Anxiety and orienting of gaze to angry and fearful faces Neuroscience research indicates that individual differences in anxiety may be attributable to a neural system for threat-processing , involving the amygdala , which modulates attentional vigilance , and which is more sensitive to fearful than angry faces . Complementary cognitive studies indicate that high-anxious individuals show enhanced visuospatial orienting towards angry faces , but it is unclear whether fearful faces elicit a similar attentional bias . This study compared biases in initial orienting of gaze to fearful and angry faces , which varied in emotional intensity , in high - and low-anxious individuals . Gaze was monitored whilst participants viewed a series of face-pairs . Results showed that fearful and angry faces elicited similar attentional biases . High-anxious individuals were more likely to direct gaze at intense negative facial expressions , than low-anxious individuals , whereas the groups did not differ in orienting to mild negative expressions . Implications of the findings for research into the neural and cognitive bases of emotion processing are discussed . 1 Introduction Research into the cognitive and neural mechanisms underlying emotion processing indicates neural circuitry , involving subcortical and cortical structures -LRB- including the amygdala and prefrontal cortex -RRB- , which is responsible for detecting threat-related cues in the environment and triggering a variety of cognitive , behavioural and physiological responses , in particular , attentional vigilance -LRB- LeDoux , 1996 ; Davis and Whalen , 2001 -RRB- . Neuroimaging studies show that the amygdala , which plays a central role in this circuitry , is reactive to threat-related cues , such as fearful faces -LRB- e.g. , Whalen et al. , 1998 -RRB- . Individual differences in the operation of this threat-processing system may underlie individual differences in vulnerability to anxiety and anxiety disorders -LRB- Davis and Whalen , 2001 -RRB- . From a clinical perspective , cognitive models of anxiety also assume the existence of a threat-processing system and propose that anxiety is characterized by cognitive biases -LRB- in particular , in stimulus evaluation and selective attention -RRB- which favour the processing of threat cues . These biases are proposed to be responsible for individual differences in anxiety vulnerability -LRB- e.g. , Beck and Emery , 1985 ; Williams et al. , 1997 ; Mogg and Bradley , 1998 -RRB- . Cognitive models , such as Beck and Emery 's -LRB- 1985 -RRB- , are important because they provide the basis for cognitive-behaviour therapy -LRB- CBT -RRB- , which aims to remove these threat-processing biases and is an effective treatment for many anxiety disorders -LRB- e.g. , Ballenger , 1999 -RRB- . Research into cognitive models of anxiety indicates that anxious individuals have an enhanced attentional bias for threat cues , compared with non-anxious individuals , and that this bias operates in early aspects of processing -LRB- e.g. , review by Mogg and Bradley , 1998 -RRB- . In order to understand the functional properties of this postulated threat-processing system , it is necessary to clarify the type of stimuli to which it is sensitive . Neuroimaging research typically indicates that the amygdala is more reactive to fearful than angry faces -LRB- e.g. , Whalen et al. , 2001 ; Blair et al. , 1999 ; Davis and Whalen , 2001 ; but see Yang et al. , 2002 -RRB- . Davis and Whalen -LRB- 2001 -RRB- suggested that fearful faces elicit more amygdala activity because they are ambiguous -LRB- i.e. they signify the presence of danger , but do not provide information about its source -RRB- and that the threat-processing system is more reactive to ambiguous -LRB- or indirect -RRB- threat cues because it is designed to promote attention to stimuli which require more detailed processing in order to determine appropriate responding -LRB- e.g. , escape or approach -RRB- . Neuroimaging evidence has been reported supporting the hypothesis of enhanced processing of ambiguous threat cues , as indexed by amygdala responses to angry and fearful faces -LRB- Adams et al. , 2003 -RRB- . Thus , according to Davis and Whalen 's -LRB- 2001 -RRB- theoretical view , fearful faces should be particularly effective in capturing attention and in eliciting vigilance . Biases in visuospatial orienting to threat faces have been investigated using visual-search , visual-probe and eye-movement paradigms . Visual-search studies require participants to search arrays of faces for discrepant emotional expressions and results have indicated faster detection of schematic angry faces , relative to positive faces -LRB- e.g. , Öhman et al. , 2001 ; Fox et al. , 2000 -RRB- . In the visual-probe task , pairs of faces -LRB- e.g. , angry face and neutral face of the same individual -RRB- are briefly presented , followed by a probe stimulus -LRB- e.g. , small dot -RRB- to which the participant responds . Response times -LRB- RTs -RRB- to probes reflect the allocation of attention to the faces , as RTs are typically faster to probes which appear in attended , rather than unattended , locations . Visual-probe studies provide evidence of enhanced attentional biases for angry faces in individuals with high non-clinical anxiety , and in anxious patients -LRB- e.g. , Bradley et al. , 1998 , 1999 ; Mogg et al. , 2004 -RRB- . They also indicate that the attentional bias increases as the intensity of the angry expression increases -LRB- Wilson and MacLeod , 2003 -RRB- . Eye-movement methodology has extended this research , as it provides a direct and ecologically-valid measure of visual orienting , and further indicates that anxious individuals have a greater bias to direct their gaze initially towards angry faces -LRB- relative to neutral or happy faces -RRB- , compared with non-anxious individuals -LRB- Bradley et al. , 2000 ; Mogg et al. , 2000 -RRB- , which suggests that the attentional bias for threat operates at a relatively early stage of visual processing and guides eye-movements . However , there has been little research using these paradigms to assess biases in visual orienting towards fearful faces . For example , one study using a visual-search task suggested that angry , but not fearful , faces preferentially attract attention -LRB- Williams et al. , 2005 -RRB- , whereas another study using a visual-probe task indicated an attentional bias for fearful faces that had been perceptually degraded -LRB- i.e. low spatial frequency ; Holmes et al. , 2005 -RRB- . Neither study examined the influence of anxiety . Consequently , the main aim of the present study was to compare biases in initial orienting of gaze towards fearful versus angry faces in high - and low-anxious individuals . A second aim was to examine the effect of manipulating the emotional intensity of the faces -LRB- cf. Blair et al. , 1999 ; Wilson and MacLeod , 2003 -RRB- on attentional responses . Neuroimaging research -LRB- Davis and Whalen , 2001 -RRB- predicts that fearful faces should elicit stronger vigilance responses than angry faces . In addition , cognitive models of anxiety predict that high-anxious individuals should show greater biases in initial orienting of gaze towards both angry and fearful faces , than low-anxious individuals . Furthermore , if biases in initial orienting to threat-related cues are primarily a function of the affective salience of the stimuli -LRB- Mogg and Bradley , 1998 -RRB- , then these biases in orienting should increase as the intensity of the fearful and angry expressions increases . 2 Method 2.1 Participants Participants were undergraduates who were screened on the trait version of the State-Trait Anxiety Inventory -LRB- STAI ; Spielberger , 1983 -RRB- ; those with trait anxiety scores of 40 or less were allocated to the low anxiety group , and those scoring 50 or more to the high anxiety group . Additional selection criteria were fluency in spoken English and visual acuity within normal limits . Nine volunteers did not complete the study due to equipment calibration problems and six participants had excessive missing eye-movement data -LRB- described later -RRB- . The final sample comprised 28 participants -LRB- 3 M , 25 F -RRB- in the low-anxious group , and 21 -LRB- 3 M , 18 F -RRB- in the high-anxious group . 2.2 Materials and apparatus Twenty-four face stimuli were selected from the NimStim Set of Facial Expressions -LRB- http://www.macbrain.org/faces/ -RRB- .1 They consisted of angry , fearful and neutral prototypical expressions posed by eight models -LRB- four female and four male numbered : 01f , 03f , 07f , 08f , 21m , 23m , 27m , 34m -RRB- . For each model and type of facial expression -LRB- fearful or angry -RRB- , the emotional face was blended with the neutral face to produce a continuum comprising five exemplars of increasing emotional intensity : i.e. neutral prototype -LRB- 0 % -RRB- , three morphed expressions -LRB- 25 % , 50 % and 75 % -RRB- and the emotional prototype -LRB- 100 % -RRB- ; see Fig. 1 . The preparation of the morphed faces used Gryphon Morph v2 .5 software -LRB- Gryphon Software Corporation , 1994 -RRB- . Each emotional face -LRB- i.e. 25 % , 50 % , 75 % or 100 % exemplar of a fearful or angry face -RRB- was paired with the neutral expression -LRB- 0 % -RRB- of the same model to create 64 face pairs for use in the attentional task . The task was administered using MEL2 software -LRB- Schneider , 1995 -RRB- , Pentium 450 MHz PC , 15 in . VGA monitor and MEL2 response box . Eye-movements were monitored with 120 Hz infrared pan/tilt eye tracking system -LRB- Model 504 , Applied Scientific Laboratories , Bedford , Massachusetts -RRB- and E5000 software -LRB- Applied Science Group , 2000 -RRB- which was run on a Pentium 333 MHz PC . Testing was conducted in a dimly lit room . 2.3 Procedure Participants completed a visual acuity check and were seated 1 m from the monitor , with the eye tracking camera positioned 50 cm in front of them , below the right eye . The equipment was calibrated by displaying the numbers 1 to 9 on the screen in a 3 × 3 array and recording the direction of gaze whilst participants looked at each number in turn . Each trial of the attentional task started with a central fixation-cross shown for 1000 ms , followed by a pair of pictures presented side by side for 500 ms. The pictures measured 90 mm × 110 mm , with their inner edges 45 mm apart . Immediately after the offset of the picture-pair , a probe -LRB- pair of dots : or . . -RRB- was presented in the position of one of the preceding pictures -LRB- visual angle of 7.7 ° between two probe positions -RRB- until a manual response . Participants were asked to press one of two keys as quickly as possible to indicate the type of probe . Inter-trial interval varied randomly between 750 and 1250 ms. Participants were instructed to keep their head still throughout the task and to look at the fixation-cross at the start of each trial . Eye-movement data were recorded from the onset of the fixation-cross until the manual response . There were eight practice trials followed by two blocks , each consisting of two buffer trials and 256 experimental trials , with a short rest-break between the blocks . Across the whole task , the 64 face pairs were presented eight times , balanced for emotional face location and probe location -LRB- left vs. right -RRB- . Trials were presented in a new random order for each participant . If eye calibration quality deteriorated during the task , the task was briefly interrupted to repeat the calibration procedure . After the attentional task , participants completed three face rating tasks : forced-choice discrimination , anger and fear ratings , which were included to check the effectiveness of the manipulation of emotional intensity . Each task presented the faces from the attentional task , one at a time , in random order . Each trial consisted of a central fixation-cross -LRB- 500 ms -RRB- , followed by a face and rating scale , displayed until a keyboard response . In the forced-choice discrimination task , participants indicated whether each face was angry or fearful . In the anger and fear rating tasks , they rated each face for how angry or fearful it appeared on a scale ranging from 0 -LRB- not at all -RRB- to 8 -LRB- extremely -RRB- . Finally , they completed questionnaires including the state and trait versions of the STAI , and short-form Social Desirability Scale -LRB- SDS ; Strahan and Gerbasi , 1972 ; the latter was included because defensiveness can have a confounding effect on measures of anxiety and attentional bias , e.g. , Eysenck , 1997 -RRB- . 2.4 Data preparation 2.4.1 Eye-movement data These were prepared using the Eyenal Data Analysis Program -LRB- Applied Science Group , 2000 -RRB- . Direction of gaze was measured in degrees approximately once every 8 ms. If eye-movements were stable within 1 ° of visual angle for 100 ms or more , this was classified as a fixation to that position . Initial fixations to the pictures were calculated if the following criteria were met : -LRB- i -RRB- gaze was fixated in the central region before picture onset , -LRB- ii -RRB- fixations occurred at least 100 ms after picture onset , and -LRB- iii -RRB- fixations were directed at either the left or right picture -LRB- > 1.3 ° wide of central fixation position on the horizontal plane -RRB- . Six participants had excessive missing data -LRB- 85 % or more of trials lacked identifiable fixations -RRB- , so were excluded from all analyses . The amount of missing data did not vary as a function of anxiety group , F -LRB- 1 , 47 -RRB- < 1.7 , emotional face type , emotional face intensity , or their interaction , F -LRB- 3 , 141 -RRB- < 1.5 , all p 's > .2 . Gaze-direction bias scores were obtained for each participant and each level of intensity of fearful and angry faces by calculating the number of trials in which the first shift of gaze was towards the emotional face , as a proportion of the number of trials in which the initial shift in gaze was made to either the emotional or neutral face -LRB- bias scores > .5 reflect an orienting bias towards the emotional face ; .5 = no bias -RRB- . 2.4.2 RT data Data from error trials -LRB- 2 % -RRB- and RTs < 200 ms or > 1250 ms -LRB- 1 % -RRB- were excluded . The groups did not differ in missing data or overall mean RT , ts < 1 . An inverse transformation was applied to the RT data to reduce the influence of skewness and outliers -LRB- Ratcliff , 1993 -RRB- . Attentional bias scores were calculated using a standard formula -LRB- e.g. , Bradley et al. , 1998 -RRB- , for each participant and each level of intensity of fearful and angry faces , by subtracting the mean RT when the emotional face and probe were in the same location from the mean RT when the emotional face and probe were in different locations . RT bias scores were corrected -LRB- reverse sign and multiply by 106 -RRB- so that they would be easier to comprehend , i.e. positive values of bias scores reflect an attentional bias for emotional faces , relative to neutral faces -LRB- 0 = no bias -RRB- . Kolmogorov -- Smirnov tests showed that the distributions of RT bias scores and gaze-direction scores were within normal limits . 3 Results 3.1 Group characteristics The high - and low-anxious groups differed significantly in trait and state anxiety -LRB- see Table 1 -RRB- , but not in social desirability scores , age or gender ratio . 3.2 Eye-movement data Gaze-direction bias scores were entered into a 2 × 2 × 4 mixed design ANOVA with anxiety group -LRB- high , low -RRB- as the between-subjects independent variable -LRB- IV -RRB- , and emotional face type -LRB- angry , fearful -RRB- and intensity of emotional face -LRB- 25 % , 50 % , 75 % , 100 % -RRB- as within-subject IVs -LRB- see Fig. 2 for means -RRB- . There were significant main effects of anxiety group , F -LRB- 1 , 47 -RRB- = 4.68 , p < .01 , , and emotion intensity , F -LRB- 3 , 141 -RRB- = 10.49 , p < .01 , , which were qualified by a significant anxiety group × emotion intensity interaction , F -LRB- 3 , 141 -RRB- = 3.02 , p < .05 , . There were no other significant results , e.g. , anxiety group × emotion intensity × type of emotional face : F < 1 . Separate ANOVAs of bias scores from each group showed a significant main effect of emotional intensity on gaze direction in the high-anxious group , F -LRB- 3 , 60 -RRB- = 9.54 , p < .001 , , but not in the low-anxious group , F -LRB- 3 , 81 -RRB- = 2.04 , p = .12 , . To clarify further the anxiety group × emotion intensity interaction , the groups were compared on their bias scores at each level of intensity -LRB- averaged across angry and fearful faces -RRB- . There was no significant difference between the high - and low-anxious groups in bias scores for 25 % negative faces , -LRB- Ms = .508 vs. .500 , t < 1 , NS , d = .11 -RRB- , 50 % negative faces -LRB- .533 vs. .533 , t < 1 , NS , d = .003 -RRB- , or 75 % negative faces -LRB- .561 vs. .528 , t -LRB- 47 -RRB- = 1.34 , NS , d = .39 -RRB- . A one-sample t-test was used to contrast the RT bias scores against a value of .5 -LRB- which indicates no bias -RRB- ; results showed that , irrespective of group , participants showed no attentional bias for 25 % negative faces -LRB- .504 , t < 1 , NS , d = .05 -RRB- , but a significant bias for 50 % negative faces -LRB- .533 , t -LRB- 48 -RRB- = 2.99 , p < .01 , d = .43 -RRB- and also for 75 % negative faces -LRB- .542 , t -LRB- 48 -RRB- = 3.39 , p < .01 , d = .48 -RRB- . For 100 % negative faces , the high-anxious group showed a significantly greater bias in orienting towards them , compared with the low-anxious group -LRB- .629 vs. .547 , t -LRB- 47 -RRB- = 3.21 , p < .01 , d = .91 -RRB- . One-sample t-tests showed a significant bias in gaze towards 100 % negative faces , relative to neutral faces , in both the high-anxious -LRB- .629 , t -LRB- 20 -RRB- = 5.66 , p < .001 , d = 1.24 -RRB- and low-anxious -LRB- .547 , t -LRB- 27 -RRB- = 3.26 , p < .01 , d = .62 -RRB- groups . Thus , the bias for participants to direct their gaze initially at prototypical -LRB- 100 % -RRB- negative faces , was significantly stronger in the high-anxious group . 3.3 Manual RT data RT bias scores were entered into a 2 × 2 × 4 mixed design ANOVA with anxiety group , type of emotional face and intensity of emotional face as IVs . There was a significant group × emotion intensity interaction , F -LRB- 3 , 141 -RRB- = 2.83 , p < .05 , , and no other significant results -LRB- e.g. , group × intensity × face type : F < 1 -RRB- . The pattern of significant findings corresponded to that in the eye movement data . The high - and low-anxious groups did not significantly differ in RT bias scores for 25 % negative faces -LRB- − 7.4 vs. 16.4 , t -LRB- 47 -RRB- = 1.40 , NS , d = .40 -RRB- , 50 % negative faces -LRB- 13.1 vs. 19.0 , t < 1 , NS , d = .11 -RRB- , or 75 % negative faces -LRB- 4.5 vs. 24.9 , t -LRB- 47 -RRB- = 1.17 , NS , d = .33 -RRB- . Contrasts of RT bias scores against zero showed that , irrespective of group , participants showed no attentional bias for 25 % negative faces -LRB- 6.2 vs. zero , t < 1 , d = .10 -RRB- , a significant bias for 50 % negative faces -LRB- 16.4 , t -LRB- 48 -RRB- = 2.12 , p < .05 , d = .30 -RRB- , and a near-significant bias for 75 % negative faces -LRB- 16.2 , t -LRB- 48 -RRB- = 1.87 , p < .07 , d = .27 -RRB- . For 100 % negative faces , there was a significant group difference in attentional bias -LRB- t -LRB- 47 -RRB- = 2.04 , p < .05 , d = .60 -RRB- . Contrasts of the bias scores against zero showed a significant attentional bias for 100 % negative faces in the high-anxious group -LRB- 37.4 , t -LRB- 48 -RRB- = 3.48 , p < .01 , d = .76 -RRB- , but not in the low-anxious group -LRB- 4.2 , t < 1 , d = .07 -RRB- . The RT bias and gaze-direction bias scores for 100 % negative faces significantly correlated with each other -LRB- r = .35 , p < .05 -RRB- . There were no significant associations between these two measures of attentional bias for negative faces at lower levels of emotion intensity -LRB- r = .11 , .10 and .13 for 25 % , 50 % and 75 % negative faces , respectively , all NS -RRB- .2 3.4 Rating tasks 3.4.1 Forced choice discrimination task A 2 -LRB- anxiety group -RRB- × 2 -LRB- emotional face type -RRB- × 4 -LRB- intensity of emotional face3 -RRB- ANOVA of the proportion of faces correctly classified as angry or fearful showed a significant main effect of intensity , F -LRB- 3 , 141 -RRB- = 173 , p < .001 , , as , unsurprisingly , more intense emotional expressions were classified more accurately . Mean proportion of correctly classified negative faces with 25 % , 50 % , 75 % and 100 % intensity was .70 , .94 , .97 and .96 , respectively . There were no other significant results -LRB- e.g. , group × intensity , F -LRB- 4 , 188 -RRB- = 1.26 , p > .25 ; group × intensity × face type : F < 1 -RRB- . 3.4.2 Anger ratings A 2 -LRB- anxiety group -RRB- × 5 -LRB- emotion intensity -RRB- ANOVA of ratings of faces from the anger continuum showed only a significant main effect of intensity , F -LRB- 4 , 188 -RRB- = 594 , p < .001 , , with no effects involving group , Fs < 1 . Mean anger ratings of 0 % , 25 % , 50 % , 75 % and 100 % angry faces were 1.6 , 2.8 , 4.8 , 6.2 and 7.0 , respectively . Bonferroni-corrected contrasts showed that each mean differed significantly from each other , p < .005 . A 2 × 5 ANOVA of anger ratings of faces from the fear continuum showed only a significant effect of intensity , F -LRB- 4 , 188 -RRB- = 11.63 , p < .001 , , with no effects involving group , Fs < 1 . Mean anger ratings of 0 % , 25 % , 50 % , 75 % and 100 % fearful faces were 1.6 , 1.1 , 0.8 , 0.8 and 0.7 , respectively . Bonferroni-corrected contrasts showed that faces with fearful content -LRB- i.e. 25 -- 100 % -RRB- were rated as less angry than neutral faces , p < .005 . 3.4.3 Fear ratings A 2 × 5 ANOVA of fear ratings of faces from the fear continuum showed a significant main effect of intensity , F -LRB- 4 , 188 -RRB- = 515 , p < .001 , , with no effects involving group -LRB- group : F < 1 ; group × intensity : F -LRB- 4 , 188 -RRB- = 1.20 , NS -RRB- . Mean fear ratings of 0 % , 25 % , 50 % , 75 % and 100 % fearful faces were 1.3 , 2.3 , 4.4 , 5.9 , and 6.6 , respectively . Bonferroni-corrected contrasts showed that each mean differed significantly from each other , p < .005 . A 2 × 5 ANOVA of fear ratings of faces from the anger continuum showed a significant main effect of emotion intensity , F -LRB- 4 , 188 -RRB- = 2.72 , p < .05 , , and no effects involving group , Fs < 1 . Mean fear ratings of the 0 % , 25 % , 50 % , 75 % and 100 % angry faces were 1.3 , 1.5 , 1.2 , 1.0 and 0.9 , respectively . Bonferroni-corrected contrasts did not reveal significant differences between these ratings . 4 Discussion The present findings indicate that fearful and angry faces elicited similar biases in visuospatial orienting . There was a greater tendency for participants to direct gaze initially towards faces with moderate or intense threat-related facial expressions -LRB- 50 -- 100 % intensity -RRB- , relative to neutral faces , whereas mild threat-related facial expressions -LRB- 25 % intensity -RRB- did not elicit a bias in initial orienting . Moreover , visuospatial orienting to both fearful and angry faces was significantly influenced by individual differences in anxiety : high-anxious individuals showed a greater tendency to direct gaze at prototypical -LRB- 100 % -RRB- threat-related faces , irrespective of whether the faces depicted fear or anger . The secondary measure of attentional bias , which was obtained from manual RTs , showed a similar pattern of results to that obtained in the eye-movement data . The enhanced attentional bias in high-anxious individuals , which was found for prototypical angry faces , is compatible with previous findings , e.g. , from visual-probe or eye-movement studies -LRB- e.g. , Mogg and Bradley , 1998 ; Mogg et al. , 2000 -RRB- . The present results further indicate that high - and low-anxious individuals did not differ significantly in visual orienting to threat-related faces which had weaker emotional expressions . This may explain why some studies might fail to find evidence of an effect of individual differences in anxiety on attentional bias , if they use less salient exemplars of emotional facial expressions than the prototypical faces used here . The finding that fearful and angry faces elicited a similar pattern of attentional bias is consistent with cognitive and neural models which posit the existence of a threat-processing system which modulates vigilance for potential sources of danger . Previous research using prototypical expressions suggests that this bias in visual orienting of gaze is specific to threat-related faces , as , for example , anxious patients have a greater bias to shift their gaze initially towards angry faces , but not happy or sad faces , compared with non-anxious controls -LRB- Mogg et al. , 2000 -RRB- . Given this previous finding , the present study did not include these additional face types ; moreover , these would have made the task too long and fatiguing for participants . The finding of an equivalent pattern of attentional bias for fearful and angry faces is not consistent with expectation from some recent neuroimaging research findings -LRB- discussed earlier -RRB- which indicated that the amygdala , which is proposed to modulate attention to threat-related cues , is more sensitive to fearful than angry faces -LRB- Davis and Whalen , 2001 -RRB- . However , this might be resolved by considering the different component processes of attention , namely , shifting versus maintenance -LRB- LaBerge , 1995 ; Serences et al. , 2005 -RRB- . Thus , the amygdala may indeed modulate attention to threat , but its level of activation may be a function of both initial orienting and maintained attention . Fearful and angry faces may have a similar capacity to attract attention initially -LRB- as indicated by the present findings -RRB- , but may differ in the extent to which they hold attention . After initial orienting to angry faces , attention may be subsequently directed away from them , e.g. , due to emotion-regulation processes -LRB- Mogg and Bradley , 1998 ; Rohner , 2002 ; Garner et al. , 2006 -RRB- ; whereas attention may be maintained longer on fearful faces if they require more detailed processing to determine appropriate responding , as suggested by Davis and Whalen -LRB- 2001 -RRB- . This hypothesis could be addressed by both cognitive and neuroimaging studies : for example , eye-movement studies , which employ longer display times to assess the time-course of attentional responses -LRB- e.g. , > 1 s -RRB- , could compare biases in both initial orienting and gaze dwell time for fearful versus angry faces .4 Neuroimaging studies could examine whether amygdala response to angry faces is positively associated with initial orienting to threat , but inversely associated with subsequent attentional avoidance ; as suggested by research indicating that amygdala activity is suppressed by inhibitory influences of the prefrontal cortex , which mediates emotion-regulatory processes including attention control -LRB- Nomura et al. , 2004 ; Ochsner and Gross , 2005 -RRB- . Thus , although many neuroimaging studies have focused largely on the role of the amygdala in emotion processing , it is important to consider it within the context of a broader network involving other structures -LRB- e.g. , prefrontal cortex , thalamus , hippocampus , periaqueductal gray , locus coeruleus -RRB- , with which it has reciprocal connections and which subserve a variety of functions , including attention , startle , escape and avoidance -LRB- Davis and Whalen , 2001 ; McNaughton and Corr , 2004 -RRB- . The results from the face rating tasks confirmed the content validity of the morphed face stimuli , which were well-differentiated in emotion intensity . The groups did not differ in their explicit ratings of the faces , which is compatible with previous research that has also failed to reveal anxiety-related differences in ratings of emotional faces , and which might be due to demand or social desirability effects obscuring anxiety-related effects on self-report data -LRB- e.g. , Wilson and MacLeod , 2003 -RRB- . These null results contrast with those from the attentional task which indicated that individual differences in anxiety played an important role in determining the processing of emotional stimuli , namely , in visual orienting . Thus , to clarify the neurocognitive mechanisms underlying emotion processing , it would seem helpful for neuroimaging studies to make greater use of tasks that are sensitive to individual differences in anxiety , such as attentional paradigms -LRB- e.g. , Monk et al. , 2006 -RRB- , and also to routinely assess individual differences in anxiety , given that this is an important determinant of attentional responses to threat .

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O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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Inhibition of the HERG potassium channel by the tricyclic antidepressant doxepin HERG -LRB- human ether-à-go-go-related gene -RRB- encodes channels responsible for the cardiac rapid delayed rectifier potassium current , IKr . This study investigated the effects on HERG channels of doxepin , a tricyclic antidepressant linked to QT interval prolongation and cardiac arrhythmia . Whole-cell patch-clamp recordings were made at 37 °C of recombinant HERG channel current -LRB- IHERG -RRB- , and of native IKr ` tails ' from rabbit ventricular myocytes . Doxepin inhibited IHERG with an IC50 value of 6.5 ± 1.4 μM and native IKr with an IC50 of 4.4 ± 0.6 μM . The inhibitory effect on IHERG developed rapidly upon membrane depolarization , but with no significant dependence on voltage and with little alteration to the voltage-dependent kinetics of IHERG . Neither the S631A nor N588K inactivation-attenuating mutations -LRB- of residues located in the channel pore and external S5-Pore linker , respectively -RRB- significantly reduced the potency of inhibition . The S6 point mutation Y652A increased the IC50 for IHERG blockade by ∼ 4.2-fold ; the F656A mutant also attenuated doxepin 's action at some concentrations . HERG channel blockade is likely to underpin reported cases of QT interval prolongation with doxepin . Notably , this study also establishes doxepin as an effective inhibitor of mutant -LRB- N588K -RRB- HERG channels responsible for variant 1 of the short QT syndrome . 1 Introduction Diverse cardiac and non-cardiac drugs are associated with prolongation of the rate-corrected QT -LRB- QTc -RRB- interval of the electrocardiogram and with a risk of the potentially fatal arrhythmia Torsade de Pointes -LRB- TdP -RRB- -LSB- 1 -- 4 -RSB- . The majority of such agents exert a common action of inhibiting the cardiac rapidly activating delayed rectifier K + current -LRB- IKr -RRB- . IKr is a major determinant of ventricular action potential repolarization and , thereby , of the QTc interval -LSB- 2,4,5 -RSB- . The pore-forming subunit of IKr channels is encoded by HERG -LRB- human ether-à-go-go-related gene -LSB- 6,7 -RSB- -RRB- . HERG channels appear to have a larger pore cavity than other -LRB- Kv -RRB- six transmembrane domain K + channels and possess particular aromatic amino-acid residues in the S6 region of the channel -LSB- 5,8 -- 10 -RSB- . These features combine to confer a high susceptibility to pharmacological blockade upon the HERG channel . Indeed , the association between drug-induced QTc interval prolongation and pharmacological blockade of HERG channels is sufficiently strong that drug-screening against recombinant HERG channels is now an important component of cardiac safety-pharmacology during drug development -LSB- 11 -- 13 -RSB- . Doxepin is a tricyclic antidepressant -LRB- TCA -RRB- structurally related to amitriptyline and imipramine that combines antidepressant and sedative actions -LSB- 14 -RSB- . Initially doxepin was suggested to have better cardiac safety than other TCA drugs -LSB- 15 -RSB- . However , a subsequent review of the clinical and animal data -LSB- 16 -RSB- and a study in depressed patients -LSB- 17 -RSB- found no evidence that doxepin had fewer cardiovascular effects than other TCAs . Moreover , similar to other TCAs , doxepin can be cardiotoxic in overdose -LSB- 14 -RSB- . Adverse cardiac effects associated with doxepin include premature ventricular complexes -LRB- PVCs -RRB- and wide QRS complexes on the electrocardiogram . Furthermore , there are a number of documented cases of QT interval prolongation with doxepin -LSB- 18 -- 20 -RSB- . For example , in overdose doxepin has been associated with a greatly prolonged QTc interval -LRB- reaching 580 ms -RRB- and TdP -LSB- 19 -RSB- , whilst QT interval prolongation and syncope have been reported for doxepin in combination with methadone and β blocker use -LSB- 20 -RSB- . At present there is no information on the basis for QTc prolongation with doxepin . However , other TCAs including imipramine and amitryptiline have been demonstrated to inhibit recombinant HERG channels -LSB- 21 -- 23 -RSB- and we hypothesised that doxepin is also likely to act as an inhibitor of HERG channel current -LRB- IHERG -RRB- . The present study was conducted to test this hypothesis and to characterise the nature of any observed IHERG blockade . 2 Methods 2.1 Maintenance of mammalian cell lines stably expressing wild-type and mutant HERG channels Experiments on wild-type HERG were performed on a cell line -LRB- Human Embryonic Kidney ; HEK 293 -RRB- stably expressing HERG -LRB- donated by Dr Craig January , University of Wisconsin -LSB- 24 -RSB- -RRB- , except for those in Fig. 8 ; these utilised a cell line stably expressing lower levels of HERG developed in this laboratory , for use with high external -LSB- K + -RSB- -LRB- for comparison with the F656A mutant , see Section 2.2 below -RRB- . Cell lines stably expressing HERG and its mutants , F656A and Y652A were maintained as described previously -LSB- 25 -RSB- . Cell lines stably expressing the S631A -LSB- 26 -RSB- and N588K -LSB- 27 -RSB- mutants were made from appropriately mutated HERG sequences using previously described methods -LSB- 25 -RSB- . Cells were passed using a non-enzymatic dissociating agent -LRB- Splitase , AutogenBioclear -RRB- and plated out onto small sterilised glass coverslips in 30 mm petri dishes containing a modification of Dulbecco 's modified Eagle 's medium with Glutamax-1 -LRB- DMEM ; Gibco , Gibco/Invitrogen , Paisley , UK -RRB- , supplemented with 10 % fetal bovine serum -LRB- Gibco -RRB- , 400 μg ml − 1 gentamycin -LRB- Gibco -RRB- and 400 μg ml − 1 geneticin -LRB- G418 ; Gibco -RRB- . Treatment of the mutant cell lines was identical to treatment of the wild-type cell line except that cultures were maintained with 800 μg ml − 1 of hygromycin . The cells were incubated at 37 °C for a minimum of two days prior to any electrophysiological study . 2.2 Experimental solutions Whole-cell patch-clamp measurements of wild-type -LRB- WT -RRB- and mutant IHERG were made at 37 ± 1 °C . Once in the experimental chamber cells were superfused with a standard extracellular Tyrode 's solution containing -LRB- in mM -RRB- : 140 NaCl , 4 KCl , 2.5 CaCl2 , 1 MgCl2 , 10 glucose , 5 HEPES , -LRB- titrated to pH 7.45 with NaOH -RRB- . Similar to other studies from our laboratory -LRB- e.g. -LSB- 28,29 -RSB- -RRB- , for experiments employing the S6 mutant F656A -LRB- which shows comparatively low levels of channel expression -LSB- 8,25 -RSB- -RRB- and its WT control , the external solution contained 94 mM KCl -LRB- the NaCl concentration was correspondingly reduced -RRB- . Experimental solutions were applied using a home-built , warmed , solution delivery system that exchanged the solution surrounding a cell in < 1 s. Doxepin powder -LRB- Sequoia Research Products and Sigma-Aldrich -RRB- was dissolved in Tyrode 's solution to produce initial stock solutions of either 10 or 50 mM , which were serially diluted to produce working solutions ranging from 0.1 μM to 1 mM . The pipette dialysis solution for IHERG measurement contained -LRB- in mM -RRB- : 130 KCl , 1 MgCl2 , 5 EGTA , 5 MgATP , 10 HEPES -LRB- titrated to pH 7.2 with KOH -RRB- -LSB- 28,29 -RSB- . Patch-pipettes were heat-polished to 2.5 -- 4 MΩ . No correction was made for the ` pipette-to-bath ' liquid junction potential , which was measured to be − 3.2 mV . 2.3 Experiments on rabbit isolated ventricular myocytes One series of experiments was performed to investigate blockade by doxepin of native IKr from adult ventricular myocytes -LRB- Results , Fig. 2 -RRB- . For these , male New Zealand white rabbits -LRB- 2 -- 3 kg -RRB- were killed humanely in accordance with UK Home Office legislation . Ventricular myocytes were then isolated by a combination of mechanical and enzymatic dispersion , using previously described methods -LSB- 30,31 -RSB- . Pipette and external solutions for IKr measurement were identical to those described above for IHERG measurement . 2.4 Electrophysiological recording and analysis Whole-cell patch-clamp recordings were made using Axopatch 200 or 200B amplifiers -LRB- Axon Instruments -RRB- and a CV201 head-stage . Between 75 and 80 % of the pipette series resistance was compensated . Voltage-clamp commands were generated using ` WinWCP ' -LRB- John Dempster , Strathclyde University -RRB- , Clampex 8 -LRB- Axon Instruments -RRB- , or ` Pulse ' software -LRB- HEKA Electronik -RRB- . Data were recorded either via a Digidata 1200B interface -LRB- Axon Instruments -RRB- or an Instrutech VR-10B interface and stored on the hard-disk of a personal computer . The voltage-protocols employed for specific experiments are described either in the relevant ` results ' text , or are shown diagrammatically on the relevant figures ; unless otherwise stated in the text , the holding membrane potential between experimental sweeps was − 80 mV . Data are presented as mean ± S.E.M. Statistical comparisons were made using , as appropriate , paired and unpaired t-tests or one-way analysis of variance -LRB- Anova -RRB- -LRB- Prism 3 or Instat , Graphpad Inc. -RRB- . P values of less than 0.05 were taken as significant ; ns = no statistically significant difference . The following equations were used for numerical analysis and graphical fits to data : The extent of IHERG inhibition by differing concentrations of doxepin was determined using the equation : where ` Fractional block ' refers to the degree of inhibition of IHERG by a given concentration of doxepin ; IHERG -- DOXEPIN and IHERG -- CONTROL represent current amplitudes in the presence and absence of doxepin . Concentration -- response data were fitted by a standard Hill equation of the form : where IC50 is -LSB- DOXEPIN -RSB- producing half-maximal inhibition of the IHERG tail and h is the Hill coefficient for the fit . Half-maximal voltage values for IHERG activation were obtained by fitting IHERG tail-voltage -LRB- I -- V -RRB- relations with a Boltzmann distribution equation of the form : where I is the IHERG tail amplitude following test potential Vm , Imax the maximal IHERG tail observed during the protocol , V0 .5 the potential at which IHERG was maximally activated , and k is the slope factor describing IHERG activation . Data from each individual experiment were fitted by this equation to derive V0 .5 and k values in ` control ' and with doxepin . The resultant mean V0 .5 and k values obtained from pooling values from each experiment were then used to calculate the mean activation relations plotted in Fig. 3 . Parameters describing voltage-dependent inactivation of IHERG were derived from fits to voltage-dependent availability plots with the equation : where ` inactivation parameter ' at any test potential , Vm , occurs within the range 1 -- 0 , V0 .5 is the voltage at which IHERG was half-maximally inactivated and k describes the slope factor for the relationship . 3 Results 3.1 Doxepin produces concentration-dependent inhibition of WT IHERG IHERG was elicited by the protocol shown in the inset of Fig. 1A , which is a standard protocol used to study IHERG pharmacology in this laboratory -LRB- e.g. -LSB- 28,29,32 -RSB- -RRB- . Membrane potential was stepped from − 80 to +20 mV for 2 s followed by a 4-s step to − 40 mV to elicit IHERG tails . A brief -LRB- 50 ms -RRB- pulse to − 40 mV preceded the step to +20 mV in order to monitor the instantaneous current without activation of IHERG -LRB- peak outward IHERG tails on repolarization to -- 40 mV were compared with the instantaneous current during the 50 ms pulse to − 40 mV , in order to measure IHERG tail amplitude -RRB- . This voltage protocol was applied repeatedly -LRB- at 20 s intervals -RRB- prior to and during the application of doxepin . Fig. 1A shows a representative recording of IHERG in control , 5 min after the addition of 10 μM doxepin and 5 min following wash-out of the drug . Doxepin produced a substantial inhibition of IHERG ; this was largely reversible upon washout -LRB- to 73 ± 9 % of control in six cells to which 10 μM doxepin was applied -RRB- . Fig. 1B shows records for the same cell before and at increasing times during doxepin exposure , indicating that blockade was maximal within 4 -- 5 min of drug application . Fig. 1C shows a plot of mean ± S.E.M. fractional block of IHERG tails by four different concentrations of doxepin , fitted by Eq . -LRB- 2 -RRB- . The IC50 for doxepin inhibition of IHERG with this protocol was 6.5 ± 1.4 μM and the Hill coefficient for the fit was 1.0 ± 0.2 . IHERG blockade by doxepin was not accompanied by statistically significant changes to the time-course of deactivation of the time-dependent component of IHERG tails on repolarization to − 40 mV : bi-exponential fitting of the time-dependent tail current decline yielded fast time-constants of deactivation -LRB- τfast -RRB- of 203 ± 15 and 273 ± 21 ms in control solution and 10 μM doxepin , respectively -LRB- n = 6 ; p > 0.05 , paired t-test -RRB- , and slow time-constants of deactivation -LRB- τslow -RRB- of 1164 ± 141 and 1499 ± 131 ms -LRB- p > 0.1 -RRB- . Doxepin did not influence significantly the relative proportions of deactivating current described by the fast and slow time-constants of deactivation -LRB- the proportion of deactivating current fitted by the τfast was ∼ 0.6 in both control and doxepin -RRB- . It has been suggested that pharmacological inhibition of IHERG by some drugs may vary between different stimulus protocols -LSB- 33 -RSB- . Therefore , we also investigated IHERG blockade by doxepin using an action potential -LRB- AP -RRB- voltage waveform -LSB- 32,34 -RSB- . The voltage-command used -LRB- shown in the lower panel of Fig. 2A -RRB- was a previously acquired , digitised AP from a rabbit ventricular myocyte . This was applied repeatedly -LRB- at 4 s intervals -RRB- from a holding potential of − 80 mV -LSB- 32 -RSB- . The upper panel of Fig. 2A shows traces in the absence and presence of doxepin . Peak outward IHERG during the repolarizing phase of the AP was inhibited 60 ± 12 % -LRB- n = 5 -RRB- by 10 μM doxepin , which does not differ significantly from the extent of IHERG tail current blockade by this concentration obtained using the protocol shown in Fig. 1 -LRB- 62 ± 4 % ; n = 6 ; p > 0.8 ; unpaired t-test -RRB- . In order to determine effects of doxepin on native IKr , three drug concentrations -LRB- 1 , 10 and 100 μM -RRB- were applied to ventricular myocytes under whole-cell patch clamp . The command protocol for these experiments -LRB- similar to -LSB- 32 -RSB- -RRB- is shown in Fig. 2B and IKr tails were monitored on repolarization from +20 to − 40 mV . Under our conditions , the outward tail currents observed on repolarization to − 40 mV were almost completely abolished by 1 μM dofetilide -LRB- 91 ± 3 % blockade ; n = 7 -RRB- , verifying that these were carried by IKr with little or no contamination from overlapping currents . Fig. 2C shows representative IKr tails on repolarization from +20 to − 40 mV in the absence and presence of 100 μM doxepin ; at this concentration the current was largely abolished . Fig. 2D contains mean ± S.E.M fractional block data for the tested doxepin concentrations , fitted by Eq . -LRB- 2 -RRB- . The derived IC50 for doxepin inhibition of native IKr tails was 4.4 ± 0.6 μM , in good agreement with the observed inhibitory potency of doxepin on IHERG . 3.2 Voltage dependence of IHERG blockade by doxepin Voltage dependence of IHERG blockade by doxepin was determined by the application , in control and doxepin-containing solutions , of a series of 2 s duration depolarizing commands to a range of test potentials up to +40 mV -LSB- 32 -RSB- . Successive command pulses were applied at 20 s intervals . Representative control currents at selected command voltages are shown in Fig. 3Ai ; currents from the same cell following equilibration in 10 μM doxepin are shown in Fig. 3Aii -LRB- upper traces ; lower traces in each panel show corresponding voltage commands -RRB- . At all test potentials , IHERG was inhibited by doxepin . For each of five similar experiments , fractional inhibition of IHERG tails following each voltage command was calculated using Eq . -LRB- 1 -RRB- ; the mean ± S.E.M. fractional block of IHERG tails is plotted against command voltage in Fig. 3B . Also shown in Fig. 3B are voltage-dependent activation relations for IHERG in control solution and in the presence of doxepin -LRB- see Section 2 -RRB- . The derived mean V0 .5 and k values were : control V0 .5 = − 21.0 ± 3.1 ; doxepin V0 .5 = − 24.0 ± 2.2 , p > 0.1 ; control k = 5.9 ± 0.2 ; doxepin k = 7.5 ± 1.7 , p > 0.3 , with the IHERG activation relations in control and doxepin closely overlying one another -LRB- Fig. 3B -RRB- . Fractional block of IHERG showed no statistically significant dependence on voltage over the range from − 40 to + 40 mV -LRB- p > 0.2 ; Anova -RRB- . The voltage dependence of IHERG availability/inactivation was assessed using a 3-step protocol similar to those used in previous IHERG investigations from our laboratory -LRB- -LSB- 32,35 -RSB- , and shown schematically as an inset to Fig. 3C -RRB- . Mean data from five experiments were corrected for deactivation -LSB- 35 -RSB- and the resulting values were plotted to give availability plots in the absence and presence of doxepin -LRB- Fig. 3C -RRB- ; the data-sets were fitted with Eq . -LRB- 4 -RRB- -LRB- see Section 2 -RRB- . In control and doxepin the inactivation V0 .5 values were , respectively , − 37.9 ± 2.4 and − 43.5 ± 4.1 mV -LRB- ns , paired t-test -RRB- , with corresponding k values of − 16.2 ± 2.4 and − 17.3 ± 8.8 mV -LRB- ns , paired t-test -RRB- . Thus , doxepin did not alter the voltage-dependence of IHERG inactivation . Although there was a trend towards an acceleration in the time-course of IHERG inactivation , this did not attain statistical significance -LRB- inactivation time -- constant of 3.3 ± 1.2 ms in control versus 1.4 ± 0.6 ms in doxepin , p > 0.1 ; obtained from exponential fits to the inactivating phase of the current elicited during the third step of the protocol , following a brief hyperpolarizing step to − 80 mV -RRB- . 3.3 Time dependence of IHERG inhibition by doxepin Gating-dependence of IHERG inhibition by doxepin was investigated further by the use of two protocols to examine the time-dependence of development of IHERG blockade . The first protocol used a sustained -LRB- 10 s -RRB- depolarizing step to 0 mV from a holding potential of − 80 mV -LRB- shown in Fig. 4A -RRB- . This protocol was applied first in the absence of doxepin to elicit control IHERG . It was then discontinued whilst cells were equilibrated in 10 μM doxepin -LRB- for 7 min -RRB- , after which it was re-applied . The first current trace recorded on resumption of stimulation was used to determine development of fractional blockade throughout the applied depolarization . Fig. 4A shows representative traces in control and doxepin ; these traces diverged rapidly following depolarization , suggesting that IHERG blockade developed rapidly with time . Fig. 4B shows plots of mean ± S.E.M. fractional block of IHERG at various time intervals throughout the applied depolarization -LRB- main panel ; n = 8 -RRB- and on an expanded time-scale to show development of blockade over the first 0.5 s -LRB- inset -RRB- . These plots show clearly that IHERG blockade developed rapidly on depolarization , with little change in blockade after 200 -- 300 ms following the onset of the voltage-command . The time-course of development of blockade was well-described by a mono-exponential fit to the data , with a rate constant -LRB- K -RRB- for the fit of 16.33 s − 1 -LRB- equivalent to a time-constant -LRB- 1/K -RRB- of 61 ms -RRB- . Although the protocol used in Fig. 4 is well suited for examining the development of IHERG inhibition over a period of seconds following membrane depolarization , it is less well suited for accurate assessment of blockade of IHERG immediately following membrane depolarization than are protocols based on tail current measurements -LSB- 29,32 -RSB- . Therefore , a second protocol was also used -LSB- 29 -RSB- . Membrane potential was held at -- 100 mV and , from this , 10 and 200 ms duration depolarizations to +40 mV were applied , each followed by a period at − 40 mV to monitor IHERG tails -LSB- 29 -RSB- . Fig. 5A shows representative currents from the same cell activated by 10 ms -LRB- Fig. 5Ai -RRB- and 200 ms -LRB- Fig. 5Aii -RRB- commands in the absence and presence of doxepin . Some IHERG tail inhibition was evident following the 10 ms duration command , with further blockade evident after the 200 ms command . Fig. 5B shows mean fractional block of IHERG tails for five similar experiments . Whilst blockade for 200 ms commands was significantly greater than that for 10 ms commands -LRB- p < 0.02 -RRB- , the occurrence of some blockade with only a very brief duration depolarizing command is concordant with either a very rapidly developing gating-dependent blockade on depolarization or with a contribution of closed-channel block to the overall effect of doxepin -LSB- 25,29,36 -RSB- . 3.4 Effect of inactivation-attenuating mutants on IHERG inhibition by doxepin In order to investigate further the gating-dependence of IHERG blockade by doxepin , experiments were performed using two inactivation-attenuating HERG mutants : S631A and N588K . Residue S631 is located towards the outer mouth of the HERG channel pore , and the S631A -LRB- serine → alanine -RRB- mutation has been reported to shift IHERG inactivation by ∼ +100 mV -LSB- 26 -RSB- . Residue N588 is located in the external S5-Pore linker of the channel , and the N588K -LRB- asparagine → lysine -RRB- mutation , which is responsible for one form of the recently identified genetic short QT syndrome -LSB- 27 -RSB- , has been reported to shift IHERG inactivation by ∼ +60 to +100 mV -LSB- 35,37 -RSB- . The effect of each mutation on the potency of IHERG blockade by doxepin was determined using the same protocol as was used to establish concentration-dependence of WT IHERG blockade -LRB- Fig. 1 -RRB- . Fig. 6Ai shows representative currents in control and 10 μM doxepin for WT -- HERG , whilst Fig. 6Aii and Aiii show similar records for S631A -- HERG and N588K -- HERG , respectively . In contrast to WT -- HERG , for both S631A -- HERG and N588K -- HERG the IHERG tail magnitude was substantially smaller than the maximal current during the voltage-command -LRB- Fig. 6Aii and Aiii , respectively -RRB- reflecting the greatly attenuated IHERG inactivation of these HERG mutants -LSB- 26,35,37 -RSB- . Both mutant channels retained the ability to be inhibited by doxepin . Exponential fits to currents activated on depolarization to +20 mV for both mutants showed similar time-courses of current activation in control solution and following equilibration with 10 μM doxepin -LRB- S631A control : 60.6 ± 5.5 ms ; doxepin : 64.5 ± 8.0 ms , p > 0.5 ; N588K control : 49.8 ± 6.2 ; doxepin : 41.7 ± 9.9 ms , p > 0.5 ; n = 5 for both -RRB- . Three doxepin concentrations -LRB- 1 , 10 and 100 μM -RRB- were tested to obtain concentration-response data . Fig. 6B shows concentration-response relations -LRB- determined using Eq . -LRB- 2 -RRB- -RRB- for IHERG tail inhibition for both S631A -- HERG and N588K -- HERG , with corresponding data for WT -- HERG plotted for comparison . Although the S631A and N588K mutations produced small and modest increases , respectively , in the IC50 derived from the fits to the concentration-response relations -LRB- from 6.6 μM for WT to 8.6 μM for S631A -- HERG and 12.6 μM for N588K -- HERG -RRB- , these differences did not attain statistical significance -LRB- p > 0.05 ; Anova -RRB- . These observations indicate the IHERG blockade by doxepin was not highly sensitive to attenuation of HERG channel inactivation . 3.5 Sensitivity of IHERG inhibition by doxepin to the S6 mutations Y652A and F656A Two aromatic amino-acid residues , Y652 and F656 , have been shown to be important components of the drug-binding site for a variety of HERG channel blockers -LSB- 5,9,38 -RSB- . Accordingly , we investigated whether or not doxepin inhibition of IHERG was sensitive to mutation of either residue , adopting a similar approach and protocols to those used in other recent IHERG pharmacology studies -LSB- 29,32,39 -- 41 -RSB- . Fig. 7 shows the effects on doxepin inhibition of IHERG of the mutation Y652A -LRB- tyrosine → alanine -RRB- . The experimental voltage protocol used -LRB- Fig. 7Aiii -RRB- was identical to that used for WT IHERG in Fig. 1 and to investigate the S631A and N588K mutants in Fig. 6 . Representative traces showing the effect of 10 μM doxepin on WT IHERG and Y652A IHERG are shown in Fig. 7Ai and Aii . WT IHERG was inhibited substantially by this doxepin concentration -LRB- Fig. 7Ai -RRB- . In contrast , inhibition of Y652A IHERG was noticeably reduced -LRB- Fig. 7Aii ; 28 ± 3 % peak tail current inhibition compared to 62 ± 4 % for WT IHERG ; p < 0.001 -RRB- . Four other doxepin concentrations -LRB- 30 , 100 , 300μM and 1 mM -RRB- were also tested in order to construct a concentration response relation for inhibition of Y652A IHERG tails -LRB- Fig. 7B -RRB- . A fit to the data with Eq . -LRB- 2 -RRB- -LRB- dashed line -RRB- yielded an IC50 for inhibition of Y652A IHERG of 27.8 ± 8.8 μM -LRB- with a Hill coefficient of 0.9 ± 0.3 -RRB- , which represented a modest , though significant -LRB- p < 0.05 -RRB- , ∼ 4.2 fold increase in IC50 over that for WT IHERG . IHERG carried by F656A -- HERG channels was studied using the protocol shown in the inset of Fig. 8Aiii and inward tails were measured at − 120 mV -LSB- 25,28,29 -RSB- . Initial experiments employed a doxepin concentration of 100 μM -LRB- shown in Fig. 1 to produce extensive inhibition of WT IHERG with a standard -LSB- K + -RSB- e -RRB- . Representative traces showing effects of this concentration on WT and F656A IHERG are shown in Fig. 8Ai and Aii . The F656A mutation produced a modest attenuation of IHERG inhibition by this concentration of doxepin . However , as observed previously for some other drugs -LRB- e.g. -LSB- 28 -RSB- -RRB- , the extent of inhibition of WT IHERG by 100 μM doxepin under conditions of high -LSB- K + -RSB- e -LRB- see Section 2 -RRB- was significantly smaller -LRB- 56 ± 5 % -RRB- than that observed with the same concentration in the experiments with a standard -LSB- K + -RSB- e in Fig. 1 -LRB- 93 ± 4 % ; p < 0.001 -RRB- . Therefore , the effects of two further concentrations of doxepin -LRB- 500 μM and 1 mM -RRB- were also examined . Fig. 8B shows bar-chart plots of the mean levels of inhibition of WT and F656A IHERG by the three doxepin concentrations . These concentrations resulted in progressive increases in the level of blockade of WT IHERG . In contrast , although Anova comparison of the data with the three drug concentrations confirmed that doxepin inhibition of F656A IHERG was concentration-dependent -LRB- p < 0.001 -RRB- , the observed concentration-dependence was unusual : there was no statistically significant difference between the observed level of blockade of F656A IHERG between 100 and 500 μM , whilst at 1 mM the observed level of inhibition of F656A IHERG was markedly increased -LRB- p < 0.001 compared to each of 100 and 500 μM , Bonferroni post-test -RRB- and approached that of WT IHERG . Taken together , the data with the three doxepin concentrations suggest that the F656A mutation exerted some influence on the ability of doxepin to inhibit IHERG , though the F656A data did not appear to follow a conventional monotonic concentration dependence . 4 Discussion Despite a strong association between TCA use and QT interval lengthening -LSB- 42 -RSB- , and although doxepin itself has been linked with both QT interval prolongation and TdP -LSB- 18 -- 20 -RSB- , the effects of this drug on HERG K + channels have not hitherto been reported . Moreover , whilst IHERG blockade has been investigated previously for the TCAs imipramine and amitriptyline -LSB- 21 -- 23 -RSB- , to our best knowledge the present study is the first in which molecular determinants of IHERG inhibition have been investigated for any member of the TCA family . 4.1 Characteristics of IHERG blockade by doxepin Previously , imipramine has been reported to inhibit IHERG recorded in experiments using a mammalian expression system with an IC50 of 3.4 μM -LSB- 23 -RSB- whilst an IC50 of 10 μM was reported for amitriptyline -LSB- 21 -RSB- . A different study of amitriptyline , using the Xenopus oocyte expression system , reported IC50 's of ∼ 3.3 -- 4.8 μM -LRB- depending on -LSB- K + -RSB- e -RRB- -LSB- 22 -RSB- . Thus , the potency of doxepin as an IHERG inhibitor found here -LRB- IC50 of 6.5 μM -RRB- is broadly comparable to that seen previously for these other two TCAs . However , in terms of the characteristics of observed IHERG blockade , doxepin appears to be closer to imipramine than to amitriptyline : amitriptyline block of IHERG has been reported to show significant voltage-dependence -LSB- 22 -RSB- whereas imipramine showed only weak voltage-dependence -LSB- 23 -RSB- . Moreover , no significant effects of doxepin on the voltage-dependence of activation or inactivation were seen in this study . Unfortunately , comparative data for the TCAs imipramine and amitriptyline are lacking -LSB- 21 -- 23 -RSB- and , therefore , a direct comparison between doxepin and these agents can not be made in this regard . However , the atypical tetracyclic antidepressant maprotiline has recently been reported to show no alteration to the voltage-dependence of activation and inactivation -LSB- 43 -RSB- , though another study contradicted this with regard to activation -LSB- 44 -RSB- . The lack of a significant leftward shift in the voltage-dependence of inactivation with doxepin in our study suggests that this agent does not act to stabilise IHERG inactivation , and the time-course of WT IHERG inactivation was not significantly accelerated by the drug . Moreover , the results with inactivation-attenuating mutations to two residues from distinct parts of the channel -LRB- S631A : outer mouth of the channel pore ; N588K : in the S5-Pore linker -RRB- provide evidence that IHERG inactivation does not play an obligatory role in doxepin 's binding to the channel . This is further supported by unaltered levels of WT IHERG blockade with progressive depolarization over voltages at which IHERG was maximally activated , but over which inactivation increased -LRB- Fig. 3C and D -RRB- . Doxepin is quite distinct in this regard from a number of other drugs , including the archetypal high affinity HERG blocking methanesulphonanilide drugs E-4031 and dofetilide , for which IHERG inactivation exerts a strong influence on blocking potency -- either as a direct consequence of inactivation-state dependent block , or due to conformational changes during inactivation facilitating optimal orientation of S6 helical residues to which drugs bind -LSB- 10,45 -- 48 -RSB- . Blockade of IHERG by imipramine has been reported to develop rapidly during a sustained depolarization , with a component of inhibition visible even for comparatively brief depolarizing voltage commands -LSB- 23 -RSB- . These features of imipramine 's action correspond well with those seen for doxepin in the present study . They are also similar to those reported for the serotonin-selective reuptake inhibitors -LRB- SSRIs -RRB- fluvoxamine and citalopram -LSB- 25,49 -RSB- , but differ significantly from the methanesulphonanilides , for which little blockade is observed immediately upon depolarization , with blockade then increasing progressively during the maintained depolarization -LSB- 24,49,50 -RSB- . The lack of a strong dependence of HERG channel blockade by doxepin on IHERG inactivation suggests that gating-dependent blockade by this drug is likely to arise predominantly to activated/open channels . Accordingly , the observed time-dependence of IHERG inhibition by doxepin in this study is consistent with either a mixed state-dependence of blockade -LRB- with components of both closed and open channel blockade -RRB- or with the presence of a very rapidly developing component of activation-dependent inhibition immediately on depolarization . For the majority of drugs that have been studied , one or both of the Y652 and F656 aromatic amino acid residues in the S6 helices of the HERG channel comprise key elements of the drug binding site -LSB- 5,9,38,51 -RSB- . For example , the Y652A and F656A mutations increased the IC50 for HERG blockade by the methanesulphonanilide MK-499 by 94-fold and 650-fold , respectively , and that for terfenadine by ∼ 100-fold -LSB- 8 -RSB- . In comparison , the ∼ 4-fold increase in IC50 for doxepin produced by the Y652A mutation in this study , is rather modest , suggesting that this residue is less influential for binding of doxepin than for either of these high affinity blockers . The unusual concentration dependence seen with the F656A mutation indicated that at a high doxepin concentration of 1 mM IHERG blockade was little affected by mutation of this residue , whilst at concentrations of 100 and 500 μM significant attenuation of blockade occurred with no significant increase in block at 500 μM compared to 100 μM . It was not possible to obtain an adequate fit of these data with Eq . -LRB- 2 -RRB- . A cautious interpretation of the lack of a conventional concentration-dependence of F656A -- HERG inhibition by doxepin is that blockade may depend partly , but incompletely , on this residue . Neither the Y652A nor F656A mutations attenuated blockade by doxepin concentrations producing very high levels -LRB- > 90 % -RRB- of blockade of WT IHERG , suggesting that neither residue is absolutely obligatory for doxepin binding to HERG channels to occur . Whilst unusual , this is not unprecedented ; IHERG block by both the SSRI fluvoxamine and the antiarrhythmic agent dronedarone has been reported to be only partially attenuated by the Y652A and F656A mutations -LSB- 25,28 -RSB- . Comparative data for other TCAs are lacking , though recently these residues have been implicated in IHERG inhibition by the tetracyclic drug maprotiline -LSB- 43,44 -RSB- ; one of these two studies obtained IC50 values for the Y652A and F656T mutants , with respective -LRB- modest -RRB- 3-fold and 7-fold increases in IC50 -LSB- 43 -RSB- . Whilst obligatory molecular determinants of doxepin-binding to HERG remain to be found , the fact that IHERG inhibition by doxepin developed progressively over several minutes following rapid external solution exchange is consistent with the drug crossing the cell membrane to reach its site of action . The reduced IHERG inhibition by doxepin of WT IHERG in the presence of raised -LSB- K + -RSB- e may also be of significance . Since IHERG inhibition by doxepin appears not to be critically dependent on channel inactivation , decreased blockade with high -LSB- K + -RSB- e is unlikely to result from any effect of -LSB- K + -RSB- e on IHERG inactivation . Rather , reduced inhibition in high -LSB- K + -RSB- e may be accounted for by an interference with drug-binding due to an electrostatic repulsion or `` knock-off '' process -LSB- 28,52 -RSB- , consistent with drug binding to the channel at a site close to the ion conduction pathway . It remains to be determined whether this site would need to reside within the channel pore . However , in a limited series of experiments using the D540K mutant -LSB- 53 -RSB- , we did not find evidence that doxepin can readily unbind on hyperpolarization-induced channel opening -LRB- data not shown -RRB- ; though not all drugs that bind within the pore exhibit marked ` untrapping ' -LSB- 54 -RSB- . Nevertheless , given the presence of a significant component of IHERG blockade with brief depolarization and incomplete attenuation of inhibition by the Y652A and F656A mutations , we can not exclude the possibilities that a proportion of the observed blockade with doxepin involves binding outside of the channel pore , or binding to closed HERG channels . 4.2 Clinical significance of IHERG blockade by doxepin TCAs are associated with QTc interval lengthening in clinical use -LSB- 42 -RSB- , and IHERG inhibition by doxepin observed in the present study is consistent both with documented cases of QT interval prolongation and TdP with doxepin -LSB- 18 -- 20 -RSB- and with QT interval increases seen in anaesthetised guinea-pigs receiving doxepin infusion -LSB- 15 -RSB- . Our study was conducted at a physiologically relevant temperature and the extent of IHERG blockade by doxepin was similar between ventricular action potential and conventional voltage step protocols . A lack of relief of IHERG blockade on membrane hyperpolarization for the D540K mutant -LRB- mentioned in Section 4.1 , above -RRB- is suggestive of relatively poor drug unbinding at negative voltages in the maintained presence of drug , whilst the data shown in Fig. 5 indicate that at 10 μM -LRB- the same concentration as used for the AP clamp experiment in Fig. 2 -RRB- doxepin was able to bind and inhibit IHERG rapidly on membrane depolarization to a positive voltage . These factors may combine to account for the similar levels of IHERG inhibition by doxepin observed with step and AP clamp protocols . Although we did not co-express HERG with MiRP1 , a putative β subunit suggested to be necessary to recapitulate native IKr -LSB- 55 -RSB- , it has recently been suggested that MiRP1 is unlikely to interact with HERG outside of the cardiac conduction system -LSB- 51 -RSB- and , additionally , the pharmacological sensitivity of HERG channels expressed in mammalian cells without MiRP1 co-expression has been found to be similar to that of native IKr -LSB- 56 -RSB- . This notion is reinforced by the close concordance of inhibitory potency of doxepin on IHERG and native IKr in our experiments -LRB- IC50 values of 6.5 and 4.4 μM , respectively -RRB- . A question therefore arises as to the relationship between the potency of IHERG/IKr blockade seen in this study and plasma concentrations of doxepin in patients . As a class , the TCAs are lipophilic and are known to become concentrated in some tissues , including the myocardium -LSB- 57 -RSB- . In the case of doxepin , one experimental study has reported doxepin concentration in cardiac muscle to be 41-fold higher than plasma levels -LSB- 58 -RSB- . This makes it difficult to extrapolate with accuracy from known plasma concentrations to likely levels of IHERG/IKr blockade by doxepin in vivo . The therapeutic plasma level of doxepin is thought to be between 50 and 250 ng/ml -LRB- 0.16 -- 0.8 μM -RRB- , although a wide variety of recommendations from university psychiatric departments and laboratories -LRB- up to 1000 ng/ml ; 3.2 μM -RRB- have been reported -LSB- 59 -RSB- . Whilst IHERG -LRB- or IKr -RRB- blockade at the lower end of this range might be anticipated to be modest , inhibition at higher concentrations would be significant and , taking into account also potential cardiac accumulation , the observed potency of IHERG inhibition by doxepin in this study is likely to be clinically relevant , particularly in overdose . Such an effect may be exacerbated in individuals exhibiting pre-existing QT interval prolongation -LRB- congenital or acquired -RRB- , electrolyte abnormalities or with impaired drug metabolism . Thus , as for other IHERG blocking medications , caution is warranted in its use in patients with pre-existing QT interval prolongation or with risk factors likely to exacerbate the effects of IHERG blocking medications . The findings of this study have further clinical relevance in a second , perhaps less expected , respect . The attenuated-inactivation N588K -- HERG mutant used in this study has been shown recently to underlie the SQT1 familial form of the recently identified genetic ` Short QT syndrome ' , which carries a risk of cardiac arrhythmia and sudden death -LSB- 27,60 -RSB- . Pharmacological approaches to correcting the QT-interval of SQT1 patients are currently very limited . These patients are comparatively insensitive to Class III IKr/HERG blocking drugs -LSB- 27,61 -RSB- and the N588K -- HERG blocking potencies of the IKr/HERG blockers E-4031and D-sotalol are reduced ∼ 12 -- 20-fold compared to their effects on WT -- HERG -LSB- 62,63 -RSB- , presumably due to a role -LRB- direct or otherwise -RRB- of channel inactivation in facilitating drug binding to the HERG channel . To date , only the Class Ia antiarrhythmic drug quinidine has been found both to inhibit N588K -- HERG effectively and to correct the QT interval in such patients -LSB- 27,62,63 -RSB- ; however , very recently , another Class Ia antiarrhythmic , disopyramide , has been shown to be effective against N588K -- HERG in vitro -LSB- 63 -RSB- . The present study identifies doxepin as both an IHERG-blocker for which channel inactivation does not play a major role in drug binding and as an additional drug that is an effective inhibitor of N588K -- HERG . Whilst the sedative effects of doxepin may make it unsuitable as a corrective treatment for SQT1 patients , our findings prompt the question as to whether chemical structures related to doxepin might feasibly offer viable IHERG blocking agents in SQT1 .

O O O B B I O O O B B B O O O O O O O O O O O B I I O O O O O O O O O O O B O O B O O O B O O B I O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O B I O O B O O O O O O O B O O O O O O O O O B O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O B O O O B O O O O O O O O B O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O B O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O

antidepressant
arrhythmia
doxepin
herg
potassium channel
qt interval
rapid delayed rectifier
short qt syndrome
torsade de pointes