Conduction and refractory disorders in the diabetic atrium

$: Conduction and refractory disorders in the diabetic atrium$
Conduction and refractory disorders in the diabetic atrium

Masaya Watanabe, Hisashi Yokoshiki, Hirofumi Mitsuyama, Kazuya Mizukami, Taisuke Ono,and Hiroyuki TsutsuiDepartment of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Submitted 5 January 2012; accepted in final form 2 May 2012

Watanabe M, Yokoshiki H, Mitsuyama H, Mizukami K, OnoT, Tsutsui H. Conduction and refractory disorders in the diabeticatrium. Am J Physiol Heart Circ Physiol 303: H86–H95, 2012. Firstpublished May 4, 2012; doi:10.1152/ajpheart.00010.2012.—Diabetesmellitus (DM) is an independent risk of atrial fibrillation. However, itsarrhythmogenic substrates remain unclear. This study sought to ex-amine the precise propagation and the spatiotemporal dispersion ofthe action potential (AP) in the diabetic atrium. DM was induced bystreptozotocin (65 mg/kg) in 8-wk-old male Wister rats. Opticalmapping and histological analysis were performed in the right atrium(RA) from control (n � 26) and DM (n � 27) rats after 16 wk.Rate-dependent alterations of conduction velocity (CV) and its heter-ogeneity and the spatial distribution of AP were measured in RA usingoptical mapping. The duration of atrial tachyarrhythmia (AT) inducedby rapid atrial stimulation was longer in DM (2.4 � 0.6 vs. 0.9 � 0.3s, P � 0.05). CV was decreased, and its heterogeneity was greater inDM than control. Average action potential duration of 80% repolar-ization (APD80) at pacing cycle length (PCL) of 200 ms from fourareas within the RA was prolonged (53 � 2 vs. 40 � 3 ms, P � 0.01),and the coefficient of variation of APD80 was greater in DM thancontrol (0.20 � 0.02 vs. 0.15 � 0.01%, P � 0.05). The ratio of APD80

at PCL shorter than 200 ms to that at 200 ms was smaller (P � 0.001),and the incidence of APD alternans was higher in DM than control(100 vs. 0%, P � 0.001). Interstitial fibrosis was greater and connexin40 expression was lower in DM than control. The remodeling of thediabetic atrium was characterized as follows: greater vulnerability toAT, increased conduction slowing and its heterogeneity, the prolon-gation of APD, the increase in spatial dispersion and frequency-dependent shortening of APD, and increased incidence of APDalternans.

atrial fibrillation; action potential duration; conduction velocity; dia-betes mellitus; optical mapping

THE NUMBER OF PATIENTS WITH diabetes mellitus (DM) has beenincreasing. DM is strongly associated with increased mortalityand an independent risk factor for various cardiovasculardiseases (2) such as myocardial infarction and atrial fibrillation(AF) (12). Especially, DM increases the incidence of strokeand death in patients with AF (14).

Recent experimental studies reported that the increased sus-ceptibility to AF in the diabetic animals (13, 26) was presum-ably due to the slowing of conduction associated with in-creased interstitial fibrosis (13) and the augmentation of theheterogeneity in the atrial effective refractory period (ERP)produced by sympathetic nerve stimulation (26). However, thearrhythmogenic mechanisms in the diabetic atrium remainundetermined. Especially, the underlying mechanisms for theelectrical alterations of diabetic atrium in response to rapidactivation need to be elucidated because most of paroxysmal

AF has been known to occur as a consequence of rapid firingfrom pulmonary veins (8).

We thus aimed to determine whether the slowing and het-erogeneity of conduction and/or the spatiotemporal dispersionof the action potential duration (APD) are increased in thediabetic atrium and these changes are augmented as the atrialactivation rate becomes faster. For this purpose, we investi-gated the precise conduction pattern and the spatiotemporalchanges in action potential (AP) at various pacing cycle lengthsin the right atrium obtained from streptozotocin (STZ)-induceddiabetic rats using an optical mapping system.

METHODS

Experimental Animals

The study was approved by our institutional animal researchcommittee and conformed to the animal care guidelines for the Careand Use of Laboratory Animals in Hokkaido University GraduateSchool of Medicine.

Diabetes was induced by intraperitoneal injection of STZ (65mg/kg) dissolved in citrate buffer in 8-wk-old male Wister rats (n �27). Age-matched control rats (n � 26) received an equivalent volumeof the citrate buffer solution alone. One week later, the diabetic statewas confirmed by blood glucose of higher than 300 mg/dl. Sixteenweeks after the administration of STZ or the citrate buffer, the tailartery blood pressure and heart rate were measured in a consciousstate by the oscillometric method (BP-98A; Softron, Tokyo, Japan).After the rats were fasted for 12 h, blood samples were collected fromthe internal jugular vein under anesthesia with inhalation of diethylether. Blood glucose was measured in both groups of rats using aglucometer (Glutest Ace R; Sanwa Kagaku Kenkyusho, Nagoya,Japan).

Langendorff Perfusion

The heart was excised from control or DM rats after anesthesiawith intraperitoneal injection of pentobarbital (50 mg/kg) and heparinsodium (400 IU/kg). The heart was mounted on a Langendorffapparatus and was retrogradely perfused with Tyrode solution (37°C)containing (in mM) 143 NaCl, 5.4 KCl, 0.33 NaH2PO4, 5 HEPES, 5.5glucose, 0.5 MgCl2, and 1.8 CaCl2 (pH 7.4 using NaOH) gassed with100% O2 until the beating rate became stable (5–10 min). Next,optical mapping (in 8 control and 10 DM rats) or AF induction (in 12control and 10 DM rats) was performed.

ERP and Susceptibility to Atrial Tachyarrhythmia

ERP and the duration of atrial tachyarrhythmia (AT) were mea-sured in 12 control and 10 DM rats. Two electrodes were attached tothe left atrial appendage (LAA) and right ventricle for the recording ofelectrocardiogram. Another electrode was attached to the right atrialappendage (RAA) for delivering programmed stimulations as a uni-polar cathode. The indifferent anodal electrode was attached to theextracardiac tissue.

The intensity of the current pulse was twice that of the threshold,and the pulse duration was 0.5 ms. ERP was measured by introducingS2 extrastimulus with 5-ms decrements following eight regulatory

Address for reprint requests and other correspondence: H. Yokoshiki, Dept.of Cardiovascular Medicine, Hokkaido Univ. Graduate School of Medicine,Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638, Japan (e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 303: H86–H95, 2012.First published May 4, 2012; doi:10.1152/ajpheart.00010.2012.

0363-6135/12 Copyright © 2012 the American Physiological Society http://www.ajpheart.orgH86

S1–S1 stimuli of 150 ms and defined as the longest S1–S2 interval atwhich S2 failed to induce a propagated response. We measured ERPtwo times on each animal, and the average of them was defined asERP of each animal. The induction of AT was performed by burstpacing five times repeatedly, at the pacing cycle length (PCL) from 70to 30 ms in 5-ms decrements for 5 s. The duration of AT was definedas the longest time for persistence deviating from sinus rhythm for�0.1 s.

Optical Mapping

We used voltage-sensitive dye imaging technology according to themethods reported previously (6). After the beating rate of the isolatedheart became stable, the perfusate was switched to Tyrode solutioncontaining 1.25 �M di-4-ANEPPS for 20 min. After that, the heartwas continuously perfused with Tyrode solution containing 10 �Mblebbistatin, a selective myosin II inhibitor, to eliminate motionartifact without any effects on the electrical parameters (7). Next, theheart was illuminated with quasimonochromatic light (530 � 20 nm)from a 150-W Halogen light source. The emitted fluorescence wascollected by an image-intensified charge-coupled device camera (Mi-CAM02; BrainVision, Tokyo, Japan) through a 590-nm long-passfilter. The optical signals were collected at 2.2-ms sampling intervals,acquired from 96 � 64 pixels over a 12.6 � 8.4-mm2 area in the rightatrium (RA). Optical recordings were obtained during incrementalstimulation in RAA at PCL of 200, 150, 100, and 80 ms and in 5-msdecrements until the pacing stimuli failed to capture one-to-onebeating of the atrium.

Data Processing

Optical signals were analyzed using custom-made software (Bra-inVision Analyzer). To extract RA from original data, the region ofinterest (ROI) was selected manually. The spatial filtering procedure(7 � 7 for activation analysis and 5 � 5 for APD analysis) wasconducted to remove the noise. Local activation was determined as thetime point of maximum change in fluorescence over time (dF/dt) foreach fluorescence signal. An activation map was constructed bycalculating the time delay of the activation at each pixel relative toactivation at the pacing site.

A conduction velocity (CV) vector map was constructed from anactivation map with custom-written software using an algorithm asdescribed previously (4). Average CV was calculated by averagingCV from each vector of the ROI. To assess the difference in theconduction heterogeneity between control and DM rats, we examinedthe phase difference (17), which indicates the maximum difference intime points of the focal activation among the neighboring pixels. Tocalculate phase difference, optical signals extracted from a rectangularROI (49 � 41 pixels, the distance between neighboring pixels �0.13mm) were compressed to 13 � 11 pixel area (the distance betweenneighboring pixels �0.52 mm). Next, the largest difference in theactivation time point between one pixel and its neighboring pixelsdivided by the distance between the pixels in a compressed area wasdefined as the phase difference at each point (17). Frequency histo-grams were constructed for the phase difference in the compressedpixel areas. The absolute heterogeneity was calculated by subtractingthe 95th percentile from the 5th percentile of the phase difference. Theheterogeneity index was determined by dividing this value by themedian of the phase difference (17).

APD at 80% repolarization (APD80) and that at 50% repolarization(APD50) were determined as the time difference between the point ofactivation [i.e., the time point of maximum upstroke fluorescencechange over time (dF/dt)] and the point at 80 and 50% repolarizationof maximum AP amplitude, respectively. To examine the spatialdispersion of APD, we measured APD80 at four area as follows: RAA,right atrial free wall (Free wall), high right atrial (HRA) wall, and lowright atrial (LRA) wall. HRA or LRA was defined as the area belowSVC or above IVC in anatomic orientation, and the area of the Free

wall was determined as one that was equally distant from RAA, HRA,and LRA (shown in Fig. 1 and see Fig. 5A).

APD restitution curves were constructed by plotting APD80

against the preceding diastolic interval during burst pacing (i.e.,dynamic restitution), and the data points were best fitted to a singleexponential (15).

We measured pairwise difference in APD80 to examine theincidence of the beat-to-beat alteration in APD, so-called “APDalternans.” APD alternans was considered to be present when therewas alternating lengthening/shortening in consecutive APs and thedifference of APD80 in consecutive beats was �4 ms and 10%,respectively (28).

Histology

Histological analysis of RA was performed in six control and sixDM rats. Right atrial tissues were dissected from the hearts and storedin neutral buffered formalin for �1 wk. The sections of the atrialtissue were stained with Masson’s trichrome. In these stained sections,the ratio of the area occupied by interstitial fibrosis was measuredusing a public domain software (Image J; NIH, Bethesda, MD).

Western Blot Analyses

Western blot analyses for connexin 40 (CX 40) and connexin43(CX 43) were performed in six control and seven DM rats. Theatrial tissues were snap-frozen, homogenized, and dissolved in 1�cell lysis buffer (Cell Signaling, Danvers, MA), supplemented with1� Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland).After centrifugation at 15,000 rpm for 20 min at 4°C, the supernatantswere separated into aliquots and stored at �80°C until the time of

Fig. 1. Photograph of right atrial preparation. SVC, superior vena cava; IVC,inferior vena cava; RAA, right atrial appendage; Free wall, right atrial freewall; HRA, high right atrial wall; LRA, low right atrial wall; RV, rightventricle.

H87CONDUCTION AND REFRACTORY DISORDERS IN DIABETIC ATRIUM

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00010.2012 • www.ajpheart.org

assay. Protein concentrations were determined using a standardizedcolorimetric assay. Proteins were fractionated by SDS-PAGE, trans-ferred to a nitrocellulose membrane, blocked with 5% nonfat milk,and blotted with anti-CX 40 (1:500; Millipore, Billerica, MA), an-ti-CX 43 (1:4,000; Sigma-Aldrich, St. Louis, MO), and anti-glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000; Cell Sig-naling) overnight at 4°C. Membranes were washed with blockingbuffer and incubated with secondary antibodies directed against theprimary and conjugated with horseradish peroxidase. Bands weredetected with the enhanced chemiluminescence assay and quantifiedusing Image J. Band intensity for the protein under question wasnormalized to the intensity of GAPDH in each lane.

Statistical Analysis

All data are presented as means � SE. Simple between-groupanalyses were conducted by Student’s t-test. When data were analyzedacross more than two cycle lengths, two-way repeated ANOVA wasused. Categorical variables were compared using the Chi square test.Differences with P � 0.05 were considered significant.

RESULTS

Animal Characteristics

Table 1 shows the characteristics of control and DM rats.The fasting blood glucose was significantly higher in DM rats.Body weight and heart weight were significantly lower in DMthan control rats. However, the ratio of heart to body weightwas significantly higher in DM rats. Systolic blood pressureand heart rate were significantly lower in DM rats.

ERP and Susceptibility to AT

ERP in RAA was significantly longer in DM (n � 10) thancontrol (n � 12) rats (49 � 4 vs. 38 � 2 ms, P � 0.05;Fig. 2A). The duration of AT was significantly longer in DMthan control rats (2.4 � 0.6 vs. 0.9 � 0.3 s, P � 0.05; Fig. 2,B and C). In addition, the average cycle length of AT sustained�0.5 ms was longer in DM (n � 7) than in control (n � 4) rats(98.1 � 7.2 vs. 67.9 � 6.5 ms, P � 0.05).

Optical Mapping

Conduction velocity. The activation maps and their CVvector maps within the RA from control and DM rats wereconstructed at PCL of 200 ms (Fig. 3A). In control rats, astimulation pulse from RAA propagated RA almost uniformly(Fig. 3A, left). In contrast, isochronal crowding, indicatingfocal conduction delay, was observed, and the direction andmagnitude of CV vector varied widely in DM rats (Fig. 3A,right). The average CVs, calculated from the conduction vectormaps, were significantly slower in DM rats at any PCL(Fig. 3B).

Phase difference. Figure 4 shows representative activationmaps (Fig. 4, left), phase difference maps (Fig. 4, middle), andphase difference histograms (Fig. 4, right) in control (Fig. 4A)and DM (Fig. 4B) rats. In control rats, focal conduction blockwas rarely seen, and the phase difference was distributedwithin 4.2 ms/mm even at the faster PCL (Fig. 4A). In contrast,it was increased in DM compared with control rats even at PCLof 200 ms (Fig. 4B). Moreover, a block line emerged at theshorter PCL, and the phase difference histogram showed widerdistribution in DM rats (Fig. 4B). In addition, most of the blocklines in DM rats were seen at the posterolateral portion of theatrial free wall that was close to the crista terminalis. Quanti-tative analysis of the phase difference demonstrated that theabsolute heterogeneity was significantly greater in DM rats atany PCL (Fig. 4C). In addition, the absolute heterogeneitybecame larger in DM rats in accordance with the shortening ofPCL. Similarly, heterogeneity index was greater at any PCL inDM rats with rate-dependent augmentation.

APD. Superimposed optical recordings of AP at PCL of 200ms from four areas, RAA, right atrial Free wall, HRA wall, and

Fig. 2. A: effective refractory period (ERP) at the right atrial appendage incontrol (n � 12) and diabetes mellitus (DM) (n � 10) rats. *P � 0.05 vs.control. B: representative recording of electrocardiogram during an atrialfibrillation (AF) episode in control (top) and DM (bottom) rats. A, atrialelectrocardiogram; V, ventricular electrocardiogram. C: summary data of theduration of atrial tacharrhythmia (AT) in control (n � 12) and DM (n � 10)rats. AF, atrial fibrillation. *P � 0.05 vs. control.

Table 1.

Control DM P Value

n 26 27Fasting blood glucose, mg/dl 141 � 5 470 � 14 �0.001Body weight, g 360 � 4 135 � 7 �0.001Heart weight, mg 969 � 22 594 � 27 �0.001Heart weight-to-body weight ratio, �103 2.7 � 0.1 4.4 � 0.1 �0.001Heart rate, beats/min 382 � 6 322 � 6 �0.001Systolic blood pressure, mmHg 118 � 2 110 � 3 �0.05

Values are means � SE; n, no. of rats. DM, diabetes mellitus.

H88 CONDUCTION AND REFRACTORY DISORDERS IN DIABETIC ATRIUM


LRA wall, are shown in Fig. 5. APD in control rats was almostsimilar among four areas (Fig. 5A). In contrast, it varied widelyamong four areas in DM rats, and its duration was longercompared with control rats (Fig. 5A). The APD80 at each areawas significantly longer in DM rats (Fig. 5B), and the averagevalues in these four areas were also longer in DM rats (53 �2 vs. 40 � 3 ms, P � 0.01). The coefficient of variation(SD/mean) of APD80 obtained from four areas was signifi-cantly greater in DM rats (Fig. 5C), indicating that the spatialdispersion of APD80 was increased in DM rats.

In addition to the spatial dispersion of APD, the rate-dependentchanges in APD were also evaluated (Fig. 6). Figure 6A showssuperimposed optical recordings of AP from RAA. While noapparent changes in APD were observed among different PCLs incontrol rats (Fig. 6A, left), it became shorter as PCL decreased inDM rats (Fig. 6A, right). The ratios of APD50 (Fig. 6B) andAPD80 (Fig. 6C) at PCLs of 150, 100, and 80 ms to those at PCLof 200 ms were significantly smaller in DM than in control rats. Inanalysis of APD restitution properties, the maximum slope of theAPD restitution curve was �1 in two control (25%) and two DM(20%) rats [P � not significant (NS)]. There was no significantdifference in the maximum restitution slope between control andDM rats (0.67 � 0.22 in control vs. 0.81 � 0.22 in DM rats, P �NS) (Fig. 6D).

AP alternans. During the continuous optical recordings,APD alternans was more frequently observed in DM than incontrol rats (0 vs. 100%, P � 0.001) (Fig. 7, A and B). In Fig.7C, the relationship between APD alternans and PCL was

shown. The average of the longest PCL, at which APD alter-nans were observed in DM rats, was 99 � 7 ms. Moreover, inDM rats, as PCL became much shorter, AP frequently divertedto complex oscillation, and finally focal conduction block wasobserved (Fig. 7B).

Histological Analysis

Masson trichrome staining of the right atrial section dem-onstrated that interstitial fibrosis was more abundant in DMthan in control rats (Fig. 8).

Western Blot Analyses

The expression of CX 40 protein in the atrial tissue wassignificantly lower in DM than control rats (Fig. 9, A and B).On the other hand, there was no significant difference in theexpression of CX 43 protein between two groups of rats (Fig.9, C and D).

DISCUSSION

The present study clearly demonstrated the important elec-trophysiological properties of the diabetic atrium comparedwith normal controls as follows. First, the susceptibility to ATwas increased. Second, conduction slowing and its heteroge-neity were increased. Third, APD was longer, and its spatialdispersion was increased. Fourth, the shortening of APD withthe decrease in PCL was augmented. Fifth, APD alternans wasmore frequently observed. Sixth, interstitial fibrosis was in-

Fig. 3. A: representative activation maps (top)and conduction velocity (CV) vector maps (bot-tom) from control (left) and DM (right) rats. Theactivation maps are shown with isochronesdrawn at 2.2-ms intervals. Pacing stimulations(�) from RAA at pacing cycle length (PCL) of200 ms spread according to the color scale barranged from 0 to 24.2 ms. The isochronalcrowding was evident within right atrium (RA)from DM rats. In CV vector map and the direc-tion and magnitude of focal CV vectors variedwidely within RA from DM rats. B: summarydata of CVs at different PCLs in control (n � 8)and DM (n � 10) rats. *P � 0.01 vs. control.



creased. Finally, CX 40 protein expression was reduced. Thisis the first study that demonstrated the rate-dependent changesin CV, its heterogeneity, and AP in the diabetic atrium. Thesechanges in electrophysiological and histological properties andgap junction protein might be the substrate for AF in thediabetic atrium.

Conduction Slowing and Heterogeneity

The present study demonstrated, for the first time, a signif-icantly increased conduction heterogeneity and conductionslowing in the RA from DM rats using optical mapping.Recently, Kato et al. (13) examined the interatrial conduction

time from RAA to the LAA during RAA pacing and reportedthe increase in the interatrial conduction time in the diabeticatrium (13). In their study, an interatrial conduction time wasdefined as a time interval for an activation to conduct fromRAA to LAA, which could be influenced by the surface areaof the atria, the direction of conduction, and focal conduc-tion blocks, as well as CV. In contrast, CV from a conductionvector map represent the average of the global conductionwithin the atrium (4), and the heterogeneity represents focalconduction block or slowing (17). In addition, the opticalmapping employed in the present study depicted the conduc-tion pattern in the global RA with high spatial resolution (4).

Fig. 4. Representative activation maps (left),phase difference maps (middle), and the cor-responding histograms (right) at PCL of 200(top) and 80 (bottom) ms from control (A) andDM (B) rats. The phase difference was morewidely distributed in DM than in control rats,especially at PCL of 80 ms. C: absolute het-erogeneity (P95 � P5, where P95 is the 95thpercentile and P5 is the 5th percentile) (left)and heterogeneity index [(P95 � P5)/P50,where P50 is the median] (right) at differentPCLs of 200, 150, 100, and 80 ms in control(n � 8) and DM (n � 10) rats. *P � 0.001 vs.control.



Therefore, our optical mapping methods would represent theprecise propagation in RA.

Previous studies reported that the conduction slowing andheterogeneity might lead to the greater propensity for AF in

transforming growth factor-1 transgenic mice (35) and thehypertensive ovine model (18). In addition, Roberts-Thomsonet al. (29) demonstrated increased conduction delay and heter-ogeneity in the left atria from patients with mitral regurgitation

Fig. 5. A: representative superimposed recordings of actionpotential from 4 areas (RAA, Free wall, HRA wall, and LRAwall) at PCL of 200 ms in control (left) and DM (right) rats.B: summary data of action potential duration at 80% repolar-ization (APD80) at 4 areas from control (n � 8) and DM (n �9) rats. C: the coefficient of variation (CoV, the ratio of SD tomean) in control (n � 8) and DM (n � 9) rats. *P � 0.05 and**P � 0.01 vs. control.

Fig. 6. A: representative superimposed recordings of actionpotential in right atrial appendage at PCL of 200 (black),150 (red), 100 (blue), and 80 (green) ms from control (left)and DM (right) rats. The ratio of action potential duration at50% repolarization (APD50, B) and that of APD80 (C) atPCL of 150, 100, and 80 ms to that at 200 ms in control(n � 8) and DM (n � 10) rats. *P � 0.001 vs. control.D: maximum slope of action potential duration (APD) res-titution curve in control (n � 8) and DM (n � 10) rats. P �not significant (NS).



and persistent AF compared with those in sinus rhythm. There-fore, increased conduction slowing and its heterogeneity in thediabetic atrium seen in this study might be one of the substratesfor AF.

In this study, we, for the first time, demonstrated thereduced expression of CX 40 protein in the diabetic atrium(Fig. 9). Prior studies reported that AF is associated with thedecrease in CX 40 and/or CX 43 protein, the principal atrialgap junction proteins, and/or the abnormal distribution ofthem (10, 16) and that those remodelings of gap junctionproteins were responsible for the conduction abnormality inAF (10). In analyses of the diabetic ventricle, the abnormaldistribution of CX 43, the principal ventricular gap junctionprotein, and the reduced conduction reserve were reported(25). Therefore, we interpret that the reduced expression ofCX 40 protein, as well as the increased interstitial fibrosis(Fig. 8), was responsible for the conduction slowing andheterogeneity in the diabetic atrium.

Prolongation of APD in Diabetic Atrium

Although the prolongation of APD in the ventricle has beenobserved in an earlier diabetic model (33), there have beenlimited data regarding APD or ERP in the diabetic atrium (27).

We demonstrated the prolongation of APD in the diabeticatrium at physiological conditions (Fig. 5). These findings areconsistent with the previous study by Pacher et al. (27) inwhich the prolongation of APD was augmented in STZ-induced diabetic rats. In contrast, other studies could not findany significant changes in ERP between control and DManimals (13, 26). One of the reasons for this discrepancy mightbe the differences in the types of diabetic model (type 1 vs.type 2) and the duration of diabetic condition of the experi-mental animals (8 vs. 16 wk).

There are several potential mechanisms responsible for theprolongation of APD in the diabetic atrium. One possiblemechanism is the attenuation of K currents in the diabeticatrium. Previous study in diabetic ventricular myocytes ob-served the attenuation of the transient outward K current andthe steady-state K current (33), which are now thought to bemajor causes for the prolongation of APD. Therefore, the samemechanism might be involved also in the diabetic atrium.Another possibility is the augmentation of Na/Ca2 ex-changer (NCX) current (1, 24). Cytosolic Ca2 ([Ca2]i) isreported to be increased in ventricular myocytes from thediabetic animals due to downregulation of sarcoplasmic retic-ulum Ca2-ATPase 2a (SERCA2a) (1, 23). The increased

Fig. 7. Representative continuous optical recordings from 4areas within RA in control (A) and DM (B) rats. In control rats,the beat-to-beat change in APD was not clear at PCLs of 80 and45 ms. In DM rats, APD alternans, defined as APD80 alterationof �10% for consecutive beats, was observed at a PCL of 100ms (top). Moreover, complex oscillation of APD as well as focalconduction delay and blocks were observed at a PCL of 55 ms(bottom). L and S indicate a long and short action potential,respectively. ¡, wavy arrow, and inverted “T” indicate a normalconduction, conduction delay, and conduction block, respectively.Abbreviations are the same as shown in Fig. 5. C: relationshipbetween APD alternans and PCL in control (n � 8) and DM (n �10) rats.



[Ca2]i is thought to augment the forward-mode NCX currents(i.e., inward currents), resulting in the prolongation of APD.

Frequency-Dependent Shortening of APD

In this study, we, for the first time, demonstrated the in-creased frequency-dependent shortening of APD in the diabeticatrium, which might be explained by the abnormal Ca2

handling in diabetes. It has been reported that reuptake of[Ca2]i is insufficient due to downregulation of SERCA2a inthe diabetic ventricle (1, 23). This reduced reuptake of [Ca2]i

might be augmented as the heart rate increases (because of thereduced diastolic interval), thereby leading to a much higher[Ca2]i level. Next, a frequency-dependent increase in [Ca2]i

would cause inactivation of the L-type calcium channel (ICa,L)and the shortening of APD.

This hypothesis might be partially supported by the recentreport by Harada et al. (9). In their study, the frequency-dependent shortening of APD was increased in the failingrabbit heart, in association with the augmented frequency-dependent reduction in ICa,L. That is because, as the PCLbecomes shorter, functional reduction of ICa,L, due probably toCa2-induced inactivation of ICa,L, occurs in the failing heart,in which reuptake of [Ca2]i is reduced like in the diabeticventricle.

Arrhythmogenic Mechanisms for Prolonged and IncreasedDispersion of APD

In the present study, we demonstrated the prolongation inthe cycle length of AT as well as increased susceptibility to ATin DM rats, which would imply that the prolongation of APDwas associated with the increased susceptibility to AT in DMrats. The prolongation of APD due to reduced K current hasbeen shown to produce early afterdepolarizations (3), therebyinducing torsades des pointes in long QT syndrome. Moreover,recent studies showed AF risk was increased in patients withcongenital long QT syndrome (11). In fact, the prolongation of

APD produced by cesium-induced loss of K channel activi-ties promoted AF in anesthetized dogs (32). The ablation ofsmall-conductance Ca2-activated K channels resulted in theprolongation of APD in mouse atrial myocytes and facilitatedinduction of AF with unknown electrophysiological mecha-nisms (20).

In addition to the prolongation of APD, spatial dispersion ofAPD was increased in DM rats, indicating that atrial refracto-riness differed within the whole atrium. Spatial heterogeneityof APD could easily produce conduction block, thereby in-creasing the vulnerability to AT (5). Moreover, the suscepti-bility to APD alternans was increased in diabetic atrium (Fig.7). APD alternans is considered to be one of the centralmechanisms of ventricular fibrillation (30). However, limited

Fig. 9. Representative Western blots for connexin 40 (CX 40) (A) and connexin43 (CX 43) (C) in control and DM rats. Relative ratios of CX 40/glyceralde-hyde-3-phosphate dehydrogenase (GAPDH, B) and CX 43/GAPDH (D) incontrol (n � 6) and DM (n � 7) rats. *P � 0.001 vs. control.

Fig. 8. A: representative Masson’s trichromestaining of the right atrial myocardium fromcontrol and DM rats. Scale bars represent 50�m. B: the relative ratio of the area occupiedby interstitial fibrosis to the total area fromcontrol (n � 6) and DM (n � 10) rats. *P �0.001 vs. control.



data have been available concerning its contribution to thearrhythmogenesis of AF (22, 34). Tsai et al. (34) recentlyreported that the thresholds of APD and calcium transientalternans were significantly decreased in the mechanicalstretched atrial myocyte monolayer, and these observationswere rescued by the overexpression of SERCA2a. SERCA2ahas been shown to be downregulated in diabetic ventricularmyocardium (1, 23). Therefore, the decrease in SERCA2amight account for the increased susceptibility to APD alternansin the diabetic atrium.

Heart failure (HF) is also known as a common cause of AFin clinical settings (21). Sanders et al. (31) reported the pro-longed atrial ERP and increased propensity for AF in patientswith HF. Lee et al. (19) reported the augmented dispersion ofthe left atrial ERP and significant increase in AF duration inrapid pacing-induced HF dogs. The electrophysiological alter-ations such as prolonged and enhanced dispersion of APD infailing atrium are similar to those of diabetic atrium observedin this study. In addition, we demonstrated the low thresholdsof APD alternans in the diabetic atrium. Therefore, theserepolarization abnormalities might serve as a central mecha-nism of the vulnerability to AT in the atrial myocardium withDM or HF, thereby contributing to the development of a noveltherapeutic strategy.

Study Limitation

First, we could not examine the maximum rate of depolar-ization. This is due to the absence of a calibration of the opticalsignals, whose change did not indicate the absolute value of thevoltage change (6). Second, we could not identify the mecha-nisms of the prolongation of APD. It should be evaluatedwhether the same mechanisms as demonstrated in the diabeticventricle (1, 24, 33) are also involved in the diabetic atrium.Third, the abnormal regulation of intracellular Ca2 transienthas been demonstrated in the diabetic ventricular myocardium(1, 23), which might be involved in APD alternans (34).Therefore, future studies are needed to examine [Ca2]i in thediabetic atrium. Finally, it remains unknown whether the elec-trophysiological changes observed in this study also occur inother types of the diabetic models or in diabetic patients.Further studies are needed to clarify these issues.

In conclusion, the diabetic atrium had increased conduc-tion slowing and its heterogeneity. APD in the diabeticatrium was significantly prolonged, and the spatial disper-sion and the frequency-dependent shortening of the APDwere increased. Moreover, the incidence of APD alternanswas higher in the diabetic atrium. These electrophysiologi-cal abnormalities might be responsible for the increasedsusceptibility to AT in DM.

ACKNOWLEDGMENTS

We thank Kaoruko Kawai, Miwako Fujii, and Akiko Aita for technicalassistance in the experiments.

GRANTS

This work was supported, in part, by a grant from Grant-in-Aid forScientific Research (23591075) from the Japan Society for the Promotion ofScience to H. Yokoshiki.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: M.W., H.Y., H.M., K.M., T.O., and H.T. conceptionand design of research; M.W. performed experiments; M.W., H.Y., H.M.,K.M., T.O., and H.T. analyzed data; M.W., H.Y., H.M., K.M., T.O., and H.T.interpreted results of experiments; M.W., H.Y., H.M., K.M., T.O., and H.T.prepared figures; M.W., H.Y., H.M., K.M., T.O., and H.T. drafted manuscript;M.W., H.Y., H.M., K.M., T.O., and H.T. edited and revised manuscript; M.W.,H.Y., H.M., K.M., T.O., and H.T. approved final version of manuscript.

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Conduction and refractory disorders in the diabetic atrium

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