K_ATP Channels and Cardiovascular Disease: Suddenly a Syndrome

Canonical ATP-sensitive potassium (K_ATP) channels are heterooctameric complexes of pore-forming Kir6 channel-forming subunits associated with regulatory SUR subunits, members of the ATP binding cassette (ABC) family of membrane proteins (Figure). Two Kir6-encoding genes, KCNJ8 (Kir6.1) and KCNJ11 (Kir6.2),^4,5 and 2 SUR genes, ABCC8 (SUR1) and ABCC9 (SUR2),^5–7 encode mammalian K_ATP subunits, but alternative RNA splicing can give rise to multiple SUR protein variants (eg, SUR2A and SUR2B) that confer distinct physiological and pharmacological properties on the channel complex.^8,9 Interestingly (Figure C), the genes for Kir6.2 and SUR1 are located next to each other on human chromosome 11p15.1,⁵ suggesting an as yet unconsidered coregulation at the gene level. In addition, the genes for Kir6.1 and SUR2 are also adjacent to one another on chromosome 12p12.1,^7,10 implicating an evolutionary duplication. In heterologous expression systems, both Kir6.2 and SUR1 subunits coassemble in a 4:4 stoichiometry⁵ (Figure A and B) to generate the functional K_ATP channel.^11–13 Similarly, biochemical studies demonstrate that the SUR2 protein variants, SUR2A and SUR2B, can also coassemble with Kir6 subunits,^4,14–16 presumably in a similar octameric arrangement.

Crystallographic studies of bacterial and eukaryotic Kir channels^17,18 demonstrate a conserved architecture of Kir channels with 2 transmembrane helices (M1, M2) bridged by an extracellular loop that generates the narrow portion of the pore and controls ion selectivity (Figure A). As with other ABCC family members, SURs contain 2 6-helix transmembrane domains, TMD1 and TMD2, but SURs also have an additional N-terminal TMD0 domain consisting of 5 transmembrane helices (Figure A), critical for Kir6.x trafficking and gating.¹⁹ SURs also contain one nucleotide binding fold (NBF1) between TMD1 and TMD2, and a second NBF (NBF2) after TMD2 in the cytoplasmic loops (Figure A). NBFs from bacterial ABC proteins crystallize as head-to-tail dimers, and this is likely the functional arrangement between NBF1 and NBF2 in SUR (Figure B).²⁰ How the Kir6 and SUR subunits are physically connected remains unknown, but electron micrography and intersubunit fluorescence resonance energy transfer studies of complete K_ATP complexes suggest an intimate packing of 4 SUR and 4 Kir6.x subunits^21,22 (Figure A).

The key regulatory features of K_ATP channels are rapid and reversible closure by cytoplasmic ATP and activation by nucleotide triphosphates and diphosphates²⁰ (Figure, B). In the absence of other nucleotides, the free ATP concentration that causes half-maximal channel inhibition is in the micromolar range. Because cellular levels of cytosolic ATP concentration are in the millimolar range (1–5 mmol/L) and change little with metabolism, ATP is probably always sufficient to almost fully inhibit channel activity. Channel activation then arises from the activating effects of Mg nucleotides, particularly MgADP, on the SUR subunit.²³ Nucleotide regulation is probably the key molecular regulator of K_ATP channel activities, although other second messenger systems and regulators²⁴ may be involved in control of channel activity and in causing channel-dependent pathologies.

It has long been recognized that K_ATP channels provide a large potential ionic conductance in the surface membranes of cardiac myocytes as well as VSM and endothelium, and perhaps in the mitochondrial inner membrane of many cells. Under normal metabolic conditions, cardiac sarcolemmal K_ATP channels are predominantly closed, and they do not significantly contribute to cell excitability. However, these channels can open when exposed to a severe metabolic stress such as anoxia, metabolic inhibition, or ischemia. In muscle cells, shortening the action potential reduces calcium entry and inhibits contractility,⁹¹ thereby reducing energy consumption, potentially protecting the cell. Such a preservation strategy is of course self-limiting because if too many myocytes stop contracting, the heart will stop pumping and the animal will die, but it has always been a reasonable, if unproven, notion that temporary protection of a small number of cells, or region of the heart, against the damage of Ca overload during ischemia, is likely to be operable.

In the vasculature, inhibition of K⁺ channel activity will tend to cause depolarization of the membrane potential, activation of L-type voltage-sensitive Ca²⁺ channels, Ca²⁺ entry, and vasoconstriction.⁹² Conversely, activation of K⁺ channels will lead to membrane hyperpolarization, decrease in voltage-dependent Ca²⁺ entry, and vasodilation.⁹² The relationship between membrane potential and Ca²⁺ influx is especially steep in smooth muscle, with membrane depolarization or hyperpolarization of only a few millivolts causing several-fold increases or decreases in [Ca²⁺]_i, respectively.^93,94 Endothelial cells lack voltage-dependent Ca channels, and Ca entry through nonselective channels is enhanced at hyperpolarized voltages, in contrast to excitable cells.^95,96 Activation of K_ATP channels will tend to hyperpolarize cells, leading to elevated [Ca²⁺]_i and elevated release of vasoactive agents, including endothelium-derived hyperpolarizing factor and endothelin. Thus, gain of or loss of K⁺ channel activity in either smooth muscle or endothelium could have profound prorelaxant or proconstrictive effects, respectively, on smooth muscle tone.

ABCC8 encodes SUR1, which is the predominant regulatory sulfonylurea receptor (SUR1) in the pancreatic β cell and is also present in the heart, predominantly in atria in rodents³⁴ but potentially more widespread in humans.³⁵ Because of its involvement in the pancreatic K_ATP channel, gain-of-function and loss-of-function mutations in this gene also underlie neonatal diabetes mellitus and congenital hyperinsulinism, respectively, but again there is no report of cardiac problems in these patients.^105–107 ABCC9 encodes the second SUR2 subunit, and this is likely to be the major SUR isoform in both cardiac and vascular muscle. There have been 2 reports of SUR2 loss-of-function mutations leading to cardiac disease, both from Terzic et al^108,109 (Table 1). In each case, the mutations are present in the C-terminal exons and will lead to a disruption of the NBF2 of SUR2A and, hence, reduce nucleotide stimulation of channel activity without affecting SUR2B. In the first report, the single patient with the mutation presented with long-standing atrial fibrillation originating in the vein of Marshall, with normal cardiac morphology and contractile features.¹⁰⁹ The patient was successfully treated by radiofrequency ablation. In the second report, 2 individuals with 2 distinct mutations presented with heart failure attributable to idiopathic dilated cardiomyopathy.¹⁰⁸ There have been no subsequent reports of similar genetic defects in the intervening 5 years, and further evidence for causality of association of similar gene variants with disease in additional cases is lacking.

Two new studies reporting multiple different ABCC9 mutations, all associated with Cantu syndrome, a distinctive multi-organ disease (Table 2), now provide a clear picture of associated outcomes and open multiple new avenues of investigation. The first study¹¹⁰ involved genetic analysis of 14 individuals with Cantu syndrome,¹¹¹ and ABCC9 coding mutations were identified in 11 of them. In 6 individuals with no affected relatives, the mutations were de novo. Two families were also reported, 1 with an affected mother and 2 affected daughters and 1 with an affected father and daughter, confirming that inheritance in this case is autosomal-dominant. No analysis of recombinant channel function was made in this first study, but the conclusion that these mutations all lead to a gain-of-channel function¹¹² is cemented by the second study,¹¹³ which identified ABCC9 coding mutations in an additional 14 of 16 identified patients. In that study, recombinant expression of mutant channel proteins clearly demonstrated a reduced sensitivity to ATP inhibition in 3 example mutants that, as discussed, will lead to enhanced K_ATP channel activity wherever the channels are located.

Cantu syndrome (MIM 239850), or hypertrichosis-osteochondrodysplasia-cardiomegaly syndrome, was first described in 1982.¹¹¹ Subsequent reports^{112,114–121} have confirmed a constellation of features in ≈30 patients (Table 2). Congenital hypertrichosis is a constant feature, with thick scalp hair and excessive hair growth on the forehead, face, back, and extremities. Generalized macrosomia is present in most cases, with large birth weights and lengths, although ultimate adult height is usually within the normal range. Macrocephaly is typically present at birth and usually persists. Multiple dysmorphic features (Table 2), including coarse facial appearance, skeletal abnormalities, and generalized osteopenia, as well as multiple additional clinical features also have been described. The cardiac features include cardiac enlargement, concentric hypertrophy of the ventricles, pulmonary hypertension, and pericardial effusion. Yet, despite the enlargement of the heart with increased muscle mass, cardiac function is typically normal, with normal ventricular contractility on imaging studies.¹¹² Cardiac muscle biopsy in 1 patient showed mild myofibrillar disorganization but normal myofibers and mitochondria on electron microscopy, and in 2 other patients cardiac biopsy result was reported as normal.^112,122 Pulmonary hypertension secondary to partial pulmonary venous obstruction has been reported in 1 case and was associated with severe mitral valve regurgitation that spontaneously resolved.¹¹⁶ Some patients have required pericardiocentesis and ultimately needed pericardial stripping to prevent reaccumulation of the pericardial effusion. A significant number of patients have had patent ductus arteriosus requiring surgical closure, as well as bicuspid aortic valves with and without stenosis. Lymphedema involving the lower extremities may develop over time and, in 1 patient, lymphangiogram demonstrated dilated lymphatic vessels in the legs with delayed lymphatic drainage.¹²³

Diazoxide, minoxidil, and other related drugs have been used since the 1960s to treat severe refractory hypertension. Multiple reports of side effects of these drugs also include pronounced hypertrichosis, pericardial effusions, and edema in treated patients.¹²⁴ One report even noted coarsening of the facial features, reminiscent of Cantu syndrome, after 8 months treament with minoxidil.¹²⁵ It was subsequently recognized that 1 major action of minoxidil is opening of K_ATP channels,^97,126,127 and this led us to note the parallels between the symptoms of minoxidil exposure and the features of Cantu syndrome, and to suggest the possibility that Cantu syndrome might be the result of K channel hyperactivity.¹¹² Teratogenic effects of minoxidil, including marked hypertrichosis, dysmorphic facial features, and low blood pressure, have been reported in the offspring of a minoxidil-treated mother.¹²⁸ In additional reported cases of minoxidil teratogenicity, 1 infant had transposition of the great vessels and pulmonary bicuspid valvular stenosis leading to neonatal death, and another infant had hypertrichosis that resolved over the first 3 months of life.¹²⁹

The 2 recent studies that describe specific mutations in the ABCC9 gene in a total of 25 of 31 Cantu syndrome patients^110,113 definitively link the gene defect to the syndrome. All reported patients had the typical Cantu syndrome phenotype (Table 2), but 6 of 31 patients had no identifiable ABCC9 mutation, suggesting that additional gene defects may be involved. Previous studies of Cantu syndrome patients have provided no definitive explanation of the underlying cause of the various features, and even now the realization of SUR2 mutations as causal does not immediately provide explanations for all features. There is strong evidence, for a physiologically important role of SUR2 in vascular relaxation, such that persistence of the patent ductus arteriosus in Cantu syndrome patients may be readily explained as a consequence of maintained vessel dilation after birth. Patency of the ductus arteriosus is controlled by many factors, the most important of which are relatively low fetal oxygen tension, prostaglandin 2, and prostacyclin 2 in the fetus. After birth, the abrupt increase in oxygen tension and decreasing prostaglandin 2 and prostacyclin 2 levels lead to inhibition of voltage-gated K channels and contraction of the smooth muscle fibers in the ductus, resulting in wall thickening and lumen obliteration. Mechanisms of persistent patent ductus arteriosus are not clear,¹³⁰ but the enhancement of a K current in smooth muscle presents an obvious potential explanation in Cantu syndrome patients. Altered vascular tone also may underlie the edema and pericardial effusion, but the reason for cardiomegaly is not obvious. Cardiomegaly reported in most cases of Cantu syndrome is attributable to increased myocardial mass (hypertrophy) with larger cardiac chambers but with normal systolic function, and this does not fit the diagnostic criteria of dilated or hypertrophic cardiomyopathy.¹³¹ As we reported, cardiomegaly in 2 related Cantu syndrome cases has been associated with high output failure¹¹² and may well be a secondary response to reduced vascular tone.¹³² Similarly, the reason for osteochondrodysplasia and facial dysmorphology is not obvious, and the mechanism by which minoxidil causes hair growth has remained controversial.¹³³ It has been speculated that by opening vascular K channels and dilation of blood vessels, the supplies of oxygen, blood, and nutrients to the hair follicle are increased, causing follicles in the telogen phase to shed and to be replaced by new thicker hairs in a new anagen phase. However, there is also evidence that SUR2 isoforms are present in follicular dermal papillae,¹³⁴ and although the new realization definitively ties the hair growth to an action on K_ATP channels, it does not immediately prove where the action is.

There is tremendous potential for modulation of K_ATP channel activity in general and, more importantly, perhaps, in a tissue-specific manner, because there is already a rich pharmacology, not only of channel inhibitors but also of KCOs. KCOs have been used in 2 major clinical settings: as insulin secretion blockers in conditions of hyperinsulinema and as antihypertensives. So far, clinical use of sulfonylureas has been limited to treatment of type 2 diabetes mellitus, and there has been debate about negative cardiovascular effects.

Minoxidil is reportedly the most active KCO at causing human hair growth, hence its commercial use in topical hair restoration products,^164,165 and it appears that most, if not all, of the effects of Cantu syndrome are replicated by high-dose minoxidil, including hypertrichosis, facial dysmorphology, and pericardial effusion.¹²⁸ Such features have been reported for other KCOs; there is 1 report of pericardial effusion as a result of diazoxide therapy¹⁶⁶ and, although not attributed to the drug by the authors, another reported case of a patient using diazoxide who experienced pericardial effusion.¹⁶⁷ Interestingly, a clinical trial for the use of nicorandil, as a SUR2A-specific activator, in the setting of acute myocardial infarction actually reported lower rates of pericardial effusion than in untreated patients.¹⁶⁸

Although there are certain dogmas in the literature regarding specificity of KCOs or inhibitors, careful binding analyses performed on cloned SURs have revealed complexities of binding and dependence on nucleotides, which makes it difficult to predict in vivo efficacies at different SUR targets. In addition, it is clear from intact cell and excised patch-clamp recordings that the ability of KCOs to activate K_ATP channel currents depends critically on the metabolic state of the intracellular milieu, making direct comparison between different studies difficult.¹⁶⁹ The ability of diverse KCOs to lower blood pressure is well-recognized, leading to their clinical use in acute and refractory hypertensive settings. Sulfonylureas inhibit K_ATP channels and have seen widespread use as glucose-lowering agents in the type 2 diabetes. There is a wide therapeutic range and the main recognized side effect is hypoglycemia, but there is a long-standing debate regarding potential cardiovascular side effects. K_ATP channel inhibitory drugs have not reached clinical acceptance in the cardiovascular arena, with the expectation being that blockade of cardiac K_ATP channels may be detrimental in conditions of myocardial ischemia, during which these channels can open and are presumed protective. This debate is still not resolved.^170,171

Given the new realization of the SUR2-dependent basis of Cantu syndrome, the opportunity immediately presents itself for the use of K_ATP channel inhibitors as a potential magic bullet therapy, because they have proven in the treatment of Kir6.2-dependent or SUR1-dependent neonatal diabetes mellitus.¹⁷² It is generally accepted that most sulfonylureas are physiologically more potent inhibitors of SUR1-dependent K_ATP than SUR2A-dependent channels, although there has been no careful comparison of effect on SUR1-dependent vs SUR2B-dependent channels. There has been a long-standing dogma that the drug HMR1098 is a cardiac-specific K_ATP blocker,^173–176 although several studies including our recent direct head-to-head comparison confirm that it is also a more effective blocker of SUR1-dependent than SUR2A-dependent K_ATP channels.^34,177,178 Relative efficacies of HMR1098 vs sulfonylureas in specific physiological conditions may be important to understand, because it is conceivable that specific K_ATP inhibitors may counteract the symptoms of Cantu syndrome without significantly affecting blood glucose control, a key issue if K_ATP channel inhibition is to be a viable treatment for the disease.

Despite almost 30 years of research, we have remained “largely in the dark regarding the true physiological determinants and relevance of sarcolemmal K_ATP activity”¹⁷⁹ until very recently. We now realize that the subunit make-up of sarcolemmal K_ATP channels can be far more complex and labile than originally thought¹⁶ and, together with the existence of mitoK_ATP, it may be reasonable to consider K_ATP channels as a family of channels.¹⁸⁰ The details of the involvement of sarcolemmal vs mitochondrial K_ATP channels in cardiovascular physiology and pathology remain unclear, but the growing association of Kir6.1 and SUR2 variants with specific electric and contractile derangements and the new clear association with a complex syndrome firmly establish the importance of appropriate activity in normal function of the heart and vasculature. In addition to consideration of potential therapeutic implications of these new findings, we can also consider the broader mechanistic implications. As discussed, key features of Cantu syndrome are consistent with activation of SUR2B-dependent K_ATP channels in the vasculature, leading to vasorelaxation. In this case, the likely associated Kir channel subunit is Kir6.1 (Figure D), and we might reasonably suggest that GOF mutations in Kir6.1 also should be associated with these, if not all, symptoms of Cantu syndrome, paralleling the similar neonatal diabetic phenotypes of Kir6.2 and SUR1 GOF mutations. A purported GOF Kir6.1 mutation is already associated with the J-wave syndrome,^100–103 and this leads to a clear inconsistency: neither J-wave abnormalities nor other arrhythmias have been reported in Cantu syndrome patients, and none of the Cantu syndrome features has been reported for early repolarization syndrome patients. It remains conceivable that Cantu syndrome features are not attributable to enhanced cell membrane K_ATP activity, but instead they are the result of Kir6-independent (ie, mitochondrial) SUR2 activity. Also unexplained thus far is how opposing effects of loss-of-function mutations^108,109 vs gain-of-function mutations^110,113 in SUR2 could give rise to myocardial electric derangements in the first case, but vascular derangements in the second case.

Finally, we should recognize that the monogenic disease-associated K_ATP mutations, which cause relatively severe changes in channel function, are likely to represent only the tip of the iceberg when it come to the disease-promoting effects of change in protein activity. Further studies of patients with some or all symptoms of Cantu syndrome will be facilitated by efforts to bring such patients together (www.cantu-syndrome.org) and will no doubt reveal new mutations in the K_ATP subunits and perhaps in proteins that regulate K_ATP synthesis, trafficking, or location. We do not yet know which of the Cantu syndrome features are the most penetrant and, hence, which of these features might appear in isolation, because the severity of the effect of a specific mutation is reduced. If one or another of the affected cardiovascular functions is the most sensitive to SUR2 gain of function, then we may find far more cases of individuals with gain-of-function variants linked to specific features such as patent ductus arteriosus, pericardial effusion, and cardiomegaly with or without high-output cardiac failure. It may require the detection of these patients with newer cardiac imaging modalities, such as strain imaging to study the heterogeneity of myocardial fiber mass and orientation, or to detect abnormal electric activation sequences at subclinical levels that may exist with SUR2 gain-of-function mutations. We are only just beginning to recognize the cellular control mechanisms that regulate K_ATP channel subunit synthesis, trafficking, and degradation.¹⁸¹ Any alterations in such mechanisms, whether genetic or environmentally based, also may give rise to disease phenotypes similar to those resulting from the mutations discussed, and ultimately may benefit therapeutically from the unique pharmacology of the sulfonylurea receptors.