO donor). As more information on KATP a finding we were able to confirm later in an in became available, we wanted to examine the role of vivo model of ischemia in dogs.16

KATP in myocardial ischemia in more detail using Since these early studies, numerous investigators more selective pharmacologic agents. When the idea have shown cardioprotective effects for KATP openers of testing KATP openers in models of myocardial in isolated heart models from several species such ischemia was suggested, we had to decide upon the as rats, rabbits, ferrets, and guinea-pigs.44–48 The best initial model to test these compounds. While profile of action of these compounds was similar these agents opened cardiac KATP , they were more and in general the cardioprotective concentrations potent as vasodilators and we realized that in- of these compounds were between 300 n and terpretation of the data from systemically treated 10 l. The cardioprotective effects observed for animals could be difficult. We therefore determined KATP openers have been observed as increased post- the effect of KATP openers in an isolated rat heart ischemic functional recovery, increased time to elec- model of ischemia and reperfusion in which direct trical uncoupling, and improved energy status, and cardioprotective activity could be ascertained. We reduced necrosis (enzymatic and histologically de- first tested pinacidil and cromakalim and found them termined).35,46,48,49 The study by Tan et al.48 showed both to protect isolated rat hearts subjected to 25 min that guinea-pig papillary muscles had significantly global ischemia followed by 30 min reperfusion.39 increased times to electrical uncoupling during hy- Protection was denoted by increased time to ischemic poxia suggesting protection of gap junctions. Recent contracture, improved post-ischemic recovery of con- studies from Ganote’s laboratory have shown KATP tractile function, reduced reperfusion contracture, openers to protect hypoxic/reperfused rabbit vent- and improved reperfusion flow. Figure 3 shows the ricular myocytes.50 This study showed that at least 682 G. J. Grover and K. D. Garlid

some of the cardioprotective effects of KATP openers Most of the studies aimed at elucidating the are exerted at the level of myocytes. Liang51 has mechanism of action of preconditioning have used also shown KATP openers to protect isolated chick a pharmacologic approach. This has led to a be- myocytes. KATP openers also appear to have some wildering array of suggested receptors, signaling efficacy in human tissue based on studies by pathways and metabolic mechanisms.61–63 The mul- Speechly-Dick et al.52 These investigators showed tiplicity of biochemical systems involved with pre- that cromakalim protected hypoxic/reoxygenated conditioning makes it difficult to understand how human atrial trabecula, suggesting KATP to be a these various pathways fit together. clinically relevant pharmacologic target. A useful organization of these findings divides the 64,65 Results with KATP openers in models of myocardial process into three components. (1) Triggers are ischemia in vivo have been somewhat more variable. metabolites and receptor agonists that are released

This may be due to the fact that most of the KATP locally during the preconditioning ischemia. These openers used in these studies were not selective for include adenosine, acetylcholine, bradykinin, ca- the heart and such activities (including hypo- techolamines, angiotensin II, and opioids. Receptor tension) could have clouded interpretation of the activation triggers a cascade that ultimately ac- data. Nevertheless, there have also been numerous tivates mediators of preconditioning. (2) Mediators 14,66,67 in vivo studies showing KATP openers to be car- include, most notably, protein kinase C, but dioprotective. Early into our investigations, we other kinases may also participate, including tyros- found it difficult to achieve cardioprotection in vivo ine protein kinase68 and mitogen-activated protein 69 with KATP openers when given systemically because kinases. There is evidence that a threshold quant- of profound hypotension. Therefore, we found it ity of various triggers must be achieved in order necessary to administer the KATP openers directly for protein kinase C activation to be sufficient for into the coronary arteries of dogs in order to observe cardioprotection.64 The proteins that are phos- cardioprotection. We found that both cromakalim phorylated have not been identified, but they pre- and pinacidil significantly reduced infarct size in sumably reside within the final common pathway dogs.40 Gross’s laboratory showed that aprikalim of protection and may include the end effector could be given systemically in dogs and a reduction itself. (3) The end effector of preconditioning is also in post-ischemic stunning observed, a result that unknown, but there is a general consensus that was confirmed by our laboratory using cromakalim this may be the KATP channel. In view of the results 37 (except it was given into the coronary arterial of Garlid et al., mitoKATP may play this role or may circulation).16,53 Later studies by several laboratories at least be downstream of the previously mentioned have shown KATP openers to reduce infarct size in mediators (it is currently unknown how mitoKATP diverse species such as dogs, pigs, and rabbits.54–57 affects protection). In keeping with the scope of this It should be stated that several investigators have review, we will limit further discussion to aspects shown KATP openers not to protect ischemic myo- involving KATP . cardium in vivo and it is presently still difficult to The studies showing KATP openers to mimic pre- reconcile these results.58,59 conditioning, which have already been described,

are consistent with the notion that KATP is crucial to preconditioning, but does not prove it. Further Role of KATP in Preconditioning proof required the use of pharmacologic blockers of this channel. Before describing some of this work, Endogenous protective mechanisms exist in many it is worthwhile to point out one interesting study 60 tissue types. Murry et al. described a phenomenon showing a role for KATP in preconditioning using an 70 in hearts in which a short bout of ischemia will agonist. Yao and Gross showed that the KATP protect a heart from a subsequent ischemic episode opener, bimakalim, significantly lowered the thresh- of more prolonged duration. Preconditioning exists old for preconditioning in a canine model of in- in many mammalian species and is found by nearly farction. This was done using a subthreshold all investigators. It is not known whether this preconditioning protocol combined with a sub- protective effect evolved specifically to increase re- threshold dose of bimakalim. This clever study sistance to ischemia, or whether protection is a strongly suggested that KATP openers protect isch- beneficial consequence of triggering a normal emic tissue in a manner which is similar to pre- physiological response. While all investigators agree conditioning. on the profound protection conferred by pre- Gross’s laboratory was the first to demonstrate conditioning, there is less agreement on the mo- that KATP blockers abolish preconditioning using lecular mechanism of protection. glyburide in a canine model.71 The structurally ATP-Sensitive Potassium Channels 683

distinct blocker 5-HD was also shown to abolish have suggested that KATP may be involved with 72 preconditioning in a similar model. KATP blockers activation of adenosine A1 receptors. It has been have been shown to abolish preconditioning in suggested that KATP openers are protecting ischemic 73 rabbits, rats, pigs, and man. The ability of KATP myocardium by increasing adenosine production blockers to abolish preconditioning in rats has been secondary to activation of ectosolic 5′-nu- variable and may be model dependent. Thus, nei- cleotidase.84 A study in hypoxic rabbit ventricular ther 5-HD or glyburide abolished preconditioning in myocytes showed that the KATP opener pinacidil was isolated rat heart;73 however, recent studies showed protective and this effect was abolished by the 50 KATP blockers to abolish preconditioning in rat hearts adenosine receptor antagonists SPT and DPCPX. in vivo.74,75 In addition to animal studies, pre- A recent collaborative study between our group conditioning has also been shown to exist in human and Grosss group showed that the protective effect tissue. Tomai et al.76 showed that glyburide ab- of bimakalim in dogs was not abolished by DPCPX, olished preconditioning in human subjects. They suggesting that this “reverse” pathway is not op- were preconditioned with short periods of ischemia erative in this model.85 It is difficult to reconcile using coronary balloon occlusion and severity of these differences at the present time. ischemia was deteremined using ST-segment shifts If one assumes that adenosine receptor activation and severity of chest pain. Glyburide abolished this is “upstream” of KATP activation during pre- protective effect. These results were confirmed by conditioning, then it becomes important to deter- Yellon’s group, showing that human atrial tra- mine the signaling pathway linking the two belcula could be preconditioned with short dur- systems. Adenosine A1 receptor stimulation inhibits ations of hypoxia and that glyburide abolished this PKA and can activate PKC, both of which are 52 protective effect. known to be involved with KATP gating. PKC ac- It was of great interest that blockers of adenosine tivation seems to be involved in the mechanism of

A1 receptors and KATP completely abolished pre- preconditioning in several species. PKC has been conditioning in similar animal models, when given shown to be involved with activation of sar- alone. It was difficult to reconcile these results colemmal KATP in patch clamp studies and PKC unless these two systems were linked. Studies by activation protects ischemic myocardium in a glyb- Kirsch et al.4 in neonatal rat cardiac myocytes, uride-reversible manner.29,52 PKC activation appears showed that adenosine A1 receptor activation also to phosphorylate a protein associated with KATP , activated KATP through a Gi coupled pathway. This resulting in activation. It should be remembered study prompted us to determine the effect of glyb- that the work relating potassium currents to PKC uride on the cardioprotective effect of an adenosine activation was done using sarcolemmal channels, 77 A1 receptor agonist, R-PIA. We found that R-PIA and the relevance to the pertinent KATP (mito- reduced infarct size in dogs and this effect was chondrial?) is not presently clear. This will be dis- completely abolished by glyburide, suggesting ad- cussed in more detail later in this review. enosine receptor activation to be “upstream” of KATP Finally, it is constructive to review briefly the activation. Similar studies were performed in pigs phenomenon of calcium preconditioning as de- showing 5-HD to abolish the protective effect of R- scribed by Meldrum et al.86,87 and Miyawaki and PIA.78 The cardioprotective effects of adenosine Ashraf.88,89 In calcium preconditioning, a transient have also been shown to be abolished by glyburide increase in intracellular calcium preconditioned rat in several species.79,80 A recent study by Cleveland et heart against subsequent global ischemia, and this al.81 showed that adenosine could protect hypoxic/ cardioprotection is associated with activation of reoxygenated human atrial trabecula and this effect protein kinase C.86,87,90 In a careful study, Kouchi 91 was abolished by glyburide, suggesting this path- et al. have shown KATP to be a common target of way to be operative in man. Interestingly, rat both ischemic and calcium preconditioning. Sig- cerebral preconditioning was abolished by glyb- nificantly, these authors showed that glyburide uride,82 suggesting that this phenomenon is im- blocked cardioprotection when administered portant in tissues other than heart. In this study, 30 min before preconditioning but not when given

KATP-mediated protection appeared to be preceded 5 min after preconditioning. These data are con- and activated by adenosine A1 receptor stimulation. sistent with data reported previously in rats in A recent study by Ford et al.83 showed that glyburide vivo.92 The time dependence of glyburide blockade failed to abolish the protective effects of adenosine is consistent with the necessity to interact with an

A1 activation, although rat hearts may not be intracellular receptor, and emphasizes the import- predictive of human tissue in this regard. ance of consdiering pharmacokinetics in such stud- It is important to note that several investigators ies. 684 G. J. Grover and K. D. Garlid

Physiologic and Molecular Mechanism sible that insufficient time for adequate drug pen- of Cardioprotection: Are Sarcolemmal etration was allowed in these studies of rapid re- perfusion. An interesting study by Gross’s KATP Involved? laboratory did show significant protection to be While there appears to be general agreement that observed in a canine infarct size model when bi- 95 KATP openers are cardioprotective, much less is makalim was given during reperfusion. When known about their mechanism of action. Current we administered a highly amphiphilic KATP opener, studies are shedding some light on this subject and BMS-180448, only during reperfusion, we did show we will briefly review what is known. While similar a weak protective effect in rats and ferrets, although stereoselective cardioprotective activity observed for bimakalim and cromakalim were without effect.96 structurally distinct KATP openers strongly suggested BMS-180448 has been shown to penetrate ischemic an involvement of KATP , further proof was required. tissue more efficiently than some other KATP open- 97 Two structurally distinct KATP blockers, sodium 5- ers. Therefore, it appears as if most of the protective hydroxydecanoate (5-HD) and glyburide were avail- effects of KATP openers occurs during ischemia, al- able to further test the hypothesis that KATP are though some effects on reperfusion injury cannot important in mediating cardioprotection. Both be ruled out at the present time. agents have been universally shown to abolish Most KATP openers are potent vasodilators and 46 the cardioprotective effects of KATP openers. In one possible mechanism for their protection was addition, glyburide abolishes the pre-ischemic cor- coronary dilation, although this was thought to be 93 onary vasodilator effects of KATP openers. 5-HD unlikely. We showed that the protective effect of seems to be more selective and was originally aprikalim was still observed even when coronary thought to be specific for inhibiting the car- flow was held constant in isolated rat hearts.42 In dioprotective effects of KATP openers and will not vivo, several investigators showed that car- abolish the effects of KATP openers on non-ischemic dioprotective doses of KATP openers had no effect on cardiac and vascular tissue.93 This will be discussed coronary collateral blood flow, although they did in more detail later. It is important to note that most enhance reperfusion blood flow.40,54 The studies 50 investigators find that KATP blockers (in reasonable by Armstrong et al. showed pinacidil to protect doses) do not further aggravate severity of ischemia, isolated cardiac myocytes, which further shows a suggesting that KATP are not sufficiently open during lack of importance of coronary flow changes. Later ischemia to contribute to protection, unless the development of KATP openers devoid of vasodilating tissue has been preconditioned.46,71,78 Blockers such activity confirmed these earlier results.36 5-HD will as glyburide and 5-HD appear to be specific in completely abolish the cardioprotective effects of blocking the cardioprotective activity of KATP open- KATP openers while being completely devoid of effects ers and have been shown to be without effect on on their coronary dilator activity, further proof for cardioprotective agents such as calcium blockers, a lack of importance for coronary dilator activity sodium blockers, Na+/H+ exchange inhibitors, and in mediating protective effects.93 94 calmodulin inhibitors. These data strongly suggest The data showing KATP openers to increase the that KATP are involved with the protective mech- time to the onset of ischemic contrcture suggested anism observed for KATP openers. Assuming that conservation of ATP during ischemia. Studies from 41 98 KATP activation is important, it is still unknown how our laboratory and McPherson et al. showed this could confer physiologic protection of ischemic KATP openers to significantly conserve myocardial or reperfused myocardium. We will now discuss ATP during ischemia. The degree of ATP con- what is known about the physiologic mechanism servation was comparable to that seen for calcium of action for cardioprotection for this class of agents. channel blockers, although KATP openers caused

One question that arose was whether KATP openers little negative inotropic effects, at least in our hands. protected during ischemia per se or during re- KATP openers also enhanced post-ischemic recovery perfusion. An increase in the time to onset of of ATP99 suggesting that mitochondria are protected contracture during ischemia strongly suggested an and electron microscopy showed this to be true.49 effect of KATP openers during ischemia (see Fig. Efficiency of oxygen utilization was significantly 46 3). Pharmacologic studies have shown that KATP enhanced by KATP openers, indicating that ischemic/ openers work best when given well before the reperfusion uncoupling of mitochondria is in- ischemic event, although this does not prove an hibited.99 40 effect during ischemia. In our hands, most KATP The results showing little cardiodepressant effects openers have weak (or non-existent) protective ef- for KATP openers, while they conserved ischemic fects when given during reperfusion, but it is pos- ATP was intriguing as significant APD shortening ATP-Sensitive Potassium Channels 685 should cause reduced contractile function. This nel patches or by measuring whole myocyte K+ 47 suggested the possibility that KATP openers were not currents. In guinea-pig papillary muscles, BMS- working through a cardioplegic mechanism and 180448 was devoid of APD shortening activity possibly not through sarcolemmal channels. A (intracellular recordings) at concentrations which study by Pignac et al.100 showed that aprikalim exerted significant cardioprotective effects, al- exerted an additional protective effect over that though the cardioprotective effects were abolished afforded by depolarizing cardioplegia in isolated by glyburide or 5-HD.104 These data suggested that rabbit hearts. We confirmed this in isolated rat compounds which are selective for the cardio- hearts and found the KATP openers cromakalim and protective site could be developed, although the BMS-180448 to exert additive protective effects nature of this site is still not clear. It is evident over hypothermic or normothermic St Thomas’ that some degree of selectivity can be observed cardioplegic solution (16 m K+).97 Interestingly, for blockers as well. 5-HD efficiently abolishes the glyburide abolished the additive protective action cardioprotective effects of all KATP openers tested, 37,93 of KATP openers and had no effect on the protective but has little effect on cardiac sarcolemmal KATP . activity of the cardioplegic solution alone. These It is also of interest that 5-HD is incapable of results suggest that KATP openers work via a mech- blocking the vasodilator effects of KATP openers. anism distinct from cardioplegia and since these As discussed earlier, KATP appear to be involved hearts are arrested in systole, the hearts were elec- in mediation of preconditioning and the potential trically quiescent and therefore APD shortening relevance of sarcolemmal KATP has also been ques- was not likely to be an important mechanism of tioned for this phenomenon. Studies have shown action for these agents. preconditioning to cause APD shortening, both dur- The suggestion of a lack of importance of APD ing and after preconditioning in some studies, al- shortening was disquieting as it suggested a lack though this does not definitively prove sarcolemmal of importance of sarcolemmal K+ currents in me- channels to be essential for preconditioning.56,105 106 diating the cardioprotective effects of KATP openers. Recent studies by Much-Ellingsen et al. showed At approximately the same time as the cardioplegic that dofetilide has no effect on preconditioning in studies were published by Pignac et al.,100 Gross rabbits. These studies suggest that preconditioning, and co-workers found that a dose of bimakalim like pharmacologic KATP opening, does not depend could be used which reduced infarct size in dogs, on activation of sarcolemmal K+ currents and an but was without effect on epicardial monophasic intracellular site of action may be relevant. A recent APD.101 We confirmed these findings by phar- study by Hamada et al.103 confirmed that pre- macologically blocking the APD shortening activity conditioning in dogs is independent of APD short- of cromakalim (epicardial monophasic APD) with ening, further suggesting sarcolemmal KATP the delayed rectifier blocker dofetilide and found activation is not the primary or sole mediator of cromakalim to nevertheless reduce infarct size.102 preconditioning. A recent study by Linz et al.107 A recent study by Hamada et al.103 showed that APD suggested that a pharmacologic blocker which is shortening is not a prerequisite for cardioprotection selective for sarcolemmal KATP did not abolish pre- induced by pinacidil in a canine model of infarction conditioning while glyburide completely abolished in anesthetized dogs. While these studies were well preconditioning. executed and represented early evidence for a sep- The question is whether sarcolemmal KATP are aration of APD shortening and cardioprotection, critical for the protective effect of KATP activation measurement of epicardial monophasic action po- (both pharmacologic or preconditioning) or tentials are a relatively crude technique for meas- whether intracellular KATP may be involved. Taken uring changes in APD, especially in the critical areas together, the data described above show a poor of the ischemic myocardium (below the epicardial correlation between sarcolemmal K+ current ac- surface). It therefore became necessary to undertake tivation and cardioprotection for KATP openers. This more detailed studies using patch clamp and well is perplexing given the near universal finding of as intracellular recording techniques. cardioprotection for structurally distinct KATP open-

We have developed pyranyl cyanoguanidine ana- ers and their inhibition by KATP blockers. It is cer- logs which retain the glyburide-reversible car- tainly possible that KATP are not involved with dioprotective activity of cromakalim while being cardioprotection, but this seems improbable. It is 36 relatively devoid of vasodilator activity. In- also possible that KATP openers may interact with terestingly, we also found that many of these agents, sarcolemmal KATP in a manner we do not understand including BMS-180448, were poor at opening car- and will affect cardioprotection even without ac- diac sarcolemmal KATP as measured in single chan- tivation of a potassium current. We were hampered 686 G. J. Grover and K. D. Garlid

117 by a lack of knowledge of the relevant car- reconstitution of a highly purified mitoKATP. Al- dioprotective binding site for the pharmaologic though it possesses unique regulatory properties, mi- openers of KATP. Binding sites for KATP openers have toKATP is regulated by every ligand that regulates been found in cardiac, smooth muscle and skeletal plasma membrane KATP channels; consequently, we muscle cell membranes, but the relationship of these infer that it belongs to the same gene family. sites to ischemia are not clear.108,109 It has also been The sole known function of the mitochondrial + 114 suggested that KATP openers may interact with an K cycle is to regulate matrix volume. The effect + intracellular KATP . We hypothesized several years of net K movement causes only minor per- ago that mitochondrial KATP might be the car- turbations of matrix pH, because of accompanying + dioprotective site of action for KATP openers. In the movement of weak acids; of matrix K con- next section the possible importance of mito- centration, because of accompanying movement of Du chondrial KATP in mediating cardioprotection will osmotically obligated water; or of (the electrical be discussed in detail. potential difference), because the fluxes are low relative to proton pumping by the electron transport chain. Thus, the K+/H+ antiporter cannot sense changes in either of its substrates. Rather, it is 2+ + Role of Mitochondrial KATP in regulated indirectly (by matrix Mg and H )to Cardioprotection sense changes in matrix volume, and consequently, volume must change before K+/H+ adjusts to equal + In the previous section, we developed the case the rate of K influx. For example, opening mitoKATP + + against the involvement of sarcolemmal KATP in the will transiently shift the balance until K /H anti- + cardioprotective effects of KATP openers. We will port catches up with the higher rate of K influx. now review what is known about the physiology, This will cause transient swelling and will result in biophysics, and pharmacology of mitochondrial KATP a higher steady-state volume for as long as mitoKATP

(mitoKATP), then we will discuss evidence for a role remains open. of mitoKATP in mediating cardioprotection.

Properties and regulation of mitoKATP

+ Physiological role of mitoKATP K flux through reconstituted mitoKATP is highly selective for K+ and unaffected by Na+ or tetra-

The mitochondrial potassium cycle consists of elec- ethylammonium. MitoKATP is not voltage-gated, and trophoretic K+ uptake and electroneutral K+ efflux the flux-voltage dependence is consistent with a chan- across the inner membrane. K+ efflux is mediated nel containing a single energy well near the center by the K+/H+ antiporter, whose existence was pre- of the membrane.117 The open channel conductance 110,111  dicted by Mitchell and first demonstrated of mitoKATP is about 16 pS in symmetric 150 m 112 + 118 nearly 20 years later. K influx is mediated by KCl. MitoKATP activity has also been studied in the mitochondrial KATP channel (mitoKATP) and by intact, respiring mitochondria. To control for the inward K+ leak due to diffusion caused by the high unavoidable coexistence of K+ diffusion, we compared electrochemical gradient favoring inward flux. The the effects of ATP on fluxes of K+ and TEA+.K+ flux redox energy consumed by the K+ cycle is the cost was inhibited by ATP to the level of TEA+ flux, of regulating matrix volume.113,114 whereas TEA+ flux was unaffected by ATP.119,120 + + A primary role of the regulated K /H antiporter MitoKATP is subject to complex regulation by meta- is to compensate for unregulated K+ leak into the bolites and pharmacological agents.117,120–122 ATP, matrix, driven by the high voltages required for ADP, and acyl CoA esters are mutually competitive + + + oxidative phosphorylation. Thus, the K /H anti- inhibitors of K flux through mitoKATP. Inhibition porter is responsible for volume homeostasis and is by each of these metabolites exhibits an absolute essential for maintaining vesicular integrity in the requirement for divalent cations. ATP or palmitoyl face of high ionic traffic across the inner membrane. CoA have no effect in the absence of Mg2+, and Mg2+ A reconstitutively active K+/H+ antiporter from has no effect in the absence of ATP or palmitoyl CoA. liver and heart mitochondria has been identified as Since palmitoyl CoA is not a Mg2+ chelator, these an 82 kD inner membane protein.115 results imply that Mg2+ ions interact independently 116 MitoKATP was discovered in 1991. Inoue et al. with mitoKATP. reported evidence from patch clamp studies of fused The high affinity of mitoKATP for ATP raised the mitoplasts at the same time that we began describing conundrum of how this channel can be opened ATP-Sensitive Potassium Channels 687 under physiological conditions. We hypothesized that which would normally be present during in vivo endogenous activators overcome the high affinity for experiments. We infer from these and other ex- ATP, and we confirmed this hypothesis by showing periments that binding of glyburide and 5-HD to that guanine nucleotides reverse the inhibition by mitoSUR is conformation-dependent and that mi- ATP, ADP or palmitoyl CoA in both mitochondria toSUR conformation is changed by binding of the and proteoliposomes.122 Guanine nucleotides are other ligands. When the appropriate ligands (e.g. competitive with ATP. GTP appears to react with a Mg2+, ATP, or cromakalim) are present in the assay high affinity (0.2 l) and a low affinity (15–20 l) medium, glyburide and 5-HD are potent, specific site, whereas GDP appears to react with two low blockers of K+ flux in respiring mitochondria, with l l l 120 affinity sites (20 ). ATP is unable to inhibit in the Ki values of about 1 and 50 , repectively. presence of physiological GTP concentrations; for l example, 20 GTP increased the K1/2 for ATP l  inhibition from 21 to 6 m . On the other hand, Molecular structure of mitoKATP the K1/2 for GTP activation of palmitoyl CoA inhibition is in the physiological range. Our present thinking We have tentatively identified two components of is that ATP does not regulate mitoKATP in vivo, and that mitoKATP—a 55 kD channel protein and a 63 kD the open/closed state of the channel is determined by sulfonylurea receptor (SUR), based on its labeling the relative occupancy of the sites by GTP or long- with bodipy-glyburide (unpublished observation). chain acyl CoA esters. We speculate that the two Thus, it appears that mitoKATP has a hetero- binding sites correspond to the nucleotide binding multimeric structure akin to that of plasma mem- folds on mitoSUR. brane KATP. Neither subunit has been cloned, but

In order to understand the role of mitoKATP in this is the focus of active research. cellular bioenergetics, it is necessary to know where An interesting observation was made by Suzuki its regulatory sites are located. Do they face the et al.,124 who showed that mitochondria are im- matrix, as suggested by Inoue et al.,116 or do they munostained with antibodies to KIR6.1, an inward face the cytosol? Do they all coexist on the same rectifying K+ channel that is known to be expressed pole of the protein? Experiments in proteoliposomes in plasma membranes. Preliminary data show that and mitochondria demonstrate that the mitoKATP these antibodies do indeed react with a mito- regulatory sites for Mg2+, ATP, GTP and palmitoyl chondrial protein; however, they do not react with CoA face the cytosol, or more precisely, the inter- any protein in the reconstitutively active purified 118 membrane space. fraction of mitoKATP . In view of the fact that the antibodies were raised to a 12 amino acid fragment of KIR6.1, it may be that they recognize a non- Pharmacological modulators of mitoKATP KATP protein with homology in a limited domain.

KATP openers reverse inhibition of mitoKATP by ATP,

ADP, and palmitoyl CoA with K1/2 values that are well within the ranges observed for plasma mem- Differential pharmacology of cardiac sarcolemmal and 123 brane KATP channels from various tissues. Thus, mitochondrial KATP l l the K1/2 values were 1 and 0.4 for cro- makalim and diazoxide, respectively.121 Similar val- We measured K+ fluxes in liposomes containing ues were obtained in intact mitochondria and in purified KATP to address the question of receptor liposomes containing reconstituted mitoKATP . subtypes within the cardiac myocyte. These pro- We have recently succeeded in removing a major tocols have several advantages, including the ability barrier to understanding the pharmacology of mi- to compare proteins from different sources using 120 toKATP inhibitors. Under most conditions, glyb- identical assay conditions. Sarcolemmal KATP and uride and 5-HD are ineffective in intact, respiring cardiac mitoKATP were purified from beef heart and mitochondria.119 We have now shown that this assayed for flux. The two preparations shared many phenomenon reflects a particular characteristic of properties in common. Flux was inhibited by ATP, mitoKATP and is due entirely to the conditions of and this inhibition was reversed by cromakalim in the experiment. Thus, in the absence of other lig- both preparations. The resulting pharmacological ands, the channel is open, but not inhibitable by open state was inhibited by glyburide in both pre- glyburide or 5-HD. Sensitivity to inhibitors requries parations. the simultaneous presence of Mg2+, ATP, and an 5-HD exhibited a profoundly differet phar- opener which may be GTP or a pharmacologic macology in the two preparations. 5-HD always opener. Of course, these ligands are precisely those inhibited mitoKATP under appropiate conditions, but 688 G. J. Grover and K. D. Garlid

+ Figure 4 Activation of K flux by diazoxide or cromakalim in KATP from bovine heart mitochondria and sarcolemma. Relative K+ flux (DJ/DJmax) is plotted v concentration of drug added to the assay. The figure shows relative fluxes from cardiac mitochondrial KATP (solid circles) and sarcolemmal channels (open circles) in response to diazoxide or cromakalim. ± l ± l Observed K1/2 values for cromakalim were 1.6 0.1 for mitochondrial channels and 18 2 for sarcolemmal ± l ± l channels. K1/2 values for diazoxide were 0.8 0.03 for mitochondrial KATP and 840 25 for sarcolemmal KATP . Figure taken from Garlid et al. (1997) with permission.

never inhibited sarcolemmal KATP under any con- Are mitoKATP the sites of cardioprotection? ditions. This failure of 5-HD to block sarcolemmal 93 KATP is consistent with previous observations. It A number of studies have demonstrated that cardiac should be pointed out that the blocking activity of sarcolemmal KATP are relatively insensitive to diazo- 23,121 5-HD in sarcolemmal channels has not been studied xide, whereas mitoKATP are sensitive in the in any detail. micromolar range.121 Accordingly, we studied the Diazoxide also exhibited a pronounced differential effect of diazoxide in an isolated rat heart model of pharmacology between sarcolemmal KATP and mi- ischemia and reperfusion. We observed con- toKATP, as shown in Figure 4. In this experiment, centration-dependent cardioprotection in the low diazoxide was 1000-times more potent in opening micromolar range with no correlation to APD short- 37 mitoKATP than in opening sarcolemmal KATP . We ening (Figs 3, 5 and 6). While diazoxide had normally observe K1/2 values for opening by diazo- little effect on APD, cromakalim did shorten APD l  xide of 0.5–0.8 with mitoKATP and 0.8–1.0 m markedly within its cardioprotective range. There- with sarcolemmal KATP. fore, diazoxide and cromakalim were found to be- ATP-Sensitive Potassium Channels 689

Figure 5 Effect of diazoxide on the release of LDH (lactate dehydrogenase) from ischemic/reperfused rat hearts. The hearts were pre-treated for 10 min with diazoxide and then were rendered totally ischemic for 25 min. This was then followed by 30 min of reperfusion without drug. LDH release was reduced in a concentration-dependent manner by diazoxide. ∗ Denotes significance (P<0.05) compared to vehicle. Figure taken from Garlid et al. (1997) with permission.

126 have similarly in terms cardioprotection and in preconditioning, PKC activity and mitoKATP . With opening mitoKATP. They differed markedly in their regard to preconditioning, it is important to note effects on APD and on the opening of reconstituted that 5-HD also completely abolishes the protective sarcolemmal KATP . Both glyburide and 5-HD com- effect of this phenomenon, further suggesting an pletely abolished the protective effects of diazoxide involvement of mitoKATP . (Fig. 6). These results appear to exclude a role for sarcolemmal KATP in ischemic protection. These differential effects of 5-HD and diazoxide KATP: Future Directions are consistent with our hypothesis that mitoKATP is the receptor for cardioprotection by KATP openers Recent years have seen an explosive growth in and for 5-HD blockade of pharmacolgical protection interest in the role of KATP in the pathogenesis of or preconditioning.37 The caveat, at present, is that myocardial ischemia. The great majority of in- the existence of a third myocardial KATP subtype, vestigators find that KATP openers exert car- with receptor properties similar to mitoKATP , has dioprotective effects in numerous models of not yet been ruled out. myocardial ischemia. This protection appears to be Liu et al.125 have recently confirmed diazoxide related to energy sparing effects which are not due cardioprotection in the isolated rabbit myocyte to reductions in cardiac work. Overall, KATP openers model and these authors support the hypothesis have an excellent cardioprotective profile which is that mitoKATP mediates cardioprotection with phar- further enhanced by the possibility that they mimic macologic KATP openers. Additional experiments on an endogenous protective mechanism. While an the same model provide evidence that protein kinase important role for KATP in ischemia seems to have C activation potentiates diazoxide opening of mi- met with general agreement, there is much that is toKATP, providing a partial linkage between ischemic still unknown. 690 G. J. Grover and K. D. Garlid

Figure 6 The effect of diazoxide or cromakalim with or without concomitant glyburide on release of LDH (lactate dehydrogenase) from ischemic/reperfused rat hearts. Both cromakalim and diazoxide significantly (∗ P<0.05) reduced reperfusion LDH release and this effect was abolished by glyburide. Figure taken from Garlid et al. (1997) with permission.

There are two major mechanistic issues: current their use. Agents have been reported which retain information suggests that mitoKATP may be the cardioprotective effects while being devoid of vaso- end effectors of cardioprotection, but further work dilator activity,36 suggesting that tissue selectivity is essential to assure ourselves that mitoKATP is is possible. In this series are found many agents indeed the protective site of action of both that are devoid of vasodilator and APD shortening pharmacologic agonists and preconditioning. Sec- activities, making them more selective for treating ondly, it is not clear how opening mitoKATP will ischemia without the potential for increasing re- exert ATP-sparing effects. It is critical to determine entrant arrhythmias, as might be seen for agents the molecular mechanism of cardioprotection pro- activating sarcolemmal currents.127 The results of vided by activation of KATP . Only when this studies designed to assess pro-arrhythmic potential mechanism is understood can we begin to un- of non-selective KATP openers have been variable, derstand the complex signaling pathways im- 128–133 but nevertheless any drug discovery effort portant in preconditioning. Understanding this should take this potential toxicity seriously and mechanism may also be important in developing attempt to minimize the effects of these agents on novel therapeutics for treating myocardial isch- APD or refractoriness. Despite the absence of APD emia. shortening or vasodilator activity, the cardio-

The therapeutic issue is that existing KATP openers protective effects of the selective compounds de- may not be optimal for treating acute myocardial scribed by Atwal et al.36 are abolished by glyburide ischemia. Most KATP openers have little tissue se- and 5-HD. It is presently unknown whether these lectivity, and they also have the propensity to cause compounds are selective for mitoKATP . Although hypotension and APD shortening, which may limit these agents are more selective for cardioprotection, ATP-Sensitive Potassium Channels 691 little improvements in cardioprotective potency References have been seen.  + A convincing demonstration that mitoKATP are 1. N A. ATP-regulated K channels in cardiac the cardioprotective site, together with cloning of muscle. Nature 1983; 305: 147–148. 2. E G, W A. The pharmacology of ATP- this channel, would set the stage for setting up sensitive potassium channels. Annu Rev Pharmacol high throughput screening. 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