Hyperglycemia aggravates atrial interstitial fibrosis, ionic remodeling [630828]

Hyperglycemia aggravates atrial interstitial fibrosis, ionic remodeling
and vulnerability to atrial fibrillation in diabetic rabbits
Hiperglisemi diyabetik tavșanlarda atriyal interstisiyel fibrosis, iyonik remodeling ve atriyal
fibrilasyon duyarlılığını arttırmaktadır
Address for Correspondence/Yaz›șma Adresi: Tong Liu, M.D., PH.D. and Guangping Li, M.D., PH.D., Department of Cardiology, Tianjin Institute of
Cardiology, Second Hospital of Tianjin Medical University 300211 Tianjin-People’s Republic of China Phone: +86 22 88328368 Fax: +86 22 28261158
E-mail: [anonimizat] (T .L.) and [anonimizat] (G. L.)
Accepted Date/Kabul Tarihi: 02.05.2012 Available Online Date/Çevrimiçi Yayın Tarihi: 08.08.2012
©Telif Hakk› 2012 A VES Yay›nc›l›k Ltd. Ști. – Makale metnine www.anakarder.com web sayfas›ndan ulaș›labilir.
©Copyright 2012 by A VES Yay›nc›l›k Ltd. – Available on-line at www.anakarder.com
doi:10.5152/akd.2012.188Changle Liu, Huaying Fu, Jian Li, Wansong Yang, Lijun Cheng, Tong Liu, Guangping Li
Department of Cardiology , Tianjin Institute of Cardiology , Second Hospital of Tianjin Medical University , Tianjin- People’s Republic of China
ABSTRACT
Objective: The purpose of this study was to investigate the effects of hyperglycemia on atrial interstitial fibrosis, ionic remodeling and vulner –
ability to atrial fibrillation (AF) in alloxan-induced diabetic rabbits.
Methods: Sixty Japanese rabbits were randomly assigned to alloxan-induced diabetic group (n=30) and control group (n=30). Ten rabbits in each group were respectively used to electrophysiological and histological study, patch-clamp study and Western blotting analysis. Langendorff perfu-sion was used to record inter-atrial conduction time (IACT), atrial effective refractory period (AERP) and dispersion (AERPD) and vulnerability to
AF . Histological study was measured by Sirius-red stain. Patch-clamp technique was used to measure action potential duration (APD) and atrial
ionic currents (INa and ICaL). Western blotting was applied to assess atrial protein expression of transforming growth factor beta 1 (TGFβ 1).
Results: Compared with control group, electrophysiological studies showed IACT was prolonged (37.91±6.81 vs. 27.43±1.63ms, p<0.01), AERPD was increased (30.37±8.33 vs. 14.70±5.16ms, p<0.01) in diabetic group. Inducibility of AF in diabetic group was significantly higher than in controls (8/10 vs. 1/10 of animals, p<0.01). Collagen volume fraction was increased (6.20±0.64% vs. 2.15±0.21%, p<0.01) in diabetic group. Patch-clamp studies demonstrated APD90 and APD50 were prolonged in diabetic rabbits (p<0.05 vs. control). The densities of INa were reduced and the densities of ICaL were increased (p<0.01 vs. control). Protein expression of TGFβ1 was increased in diabetic group (p<0.001 vs. control).Conclusion: Our study suggests that hyperglycemia contributes to atrial interstitial fibrosis, ionic remodeling and vulnerability to AF in diabetic rabbits, resulting in atrial structural remodeling and electrical remodeling for the development and perpetuation of AF .
(Anadolu Kardiyol Derg 2012; 12: 543-50)
Key words: Hyperglycemia, atrial interstitial fibrosis, ionic remodeling, atrial fibrillation, animals Original Investigation Özgün Arașt›rma 543
ÖZET
Amaç: Bu çalıșmanın amacı, alloksan etkisi ile olușturulmuș diyabetik tavșanlarda atriyal intersitisyel fibrosis, iyonik remodeling ve atriyal fibrilas-
yona (AF) duyarlılığa hipergliseminin etkisini araștırmaktır.
Yöntemler: Altmıș Japon tavșanı alloksan etkisi ile olușturulmuș diyabetik grup (n=30) ve kontrol grup (n=30) olarak geliși güzel belirlendi. Herbir
grupta 10 tavșan sırasıyla elektrofizyolojik ve histolojik çalıșma, “patch-klemp” çalıșma ve Western blotting analizi kullanıldı. İnter-atriyal iletim zamanı (İAİZ), atriyal efektif refraktör dönem (AERP), dağılım (AERD) ve AF duyarlılığını kaydetmek için Langendorff perfüzyonu kullanıldı. Histolojik çalıșma
Sirius-kırmızı boyama ile değerlendirildi. Aksiyon potansiyel süresi (APS) and atriyal iyonik akımları (INa and ICaL) ölçmek için “patch-klemp” tekniği
kullanıldı. Dönüștürücü büyüme faktörü beta 1 (DBFβ 1)’in atriyal protein ekspresyonunu değerlendirmek için Western blotting uygulandı.
Bulgular: Kontrol grubu ile karșılaștırıldığında, diyabetik grupta elektrofizyolojik çalıșmalar İAİZ’nın uzadığını (37.91±6.81 ve 27.43±1.63 ms, p<0.01),
AERD’nin yükseldiğini gösterdi (30.37±8.33 ve 14.70±5.16 ms, p<0.01). Diyabetik grupta AF’nin indüklenebilmesi kontrol grubundan önemli derecede
yüksekti (hayvanların 8/10 ve 1/10'u, p<0.01). Diyabetik grupta kollajen hacim fraksiyonu artmıștı (%6.20±0.64 ve %2.15±0.21, p<0.01). “Patch-klemp” çalıșmaları, diyabetik tavșanlarda APS90 ve APS50’lerin uzadığını gösterdi (p<0.05 ve kontrol). INa yoğunluğu azaldı, ICaL yoğunluğu arttı (p<0.01 ve
kontrol). DBFβ 1’nin protein ekspresyonu diyabetik grupta arttı (p<0.001 ve kontrol).

Introduction
Atrial fibrillation (AF) is known to induce atrial electrical
remodeling (AER), which includes shortening, maladaptation, and
increased dispersion of atrial effective refractory period (AERP) as well as decreased atrial conduction velocity (1, 2). Intra-
cellular calcium overload and inhibition of the sodium pump have
been regarded as the major ion mechanisms of the initiation and maintenance of AF (3, 4). Short-term atrial remodeling primarily involves functional changes of L-type Ca
2+ channel and subse-
quent inactivation of voltage-dependent L-type Ca2+ current (ICaL)
(5). The slowing of intra-atrial conduction is considered to be one of the key factors for the formation of reentry, which is necessary for the induction of AF . Also, Na
+ current (INa) is essential for
conduction of the electrical impulses (6).
Diabetes mellitus (DM) is one of the strongest independent
risk factors for subsequent AF (7, 8) and has pathophysiological
links with AF (9), but the exact mechanisms that underlying the
relationship between DM and AF remain speculative. A recent meta-analysis (10) indicated that individuals with DM had an approximate 40% greater risk of AF compared with unaffected individuals. The pathophysiology of diabetic cardiomyopathy is
multifactorial, and major reason has been attributed to persistent
hyperglycemia (11). Hayden et al. (12) showed that high glucose levels have been shown to induce myocardial fibrosis in rats.
Molecular mechanisms for DM-induced electrical and struc-
tural remodeling is still unknown.
The aim of this study was to expound the effects of hypergly-
cemia on atrial interstitial fibrosis, ionic remodeling and vulner –
ability to AF .
Methods
Study design and preparation of the rabbit DM modelThis study was designed as randomized experimental study.
Sixty Japanese rabbits of either sex, weighing between 1.7 and 2.5kg were randomly assigned to alloxan- induced DM group
(n=30) and control group (n=30). Ten rabbits in each group were
respectively used to electrophysiological and histological study, patch-clamp study and Western blotting analysis. Experimental animal application approvals by the Experimental Animal Administration Committee of Tianjin Medical University and
Tianjin Municipal Commission for Experimental Animal Control
were obtained, which followed the guidelines established by the
U.S. National Institutes of Health.
In DM group, alloxan monohydrate (Sigma, Saint Louis, USA)
was dissolved in sterile normal saline to achieve a concentra-tion of 5% (W/V), and 150mg/kg was immediately administered
intravenously via the marginal ear vein. The diabetic state was
examined 48h later by quantitative determination of blood glu-
cose levels of ≥14mmol/L (once) or ≥11mmol/L (twice). Then,
blood glucose concentration had been monitored weekly with glucometer Optium Xceed (Abbott, Bedford, USA) for 8 weeks.
Hemodynamic and echocardiographic examination
At 8 weeks, the right carotid artery of the rabbit was iso-
lated surgically with a median incision in the neck. Cannula was inserted into the right carotid artery to monitor and record aortic systolic and diastolic blood pressure (SBP and
DBP). The cannula was moved through the aortic valve into
left ventricle to measure left ventricle end-diastolic pressure
(LVEDP). All the rabbits (n=20) for electrophysiological and
histological studies underwent transthoracic echocardiogra-
phy to measure left atrial diameter (LAD) and left ventricular ejection fraction (LVEF) with standard method.
Electrophysiological studies
A median sternotomy was performed, and the heart was
quickly excised and placed in perfusion fluid (4°C). The aorta was cannulated and connected to Langendorff perfusion system
filled with Tyrode’s solution saturated with 95% O
2 and 5% CO2.
The heart was perfused at 25 mL/min and the perfusion pressure
was maintained at 80 mmHg. Tyrode’s solution contained
(mmol/L) NaCl 130, KCl 5.6, NaHCO3 24.2, CaCl2 2.2, MgCl2 0.6,
NaH2PO4 1.2, and Glucose 12, pH 7.40 (adjusted with NaOH).
Four sets of silver bipolar electrodes were placed in the high
right atrium (HRA), high left atrium (HLA), low left atrium (LLA)
and right ventricle (RV). Wenckebach cycle length of atrial-ven-
tricular conduction was measured by right atrial incremental pacing. The inter-atrial conduction time (IACT) was measured
during 250 ms pacing of HRA. The atrial effective refractory period (AERP) was evaluated using programmed extra-stimuli,
which defined as the longest S1S2 interval that failed to capture.
AF vulnerability was tested by the burst pacing (50 ms) for 1s, 5
times at 30s interval. The appearance of AF was defined as
rapid, irregular atrial response longer than 1000 ms.
Histological examination
Following electrophysiological study, isolated left atrium (LA)
was placed in 4% paraformaldehyde, embedded in paraffin, and cut into 4μm cross-sections. Sirius-red (SR) (Sigma) stain was
used to evaluate interstitial fibrosis (n=10 for each group).
Micrographs were digitized using Photoshop 7.0 (Adobe, San
Jose, CA), and areas of fibrosis were analyzed using Image Pro Plus 4.5 Scion image software (Scion Co., Frederick, MD).Sonuç: Çalıșmamız, hipergliseminin diyabetik tavșanlarda AF gelișmesi ve tekrarlamasında etkili atriyal yapısal ve elektriksel remodeling'le sonuçla-
nan AF duyarlılığı, atriyal intersitisyel fibrozis ve iyonik remodelinge katkıda bulunduğunu düșündürmektedir.
(Anadolu Kardiyol Derg 2012; 12: 543-50)
Anahtar kelimeler: Hiperglisemi, atriyal intersitisyel fibrozis, iyonik remodeling, atriyal fibrilasyon, hayvanlarLiu et al.
Atrial fibrillation in diabetic rabbitsAnadolu Kardiyol Derg
2012; 12: 543-50 544

Whole-cell patch-clamp studies
In our study, experimental procedures were complied with
previously developed methods (13-15). Another twenty rabbits were used for whole-cell patch-clamp studies. LA was used for the isolation of single myocyte by 15-min perfusion with the solu-tion containing collagenase (315 U/mL, CLS II, Worthington Biochemical) and 0.2% bovine serum albumin (BSA) (Sigma). The whole-cell patch-clamp technique was used with an Axopatch 200B amplifier (Axon Instruments) to record ionic cur –
rents in voltage-clamp mode and AP in current-clamp mode. Membrane capacitance averaged 63.76±10.49 pF in the cells from control group (n=35 cells), 65.75±12.58 pF in DM group (n=37 cells). To control for cell-size variability, currents were expressed as densities (pA/pF). Voltage command pulses were generated by a 12-bit digital-to-analog (D/A) converter (Digidata 1200, Axon Instruments) controlled by pClamp 10.0 software. The action potentials (APs) were recorded at cycle lengths 60 bpm, 120 bpm, 180 bpm, 240 bpm and 300 bpm. APD stabilized within 5 action potentials at each cycle length, and steady state APD was mea-sured to 50% (APD50) and 90% (APD90) of full repolarization. The recordings were low pass-filtered at 0.5kHz and the sampling frequency was 0.5 Hz for recording INa, and was 0.2 Hz for recording ICaL. The current-voltage relation was determined in the extracellular solution over a voltage range of -80 to +60 mV increased in 10-mV steps from a hold potential (HP) of -90mV for INa and -40 to +60 mV increased in 10-mV steps from a HP of -50mV for ICaL.
Western blotting analysisTen rabbits from each group were prepared for Western blotting
analysis. Protein of LA tissue was extracted by total protein extrac-tion buffer. An equal amount of protein was loaded onto a 15% SDS denaturing polyacrylamide gel, separated by electrophoresis,
transferred onto PVDF membrane (Merck Millipore, USA) and incu-
bated with the specific primary antibody overnight at 4˚C. Protein levels of transforming growth factor beta 1 (TGFβ 1) were expressed
as ratio to levels of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). The membranes were then washed and subsequently
incubated with the secondary antibody conjugated to horseradish peroxidase (HRP). Protein was visualized using enhanced chemilu-minescence. The anti-GAPDH and anti-TGFβ 1 antibodies were
purchased from Abcam Inc. (Cambridge, UK). The resulting bands were quantified using GeneTools software (Gene, USA).
Statistical analysisStatistical analysis was performed using SPSS 13.0 (SPSS
Inc., Chicago, IL, USA). Continuous variables are expressed as mean±1 SD and categorical variables are presented as percent-ages. Kolmogorov-Smirnov test was used to test the distribution of numeric variables and variables with normal distribution were compared with Student t-test and those without normal distribu-tion were compared with Mann-Whitney U test. The incidence of AF was analyzed by Fisher’s exact test. A two-tailed p<0.05
was considered significant.Results
Hemodynamic and echocardiographic parameters
As shown in Table 1, there are no significant differences in
heart rate or SBP between both groups at baseline (p>0.05). The DBP and LVEDP were slightly increased in DM group, however,
no significant differences were found in each group (p>0.05).
LAD was significantly increased in DM group (p<0.001), but there are no significant differences in LVEF between both groups
(p>0.05).
Electrophysiological studiesAs shown in Table 2, IACT in DM group was significantly
prolonged compared with controls (p<0.01). AERPs in DM group
were slightly prolonged, but no significant differences were
observed between both groups. AERPD was significantly
increased in DM group (p<0.01). Figure 1 shows the inducibility
of AF in both groups. AF was induced in 8/10 (80%) of animals in
Variables Control DM *p
(n=10) (n=10)
Males/females, n 5/5 5/5 1.00
Weight, kg 2.17±0.15 2.29±0.33 0.30
Glucose, mmol/L 5.96±0.71 18.47±2.01 <0.001
Insulin, mU/L 21.59±8.05 13.78±3.12 <0.05
HR, bpm 167.20±15.44 156.45±26.05 0.27
SBP , mmHg 126.1±8.68 118.75±7.50 0.06
DBP , mmHg 96.60±6.60 100.40±6.53 0.21
LVEDP , mmHg -8.50±2.80 -5.80±4.91 0.15
LAD, mm 6.89±0.53 8.21±0.67 <0.001
LVEF , % 72.80±6.08 70.21±6.34 0.36
Values are presented as mean±SD
*Student t-test, Mann-Whitney U test
DBP – diastolic blood pressure, DM – diabetes mellitus, HR – heart rate, LAD – left atrial
diameter, LVEDP – left ventricular end-diastolic pressure, LVEF – left ventricular ejection fraction, SBP – systolic blood pressureTable 1. Baseline characteristics
Variables Control DM *p
(n=10) (n=10)
AVWCL, ms 167.90±7.32 181.82±27.40 0.138
IACT , ms 27.43±1.63 37.91±6.81 <0.01
HRAERP , ms 81.50±4.10 92.40±21.08 0.125
HLAERP , ms 84.25±6.64 93.70±18.01 0.136
LLAERP , ms 85.40±5.94 94.13±19.02 0.182
AERPD, ms 14.70±5.16 30.37±8.33 <0.01
Data are presented as mean±SD*Student t-test
AERPD – atrial effective refractory periods dispersion, AVWCL – Wenckebach cycle
length of A-V conduction, DM – diabetes mellitus, HLAERP – high left atrium effective
refractory period, HRAERP – high right atrium effective refractory period, IACT – inter –
atrial conduction time, LLAERP – low left atrium effective refractory periodTable 2. Electrophysiological studies measurementsLiu et al.
Atrial fibrillation in diabetic rabbitsAnadolu Kardiyol Derg
2012; 12: 543-50 545

DM group, whereas 1/10 (10%) of animals in control group
(p<0.01). Average duration of AF was 1180.63±93.78ms.
Atrial interstitial fibrosis
Figure 2 shows the representative histological sections of the
LA free wall from control group (A) (× 200; SR stain) and DM group
(B) (× 200; SR stain). Connective tissue was shown crimson in DM
group, atrial tissue from control group appeared normal. Mean percentage of left atrial interstitial fibrosis shows 2.15±0.21% in
control group vs. 6.20±0.64% in DM group (p<0.01); Data are pre-sented as mean±SD (C).
Action potential changes
AP measurements were begun 5 min after cell rupture. In cells
used for AP studies, the resting potential averaged respectively -62.8±2.4 mV in controls (n=10 cells) and -60.3±2.2 mV in DM (n=10
cells). In both groups, APD90 and APD50 decreased as stimulation frequency increased. DM substantially altered APD90 and APD50
compared with controls, mean values of APD are shown for two groups in Table 3. In addition to reducing APD90, DM caused no
significant reductions in rate-dependent APD90 changes.
ICaL and INa changes (Figures 3, 4)Figure 3A illustrates overall results for ICaL densities, peak
ICaL density is shown as a function of test potential (TP). DM
was associated with a increase in ICaL density. Table 4 shows
maximum peak ICaL density averaged 9.60±3.59 pA/pF in DM
group (n=10 cells) compared with 4.79±1.28 pA/pF in control
group (n=10 cells) (p<0.01). In Figure 4A, B, representative
recordings of ICaL from a cell of each group are displayed.
Figure 1. AF inducibility in both groups: A- AF episode induced by burst pacing in DM group, B- inducibility of AF in both groups, bars indicate
percentage of inducibility of AF
*p<0.01 vs. corresponding value in control group
AF – atrial fibrillation, DM – diabetes mellitus
Figure 2. Histological examinations on atrial fibrosis in both groups. A) SR stain of control group, B) SR stain of DM group, C) comparison of the
mean percentage of atrial fibrosis
*p<0.01 vs. corresponding value in control group
DM – diabetes mellitus, SR – Sirius-redLiu et al.
Atrial fibrillation in diabetic rabbitsAnadolu Kardiyol Derg
2012; 12: 543-50 546

Depolarizing upon 200-ms pulses from an HP of -50mV to volt-
ages ranging from -40 mV to +60 mV elicited typical ICaL (Fig. 4E).
Figure 3B illustrates overall results of INa densities, peak
INa density is shown as a function of TP . DM was associated
with a decrease in INa density. Table 4 shows maximum peak
INa density averaged 76.90±12.53 pA/pF in DM group (n=17 cells)
compared with 119.33±17.58 pA/pF in control group (n=15 cells) (p<0.01). Although INa density decreased in DM group, the form
of the I-V curve did not change. INa was significantly reduced at voltages ranging from -60mV to +20mV . In Figure 4C-D, represen-
tative recordings of INa from a cell of each group are displayed.
INa was measured upon 50-ms pulses from an HP of -90mV to voltages ranging from -80mV to +60mV (Fig. 4F).
Western blotting analysis
Figure 5 shows a product obtained by western blotting for
TGFβ1. Figure 5A shows the upper band in lines corresponds to TGFβ1 protein product, and the lower band is GAPDH protein
product. Figure 5B shows TGFβ 1 protein expression was
increased significantly in DM group (p<0.001).

Discussion
The major findings of our study are as follows: (i)
Electrophysiological changes in DM group included prolonged
IACT and increased AERPD; (ii) Increased inducibility of AF and
LA interstitial fibrosis are evident and may constitute a substrate for the development of AF; (iii) APD90 and APD50 of atrial myo-cytes were prolonged in diabetic rabbits. The densities of reduced INa and increased ICaL in the atria were associated
with DM ionic remodeling; (iv) DM increased fibrosis -related
TGFβ1 proteins in rabbit atria. Thus, this study provides patho-
physiological insights for the mechanisms of atrial electrical and
structural remodeling in the setting of DM.
Recent epidemiological studies have shown that DM may
exert a pro-arrhythmic substrate of AF (16-18). Type-1 diabetes,
chronic hyperglycemia and insulin resistance might be the
mechanisms responsible for the observed increased risk of AF .
Hyperglycemia and AF have been studied by others in more detailed experiments in mice, but not in rabbits. In keeping with previous findings (19), we showed that atrial electromechanical function were associated with increased IACT measured in
Langendorff perfused rabbit hearts. Kato et al. (20) showed that
Figure 3. A-I-V curves of ICaL obtained from both groups, B-I-V curves of INa obtained from both groups, current densities as function of test
potential (TP)
DM – diabetes mellitus, ICaL – ionic calcium L type current, INa – ionic sodium current
Variables Control DM *p
(n=10) (n=10)
ICaL, pA/pF -4.79±1.28 (10 cells) -9.60±3.59 (10 cells) <0.01
INa, pA/pF -119.33±17.58 (15 cells) -76.90±12.53 (17 cells) <0.01
Data are presented as mean±SD
*Student t-test
DM – diabetes mellitus, ICaL – ionic calcium L type current, INa – ionic sodium current Table 4. Current densities of ICaL and INa
Variables Control DM *p
(n=10) (n=10)
60 bpm APD90, ms 140.13±27.17 210.03±54.30 <0.01
APD50, ms 43.94±10.15 88.08±22.65 <0.01
120 bpm APD90, ms 132.30±22.41 206.69±45.89 <0.05
APD50, ms 41.65±15.32 74.38±33.56 <0.05
180 bpm APD90, ms 130.67±16.33 190.20±30.20 <0.05
APD50, ms 36.23±12.39 65.99±20.20 <0.05
240 bpm APD90, ms 123.60±13.09 168.02±29.73 <0.05
APD50, ms 31.49±7.42 62.43±18.64 <0.01
300 bpm APD90, ms 115.21±7.68 150.10±20.26 <0.05
APD50, ms 27.33±6.46 53.24±15.37 <0.01
Values are presented as mean±SD
*Student t-test
60, 120, 180, 240 and 300 bpm – stimulating frequency
APD – action potential duration, DM – diabetes mellitusTable 3. Changes in APD90 and APD50 in cells from both groupsLiu et al.
Atrial fibrillation in diabetic rabbitsAnadolu Kardiyol Derg
2012; 12: 543-50 547

inter-atrial conduction delay and increased fibrotic deposition in
atrium play a major role in producing atrial arrhythmogenicity,
which indicates that atrial structural remodeling characterized by extensive interstitial fibrosis may be one of the major mecha-
nisms of AF development in DM. The present study showed that
atrial fibrosis along with increased IACT and LAD was related to
increased inducibility of AF . AERPD was increased in DM rab-
bits, which suggesting that heterogeneity of repolarization is an important electrophysiological change in this setting.
One of the hallmarks of diabetic cardiomyopathy is the extra-
cellular matrix (ECM) remodeling leading to the increased fibro-
sis of myocardium. TGFβ1 cascade is most probably involved in
this process, which includes disproportionate increase in colla-gen and excessive ECM deposition due to enhanced expression
of TGFβ1 (21). We hypothesize that TGFβ1 might involve in the
pathogenesis of AF . Western blotting results revealed that TGFβ1 elevated in LA tissue of DM rabbits had positive correlation with
atrial fibrosis. However, the precise nature of molecular compo-nents activated by TGFβ1 leading to fibrosis is not known and needs further studies.
Shortening of APD is the major characteristic in atrial myo-
cytes during AF . By promoting the formation and maintenance of multiple wavelets, the reductions in refractory period and APD are associated with the onset and maintenance of AF (1, 22). Both clinical (23, 24) and experimental (25) studies consistently point to an important down-regulating effect of AF on ICaL, also down-regulation of ICaL as one of the arrhythmogenic AP abnor –
malities was associated with AF maintenance. ICaL plays a sig-nificant role in maintaining the plateau in atrial myocytes (13), therefore, it is a candidate to underlie the APD changes caused by DM. Depression of ICaL was responsible for much of the APD shortening in DM rabbits, and blockade of INa caused little fur –
Figure 4. Voltage-dependent ICaL and INa races recorded in a representative myocyte, A) ICaL in control group, B) ICaL in DM group, C) INa in
DM group, D) INa in control group, E) voltage protocol for recording ICaL, F) voltage protocol for recording INa
DM – diabetes mellitus, ICaL – ionic calcium L type current, INa – ionic sodium currentLiu et al.
Atrial fibrillation in diabetic rabbitsAnadolu Kardiyol Derg
2012; 12: 543-50 548

ther change in APD (26). In our study, based on the presence of
atrial ion current changes, eight weeks of hyperglycemia is suf-ficient to cause atrial ionic remodeling.
Cardiac voltage-gated Na+ channel (Nav) conducts the inward
INa which is an important determinant of conduction velocity (CV) in cardiac cells. INa plays a central role in the generation of AP and contributes to control APD (27). The Nav1.5 α subunit is the
principal component of INa channel forming the pore and all essential gating elements, which is sufficient by itself for generat-ing INa in heterologous expression systems. Previous studies
showed prolongations of IACT implying slowed atrial conduction
in different animal’s models (27, 28) and patients (29, 30) with AF . The slowing of IACT is considered to be one of the most important players for the initiation of reentry in addition to the reduction in AERP and AERP rate adaptation, also the induction of AF . A sig-
nificantly wider zone of IACT delay was observed in patients with
paroxysmal AF compared with controls (31). The decrease of INa density and gene expression may relate to intra-cellular calcium overload which is regarded as a major mechanism of electrical remodeling (3, 32). Increased cytosolic Ca
2+ can decrease INa
density by means of down-regulating mRNA expression encoding Nav (33). The number of wavelets in atrium is strongly affected by the wavelength which is defined as the distance traveled by the
activation wave during functional refractoriness and which is
determined by the local AERP and CV . Slowing conduction results in shorter wavelength and increases the number of wavelets that could coexist in the given atrial dimension, which increases the likelihood that AF would sustain itself.
Further studies on atrial electrophysiological function may
own special value to assess the impact of novel therapeutic drugs of anti-remodeling effects, such as pioglitazone (34, 35) or
candesartan (36), on prevention and treatment of AF .
Study limitationsOne of limitations of the study is that only the LA in whole cell
electrophysiological study was analyzed, the response of the
right atrium to hyperglycemia is still unknown. Furthermore, we
did not assess the possible paradoxical response of AERP to increased heart rate, which is another characteristic of electri-
cal remodeling. Finally, the molecular mechanisms for
DM-induced electrical and structural remodeling should be further investigated in future studies.
Conclusion
Our study demonstrated that, hyperglycemia contributes
to aggravate atrial interstitial fibrosis, ionic remodeling and
vulnerability to AF in diabetic rabbits, which resulting in
effects on atrial structural remodeling and electrical
remodeling for the development and perpetuation of AF .
Hence, counterbalancing the hyperglycemia actions may
represent a novel pathway to prevent atrial remodeling, and perhaps an important therapeutic approach to the prevention
of AF .
Conflict of interest: None declared.
Acknowledgments
This work was supported by grants (30900618 to T .L.) from the
National Natural Science Foundation of China.
Figure 5. DM increases TGFβ1 protein expression in rabbit atria
*p<0.001 vs. corresponding value in control group
DM – diabetes mellitus, TGFβ1 – transforming growth factors β1Liu et al.
Atrial fibrillation in diabetic rabbitsAnadolu Kardiyol Derg
2012; 12: 543-50 549

Authorship contributions: Concept – G.L.; Design – T .L.;
Supervision – C.L.; Resource – G.L.; Materials – J.L.; Data collec-
tion&/or Processing – C.L., H.F .; Analysis &/or interpretation – H.F .; Literature search – W.Y .; Writing – C.L.; Critical review – T .L.; Other – L.C.
References
1. Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillation
begets atrial fibrillation. A study in awake chronically instrumented goats. Circulation 1995; 92: 1954-68. [CrossRef]
2. Nattel S, Shiroshita-Takeshita A, Cardin S, Pelletier P . Mechanisms of atrial remodelling and clinical relevance. Curr Opin Cardiol 2005; 20: 21-5.
3. Goette A, Honeycutt C, Langberg JJ. Electrical remodeling in atrial
fibrillation. Time course and mechanisms. Circulation 1996; 94:
2968-74. [CrossRef]
4. De Mello WC. Angiotensin (1-7) re-establishes impulse conduction in cardiac muscle during ischaemia-reperfusion. The role of the sodium pump. J Renin Angiotensin Aldosterone Syst 2004; 5: 203-8. [CrossRef]
5. Shinagawa K, Mitamura H, Ogawa S, Nattel S. Effects of inhibiting Na+/
H+- exchange or angiotensin converting enzyme on atrial tachycardia-
induced remodeling. Cardiovasc Res 2002; 54: 438-46. [CrossRef]
6. Gaspo R, Bosch RF , Bou-Abboud E, Nattel S. Tachycardia-induced
changes in sodium current in a chronic dog model of atrial
fibrillation. Circ Res 1997; 81: 1045-52. [CrossRef]
7. Movahed MR, Hashemzadeh M, Jamal MM. Diabetes mellitus is a
strong, independent risk for atrial fibrillation and flutter in addition to
other cardiovascular disease. Int J Cardiol 2005; 105: 315-8. [CrossRef]
8. Levy S. Atrial fibrillation, the arrhythmia of the elderly, causes and
associated conditions. Anadolu Kardiyol Derg 2002; 2: 55-60.
9. Lip GY , Varughese GI. Diabetes mellitus and atrial fibrillation:
perspectives on epidemiological and pathophysiological links. Int J
Cardiol 2005; 105: 319-21. [CrossRef]
10. Huxley RR, Filion KB, Konety S, Alonso A. Meta-analysis of cohort
and case-control studies of type 2 diabetes mellitus and risk of
atrial fibrillation. Am J Cardiol 2011; 108: 56-62. [CrossRef]
11. Boudina S, Abel ED. Diabetic cardiomyopathy revisited. Circulation
2007; 115: 3213-23. [CrossRef]
12. Hayden MR, Chowdhury N, Govindarajan G, Karuparthi PR, Habibi J, Sowers JR. Myocardial myocyte remodeling and fibrosis in the cardiometabolic syndrome. J Cardiometab Syndr 2006; 1: 326-33.
[CrossRef]
13. Yue L, Feng J, Li GR, Nattel S. Transient outward and delayed
rectifier currents in canine atrium: properties and role of isolation methods. Am J Physiol 1996; 270: H2157-68.
14. Li D, Melnyk P , Feng J, Wang Z, Petrecca K, Shrier A, et al. Effects of experimental heart failure on atrial cellular and ionic
electrophysiology. Circulation 2000; 101: 2631-8. [CrossRef]
15. Karmazínová M, Lacinová L. Measurement of cellular excitability by
whole cell patch clamp technique. Physiol Res 2010; 59 Suppl 1: S1-7.
16. Watanabe H, Tanabe N, Watanabe T , Darbar D, Roden DM, Sasaki
S, et al. Metabolic syndrome and risk of development of atrial
fibrillation: the Niigata preventive medicine study. Circulation 2008;
117: 1255-60. [CrossRef]
17. Nichols GA, Reinier K, Chugh SS. Independent contribution of
diabetes to increased prevalence and incidence of atrial fibrillation. Diabetes Care 2009; 32: 1851-6. [CrossRef]
18. Dublin S, Glazer NL, Smith NL, Psaty BM, Lumley T , Wiggins KL, et al. Diabetes mellitus, glycemic control, and risk of atrial fibrillation.
J Gen Intern Med 2010; 25: 853-8. [CrossRef]19. Karaca M, Kınay O, Nazlı C, Biçeroğlu S, Vatansever F , Ergene AO.
The time interval from the initiation of the P-wave to the start of left
atrial appendage ejection flow: does it reflect interatrial conduction time? Echocardiography 2007; 24: 810-5. [CrossRef]
20. Kato T , Yamashita T , Sekiguchi A, Sagara K, Takamura M, Takata S,
et al. What are arrhythmogenic substrates in diabetic rat atria? J
Cardiovasc Electrophysiol 2006; 17: 890-4. [CrossRef]
21. Kaminski KA, Szepietowska B, Bonda T , Kozuch M, Mencel J,
Malkowski A, et al. CCN2 protein is an announcing marker for cardiac remodeling following STZ- induced moderate
hyperglycemia in mice. Pharmacol Rep 2009; 61: 496-503.
22. Van Wagoner DR, Pond AL, McCarthy PM, Trimmer JS, Nerbonne JM.
Outward K+ current densities and Kv1.5 expression are reduced in
chronic human atrial fibrillation. Circ Res 1997; 80: 772-81. [CrossRef]
23. Bosch RF , Zeng X, Grammer JB, Popovic K, Mewis C, Kühlkamp V .
Ionic mechanisms of electrical remodeling in human atrial
fibrillation. Cardiovasc Res 1999; 44: 121-31. [CrossRef]
24. Van Wagoner DR, Pond AL, Lamorgese M, Rossie SS, McCarthy
PM, Nerbonne JM. Atrial L-type Ca2+ currents and human atrial
fibrillation. Circ Res 1999; 85: 428-36. [CrossRef]
25. Yue L, Feng J, Gaspo R, Li G, Wang Z, Nattel S. Ionic remodeling
underlying action potential changes in a canine model of atrial
fibrillation. Circ Res 1997; 81: 512-25. [CrossRef]
26. Saffitz JE. Douglas P . Zipes Lecture. Biology and pathobiology of
cardiac connexins: from cell to bedside. Heart Rhythm 2006; 3: 102-7.
[CrossRef]
27. Morillo CA, Klein GJ, Jones DL, Guiraudon CM. Chronic rapid atrial
pacing. Structural, functional, and electrophysiological
characteristies of a new model of sustained atrial fibrillation.
Circulation 1995; 91: 1588-95. [CrossRef]
28. Elvan A, Wylie K, Zipes DP . Pacing-induced chronic atrial fibrillation
impairs sinus node function in dogs: electrophysiological
remodeling. Circulation 1996; 94: 2953-60. [CrossRef]
29. Kumagai K, Akimitsu S, Kawahira K, Kawanami F , Yamanouchi Y ,
Hiroki T , et al. Electrophysiological properties in chronic lone atrial
fibrillation. Circulation 1991; 84: 1662-8. [CrossRef]
30. Cosio FG, Palacios J, Vidal JM, Cocina EG, Gómez-Sánchez MA,
Tamargo L. Electrophysiologic studies in atrial fibrillation. Slow
conduction of premature impulses: a possible manifestation of the
background for reentry. Am J Cardiol 1983; 51: 122-30. [CrossRef]
31. Shimizu A, Fukatani M, Tanigawa M, Mori M, Hashiba K. Intra-
atrial conduction delay and fragmented atrial activity in patients
with paroxysmal atrial fibrillation. Jpn Circ J 1989; 53: 1023-30.
[CrossRef]
32. Yue L, Melnyk P , Gaspo R, Wang Z, Nattel S. Molecular mechanisms
underlying ionic remodeling in a dog model of atrial fibrillation. Circ
Res 1999; 84: 776-84. [CrossRef]
33. Duff HJ, Offord J, West J, Catterall WA. Class I and IV antiarrhythmic drugs
and cytosolic calcium regulate mRNA encoding the sodium channel alpha
subunit in rat cardiac muscle. Mol Pharmacol 1992; 42: 570-4.
34. Kume O, Takahashi N, Wakisaka O, Nagano-Torigoe Y , Teshima Y ,
Nakagawa M, et al. Pioglitazone Attenuates inflammatory atrial
fibrosis and vulnerability to atrial fibrillation induced by pressure
overload in rats. Heart Rhythm 2011; 8: 278-85. [CrossRef]
35. Xu D, Murakoshi N, Igarashi M, Hirayama A, Ito Y , Seo Y , et al. PPAR-γ
Activator pioglitazone prevents age-related atrial fibrillation
susceptibility by improving antioxidant capacity and reducing apoptosis
in a rat model. J Cardiovasc Electrophysiol 2012; 23:209-17. [CrossRef]
36. Doğan A, Akçay S. The effect of inhibition of renin-angiotensin system on cardioversion success and recurrences ofatrial fibrillation. Anadolu Kardiyol Derg 2009; 9: 505-11. Liu et al.
Atrial fibrillation in diabetic rabbitsAnadolu Kardiyol Derg
2012; 12: 543-50 550

Copyright of Anatolian Journal of Cardiology / Anadolu Kardiyoloji Dergisi is the property of Aves Yayincilik
Ltd. STI and its content may not be copied or emailed to multiple sites or posted to a listserv without the
copyright holder's express written permission. However, users may print, download, or email articles for
individual use.