Cardiac KATP Research Articles

Caveolin-3 negatively regulates endocytic recycling of cardiac KATP channels.

Sarcolemmal ATP-sensitive potassium (KATP) channels play a vital role in cardioprotection. Cardiac KATP channels are enriched in caveolae and physically interact with the caveolae structural protein caveolin-3 (Cav3). Disrupting caveolae impairs the regulation of KATP channels through several signaling pathways. However, the direct functional effect of Cav3 on KATP channels is still poorly understood. Here, we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and showed that Cav3 greatly reduced KATP channel surface density and current amplitude in a caveolae-independent manner. A screen of Cav3 functional domains revealed that a 25 amino acid region in the membrane attachment domain of Cav3 is the minimal effective segment (MAD1). The peptide corresponding to the MAD1 segment decreased KATP channel current in a concentration-dependent manner with an IC50 of ∼5 μM. The MAD1 segment prevented KATP channel recycling, thus decreasing KATP channel surface density and abolishing the cardioprotective effect of ischemic preconditioning. Our research identified the Cav3 MAD1 segment as a novel negative regulator of KATP channel recycling, providing pharmacological potential in the treatment of diseases with KATP channel trafficking defects.NEW & NOTEWORTHY Cardiac KATP channels physically interact with caveolin-3 in caveolae. In this study, we investigated the functional effect of caveolin-3 on KATP channel activity and identified a novel segment (MAD1) in the C-terminus domain of Caveolin-3 that negatively regulates KATP channel surface density and current amplitude by impairing KATP channel recycling. The peptide corresponding to the MAD1 segment abolished the cardioprotective effect of ischemic preconditioning.

Rab35 GTPase positively regulates endocytic recycling of cardiac KATP channels

ABSTRACT ATP-sensitive K+ (KATP) channel couples membrane excitability to intracellular energy metabolism. Maintaining KATP channel surface expression is key to normal insulin secretion, blood pressure and cardioprotection. However, the molecular mechanisms regulating KATP channel internalization and endocytic recycling, which directly affect the surface expression of KATP channels, are poorly understood. Here we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and characterized Rab35 GTPase as a key regulator of KATP channel endocytic recycling. Electrophysiological recordings and surface biotinylation assays showed decreased KATP channel surface density with co-expression of a dominant negative Rab35 mutant (Rab35-DN), but not other recycling-related Rab GTPases, including Rab4, Rab11a and Rab11b. Immunofluorescence images revealed strong colocalization of Rab35-DN with recycling Kir6.2. Rab35-DN minimized the recycling rate of KATP channels. Rab35 also regulated KATP channel current amplitude in isolated adult cardiomyocytes by affecting its surface expression but not channel properties, which validated its physiologic relevance and the potential of pharmacologic target for treating the diseases with KATP channel trafficking defects.

Association Between Specificity of Sulfonylureas to Cardiac Mitochondrial KATP Channels and the Risk of Major Adverse Cardiovascular Events in Type 2 Diabetes.

Previous studies have revealed an intraclass difference in major adverse cardiovascular events (MACE) among sulfonylureas. In vitro and ex vivo studies reported several sulfonylureas to exhibit high-affinity blockage of cardiac mitochondrial ATP-sensitive potassium (mitoKATP) channels and could interfere with ischemic preconditioning, the most important mechanism of self-cardiac protection. However, no studies have examined whether these varying binding affinities of sulfonylureas could account for their intraclass difference in MACE. We compared mitoKATP channel high-affinity versus low-affinity sulfonylureas regarding the MACE risk in real-world settings. Using the Taiwan nationwide health care claims database, patients with type 2 diabetes initiating sulfonylurea monotherapy between 2007 and 2016 were included in the cohort study. A total of 33,727 new mitoKATP channel high-affinity (glyburide and glipizide) and low-affinity (gliclazide and glimepiride) sulfonylurea users, respectively, were identified after 1:1 propensity score matching. Cox proportional hazard models were used to estimate adjusted hazard ratios (aHRs) and 95% CI. MitoKATP channel high-affinity sulfonylureas were associated with a significantly increased risk of three-point MACE (aHR 1.21 [95% CI 1.03-1.44]), ischemic stroke (aHR 1.23 [95% CI 1.02-1.50]), and cardiovascular death (aHR 2.61 [95% CI 1.31-5.20]), but not with that of myocardial infarction (aHR 1.04 [95% CI 0.75-1.46]). The duration-response analyses revealed the highest MACE risk to be within 90 days of therapy (aHR 4.67 [95% CI 3.61-6.06]). Cardiac mitoKATP channel high-affinity sulfonylureas were associated with an increased MACE risk compared with low-affinity sulfonylureas in a nationwide population with diabetes.

ATP-sensitive Potassium Channel Subunits in Neuroinflammation: Novel Drug Targets in Neurodegenerative Disorders.

Arachidonic acids and its metabolites modulate plenty of ligand-gated, voltage-dependent ion channels, and metabolically regulated potassium channels including ATP-sensitive potassium channels (KATP). KATP channels are hetero-multimeric complexes of sulfonylureas receptors (SUR1, SUR2A or SUR2B) and the pore-forming subunits (Kir6.1 and Kir6.2) likewise expressed in the pre-post synapsis of neurons and inflammatory cells, thereby affecting their proliferation and activity. KATP channels are involved in amyloid-β (Aβ)-induced pathology, therefore emerging as therapeutic targets against Alzheimer's and related diseases. The modulation of these channels can represent an innovative strategy for the treatment of neurodegenerative disorders; nevertheless, the currently available drugs are not selective for brain KATP channels and show contrasting effects. This phenomenon can be a consequence of the multiple physiological roles of the different varieties of KATP channels. Openings of cardiac and muscular KATP channel subunits, are protective against caspase-dependent atrophy in these tissues and some neurodegenerative disorders, whereas in some neuroinflammatory diseases, benefits can be obtained through the inhibition of neuronal KATP channel subunits. For example, glibenclamide exerts an anti-inflammatory effect in respiratory, digestive, urological, and central nervous system (CNS) diseases, as well as in ischemia-reperfusion injury associated with abnormal SUR1-Trpm4/TNF-α or SUR1-Trpm4/ Nos2/ROS signaling. Despite this strategy being promising, glibenclamide may have limited clinical efficacy due to its unselective blocking action of SUR2A/B subunits also expressed in cardiovascular apparatus with pro-arrhythmic effects and SUR1 expressed in pancreatic beta cells with hypoglycemic risk. Alternatively, neuronal selective dual modulators showing agonist/antagonist actions on KATP channels can be an option.

Sexual dimorphism in vascular ATP-sensitive K+ channel function supporting interstitial via convective and/or diffusive O2 transport.

Inhibition of pancreatic ATP-sensitive K+ (KATP ) channels is the intended effect of oral sulphonylureas to increase insulin release in diabetes. However, pertinent to off-target effects of sulphonylurea medication, sex differences in cardiac KATP channel function exist, whereas potential sex differences in vascular KATP channel function remain unknown. In the present study, we assessed vascular KATP channel function (topical glibenclamide superfused onto fast-twitch oxidative skeletal muscle) supporting blood flow and interstitial O2 delivery-utilization matching ( is) during twitch contractions in male, female during pro-oestrus and ovariectomized female (F+OVX) rats. Glibenclamide decreased blood flow (convective O2 transport) and interstitial in male and female, but not F+OVX, rats. Compared to males, females also demonstrated impaired diffusive O2 transport and a faster fall in interstitial . Our demonstration, in rats, that sex differences in vascular KATP channel function exist support the tentative hypothesis that oral sulphonylureas may exacerbate exercise intolerance and morbidity, especially in premenopausal females. Vascular ATP-sensitive K+ (KATP ) channels support skeletal muscle blood flow ( ), interstitial O2 delivery ( )-utilization ( ) matching (i.e. interstitial-myocyte O2 flux driving pressure; is) and exercise tolerance. Potential sex differences in skeletal muscle vascular KATP channel function remain largely unexplored. We hypothesized that local skeletal muscle KATP channel inhibition via glibenclamide superfusion (5mg kg-1 GLI; sulphonylurea diabetes medication) in anaesthetized female Sprague-Dawley rats, compared to males, would demonstrate greater reductions in contracting (1Hz, 7V, 180s) fast-twitch oxidative mixed gastrocnemius (97% type IIA+IID/X+IIB) (15μm microspheres) and is (phosphorescence quenching), resulting from more compromised convective ( ) and diffusive ( ) O2 conductances. Furthermore, these GLI-induced reductions in ovary-intact females measured during pro-oestrus would be diminished following ovariectomy (F+OVX). GLI similarly impaired mixed gastrocnemius in both males (↓28%) and females (↓33%, both P<0.032) via reduced (male: ↓31%, female: ↓35%, both P<0.020), (male: 5.6±0.5 vs. 4.0±0.5, female: 6.4±1.1 vs. 4.2±0.6mL O2 min-1 100g tissue-1 , P<0.022) and the resulting is, with females also demonstrating a reduced (0.40±0.07 vs. 0.30±0.04mL O2 min-1 100g tissue-1 , P<0.042) and a greater GLI-induced speeding of is fall (mean response time: Sex×Drug interaction, P=0.026). Conversely, GLI did not impair the mixed gastrocnemius of F+OVX rats. Therefore, in patients taking sulphonylureas, these results support the potential for impaired vascular KATP channel function to compromise muscle and therefore exercise tolerance. Such an effect, if present, would likely contribute to adverse cardiovascular events in premenopausal females more than males.

Novel cholesterol-dependent regulation of cardiac KATP subunit expression revealed using histone deacetylase inhibitors.

We recently discovered that the histone deacetylase inhibitor, trichostatin A (TSA), increases expression of the sulfonylurea receptor 2 (SUR2; Abcc9) subunit of the ATP‐sensitive K+ (KATP) channel in HL‐1 cardiomyocytes. Interestingly, the increase in SUR2 was abolished with exogenous cholesterol, suggesting that cholesterol may regulate channel expression. In the present study, we tested the hypothesis that TSA increases SUR2 by depleting cholesterol and activating the sterol response element binding protein (SREBP) family of transcription factors. Treatment of HL‐1 cardiomyocytes with TSA (30 ng/ml) caused a time‐dependent increase in SUR2 mRNA expression that correlates with the time course of cholesterol depletion assessed by filipin staining. Consistent with the cholesterol‐dependent regulation of SREBP increasing SUR2 mRNA expression, we observe a significant increase in SREBP cleavage and translocation to the nucleus following TSA treatment that is inhibited by exogenous cholesterol. Further supporting the role of SREBP in mediating the effect of TSA on KATP subunit expression, SREBP1 significantly increased luciferase reporter gene expression driven by the upstream SUR2 promoter. Lastly, HL‐1 cardiomyocytes treated with the SREBP inhibitor PF429242 significantly suppresses the effect of TSA on SUR2 gene expression. These results demonstrate that SREBP is an important regulator of KATP channel expression and suggest a novel method by which hypercholesterolemia may exert negative effects on the cardiovascular system, namely, by suppressing expression of the KATP channel.

EFFECT OF REGULAR EXERCISE ON EXPRESSION OF K ATP CHANNELS IN HEART OF DIABETIC RATS

There is abundant evidence that ATP sensitive potassium (KATP) channels play cytoprotective role in cardiac myocytes, allowing the cell to couple metabolic state to electrical activity of the cell membrane. In cardiac myocytes Kir6.2/SUR2A are the major subunits expressed. However, expression or function of KATP channels has been found to be impaired in the presence of persistent hyperglycemia in diabetes mellitus (DM). While regular exercise can improve hyperglycemic status in DM, its impact on the expression of KATP channels subunits in heart is unknown. PURPOSE: To assess the effect of regular exercise on expression of KATP channel subunits in heart of streptozotocin-induced diabetic rats. METHODS: Male Wistar rats (25 days old) were randomly divided into four groups, among them: sedentary control, trained control, sedentary diabetic, trained diabetic. Diabetes was induced by a single streptozotocin injection (100 mg/kg body weight), animals with fasting blood glucose levels ≥ 300 mg/dL were considered as diabetic. Groups with training program performed exercise on a treadmill (30 minutes daily, 5 days/week) for 8 weeks. At the end of the intervention, two subunits of cardiac KATP channel (SUR2A and Kir 6.2) were analyzed as indicators and quantitative analysis of these subunits was achieved with real-time RT–PCR. RESULTS: In control conditions, the regular exercise reduced Kir6.2 subunit mRNA levels significantly (76%; p=0.045) in heart. In diabetes, reduced Kir6.2 expression was also observed, and there was no difference in expression levels between sedentary diabetic and trained diabetic groups (P > 0.05). Otherwise, relative mRNA expression of the subunit SUR2A was increased in both sedentary diabetic and trained diabetic groups (80.33% and 86.08%, respectively). CONCLUSION: Collectively, our data demonstrate that the regular exercise modifies expression of KATP channel subunits of heart only in control conditions. However, the gene expression patterns of KATP channel subunits are different during diabetes, by increased SUR2A and decreased Kir6.2, which was not modified by exercise. These results may provide an opportunity to understand mechanisms leading to diabetic cardiomyopathy during stress and exercise in DM.

Functional modulation of sarcolemmal KATP channels by atrial natriuretic peptide-elicited intracellular signaling in adult rabbit ventricular cardiomyocytes.

ATP-sensitive potassium (KATP) channels couple cell metabolic status to membrane excitability and are crucial for stress adaptation and cytoprotection in the heart. Atrial natriuretic peptide (ANP), a cardiac peptide important for cardiovascular homeostasis, also exhibits cytoprotective features including protection against myocardial ischemia-reperfusion injuries. However, how ANP modulates cardiac KATP channels is largely unknown. In the present study we sought to address this issue by investigating the role of ANP signaling in functional modulation of sarcolemmal KATP (sarcKATP) channels in ventricular myocytes freshly isolated from adult rabbit hearts. Single-channel recordings were performed in combination with pharmacological approaches in the cell-attached patch configuration. Bath application of ANP markedly potentiated sarcKATP channel activities induced by metabolic inhibition with sodium azide, whereas the KATP-stimulating effect of ANP was abrogated by selective inhibition of the natriuretic peptide receptor type A (NPR-A), cGMP-dependent protein kinase (PKG), reactive oxygen species (ROS), extracellular signal-regulated protein kinase (ERK)1/2, Ca2+/calmodulin-dependent protein kinase II (CaMKII), or the ryanodine receptor (RyR). Blockade of RyRs also nullified hydrogen peroxide (H2O2)-induced stimulation of sarcKATP channels in intact cells. Furthermore, single-channel kinetic analyses revealed that ANP enhanced the function of ventricular sarcKATP channels through destabilizing the long closures and facilitating the opening transitions, without affecting the single-channel conductance. In conclusion, here we report that ANP positively modulates the activity of ventricular sarcKATP channels via an intracellular signaling mechanism consisting of NPR-A, PKG, ROS, ERK1/2, CaMKII, and RyR2. This novel mechanism may regulate cardiac excitability and contribute to cytoprotection, in part, by opening myocardial KATP channels.

ATP- and voltage-dependent electro-metabolic signaling regulates blood flow in heart

Local control of blood flow in the heart is important yet poorly understood. Here we show that ATP-sensitive K+ channels (KATP), hugely abundant in cardiac ventricular myocytes, sense the local myocyte metabolic state and communicate a negative feedback signal-correction upstream electrically. This electro-metabolic voltage signal is transmitted instantaneously to cellular elements in the neighboring microvascular network through gap junctions, where it regulates contractile pericytes and smooth muscle cells and thus blood flow. As myocyte ATP is consumed in excess of production, [ATP]i decreases to increase the openings of KATP channels, which biases the electrically active myocytes in the hyperpolarization (negative) direction. This change leads to relative hyperpolarization of the electrically connected cells that include capillary endothelial cells, pericytes, and vascular smooth muscle cells. Such hyperpolarization decreases pericyte and vascular smooth muscle [Ca2+]i levels, thereby relaxing the contractile cells to increase local blood flow and delivery of nutrients to the local cardiac myocytes and to augment ATP production by their mitochondria. Our findings demonstrate the pivotal roles of local cardiac myocyte metabolism and KATP channels and the minor role of inward rectifier K+ (Kir2.1) channels in regulating blood flow in the heart. These findings establish a conceptually new framework for understanding the hugely reliable and incredibly robust local electro-metabolic microvascular regulation of blood flow in heart.

ATP‐sensitive K+ Channel Inhibition via Glibenclamide Impairs Maximal Aerobic Capacity and Critical Speed of Healthy Rats without Compromising Cardiac Function

Glibenclamide (GLI) enhances insulin release for Type II diabetes patients by inhibiting pancreatic ATP‐sensitive K+ (KATP) channels. However, systemic KATP channel inhibition decreases maximal aerobic capacity (V̇O2max) during treadmill running in rats; where V̇O2max is determined by cardiac output (Q̇) and O2 utilization within contracting skeletal muscle (arterial‐venous O2 difference). In vitro studies demonstrate that KATP channels enhance isolated cardiomyocyte and vascular smooth muscle relaxation. In vivo studies show that GLI induces vasoconstriction, decreases blood flow, and impairs O2 delivery‐utilization matching in contracting skeletal muscle. Whether high‐intensity exercise tolerance (i.e. the speed‐duration relationship with critical speed, CS, and distance covered above CS, D′) is dependent upon cardiac and/or peripheral (i.e., vascular) KATP channel function in vivo is unknown.PURPOSETo determine the role of KATP channels in establishing cardiac function (see below), maximal aerobic capacity (V̇O2max) and sustaining submaximal exercise tolerance (CS). We hypothesized that acute KATP channel inhibition via GLI would decrease V̇O2max and CS, but not decrease cardiac function.METHODSTen adult female Sprague‐Dawley rats were assessed during the proestrus cycle in randomized order with and without GLI. GLI (10 mg kg−1 in DMSO i.p.) was administered 30–60 min prior to resting Doppler echocardiography and exercise tests. Doppler echocardiography measured Q̇, heart rate (HR), stroke volume (SV), ejection fraction (EF), fractional shortening (FS) and rates of left ventricular contraction and relaxation. V̇O2max and CS tests utilized a motorized treadmill at a 5% incline. V̇O2max was measured via plexiglass metabolic chamber connected to an O2 analyzer with increasing treadmill speed (5–10 m min−1) every minute until V̇O2 plateaued with increases in treadmill speed. Multiple (i.e., ≥5) constant speed runs to exhaustion were used to resolve CS and D′.RESULTSAt rest GLI did not alter SV, EF, FS, nor rates of left ventricular contraction and relaxation compared to control (p&gt;0.05 for all). However, GLI did reduce HR (321 ± 8 vs 300 ± 7 b min−1) and V̇ (217 ± 23 vs 192 ± 13 mL min−1; p&lt;0.05 for both). Despite HR and Q̇ reductions at rest, neither were significantly correlated with decreases in V̇O2max or CS (p&gt;0.05 for both). GLI significantly reduced V̇O2max (69 ± 2 vs 72 ± 1 mL O2 kg−1 min−1), CS (32 ± 1 vs 36 ± 1 m min−1; n=8), and D′ (100 ± 6 vs 86 ± 8 m; n=7) compared to control (all p&lt;0.05).CONCLUSIONSConsistent with our hypothesis, KATP channels are vital in establishing maximal aerobic capacity (GLI decreased V̇O2max ~4%) and exercise tolerance (GLI decreased CS ~12%). Therefore, we speculate that HR regulation via KATP channel activity is overridden during exercise. These data support that vascular KATP channel function is important for matching O2 delivery to O2 requirements within skeletal muscles and this plays a pivotal role in determining exercise tolerance. Thus GLI treatment may potentiate the exercise intolerance of diabetic and other cardiovascular disease patients.Support or Funding InformationHL108328This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of the C-terminus of SUR in the differential regulation of β-cell and cardiac KATP channels by MgADP and metabolism.

Key points β‐Cell KATP channels are partially open in the absence of metabolic substrates, whereas cardiac KATP channels are closed.Using cloned channels heterologously expressed in Xenopus oocytes we measured the effect of MgADP on the MgATP concentration–inhibition curve immediately after patch excision.MgADP caused a far more striking reduction in ATP inhibition of Kir6.2/SUR1 channels than Kir6.2/SUR2A channels; this effect declined rapidly after patch excision.Exchanging the final 42 amino acids of SUR was sufficient to switch the Mg‐nucleotide regulation of Kir6.2/SUR1 and Kir6.2/SUR2A channels, and partially switch their sensitivity to metabolic inhibition.Deletion of the C‐terminal 42 residues of SUR abolished MgADP activation of both Kir6.2/SUR1 and Kir6.2/SUR2A channels.We conclude that the different metabolic sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A channels is at least partially due to their different regulation by Mg‐nucleotides, which is determined by the final 42 amino acids. ATP‐sensitive potassium (KATP) channels couple the metabolic state of a cell to its electrical activity and play important physiological roles in many tissues. In contrast to β‐cell (Kir6.2/SUR1) channels, which open when extracellular glucose levels fall, cardiac (Kir6.2/SUR2A) channels remain closed. This is due to differences in the SUR subunit rather than cell metabolism. As ATP inhibition and MgADP activation are similar for both types of channels, we investigated channel inhibition by MgATP in the presence of 100 μm MgADP immediately after patch excision [when the channel open probability (P O) is near maximal]. The results were strikingly different: 100 μm MgADP substantially reduced MgATP inhibition of Kir6.2/SUR1, but had no effect on MgATP inhibition of Kir6.2/SUR2A. Exchanging the final 42 residues of SUR2A with that of SUR1 switched the channel phenotype (and vice versa), and deleting this region abolished Mg‐nucleotide activation. This suggests the C‐terminal 42 residues are important for the ability of MgADP to influence ATP inhibition at Kir6.2. This region was also necessary, but not sufficient, for activation of the KATP channel in intact cells by metabolic inhibition (azide). We conclude that the ability of MgADP to impair ATP inhibition at Kir6.2 accounts, in part, for the differential metabolic sensitivities of β‐cell and cardiac KATP channels.

Ectopic overexpression of Kir6.1 in the mouse heart impacts on the life expectancy

We recently reported the reduced ATP-sensitive potassium (KATP) channel activities in the transgenic mouse heart overexpressing the vascular type KATP channel pore-forming subunit (Kir6.1). Although dysfunction of cardiac KATP channel has been nominated as a cause of cardiomyopathy in human, these transgenic mice looked normal as wild-type (WT) during the experiment period (~20 weeks). Extended observation period revealed unexpected deaths beginning from 30 weeks and about 50% of the transgenic mice died by 55 weeks. Surface ECG recordings from the transgenic mice at rest demonstrated the normal sinus rhythm and the regular ECG complex as well as the control WT mice except for prolonged QT interval. However, the stress ECG test with noradrenaline revealed abnormal intraventricular conduction delay and arrhythmogeneity in the transgenic mouse. Fibrotic changes in the heart tissue were remarkable in aged transgenic mice, and the cardiac fibrosis developed progressively at least from the age of 30 weeks. Gene expression analyses revealed the differentiation of cardiac fibroblasts to myofibroblasts with elevated cytokine expressions was initiated way in advance before the fibrotic changes and the upregulation of BNP in the ventricle. In sum, Kir6.1TG mice provide an electro-pathological disease concept originated from KATP channel dysfunction.

Differential Metabolic and Nucleotide Sensitivity of Beta-Cell and Cardiac KATP Channels

Differential Metabolic and Nucleotide Sensitivity of Beta-Cell and Cardiac KATP Channels

The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

ATP-sensitive K+ (KATP) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of KATP channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of KATP channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate KATP channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of KATP channel trafficking to increase surface KATP channel density. EHD2 had no effect on cardiac Na+ channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on KATP channel surface expression. EHD2 did not directly affect KATP channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing KATP channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated KATP channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with KATP channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

The mechano-sensitivity of cardiac ATP-sensitive potassium channels is mediated by intrinsic MgATPase activity

Cardiac ATP-sensitive K+ (KATP) channel activity plays an important cardio-protective role in regulating excitability in response to metabolic stress. Evidence suggests that these channels are also mechano-sensitive and therefore may couple KATP channel activity to increased cardiac workloads. However, the molecular mechanism that couples membrane stretch to channel activity is not currently known. We hypothesized that membrane stretch may alter the intrinsic MgATPase activity of the cardiac KATP channel resulting in increased channel activation. The inside-out patch-clamp technique was used to record single-channel and macroscopic recombinant KATP channel activity in response to membrane stretch elicited by negative pipette pressure. We found that stretch activation requires the presence of the SUR subunit and that inhibition of MgATPase activity with either the non-hydrolysable ATP analog AMP-PNP or the ATPase inhibitor BeFx significantly reduced the stimulatory effect of stretch. We employed a point mutagenic approach to determine that a single residue (K1337) in the hairpin loop proximal to the major MgATPase catalytic site in the SUR2A subunit is responsible for the difference in mechano-sensitivity between SUR2A and SUR1 containing KATP channels. Moreover, using a double cysteine mutant substitution in the hairpin loop region revealed the importance of a key residue-residue interaction in this region that transduces membrane mechanical forces into KATP channel stimulation via increases in channel MgATPase activity. With respect to KATP channel pharmacology, glibenclamide, but not glicalizide or repaglinide, was able to completely inhibit KATP channel mechano-sensitivity. In summary, our results provide a highly plausible molecular mechanism by which mechanical membrane forces are rapidly converted in changes in KATP channel activity that have implications for our understanding of cardiac KATP channels in physiological or pathophysiological settings that involve increased workload.

Cardiac KATP channel modulation by 16Hz magnetic fields - A theoretical study.

Heart exposure to 16Hz magnetic fields (MFs) was shown to be cardio-protective for diseased hearts; still, the mechanism of this effect is unknown. We hypothesize that a possible one mechanism is an increased trans-membrane KATP channel open probability due to modulation of the degree of dissociation between K+ ions, having a resonance frequency of 16Hz, and the channel selectivity filter. The Fan-Makielski Markovian KATP channel model was adopted, and the MF bio-effect was manifested by modulating the open probability of the channel using the predictive MF bio-effect parameter based on Binhi's quantum mechanics model. The model was integrated in a ventricular single cell model and the MF effect on the calcium transients [Ca2+] was assessed. Periodic pacing (Cycle Length CL=1sec) was applied and a 16Hz or 32Hz MF was turned on at t=0 for 10min. MF exposure gradually decreased [Ca2+] due to KATP channel opening, more strongly at 16Hz. Additionally, a small negative diastolic shift was observed. These numerical results demonstrated similarity to published experimental data using similar 16Hz MF exposure. We conclude that 16Hz MF exposure increases the KATP channel open probability, lowering the cellular calcium load. Our model could be integrated in a tissue model to predict optimal MF parameters for future cardiac therapy devices.

A heme-binding domain controls regulation of ATP-dependent potassium channels

Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

Nandrolone decanoate negatively reverses the beneficial effects of exercise on cardiac muscle via sarcolemmal, but not mitochondrial K(ATP) channel.

ATP-sensitive potassium channels are supposed to have a substantial role in improvement of cardiac performance. This study was performed to evaluate whether nandrolone decanoate (ND) and (or) exercise training could affect the expression of cardiac K(ATP) channel subunits. Thirty-five male albino Wistar rats were randomly divided into 5 groups, including sedentary control (SC), sedentary vehicle (SV), sedentary ND (SND), exercise control (EC), and exercise and ND (E+ND). Exercise training was performed on a treadmill 5 times per week. ND was injected (10 mg/kg/week, i.m.) to the rats in the SND and E+ND groups. Following cardiac isolation, the expression of both sarcolemmal and mitochondrial subunits of K(ATP) channel was measured using Western blot method. The expression of sarcolemmal, but not mitochondrial, subunits of K(ATP) channel (Kir6.2 and SUR2) of EC group was significantly higher compared with SC group while ND administration (SND group) did not show any change in their expression. In the E+ND group, ND administration led to decrease of the over-expression of sarcolemmal Kir6.2 and SUR2 which was previously induced by exercise. There was no significant association between the mitochondrial expression of either Kir6.2 or SUR2 proteins and administration of ND or exercise. Supra-physiological dosage of ND negatively reverses the effects of exercise on the cardiac muscle expression of sarcolemmal, but not mitochondrial, K(ATP) channel subunits.

Kir6.2 activation by sulfonylurea receptors: a different mechanism of action for SUR1 and SUR2A subunits via the same residues.

ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, and the inward rectifier K+ channel Kir6.2. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6.2). SUR modulates channel gating in response to the binding of nucleotides or drugs and Kir6.2 conducts potassium ions. The activity of K-ATP channels varies with their localization. In pancreatic β-cells, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytes SUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2. Three residues (E1305, I1310, L1313) located in the linker region between transmembrane domain 2 and nucleotide-binding domain 2 of SUR2A were previously found to be involved in the activation pathway linking binding of openers onto SUR2A and channel opening. To determine the role of the equivalent residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter multidrug resistance-associated protein 1 (MRP1), and used patch clamp recordings on Xenopus oocytes to assess the functionality of SUR1/MRP1 chimeric K-ATP channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display unexpected side-chain specificities which could account for the contrasted properties of pancreatic and cardiac K-ATP channels.

Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis.

Histone deacetylase inhibitors (HDIs) are under investigation for the treatment of a number of human health problems. HDIs have proven therapeutic value in refractory cases of cutaneous T-cell lymphoma. Electrocardiographic ST segment morphological changes associated with HDIs were observed during development. Because ST segment morphology is typically linked to changes in ATP sensitive potassium (KATP) channel activity, we tested the hypothesis that HDIs affect cardiac KATP channel subunit expression. Two different HDIs, romidepsin and trichostatin A, caused ~20-fold increase in SUR2 (Abcc9) subunit mRNA expression in HL-1 cardiomyocytes. The effect was specific for the SUR2 subunit as neither compound causes a marked change in SUR1 (Abcc8) expression. Moreover, the effect was cell specific as neither HDI markedly altered KATP subunit expression in MIN6 pancreatic β-cells. We observe significant enrichment of the H3K9Ac histone mark specifically at the SUR2 promoter consistent with the conclusion that chromatin remodeling at this locus plays a role in increasing SUR2 gene expression. Unexpectedly, however, we also discovered that HDI-dependent depletion of cellular cholesterol is required for the observed effects on SUR2 expression. Taken together, the data in the present study demonstrate that KATP subunit expression can be epigenetically regulated in cardiomyocytes, defines a role for cholesterol homeostasis in mediating epigenetic regulation and suggests a potential molecular basis for the cardiac effects of the HDIs.