International Journal of Cardiology 165 (2013) 299–307

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International Journal of Cardiology

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Rosiglitazone induces arrhythmogenesis in diabetic hypertensive rats with calciumhandling alteration

Ting-I Lee a,c, Yao-Chang Chen d, Yu-Hsun Kao a, Fone-Ching Hsiao a,e, Yung-Kuo Lin a,b, Yi-Jen Chen a,b,⁎a Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwanb Division of Cardiovascular Medicine, Wan Fang Hospital, Taipei Medical University, Taiwanc Division of Endocrinology and Metabolism, Wan Fang Hospital, Taipei Medical University, Taiwand Department of Biomedical Engineering, National Defense Medical Center, Taiwane Division of Endocrinology and Metabolism, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taiwan

⁎ Corresponding author at: Graduate Institute of Clinicaversity, 250Wu-Xing Street, Taipei 110-31, Taiwan. Tel.: +29339378, +886 2 28359946.

E-mail address: a9900112@ms15.hinet.net (Y.-J. Che

0167-5273/$ – see front matter © 2011 Elsevier Irelanddoi:10.1016/j.ijcard.2011.08.072

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 13 July 2011Accepted 20 August 2011Available online 13 September 2011

Keywords:Calcium handlingCardiomyocytesDiabetes mellitusHypertension

Background: Diabetes and hypertension have significant effects on cardiac calcium (Ca2+) regulation, whichplays an essential role in determining cardiac function. The effect of peroxisome proliferator-activated recep-tor (PPAR)-γ agonists on Ca2+ regulation in the cardiomyocytes is unclear.Objective: The purpose of this study was to investigate the effects of hypertension, diabetes, and PPAR-γ ago-nist-rosiglitazone on the regulation of Ca2+ and the electrophysiological characteristics of isolated ventricu-lar myocytes.Methods: The indo-1 fluorometric ratio technique and whole-cell patch clamp were used to investigate intra-cellular Ca2+ (Ca2+i), action potentials, and ionic currents in ventricular myocytes from rats of Wistar–Kyoto(WKY), diabetic WKY (induced by streptozotocin), diabetic WKY treated with rosiglitazone (5 mg/kg), spon-

taneously hypertensive rats (SHR), diabetic SHR, and diabetic SHR treated with rosiglitazone. Western blotwas used to evaluate protein expressions of sarcoplasmic reticulum ATPase (SERCA2a), Na+–Ca2+ exchanger(NCX), and ryanodine receptor (RyR).Results: Diabetic WKY and diabetic SHR had smaller sarcoplasmic reticulum Ca2+ contents, and Ca2+i tran-sients with a prolonged decay portion, down-regulated SERCA2a, NCX, and RyR protein expressions andsmaller L-type Ca2+ currents than non-diabetic WKY and SHR, respectively. The Ca2+ dysregulations in dia-betes were attenuated in rats treated with rosiglitazone. Diabetes and hypertension both prolonged the ac-tion potential duration which were enhanced by the use of rosiglitazone, and induced the genesis oftriggered activity.Conclusions: Diabetes and hypertension modulate Ca2+ handling. Rosiglitazone significantly changed the Ca2+

regulation and electrophysiological characteristics, and may contain an arrhythmogenic potential in diabeteswith hypertension.

© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

A 1.5–2-fold increase in morbidity was found in diabetic patientscompared to the general population [1], and cardiovascular diseaseis the leading cause of diabetes-related morbidity and mortality [2].Moreover, hypertension is a common comorbidity in diabetic pa-tients, and plays an important role in determining their prognosis[3]. However, the mechanisms and functional consequences underly-ing the pathological effects of hypertension in diabetes have not beenfully elucidated. Hypertension was shown to induce cardiac diastolicdysfunction and mechanical stress [4]. Since the mechanoelectrical

l Medicine, Taipei Medical Uni-886 2 27390500; fax: +886 2

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feedback in cardiac enlargement alters the cardiac electrophysiologyleading to cardiac arrhythmia [5], hypertension may interact with di-abetes to enhance cardiac dysfunction.

Calcium (Ca2+) influx triggers a large Ca2+ release from the sarco-plasmic reticulum (SR), thus activating further ionic currents in late di-astole which contributes to the contractility of cardiomyocytes [6]. Theamount of Ca2+ released from the SR depends not only on the triggerbut also on the SR Ca2+ content and intracellular Ca2+ (Ca2+i) concen-tration [7]. SR Ca2+ content ismostly controlled by the balance betweenCa2+ uptake, via SR Ca2+-ATPase (SERCA2a), and release largely fromthe ryanodine receptor (RyR). However, it is not clear whether hyper-tension and diabetes have significant effects on Ca2+ regulation in ven-tricular myocytes.

Peroxisome proliferator-activated receptors (PPARs) are nucleartranscription factors with three isoforms (α, γ, and δ) that play criti-cal roles in cardiac function [8]. PPAR-γ regulates glucose metabolism

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and lipogenesis and was demonstrated to have anti-inflammatory,antihypertrophic, and antiatherosclerotic effects [9]. However, highdose of PPAR-γ agonist was shown to induce cardiac dysfunctionwith marked changes in the utilization of free fatty acids and glucose[10]. Moreover, increasing incidences of myocardial infarction andmortality from cardiovascular complications were also found withthe use of PPAR-γ agonists in diabetic patients [11]. The mechanismof the cardiac effects of PPAR-γ agonists causing the increased inci-dence of myocardial dysfunction remains unclear. It is therefore im-portant to investigate the effects of PPAR-γ agonists under differentpathological conditions. Previous study had shown that PPAR-γ li-gandmay have direct electromechanical effects in isolated cardiac tis-sues [12]. However, information regarding the role of PPAR-γagonists in the cardiac electrophysiological characteristics is limited.In addition, the effects of PPAR-γ agonists on the regulation of Ca2+

in cardiomyocytes are unclear. Therefore, the purpose of this studywas to investigate whether diabetes with or without hypertensionhas significant effects on the regulation of Ca2+ in ventricular cardio-myocytes, and to evaluate the electrophysiological effects of rosiglita-zone in these cardiomyocytes.

2. Materials and methods

2.1. Animal and tissue preparations

This investigation was approved by the Institutional Animal Care and Use Commit-tee of Taipei Medical University (IACC no. LAC-97-0076) and complied with the Guidefor the Care and Use of Laboratory Animals and the Guide for the Care and Use of Labora-tory Animals published by the US National Institutes of Health (NIH Publication no.85-23, revised 1996). The rats were divided into spontaneously hypertensive rats(SHR), diabetic SHR, diabetic SHR treated with rosiglitazone, Wistar–Kyoto (WKY, con-trol group), diabetic WKY, and diabetic WKY treated with rosiglitazone groups. Fortytwo male SHR andWKY (10 weeks old) received intraperitoneal injection of streptozo-tocin (65 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) to induce diabetes with a fastingplasma glucose of ≥15 mmol/L, measured with a glucometer (Ascensia Elite, BayerHealth Care, Mishawaka, IN, USA) as described previously [13]. The rats were housedin standard environmental conditions and maintained on commercial rat chow andtap water ad libitum. At 12 weeks of age, 20 diabetic SHR and diabetic WKY were trea-ted with rosiglitazone (5 mg/kg, GlaxoSmith-Kline Pharmaceutical, Philadelphia,Puerto Rico) [14] and the SHR, diabetic SHR, WKY, and diabetic WKY (20 in eachgroup) were treated with placebo daily by oral gavage for 2 weeks. The rats were sacri-ficed at 14 weeks old. Each heart was rapidly excised, weighed, and dissected. Cardiactissues were rinsed in a cold physiological saline solution. Transverse tissue piecesfrom the ventricles were snap-frozen in liquid nitrogen for protein isolation.

2.2. Western blot analysis of Ca2+ regulatory proteins

Equal amounts of proteins were resolved by sodium dodecylsulfate polyacryl-amide gel electrophoresis as described previously [13]. Blots were probed with anti-bodies against SERCA2a (Santa Cruz Biotechnology, CA, USA), Na+–Ca2+ exchanger(NCX, Swant, Bellizona, Switzerland), RyR (Affinity Bioreagents, Golden, CO, USA),and secondary antibodies conjugated with horseradish peroxidase (Leinco Technology,St. Louis, MO, USA). Bound antibodies were detected with an enhanced chemilumines-cence detection system (Millipore, St. Louis, MO, USA) and analyzed with AlphaEaseFCsoftware (Alpha Innotech, San Leandro, CA, USA). Targeted bands were normalized tocardiac α-sarcomeric actin (Sigma-Aldrich, St. Louis, MO, USA) to confirm equal pro-tein loading.

2.3. Measurement of Ca2+i

Ventricularmyocyteswere enzymatically dissociatedwith collagenase (type I, Sigma,St, Lou) and protease (type XIV, Sigma) as described previously [15]. The Ca2+i wasrecorded using fluorometric ratio technique (indo-1 fluorescence) in single myocytes.The fluorescent indicator, indo-1, was loaded by incubating myocytes at room tem-perature for 20–30 min with 10 μM of indo-1/AM (Sigma). Myocytes were then per-fused with a normal bath solution at 35±1 °C for at least 20 min to wash outextracellular indicator and allow the intracellular deesterification of indo-1. Thebackground and cell autofluorescences were canceled out by zeroing the output ofphotomultiplier tubes using cells without indo-1 loading. The experiments were per-formed at 35±1 °C.

An ultraviolet light of 360 nm with a monochromator was used to excite indo-1from a xenon arc clamp controlled by a microfluorometric system (OSP100-CA, Olym-pus, Tokyo, Japan), and the excitation light beam was directed into an inverted micro-scope (IX-70; Olympus). The emitted fluorescence signals from indo-1/AM-loadedmyocytes were digitized at 200 Hz. The ratios of the fluorescence emissions at410 nm and 485 nm (R410/485) were recorded, and R410/485 was used as the index of

Ca2+i. This approach avoided uncertainties from calibrating the fluorescent Ca2+

indicators. The Ca2+i transient, peak systolic Ca2+i, diastolic Ca2+i, and decay portionof the Ca2+i transient were measured during a 2-Hz field-stimulation with 10-mstwice-threshold strength square-wave pulses. The Ca2+i transient was determined bya monoexponential least-squares fit. The Ca2+i transient was calculated from the dif-ference of the peak systolic Ca2+i and diastolic Ca2+i. The fluorescence ratio data wasprocessed and stored in a computer using software (OSP-SFCA; Olympus). The SRCa2+ content was estimated by adding 20 mM of caffeine after electrical stimulationat 2 Hz for at least 30 s. The total SR Ca2+ content was measured from the peak ampli-tude of caffeine-induced Ca2+i transients.

2.4. Electrophysiological study

A whole-cell patch-clamp was performed in the ventricular myocytes of rats usingAxopatch 1D amplifier (Axon Instruments, Foster City, CA, USA) at 35±1 °C. Borosili-cate glass electrodes (o.d., 1.8 mm) were used with tip resistances of 3–5 MΩ. Beforethe formation of the membrane-pipette seal, the tip potentials were zeroed in Tyrode'ssolution. Junction potentials between the bath and pipette solution (9 mV) were cor-rected for action potential (AP) recordings. The APs driven at 1 Hz were recorded inthe current-clamp mode, and ionic currents were measured in the voltage-clampmode. A small hyperpolarizing step from a holding potential of −50 mV to a test po-tential of −55 mV for 80 ms was delivered at the beginning of each experiment. Thearea under the capacitative currents was divided by the applied voltage step to obtainthe total cell capacitance. Normally, 60%–80% series resistance was electronically com-pensated. Micropipettes were filled with solutions containing (in mM): CsCl 130,MgCl2 1, Mg2ATP 5, HEPES 10, EGTA 10, NaGTP 0.1, and Na2 phosphocreatine 5, titratedto pH of 7.2 with CsOH for L-type Ca2+ current (ICa-L); NaCl 20, CsCl 110, MgCl2 0.4,CaCl2 1.75, tetraethylammonium 20, BAPTA 5, glucose 5, Mg2ATP 5, and HEPES 10, ti-trated to pH of 7.25 for NCX current; and KCl 20, K aspartate 110, MgCl2 1, Mg2ATP5, HEPES 10, EGTA 0.5, LiGTP 0.1, and Na2 phosphocreatine 5, (pH of 7.2 with KOH)for AP. Voltage command pulses were generated by 12-bit digital-to-analog convertercontrolled using pCLAMP software (Axon Instruments). Recordings were lowpass-filtered at half the sampling frequency.

ICa-L was measured as an inward current during depolarization from a holding po-tential of −50 mV to testing potentials ranging from −40 to +60 mV in 10-mV stepsfor 300 ms at a frequency of 0.1 Hz. The NaCl and KCl in the external solution were re-spectively replaced by tetraethylammonium chloride and CsCl. The NCX current waselicited by depolarizing pulses between −100 and +100 mV from holding potentialof −40 mV for 300 ms at a frequency of 0.1 Hz. The amplitudes of NCX currentswere measured as 10-mM nickel-sensitive currents. The external solution (mM) con-sisted of NaCl 140, CaCl2 2, MgCl2 1, HEPES 5, and glucose 10 with pH adjusted to 7.4and contained 10 μM strophanthidin (to block the Na+/K+ pump), 10 μM nitrendipine(dihydropyrdine antagonist) and 100 μM niflumic acid (to block Ca2+-activated Cl−

currents).

2.5. Statistical analysis

All quantitative data are expressed as the mean±S.E.M. Statistical significance be-tween different groups was determined by unpaired t-test or two-way analysis of var-iance (ANOVA) with Fisher's least significant difference for post-hoc test analysis ofmultiple comparisons as appropriate. A value of pb0.05 was considered statisticallysignificant.

3. Results

3.1. Effects of diabetes, hypertension, and rosiglitazone on Ca2+i

Fig. 1 shows tracings of Ca2+i transients in the ventricular myo-cytes of WKY, diabetic WKY, diabetic WKY treated with rosiglitazone,SHR, diabetic SHR, and diabetic SHR treated with rosiglitazone. Asshown in Fig. 1A and B, diabetic WKY ventricular myocytes had a sig-nificantly smaller Ca2+i transients (0.21±0.01 vs. 0.25±0.01,pb0.005), peak systolic Ca2+i, and diastolic Ca2+i than WKY ventric-ular myocytes, which were attenuated after rosiglitazone treatment.Similarly, diabetic SHR ventricular myocytes showed smaller Ca2+i

transients (0.17±.01 vs. 0.22±0.01, pb0.005), systolic Ca2+i, and di-astolic Ca2+i than SHR, which were attenuated after rosiglitazonetreatment. Moreover, Ca2+i transients, and peak systolic Ca2+i inSHR, diabetic SHR, and rosiglitazone-treated diabetic SHR were smal-ler than in WKY, diabetic WKY, and rosiglitazone-treated diabeticWKY, respectively.

As shown in Fig. 1B, diabetic WKY ventricular myocytes had a sig-nificantly prolonged Ca2+ decay than WKY (47 ms±4 vs. 34 ms±3,pb0.05), which was attenuated after rosiglitazone treatment. Similar-ly, diabetic SHR ventricular myocytes had a significantly prolonged

Fig. 1. Effects of diabetes, hypertension, and a PPAR-γ agonist, rosiglitazone, on intracellular Ca2+ (Ca2+)i transients in the ventricular myocytes from Wistar–Kyoto (WKY), diabetic WKY, rosiglitazone-treated diabetic WKY, spontaneouslyhypertensive rats (SHR), diabetic SHR, and rosiglitazone-treated diabetic SHR. A. Representative tracing of the Ca2+i transient of ventricular myocytes recorded from different groups. B. Mean values of the Ca2+i transient fromWKY (n=43),diabetic WKY (n=21), rosiglitazone-treated diabetic WKY (n=50), SHR (n=49), diabetic SHR (n=61), and rosiglitazone-treated diabetic SHR (n=67). *pb0.05 vs. respective WKY with and without diabetes or diabetes treated withrosiglitazone.

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Ca2+ decay than SHR (59 ms±3 vs. 52 ms±2, pb0.05), which wasalso attenuated after rosiglitazone treatment. Moreover, Ca2+ decayin SHR, diabetic SHR, and rosiglitazone-treated diabetic SHR was lon-ger than in WKY, diabetic WKY, and rosiglitazone-treated diabeticWKY, respectively.

3.2. Effects of diabetes, hypertension, and rosiglitazone on Ca2+ stores

Fig. 2 shows tracings of Ca2+i stores in ventricular myocytes ofWKY, diabetic WKY, diabetic WKY treated with rosiglitazone, SHR, di-abetic SHR, and diabetic SHR treated with rosiglitazone. As shown inFig. 2A and B, diabetic WKY ventricular myocytes had a significantlysmaller Ca2+ stores than WKY ventricular myocytes (0.57±0.05 vs.0.89±0.03, pb0.005), which was attenuated after rosiglitazone

Fig. 2. Effects of diabetes, hypertension, and rosiglitazone on Ca2+ stores measured from cWKY, rosiglitzone-treated diabetic WKY, SHR, diabetic SHR, and rosiglitazone-treated diabefrom different groups. B. Mean values of the Ca2+ stores from WKY (n=43), diabetic W(n=61), and rosiglitazone-treated diabetic SHR (n=67). *pb0.05 vs. respective WKY with

treatment. Similarly, diabetic SHR ventricular myocytes had smallerCa2+ stores than SHR (0.35±0.03 vs. 0.53±0.032, pb0.005) whichwas also attenuated after rosiglitazone treatment. Moreover, Ca2+

stores in SHR, diabetic SHR, and rosiglitazone-treated diabetic SHRwere smaller than in WKY, diabetic WKY, and rosiglitazone-treateddiabetic WKY, respectively.

3.3. Effects of diabetes, hypertension, and rosiglitazone on SERCA2a, NCX,and RyR protein expressions

As shown in Fig. 3, we compared the molecular changes in Ca2+

regulatory proteins in ventricular myocytes of WKY, diabetic WKY,diabetic WKY treated with rosiglitazone, SHR, diabetic SHR, and dia-betic SHR treated with rosiglitazone by Western blot analyses.

affeine (20 mM)-induced Ca2+ transients in ventricular myocytes from WKY, diabetictic SHR. A. Representative tracing of the Ca2+ stores of ventricular myocytes recordedKY (n=21), rosiglitazone-treated diabetic WKY (n=50), SHR (n=49), diabetic SHRand without diabetes or diabetes treated with rosiglitazone.

Fig. 3. Cardiac sarcoplasmic reticulum ATPase (SERCA2a), Na+–Ca2+ exchanger (NCX),and ryanodine receptor (RyR) in ventricle myocytes from WKY, diabetic WKY, rosigli-tazone-treated diabetic WKY, SHR, diabetic SHR, and rosiglitazone-treated diabeticSHR. Representative immunoblot and average data of SERCA2a, NCX and RyR fromWKY (n=7), diabetic WKY (n=6), rosiglitazone-treated diabetic WKY (n=6), SHR(n=6), diabetic SHR (n=7), and rosiglitazone-treated diabetic SHR (n=6). Densi-tometry was normalized to α-sarcomeric actin as an internal control. *pb0.05 vs. re-spective WKY with and without diabetes or diabetes treated with rosiglitazone.

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SERCA2a protein expression was down-regulated in diabetic WKYcompared to WKY ventricular myocytes by 62%; which was attenuat-ed after rosiglitazone treatment. Similarly, diabetic SHR ventricularmyocytes had a decrease protein expression compared to SHR by70%; which was also attenuated by rosiglitazone treatment. More-over, SERCA2a protein expressions in SHR, diabetic SHR, androsiglitazone-treated diabetic SHR were decreased compared toWKY, diabetic WKY, and rosiglitazone-treated diabetic WKY rats,respectively.

Fig. 3 also shows that NCX protein expression was down-regulatedin diabetic WKY compared to WKY ventricular myocytes by 71%,which was attenuated after rosiglitazone treatment. Similarly, diabeticSHR ventricular myocytes had a lower protein levels compared to SHRby 74%, which was also attenuated after rosiglitazone treatment.However, NCX protein expression in SHR, diabetic SHR, androsiglitazone-treateddiabetic SHRwere respectively similar toWKY, di-abetic WKY, and rosiglitazone-treated diabetic WKY.

RyR protein expressions (Fig. 3) decreased in diabetic WKY and dia-betic SHR by 46% and 87% when respectively compared to WKY and

SHR, which were attenuated after rosiglitazone treatment. Moreover,RyR protein expressions in SHR, diabetic SHR, and rosiglitazone-treateddiabetic SHR were decreased compared to WKY, diabetic WKY, androsiglitazone-treated diabetic WKY, respectively.

3.4. Effects of diabetes, hypertension, and rosiglitazone on the action po-tential duration of ventricular myocytes

Fig. 4A shows tracings of APs in ventricular myocytes from WKYand SHR. The action potential amplitude (Fig. 4C) was significantlydelayed in ventricular myocytes from SHR, diabetic SHR, and diabeticSHR treated with rosiglitazone as respectively compared to thosefrom WKY, diabetic WKY, and diabetic WKY treated with rosiglita-zone. Fig. 4C shows that action potential duration at 20% (APD20),APD50, and APD90 of diabetic WKY and diabetic WKY treated withrosiglitazone were prolonged when compared with WKY ventricularmyocytes. Similarly, ventricular myocytes from diabetic SHR and dia-betic SHR treated with rosiglitazone had longer durations when com-pared to SHR at APD20, APD50, and APD90. Moreover, APs ofventricular myocytes from SHR, diabetic SHR, and diabetic SHR trea-ted with rosiglitazone were significantly longer than those of WKY,diabetic WKY, and diabetic WKY treated with rosiglitazone at APD50

and APD90, respectively.Diabetic SHR treated with rosiglitazone had 27% delayed depolar-

ization (DAD) (4 of 15, p=0.07) and 33% higher early depolarization(EAD) (5 of 15, pb0.005) compared to diabetic WKY rats treated withrosiglitazone (Fig. 4B).

3.5. Effects of diabetes, hypertension, and rosiglitazone on ICa-L

Fig. 5 shows tracings of the ICa-L in ventricular myocytes of WKY,diabetic WKY, and diabetic WKY treated with rosiglitazone. The cur-rent density of the ICa-L in diabetic WKY treated with rosiglitazonewas significantly larger than that of WKY and diabetic WKY ventricu-lar myocytes. Compared to WKY and diabetic WKY ventricular myo-cytes, diabetic WKY treated with rosiglitazone had a larger peakICa-L. Similarly, SHR treated with rosiglitazone ventricular myocyteshad a larger peak ICa-L (elicited from −10 to +60 mV) than SHRand diabetic SHR.

3.6. Effects of diabetes, hypertension, and rosiglitazone on NCX current

Fig. 6 shows tracings and I–V relationship of the nickel-sensitiveNCX currents of ventricular myocytes from SHR, diabetic SHR, and di-abetic SHR treated with rosiglitazone. Diabetic SHR had smallernickel-sensitive NCX currents than those of SHR ventricular myocytesand was attenuated after rosiglitazone treatment.

4. Discussion

The major finding of this study is that diabetes and diabetic hyper-tensive hearts were associated with abnormalities in intramyocyteCa2+ regulation. We observed prolonged Ca2+ decay, reducedintra-SR Ca2+ stores, and decreased Ca2+i transient amplitudes andSERCA2a, NCX, and RyR protein expressions suggesting a decreasein SERCA2a activity. Rosiglitazone treatment changed the Ca2+ regu-lation in diabetic hearts with or without hypertension. To the best ofour knowledge, this is the first time to demonstrate that rosiglitazonehas direct pro-arrhythmic potential through an electrophysiologicstudy in which rosiglitazone-treated hypertensive diabetic heartswere more prone to DAD and EAD.

Similar to results of previous studies, we found that ventricularmyocytes of diabetes and hypertensive rats had a reduced Ca2+i tran-sient amplitude, prolonged transient decay, and reduced Ca2+ stores[16]. The decrease in Ca2+i transients may contribute to a decreasedSR Ca2+ content through impaired excitation–contraction coupling

Fig. 4. Effects of diabetes, hypertension, and rosiglitazone on the action potential (AP) of ventricular myocytes. A. Representative tracing of APs recorded from WKY, diabetic WKY,rosiglitazone-treated diabetic WKY, SHR, diabetic SHR, and rosiglitazone-treated diabetic SHR. B. Representative tracings of delayed depolarization and early depolarization fromrosiglitazone-treated diabetic SHR. C. Mean values of the action potential amplitude, AP duration at 20% (APD20), APD50, and APD90 of ventricular myocytes from WKY (n=15),diabetic WKY (n=14), rosiglitazone-treated diabetic WKY (n=14), SHR (n=16), diabetic SHR (n=13), and rosiglitazone-treated diabetic SHR (n=19). *pb0.05 vs. respectiveWKY with and without diabetes or diabetes treated with rosiglitazone.

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efficiency that impairs the heart's function. A prolonged Ca2+i tran-sient decay associated with depletion of the SR Ca2+ content wasfound in hearts of diabetic and hypertensive rats. The increase inthe Ca2+i transient decay may indicate a decrease in the rate of cyto-plasmic Ca2+ removal due to derangement in either the SERCA pumpor the NCX [16,17]. Slowed cytosolic Ca2+ removal suggests that a re-duction in SERCA2a activity which might be a contributing factor tothe impaired relaxation of myocytes [18]. A lower SR Ca2+ load mayhave reinforced these defects in Ca2+ release channel properties,and this impaired reuptake may be a major determinant of diastolicdysfunction in diabetic hearts. A markedly prolonged Ca2+i transientdecay associated with depletion of SR Ca2+ content was found in di-abetes with hypertensive hearts. These results suggest severe impair-ment in the regulation of Ca2+ in the model of diabetes with ahypertensive heart, which also correlates with the clinical finding of

more severe diastolic and systolic functions in diabetic patients com-plicated with hypertension [19].

In this study, we found that the decreases of Ca2+i transients andCa2+ stores with prolonged Ca2+i decay in diabetes and diabetic hy-pertensive hearts were attenuated by rosiglitazone treatment. Theseeffects may have been due to enhancement of SERCA2a activitywhich may have increased the reuptake of Ca2+i after Ca2+i release.

We further elucidated the molecular mechanism underlying theimpaired handling of SR Ca2+ by immunoblotting to determine thecontent of the SERCA pump. SERCA2a protein expression wasdown-regulated in hearts of diabetic and hypertensive rats, and wasmarkedly reduced in hearts of diabetic rats with hypertension. Thiseffect can possibly be attributed to the anomalous in SR pump activity[20] and SR Ca2+ storage [21], and a reduction in NCX activity [22]and RyR protein expressions [23]. The effect of NCX to diabetes is

Fig. 5. Effects of diabetes, hypertension, and rosiglitazone on the L-type calcium channel (ICa-L) of the ventricular myocytes. Representative tracings of current and I–V relationshipof ICa-L of ventricular myocytes from WKY (n=18), diabetic WKY (n=21), and rosiglitazone-treated diabetic WKY (n=15), SHR (n=10), diabetic SHR (n=15), and rosiglitazo-ne-treated diabetic SHR (n=21). *pb0.05 vs. respective WKY or SHR, #pb0.05 vs. respective diabetic rats treated with rosiglitazone.

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controversial with reports of increased or unchanged NCX activity ordecreased protein expression of NCX [16,24]. Makino et al. found thatdiabetes-mediated alterations in the phospholipid composition of thesarcolemmal membrane contributed to a decline in NCX activity [24].The degree of unsaturated fatty acid and lysophosphatidylcholinecontents increases in diabetic hearts, an effect known to depress

Fig. 6. Effects of diabetes, hypertension, and rosiglitazone on the NCX current of the ventricuventricular myocytes from SHR (n=10), diabetic SHR (n=15), and rosiglitazone-treated dzone, respectively. ∫pb0.05 vs. diabetic SHR.

NCX activity [25]. The decrease in NCX protein expression in diabetesand diabetic hypertensive hearts may imply a decrease in the NCX ac-tivity due to derangement of lipid metabolism during hyperglycemia.Furthermore, a decrease in RyR protein expression was also found indiabetes and hypertensive hearts, and it was markedly reduced inhearts of hypertensive rats with diabetes. Dysfunction of the RyR

lar myocytes. Representative tracings of current and I–V relationship of NCX current ofiabetic SHR (n=16). *pb0.05 vs. SHR, #pb0.05 vs. diabetic SHR treated with rosiglita-

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induced by diabetes may be caused by the formation of disulfidebonds between adjacent sulfhydryl groups, or an increase in ad-vanced glycation end products [26], thereby resulting in loss of cardi-ac function. The decrease in the protein expressions of SERCA2a, NCX,and RyR could also be due to compensatory hypertrophy of ventricu-lar myocytes.

In contrast, increases in protein expressions of SERCA2a, NCX, andRyR after rosiglitazone treatment were found in diabetes and hyper-tensive hearts. These effects may have been due to enhanced contrac-tility following rosiglitazone treatment caused by an increase in theamplitude of Ca2+i transients resulting from an increased RyR geneexpression and a quick return of Ca2+ to resting levels because of in-crements in SERCA2 and NCX gene expressions [27].

In spite of the abundant electrophysiological data on ventricularmyocytes of diabetic rats, limited data is available on the ventricularmyocytes of hypertensive rats with diabetes. In this study, we foundthat the APD in hearts of diabetic rats and hypertensive rats with di-abetes was prolonged. However, it is not clear whether rosiglitazonecould change the AP morphology and cardiac electrophysiology in di-abetic rats with or without hypertension. In this study, we found thatdiabetes and diabetic hypertensive hearts had a greater prolongedAPD after rosiglitazone treatment, suggesting that rosiglitazone altersthe electrophysiology of diabetic and hypertensive hearts with diabe-tes. Diabetic patients exhibit a high incidence of diabetic cardiomyop-athy characterized by complex changes in the mechanical andelectrical properties of the heart [28]. Prolongation of the APD mayaggravate reductions in diastolic filling and stroke volume at highheart rates [29].

Both diabetes and hypertension can significantly increase the riskof cardiac arrhythmias [30]. In this study, we evaluated the effect ofrosiglitazone on ventricular electrical activity and found enhancedarrhythmogenic activity with increases in the DAD and EAD in hyper-tensive hearts with diabetes treated with rosiglitazone. Previousstudies showed that PPAR-γ agonist inhibits Ca2+ currents and tissuecontractility in vascular smooth muscles [31]. However, our studydemonstrated that the ICa-L current was significantly larger after rosi-glitazone treatment in diabetic rats with and without hypertension.An enhanced ICa-L current can prolong the APD and increase Ca2+i

loading, which may enhance the occurrence of EAD and DAD.The increased incidence of EAD and DAD in rosiglitazone-treated

diabetic SHR led us to further investigate the NCX current in theSHR group. The larger NCX current activity in rosiglitazone-treateddiabetic SHR may also have contributed to maintaining high Ca2+i

in ventricular myocytes. Moreover, an increase in the NCX proteinalso suggests that increased NCX activity may induce the occurrenceof triggered activities. The results of this study showed that the NCXcurrents of the ventricular myocytes significantly increased after rosi-glitazone treatment. The NCX currents can cause a transient inwardcurrent and DAD in cardiomyocytes. Based on these results, we sug-gest that rosiglitazone may alter NCX currents and change the tran-sient inward current thereby enhancing arrhythmogenicity.

5. Conclusions

In summary, our study suggests that diabetes and hypertensionmodulate the regulation of Ca2+ handling. PPAR-γ agonist, rosiglita-zone, when used in diabetic with hypertension, significantly changedthe Ca2+ homeostasis and electrophysiological characteristics, whichmay possess an arrhythmogenic potential.

Acknowledgements

The present work was supported by grants from Taipei MedicalUniversity-Wan Fang Hospital (100swf-02, 100wf-eva-01), NationalScience Council, Taiwan (NSC99-2314-B-016-034-MY3, 99-2628-B-038-011-MY3).

The authors of this manuscript have certified that they complywith the Principles of Ethical Publishing in the International Journalof Cardiology.

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