The voltage-gated KV7 (KCNQ) potassium channels are activated by ischemia and involved in hypoxic vasodilatation. We investigated the effect of KV7 channel modulation on cardiac ischemia and reperfusion injury and its interaction with cardioprotection by ischemic preconditioning (IPC). Reverse-transcription polymerase chain reaction revealed expression of KV7.1, KV7.4, and KV7.5 in the left anterior descending rat coronary artery and all KV7 subtypes (KV7.1–KV7.5) in the left and right ventricles of the heart. Isolated hearts were subjected to no-flow global ischemia and reperfusion with and without IPC. Infarct size was quantified by 2,3,5-triphenyltetrazolium chloride staining. Two blockers of KV7 channels, XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone] (10 µM) and linopirdine (10 µM), reduced infarct size and exerted additive infarct reduction to IPC. An opener of KV7 channels, flupirtine (10 µM) abolished infarct size reduction by IPC. Hemodynamics were measured using a catheter inserted in the left ventricle and postischemic left ventricular recovery improved in accordance with reduction of infarct size and deteriorated with increased infarct size. XE991 (10 µM) reduced coronary flow in the reperfusion phase and inhibited vasodilatation in isolated small branches of the left anterior descending coronary artery during both simulated ischemia and reoxygenation. KV7 channels are expressed in rat coronary arteries and myocardium. Inhibition of KV7 channels exerts cardioprotection and opening of KV7 channels abrogates cardioprotection by IPC. Although safety issues should be further addressed, our findings suggest a potential role for KV7 blockers in the treatment of ischemia-reperfusion injury.
Reperfusion after a coronary artery occlusion causes myocardial injury beyond the ischemic injury. Cardiac ischemia-reperfusion (IR) injury is modifiable by ischemic preconditioning (IPC), by which brief, nonlethal episodes of ischemia generate a protective phenotype and leave the heart resistant to IR injury (Braunwald and Kloner, 1985; Murry et al., 1986). Despite identification of several protective signaling pathways, pharmacological interventions addressing specific components of these pathways generally show lower protective capacity than IPC (Heusch et al., 2015). Thus, IPC stimulates a variety of protective mechanism ranging from modification of vascular function over anti-inflammatory mechanisms to activation of cardiac intracellular survival signaling pathways (Ferdinandy et al., 2014; Lecour et al., 2014; Sivaraman and Yellon, 2014; Heusch et al., 2015).
Voltage-gated potassium channels (KVs) are the largest and most diverse group of potassium channels with a range of biologic functions (Gutman et al., 2005). The KV7 family (KCNQ) is important for regulation of the membrane potential of excitable tissues (Mackie and Byron, 2008). KV7.1 was first recognized for its role in repolarization of cardiomyocytes (Barhanin et al., 1996; Sanguinetti et al., 1996). A cytoprotective role has been suggested for KV7 channels, because blocking KV7 channels preserved neuronal cell viability (Zhou et al., 2011) and protected sympathetic neurons from nerve growth factor deprivation (Xia et al., 2002). Furthermore, KV7.1, KV7.4, and KV7.5 channels have emerged as important regulators of vascular tone (Hedegaard et al., 2014; Stott et al., 2014) and of hypoxic as well as agonist-induced vasodilatation. Adenosine and hydrogen sulfide are cardioprotective and both induce vasodilatation involving KV7 channel opening (Elrod et al., 2007; Rossoni et al., 2008; Osipov et al., 2009; Schleifenbaum et al., 2010; Szabó et al., 2011; Khanamiri et al., 2013; Hedegaard et al., 2014). Therefore, modulation of KV7 channels could be involved in cardiac IR injury.
Activation of other K channels is cardioprotective (Liu et al., 1998; Xu et al., 2002; Kristiansen et al., 2005; Bentzen et al., 2009) and involved in protection by IPC (Fryer et al., 2000), but the role of the KV7 channels in the heart is largely unknown. The aim of this study was to investigate the effect of KV7 channels on IR injury and to evaluate the hypothesis that cardioprotection by IPC involves KV7 channels. To achieve this goal, we studied 1) the expression of the KV7 subtypes in the myocardium and coronary arteries, 2) the effect of KV7 channel modulation by XE991 and linopirdine (KV7 channel inhibitors with different molecular structures and potencies) and flupirtine (KV7 channel activator) on IR injury, 3) the effect of KV7 modulation combined with IPC on IR injury, and 4) the influence of KV7 inhibition during simulated ischemia and reoxygenation in isolated coronary arteries.
Materials and Methods
Animals were handled in accordance with national and institutional guidelines for animal research and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1996). The experimental work was approved by the Danish Animal Experiments Inspectorate (license no. 2013-15-2934-00876). Male Wistar rats (Taconic, Ry, Denmark) were used at 9–11 weeks of age. They were fed an Altromin 1324 diet (Altromin, Lage, Germany) and housed under controlled conditions with 12-hour/12-hour light/dark cycles.
XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone], linopirdine ([3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one]) and flupirtine (ethyl[2-amino-6-[(4-fluorobenzyl)methylamino]pyridin-3-yl]carbamate) were purchased from Sigma (St. Louis, MO).
Reverse-Transcription Polymerase Chain Reaction.
Wistar rats were euthanized by cervical dislocation. Branches of rat left anterior descending coronary arteries (LADs), samples of left and right ventricles, brain, and kidney were kept in RNAlater (Qiagen, Copenhagen, Denmark). The samples were homogenized for 3 minutes in TissueLyser (Qiagen). The samples were processed for reverse-transcription polymerase chain reaction as previously described (Hedegaard et al., 2014). The primers and expected product size are listed in Table 1. To determine the identity of the amplification products, the agarose gel bands were excised and the DNA was purified using a QIAquick gel extraction kit (Qiagen). The purified polymerase chain reaction product was sequenced by Eurofins MWG Operon (Ebersberg, Germany).
Isolated Perfused Rat Heart Model.
We evaluated the effect of the KV7 modulators (XE991, linopirdine, and flupirtine) and the interaction with IPC on IR injury in isolated, constant pressure–perfused rat hearts (Fig. 1A). In addition, the effect of IPC and XE991 was evaluated in rat hearts subjected to flow-controlled perfusion (Fig. 1B). A concentration of 10 µM XE991 and 10 µM linopirdine was selected based on own previous results (Hedegaard et al., 2014, 2016; Engholm et al., 2015). XE991 and linopirdine are reported to have IC50 values of approximately 1–3 µM against KV7 channels (Wang et al., 1998; Søgaard et al., 2001; Ng et al., 2011). XE991 inhibits KV7.1–KV7.4 with IC50 values of approximately 1–5 μM (Yeung et al., 2007, 2008a) and KV7.5 with an IC50 value of approximately 60 μM (Jensen et al., 2005; Yeung et al., 2008a). Although XE991 and linopirdine show selectivity toward the KV7 channels, they have been reported to inhibit other KV channels at higher concentrations (Schnee and Brown, 1998; Wang et al., 1998; Wladyka and Kunze, 2006).
The rats were anesthetized with midazolam (0.5 mg/kg body weight; Dormicum, Matrix Pharmaceuticals, Herlev, Denmark), fentanylcitrate (0.158 mg/kg body weight), and fluanisone (0.5 mg/kg body weight; Hypnorm, Vetapharma Ltd., Leeds, UK) by subcutaneous injection, and adequate depth of anesthesia was ensured by the absence of the toe pinch withdrawal reflex before excision of the heart and Langendorff perfusion. The anesthetized rat was placed supine and a laparotomy and thoracotomy was performed. The heart and femoral vein were exposed and heparin (1000 IU/kg) was administered intravenously, followed by insertion of a custom-made cannula in the ascending aorta and immediate initiation of retrograde perfusion of the heart. The heart was perfused with a Krebs-Henseleit (KH) solution (containing118 mM NaCl2, 4.9 mM KCl, 27.2 mM NaHCO3, 1.2 mM MgCl2, 1.0 mM KH2PO4, 2.0 mM CaCl2, and 11.0 mM glucose) as described in detail previously (Povlsen et al., 2014). The perfusion protocol consisted of 15 minutes of stabilization; 25 minutes of infusion with KV7 channel blockers or openers, respectively; 40 minutes of global no-flow ischemia; and 120 minutes of reperfusion.
Hearts were subjected to either pressure-regulated perfusion (eight groups) with a constant pressure of 80 mm Hg or flow-regulated perfusion (three groups) with a coronary flow rate of 14 ml/min during stabilization and 12 ml/min during early reperfusion. During flow-regulated perfusion, the coronary flow was decreased by 1 ml/min for every 30 minutes in reperfusion to mimic the coronary flow decline in the pressure-regulated Langendorff hearts (Fig. 1). The coronary flow was measured in real time by an inline flowmeter (Transonic, Ithaca, NY).
IPC was induced by two cycles of 5 minutes of global ischemia and 5 minutes of reperfusion prior to sustained ischemia. Left ventricular function was continuously monitored by an intraventricular fluid-filled latex balloon connected to a pressure transducer (Hugo Sachs Elektronik, March-Hugstetten, Germany). Diastolic pressure was set to 5–8 mm Hg. Data were acquired and analyzed with Notochord-hem software (Notocord, Croissy-Sur-Seine, France).
At the end of the perfusion protocol, the heart was frozen at −80°C for 20 minutes and subsequently sliced into five transverse sections, which were submerged in 2,3,5-triphenyltetrazoliumchloride (Sigma) solution (1% in a phosphate buffer) to delineate areas of infarction (pale) from vital heart tissue (red). Infarct size (IS) and area at risk (AAR) were assessed using ImageJ image analysis software (National Institutes of Health, Bethesda, MD). The entire left ventricle constituted the AAR. Measurements were weighted with the weight of each individual slice and the IS/AAR ratio was calculated.
Recording of Arterial Reactivity.
Distal segments (approximately 2 mm) of LADs were carefully dissected free of the surrounding myocardium and cut into a maximum of four segments, and each of the segments was allocated to different treatments. Therefore, each experiment equals one animal. The segments were mounted in microvascular myographs (Danish MyoTechnology, Aarhus, Denmark) as previously described (Troelsen et al., 2015). The myograph bath contained physiological salt solution (119 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4⋅7H2O, 25 mM NaHCO3, 1.2 mM KH2PO4, 0.027 mM EDTA, 10 mM glucose, and 1.6 mM CaCl2, adjusted to pH 7.4), bubbled with air (21% O2, 5% CO2 in N2) and maintained at 37°C. The vessels were stretched to give an equivalent transmural pressure of 100 mm Hg. Preparation viability was examined by exposing arteries to physiological salt solution containing 125 mM K (NaCl was replaced by KCl on an equimolar basis). Endothelial function was evaluated as relaxation to acetylcholine (10 µM) in U46619 [(5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid] (10–100 nM)–contracted arteries. Appropriate oxygen concentration was obtained by gassing the organ bath through pimpstones allowing rapid equilibration of the oxygen tension with air or 5% CO2 in N2, which resulted in 1% O2 in the organ bath (Hedegaard et al., 2011). The arteries were incubated with vehicle or XE991; after 30 minutes, they were contracted with vasopressin (10–100 nM) to obtain similar contraction levels. Vehicle- and XE991-treated arteries were exposed to 20 minutes of air, whereas arteries exposed to ischemic preconditioning were exposed two times to 5 minutes of simulated ischemia (1% O2) interspersed by 5 minutes of air prior to 40 minutes simulated ischemia (1% O2) and reoxygenation. Vasodilatation is expressed as the percentage of the contraction to vasopressin.
Calculations and Statistical Analyses.
All data are reported as means ± S.E.M. Normal distribution of the data was assessed by the D'Agostino and Pearson omnibus normality test. IS and coronary artery tone were evaluated by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for pairwise comparison when appropriate. Hemodynamic variables and coronary flow rate were evaluated by two-way ANOVA followed by a Bonferroni post hoc test when appropriate. The effect of KV7 modulation on coronary flow and heart rate was evaluated after 5 minutes of KV7 modulation and before IPC treatment (20 minutes into the perfusion protocol, see Fig. 1) and compared with baseline (15 minutes into the perfusion protocol). Hearts were grouped according to dimethylsulfoxide or KV7 modulation regardless of subsequent allocation to IPC or time-matched control perfusion (non-IPC). Calculations were performed using GraphPad Prism (GraphPad Software, La Jolla, CA). P < 0.05 was considered statistically significant.
KV7 Expression in Cardiac Tissue.
KCNQ1, KCNQ4, and KCNQ5 (KV7.1, KV7.4, and KV7.5; Fig. 2, A, D, and E) were expressed in the rat distal LAD artery, whereas no expression of KCNQ2 and KCNQ3 (KV7.2 and KV7.3; Fig. 2, B and C) was found. All five subtypes were identified in the brain and in the left and right ventricles of the rat heart. KCNQ1, KCNQ3, KCNQ4, and KCNQ5 (KV7.1, KV7.3, KV7.4, and KV7.5) were found in the kidney. In the reverse transcription–negative samples, we did not observe bands of the same size as in the reverse transcription–positive samples, showing that genomic DNA is not responsible for the bands observed in the samples.
Effect of IPC and KV7 Modulation on IS.
The effect of IPC and KV7 modulation on IS in the pressure-regulated hearts is shown in Fig. 3. IPC reduced IS to 42% ± 3% from 76% ± 3% in the control group (P < 0.001). The KV7 inhibitors XE991 and linopirdine reduced IS to 56% ± 5% and 56% ± 4% (P = 0.02), respectively. The combination of IPC and XE991 yielded additional IS reduction to 16% ± 1% (P < 0.0001 versus control, P = 0.0006 versus IPC). An almost similar reduction in IS to 24% ± 5% was achieved with IPC plus linopirdine (P < 0.0001 versus control, P = 0.04 versus IPC). The KV7 activator, flupirtine, did not affect IS (P = 0.13) but abrogated IS reduction by IPC (P < 0.0001).
In the initial 15 minutes of reperfusion, we achieved a mean flow rate of 12.2 ± 0.2 ml/min in the flow-regulated control group versus 14.0 ± 0.8 ml/min in the pressure-regulated control hearts (P = 0.04). In the flow-controlled XE991 and flow-controlled IPC groups, we achieved similar flow rates of 12.1 ± 0.2 ml/min and 12.0 ± 0.3 ml/min, respectively (ANOVA P = 0.46). The fixed flow rate resulted in a 43% reduction in IS from 76% ± 3% in the pressure-regulated control group to 43% ± 6% in the flow-regulated control group (P < 0.001).
IPC reduced IS in flow-regulated hearts from 43% ± 6% to 24% ± 4% (P = 0.03). There was an almost similar relative, but statistically insignificant, IS reduction by XE991 in the flow-regulated hearts from 43% ± 6% to 35% ± 5% (P = 0.46) compared with the IS reduction by XE991 in the pressure-regulated hearts.
Effect of IPC and KV7 Modulation on Hemodynamics.
Hemodynamic results are shown in Table 2. Besides a slightly lower baseline coronary flow rate in the IPC plus XE991 group (P = 0.04), baseline hemodynamic variables did not differ between the study groups. IPC improved recovery of left ventricular developed pressure (LVDP) during reperfusion (P = 0.004). A similar finding was achieved by XE991 (P = 0.025), whereas the improvement of LVDP was not statistically significant in the linopirdine group (P = 0.13). The most pronounced improvements of postischemic LVDP were observed in the groups receiving the combination of IPC and KV7 inhibition (P < 0.001 versus control for both), further establishing the additional cardioprotective effects by combining the two treatments (Table 2). Left ventricular end diastolic pressure during reperfusion was significantly lower in hearts receiving the combined treatment of KV7 channel blockage and IPC, but only insignificantly lowered by XE991 (P = 0.18), linopirdine (P = 0.06), and IPC (P = 0.51) alone. Flupirtine alone neither affected LVDP nor diastolic pressure during reperfusion. However, the beneficial effect of IPC on hemodynamic recovery was abrogated by flupirtine. In the hearts subjected to flow-regulated perfusion, we observed a similar tendency of improved recovery of LVDP by IPC (P = 0.06) and XE991 (P = 0.12) and reduced postischemic left ventricular end diastolic pressure by IPC (P = 0.01) and XE991 (P = 0.12).
XE991 reduced coronary flow rate by 2.9 ± 0.3 ml/min (P < 0.0001), whereas the linopirdine and flupirtine left coronary flow rate was unchanged (Fig. 4A). XE991 and linopirdine reduced the heart rate by 32 ± 8 beats/min (P = 0.0015) and 23 ± 8 beats/min (P = 0.025), respectively (Fig. 4B). No change in heart rate was observed with flupirtine.
Effect of IPC and XE991 on Isolated Coronary Arteries.
Simulated ischemia (1% O2) during IPC and prolonged index ischemia relaxed vasopressin contracted arteries. Reoxygenation (21% O2) initially increased relaxation followed by sustained contraction as seen on the original trace (Fig. 5A). XE991 increased baseline vascular tonus (0.12 ± 0.05 mN versus −0.01 ± 0.02 mN in controls; P = 0.04), but vasoconstriction by vasopressin did not differ between XE991-treated and control arteries (2.0 ± 0.4 mN and 2.0 ± 0.3 mN; P = 0.96). During simulated ischemia, XE991 showed a tendency toward reduction of maximal dilatation in control conditions (P = 0.15, Fig. 5B), and reduced dilatation in IPC-treated arteries (P = 0.05). During reoxygenation, IPC increased dilatation (P = 0.001, Fig. 5C), and XE991 inhibited dilatation by IPC (P = 0.04, Fig. 5C).
The main findings of this study are that all KV7 channel subtypes are present in the myocardium of Wistar rats and that inhibition of KV7 channels is cardioprotective. Both the findings that KV7 channel opening abrogates cardioprotection by IPC as well as the additive protection by KV7 channel inhibition and IPC suggests partially separate pathways of cardioprotection.
Cardioprotection by KV7 Inhibition and IPC.
Expression of KV7 subtypes was previously observed in heart tissue from different species, including KV7.1–KV7.5 in zebrafish (Wu et al., 2014), KV7.1, KV7.4, and KV7.5 in rats (Morales-Cano et al., 2015), and KV7.1– KV7.3 and KV7.5 in humans (Hedegaard et al., 2014). We found expression of all KV7 subtypes in the left and right ventricles of the rat heart. KV7.1, KV7.4, and KV7.5 channels have previously been identified in rat (Khanamiri et al., 2013; Morales-Cano et al., 2015) and porcine (Hedegaard et al., 2014) coronary arteries as well as in extracardiac vessels (Yeung et al., 2007, 2008b; Mackie and Byron, 2008). In mice, KV7.1 and KV7.4 were found in coronary arteries (Lee et al., 2015) and KV7.1, KV7.4, and KV7.5 were found in aorta (Yeung et al., 2007). In human systemic arteries, all but KV7.2 have been observed (Ng et al., 2011). This study confirms expression of the subtypes previously observed in other arteries (e.g., KV7.1, KV7.4, and KV7.5). Blocking KV7 channels with the pan-KV7 blockers XE991 and linopirdine protected against IR injury and this protective effect could be mediated by direct effects on the cardiomyocytes or effects secondary to the vascular effects.
Reduction in heart rate followed by concomitant alterations in cardiac metabolism may explain the cardioprotective effect. However, the effects on heart rate by linopirdine and XE991 were minor and we did not observe any differences in heart rate between the groups in the important phase of early reperfusion. In fact, the mean heart rate was above control level in the linopirdine group. Hence, heart rate alterations seem unlikely to contribute to the cardioprotection elicited by the KV7 channel blockers.
Previous results from KV7 channel inhibition in nerve tissue have indicated antiapoptotic effects of XE991 and linopirdine (Xia et al., 2002; Zhou et al., 2011). A proposed mechanism is inhibition of cell body shrinkage induced by K+ efflux. Cell volume is mainly controlled by ion fluxes across the plasma membrane, and cell shrinkage is an early event in the apoptotic cascade. Thus, when the counteracting Na/K ATPase is impaired (e.g., during ischemia and reperfusion) (Song and Yu, 2014), excessive efflux mediated by overactive K+ channels may lead to uncontrolled cell shrinkage and apoptosis (Bortner et al., 1997; Maeno et al., 2000).
In agreement with previous studies (Murry et al., 1986; Povlsen et al., 2014), a protective effect of IPC was found in our study. ATP-sensitive K channels have been suggested to be involved in the cardioprotective effects of IPC (Liu et al., 1998; Kristiansen et al., 2005), whereas the role of KV7 channels has not previously been investigated. In this study, an opener of KV7.2–KV7.5 (flupirtine) abrogated the effect of IPC. This could suggest that inhibition of KV7 channels is involved in the cardioprotective effect of IPC. One may speculate whether IPC-induced activation of protein kinase C (PKC) leads to inhibition of the KV7 channels (Brueggemann et al., 2014), but PKC phosphorylation of the KV7 channels will only modulate the channel gating. The sensitivities of KV7.1, KV7.2, and KV7.2 plus KV7.3 for XE991 vary from 0.6 to 0.8 µM, whereas the sensitivity is 3–5 µM for KV7.4 channels (Wang et al., 1998; Søgaard et al., 2001). Therefore, 10 µM XE991 will cause approximately 60%–75% inhibition of the KV7.4 channels and 10 µM linopirdine approximately 50% inhibition of KV7.4. That IPC through PKC modulate the KV7 channels but XE991 and linopirdine cause direct inhibition may explain the additive effect of these KV7 channel blockers to IPC in reducing myocardial IS.
Effect on Coronary Flow of KV7 Channel Inhibition.
In agreement with previous findings, we found that pan-KV7 inhibition by XE991 induces coronary vasoconstriction and reduces hypoxic vasodilatation in isolated arteries. XE991 induced vasoconstriction during both the simulated ischemic phase and the reoxygenation phase. In accordance with these findings, XE991 reduced coronary flow rate in the constant pressure perfused hearts. The coronary flow reduction by XE991 administration was not observed with linopirdine. The different effect of the two blockers may be explained by their different inhibitory effects. In rat and mouse intrapulmonary arteries, XE991 is significantly more potent than linopirdine (Joshi et al., 2006). Moreover, XE991 and linopirdine display different selectivity toward the KV7 channel subtypes (Jensen et al., 2005; Yeung et al., 2007, 2008a) in which XE991 has an IC50 against KV7.5 of 60 µM (Jensen et al., 2005; Yeung et al., 2008a), indicating that it is unlikely to be the KV7.5 subtype involved in the cardioprotective effect. XE991 may inhibit other K+ channels, such as KV1.2/KV1.5 and KV2.1/KV9.3 (Zhong et al., 2010). Consequently, an effect mediated by channels other than KV7 cannot be excluded.
The absence of coronary flow reduction by linopirdine suggests that the protection by KV7 inhibition is independent of the vasomotor actions. This interpretation is supported by the results from the flow-controlled Langendorff hearts. Although XE991 failed to reduce IS significantly in the flow-controlled experiments, we observed a similar relative IS reduction in the flow- and pressure-controlled experiments. The lack of significance in the flow-controlled experiments is likely caused by the reduced absolute IS reduction combined with a relatively large variability in the flow-controlled hearts. We found that 100 µM XE991 or linopirdine increased survival in a myocardial cell line (HL-1 cells) subjected to simulated ischemia and reperfusion (Supplemental Fig. 1), further supporting that the cardioprotection is mediated by an effect on the cardiomyocytes rather than the conduction system or secondary vascular effects.
Switching from pressure-regulated to flow-regulated perfusion reduced IS per se. This difference could reflect a relatively gentle initial reperfusion in the flow-regulated hearts compared with the pressure-regulated hearts. Gentle reperfusion exerts well known cardioprotective effects that were first demonstrated by Okamoto et al.  and are suggested to be the mechanistic basis of cardioprotection by ischemic postconditioning . We cannot exclude that the flow reduction mediated by XE991 contributes to the cardioprotective effects, but other mechanisms are prevailing as discussed above.
Potential Clinical Implications and Limitations.
Linopirdine has previously been tested for safety and tolerance as a potential therapeutic agent for treatment of Alzheimer’s disease. Volunteers received single oral doses in a range from 0.5 to 55 mg, which was rapidly absorbed and eliminated. It was well tolerated and the most frequent adverse event was headache. Results of electroencephalograms, ECGs, and clinical laboratory evaluations were not affected (Pieniaszek et al., 1995). Another study administering single oral doses of 30 mg linopirdine observed electroencephalography changes suggesting vigilance-improving properties and no abnormalities in ECG or laboratory evaluations, indicating a good tolerability (Saletu et al., 1989). Although the highest plasma levels were approximately 1.5 µM in those experiments compared with 10 µM of linopirdine in our in vitro experiments, this indicates a potential for clinical use of KV7 blockers.
Flupirtine has been used in Europe for the last 25 years for the management of pain after surgery, trauma, dental extraction, pain associated with muscle spasm, cancer, degenerative joint diseases, and conditions such as headache and dysmenorrhea. It is devoid of adverse effects of routinely used analgesic drugs but is equally efficacious in reducing pain sensation (Harish et al., 2012). Moreover, the selective cyclooxygenase inhibitor, celecoxib, was found to enhance Kv7.2–7.4, Kv7.2/7.3, and Kv7.3/7.5 currents, but it inhibits Kv7.1 and Kv7.1/KCNE1 currents (Macías et al., 2010). Retigabine, another opener of KV7 channels, is used for treatment of refractory partial-onset seizures (Harris and Murphy, 2011) but differs from flupirtine because retigabine at high concentrations (approximately 10–100 µM) also block other voltage-gated K channels (Rundfeldt, 1997; Brown and Passmore, 2009). Our results thus far suggest that patients using flupirtine may not benefit from IPC in the heart, although further investigation will be required to clarify whether this is also the case for celecoxib and retigabine in IR,
The loss of KV7.2 and KV7.3 function enhances the susceptibility to seizures (Qiu et al., 2007), and development of a drug only targeting peripheral KV7 channels could be of clinical interest. KV7.4 and KV7.5 channels form heterodimers at least in the vasculature (Chadha et al., 2014), and future studies should examine the effect of targeting these heterodimers. We administered all drugs in the perfusion buffer both prior to and after ischemia in our in vitro experiments. We are therefore not able to distinguish preischemic from ischemic and postischemic effects of KV7 channel modulation. Possible in vivo interactions have not been investigated.
KV7 channels are present with variable expression in coronary arteries and myocardium. Inhibition of KV7 channels exerts cardioprotection independent of coronary flow changes and opening of KV7 channels abrogates cardioprotection by IPC. Our findings indicate that KV7 blockers have a direct cardioprotective effect suggesting a potential for those in the treatment of IR injury.
The authors thank Casper Carlsen Elkjær, Anja Helveg Larsen, and Susie Mogensen for excellent technical assistance. HL-1 cells were kindly provided by Dr. W.C. Claycomb.
Participated in research design: Hedegaard, Johnsen, Simonsen, Bøtker.
Conducted experiments: Johnsen, Hedegaard, Povlsen, Jespersen, Shanmuganathan, Laursen.
Performed data analysis: Johnsen, Hedegaard, Kristiansen.
Wrote or contributed to the writing of the manuscript: Hedegaard, Johnsen, Simonsen, Bøtker.
- Received November 16, 2015.
- Accepted February 5, 2016.
↵* E.R.H. and J.J. contributed equally to this work and are co-first authors.
The research was supported by the Danish Heart Foundation, the Helga and Peter Kornings Foundation, the Karen Elise Jensens Foundation, the Fondation Leducq [Grant 06CVD], the Danish Research Council for Strategic Research [Grant 11-115818], and the Danish Research Council [Grant 11-108354].
- area at risk
- analysis of variance
- ischemic preconditioning
- ischemia and reperfusion
- infarct size
- voltage-gated potassium channel
- left anterior descending coronary artery
- left ventricular developed pressure
- protein kinase C
- (5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics