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DOI: 10.1161/CIRCRESAHA.108.173740 2008;102;e86-e100; originally published online Apr 24, 2008; Circ. Res.
Walker and Guy Salama Carl Sims, Steven Reisenweber, Prakash C. Viswanathan, Bum-Rak Choi, William H.
Long QT Type 2Determinants of Arrhythmia Phenotype in Rabbit Hearts With Drug-Induced Sex, Age, and Regional Differences in L-Type Calcium Current Are Important
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Sex, Age, and Regional Differences in L-Type CalciumCurrent Are Important Determinants of Arrhythmia
Phenotype in Rabbit Hearts With Drug-InducedLong QT Type 2
Carl Sims, Steven Reisenweber, Prakash C. Viswanathan, Bum-Rak Choi,William H. Walker, Guy Salama
Abstract—In congenital and acquired long QT type 2, women are more vulnerable than men to Torsade de Pointes. Inprepubertal rabbits (and children), the arrhythmia phenotype is reversed; however, females still have longer actionpotential durations than males. Thus, sex differences in K� channels and action potential durations alone cannot accountfor sex-dependent arrhythmia phenotypes. The L-type calcium current (ICa,L) is another determinant of action potentialduration, Ca2� overload, early afterdepolarizations (EADs), and Torsade de Pointes. Therefore, sex, age, and regionaldifferences in ICa,L density and in EAD susceptibility were analyzed in epicardial left ventricular myocytes isolated fromthe apex and base of prepubertal and adult rabbit hearts. In prepubertal rabbits, peak ICa,L at the base was 22% higherin males than females (6.4�0.5 versus 5.0�0.2 pA/pF; P�0.03) and higher than at the apex (6.4�0.5 versus 5.0�0.3pA/pF; P�0.02). Sex differences were reversed in adults: ICa,L at the base was 32% higher in females than males(9.5�0.7 versus 6.4�0.6 pA/pF; P�0.002) and 28% higher than the apex (9.5�0.7 versus 6.9�0.5 pA/pF; P�0.01).Apex–base differences in ICa,L were not significant in adult male and prepubertal female hearts. Western blot analysisshowed that Cav1.2� levels varied with sex, maturity, and apex–base, with differences similar to variations in ICa,L;optical mapping revealed that the earliest EADs fired at the base. Single myocyte experiments and Luo–Rudysimulations concur that ICa,L elevation promotes EADs and is an important determinant of long QT type 2 arrhythmiaphenotype, most likely by reducing repolarization reserve and by enhancing Ca2� overload and the propensity for ICa,L
reactivation. (Circ Res. 2008;102:e86–e100.)
Key Words: cardiac voltage-gated calcium current � ICa,L, sex differences � QT interval � ion channel expression� Torsade de Pointes
Women have longer rate-corrected QT intervals and areespecially prone to QT prolongation and Torsade de
Pointes (TdP) after treatment with drugs that inhibit K�
channels.1,2 A number of studies have shown an increase ofTdP in women versus men following an exposure to agentsknown to block the K� channel HERG and inhibit the rapidcomponent of the delayed rectifying current, IKr.1,3–5 Theincrease in vulnerability to sudden death in women has beenreported for cardiac1,5 and noncardiac drugs.6 These sexdifferences result most likely from the regulation of ionicchannel expression by sex steroids.7 In the congenital form oflong QT type 2 (LQT2), the underlying genetic defects ofHERG reduces IKr (loss of function) and may be asymptom-atic in some conditions but in the presence of a mild block ofIKr tend to precipitate TdP in women more frequently than inmen.8
In rabbit models of drug-induced LQT2, adult females hadsignificantly lower IKr and, perhaps, inward rectifying K�
current, which contributed to their longer QT interval andgreater arrhythmia vulnerability compared with their malecounterpart.9 The present consensus is that normal femalehearts express fewer functional K� channels, resulting inlonger action potential (AP) durations (APDs), and, whentreated with agents that inhibit IKr, adult females have agreater vulnerability to early afterdepolarizations (EADs) andTdP. The concept of “repolarization reserve” emerged toexplain the greater vulnerability of women to TdP; accordingto this concept, K� channel inhibition prolongs APDs moremarkedly in females than males.
In prepubertal rabbit hearts with drug-induced LQT2, weshowed that sex differences in arrhythmia phenotype arereversed, with males being highly vulnerable to IKr blockade
Original received November 12, 2007; resubmission received February 11, 2008; revised resubmission received March 12, 2008; accepted April 15,2008.
From the Department of Cell Biology and Physiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pa. Present address for C.S.:Department of Biological Sciences, Youngstown State University, Ohio. Present address for B.-R.C.: Cardiovascular Research Center, Rhode IslandHospital & Brown Medical School, Providence.
Correspondence to Guy Salama, PhD, University of Pittsburgh, School of Medicine, Department of Cell Biology and Physiology, S312 BiomedicalScience Tower, 3500 Terrace St, Pittsburgh, PA 15261. E-mail [email protected]
compared with females. In prepubertal (before the surge ofsex hormones) rabbits (�42 days old), female hearts hadlonger APDs than males, yet the potent IKr blocker E4031failed to elicit EADs and TdP despite a marked prolongationof APDs of more than 1 second. Findings in prepubertalrabbit hearts seemed to differ from human data from childrenwith congenital forms of LQT2.10,11 Analysis of humanregistry data revealed that adult females with congenitalLQT2 had a significantly higher risk of cardiac events(syncope, aborted cardiac arrest) and that, in prepubertalchildren (�14 years old), girls had an equal likelihood ofcardiac events as in boys.10 However, a closer scrutiny of thedata revealed that boys had a 3-fold greater likelihood of alethal arrhythmia.10 Thus, the lethality of LQT2 arrhythmiasin boys trumps the number of cardiac events and indicatesthat the arrhythmia phenotype is reversed in children com-pared with adults. Thus, the arrhythmia phenotype found inadult and prepubertal rabbit hearts with drug-induced LQT2are congruent with that found for LQT2 in humans.
Interestingly, APDs were longer in prepubertal female thanmale rabbits, yet E4031 elicited TdP within minutes in malehearts but merely prolonged APDs in female hearts.11 Thus,factors other than K� currents and APD prolongation must beconsidered to predict the arrhythmia phenotype; namelyfactors that the propensity to early afterdepolarizations(EADs).
The L-type Ca2� channel is a major regulator of cardiacCa2� homeostasis and has been implicated in the genesis ofEADs and TdP.12 The classic hypothesis of EAD genesissuggests that they arise from reactivation of ICa,L.13,14 Evi-dence for this mechanism has come from experimentalreactivation of ICa,L with Bay K486413 and a theoreticalmodel.15 Another hypothesis of EAD formation proposes thatAPD prolongation promotes cellular Ca2� overload, trigger-ing spontaneous Ca2� release from the sarcoplasmic reticu-lum (SR),16 enhancing the turnover rate of the Na�/Ca2�
exchanger (NCX) and its depolarizing current, INCX,12,17,18 whichmay reactivate ICa,L. In the classic hypothesis, the EADvoltage depolarization precedes the rise of intracellular freeCa2�, [Ca2�]i, whereas [Ca2�]i precedes EADs in the alterna-tive mechanism. Compelling support for the second hypoth-esis comes from simultaneous maps of APs and [Ca2�]i inwhich E4031-induced EADs generated a rise of [Ca2�]i ofsuch magnitude and kinetics that it was most likely producedby spontaneous SR Ca2� release.17 Nevertheless, both mech-anisms implicate ICa,L as a trigger of EADs.
Studies of the genomic effects of estrogen on the expres-sion of cardiac Ca2� channels and ICa,L have yielded contra-dictory results. In papillary muscles of female rabbits, ovari-ectomy increased and estrogen replacement (7 days)decreased isometric force. Estrogen reduced 3H-nitrendipinebinding in plasma membrane preparations compared withovariectomy and control groups, yet peak L-type calciumcurrents (ICa,L) was not significantly different for the 3treatment groups.19 In contrast, Pham et al reported higherICa,L density on the epicardium of adult female rabbit heartscompared with males and no sex differences on the endocar-
dium such that female hearts, but the authors did not examineapex–base differences in ICa,L.20 In rat hearts, Western blotsindicated that females had higher levels of ryanodine recep-tor, Cav1.2 (the � subunit of the L-type Ca2� channel protein),and NCX proteins, yet their mRNA levels were lower thanmales.21
New Zealand rabbits offer significant advantages as amodel of human LQT2 and to investigate sex differences inarrhythmia phenotype. (1) Rabbit cardiac APs and ioniccurrents (in particular K� currents: IK1, Ito, IKr, and IKs) aresimilar to human APs, with similar responses to blockers ofK� currents.22,23 (2) Sex differences in arrhythmia phenotypeare similar in rabbits and men.11 (3) Numerous studies haveused rabbit models of drug-induced LQT to investigate thefactors that precipitate TdP.24–26 (4) Rabbits are “reflexovulators” with estrogen levels that remain elevated untilmating,27 which avoids estrogen oscillations that occur inmost mammals during the estrus cycle and thereby minimizesestrogen-dependent genomic variations of ion channelexpression.
Here, we investigated sex, age, and regional differences involtage-gated Ca2� channels by measuring ICa,L density usingthe whole-cell voltage-clamp technique; by analyzingCav1.2� protein levels using Western blots; by analyzingmRNA levels using real-time PCR; by correlating the re-gional elevation of ICa,L to the origin of the earliest EADs andto the LQT2 arrhythmia phenotype by optical mapping; andby showing that adult female and prepubertal male myocyteswere more prone to fire EADs using experimental andsimulation techniques. These findings provide new insight onthe mechanisms underlying the firing of EADs and on sexand age differences in arrhythmia phenotype in LQT2.
Materials and Methods
Arrhythmia Phenotype in Langendorff Model ofDrug-Induced LQT2New Zealand White rabbits were anesthetized with pentobarbital (50mg/kg) and injected with heparin (200 U/kg IV). Hearts were excisedand perfused in a Langendorff apparatus with a Tyrode’s solutioncontaining (in mmol/L): 130 NaCl, 24 NaHCO3, 1.0 MgCl2, 1.2NaHPO4, 4.0 KCl, 50 dextrose, 1.25 CaCl2 gassed with 95% O2 and5% CO2 (pH 7.4). Perfusion pressure was adjusted to 60 to70 mm Hg by controlling the flow rate of the perfusion. Hearts wereplaced in a specially designed chamber to reduce movement artifactsand control the temperature in the medium bathing the heart using afeedback control device to maintain temperature at 37.0�0.2°C.28
Hearts were stained with the voltage-sensitive dye di-4-ANEPPS (25�L of 1 mg/mL DMSO) (Molecular Probes, Eugene, Ore) byinjecting the dye through a port in the bubble trap (or a compliancechamber) located above the aortic cannula to the heart.18 Hearts werethen perfused with the IKr blocking agent E4031 {(1-[2-(6-methyl-2-pyridyl)-ethyl]-4-(4- methylsulfonylaminobenzoyl) piperidine;0.5 �mol/L} to produce a drug-induced LQT2 and allowed to beat attheir intrinsic rate, as previously described.11 Four groups of rabbitswere tested: (1) adult males (n�8) and (2) adult females (n�8) 3 to4 month old; and (3) prepubertal males (n�10) and (4) prepubertalfemales (n�18) 6 weeks old weighing �1.5 kg. For each Langen-dorff heart, the arrhythmia phenotype was determined by treating theheart with E4031 and tracking the emergence of EADs and TdP,
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which typically occurred within 5 minutes or failed to occur for morethan 30 minutes. Thus, the protocol using E4041 at 0.5 �mol/Lprovided “yes” or “no” assay of arrhythmia phenotype.
Regional Distribution of the Earliest EADsOn perfusion with E4031, APs and EADs were monitored by opticalmapping to identify the locations on the heart that fired the earliestEADs that progressed to TdP. In cases where EADs appeared atseveral sites on the epicardium, the earliest EAD was identified fromthe temporal delays between all sites that fired an EAD. Precautionswere taken to ensure that EAD signals represented electric eventsand not motion artifacts. The earliest EAD had to occur synchro-nously with a voltage change measured by surface EKG recordingsand had to propagate to adjacent regions of the heart for at least�3 mm or 3 pixels. In most cases, the first EADs appeared at thebase exclusively and propagated out but did not reach the apexregions. In other cases, EADs appeared first at the base andpropagated to the apex; in those cases, an activation map wasgenerated to ascertain the origin of the EAD wave. Cumulative plotsof the sites that fired the earliest EADs were generated separately foradult female and prepubertal male rabbit hearts to determine whetherEADs were more likely to start from basal or apical regions of theventricles. A 1-tailed “binomial test” was used as a test for thestatistical significance of deviation from the null hypothesis (P�0.5,or 50% probability) that EADs were equally likely to occur at thebase or the apex. Statistical significance for EADs firing from apreferential location is reached when the binomial test rejects the nullhypothesis with P�2%. The probability of firing the earliest EAD atthe apex is given by:
P � �(CNj) � (1/2)N
where the sum is taken from j�0 to a, a is the number of experimentsin which EADs fire first at the apex, N is the total number ofexperiments in which EADs were measured, and CN
a is the combi-nation of “a” out of “N.”
Cell IsolationVentricular myocytes were isolated from either prepubertal (30- to49-day-old) or adult (3-month-old) male and female New ZealandWhite rabbits by a modification of a previously described method.29
Briefly, rabbits were anesthetized with pentobarbital (50 mg/kg) andinjected with heparin (200 U/kg IV). The hearts were excised andperfused via the aorta with a physiological salt solution (PSS)containing (in mmol/L): 140 NaCl, 5.4 KCl, 1.5 CaCl2, 2.5 MgCl2,11 glucose, and 5.5 HEPES (pH 7.4). Hearts were then perfused withCa2�-containing PSS for 5 minutes, followed by perfusion withnominally Ca2�-free PSS for 10 minutes, after which collagenasetype 2 (Worthington; at 0.60 mg/mL) was added to Ca2�-free PSS for15 minutes of digestion at 35°C. The ventricles were removed andplaced in a high potassium buffer containing (in mmol/L): 110K-glutamate, 10 KH2PO4, 25 KCl, 2 MgSO4, 20 taurine, 5 creatine,0.5 EGTA, 20 glucose, and 5 HEPES (pH 7.4). Sections ofepicardium approximately 1 mm in depth were surgically removedfrom the apex and base regions of the left ventricle, and cell isolationwas performed separately for each region.20 Myocytes from the apexwere taken from 3 to 6 mm from the very bottom of the heart, thosefrom the base were taken from 1 to 4 mm below the left atrium, andno cells were studied from a 3 to 4 mm region in the middle of theheart. The tissues were minced, and the single myocytes wereobtained by filtering through a 100-�m nylon mesh. Cells wereallowed to settle, the supernatant was aspirated, and the pellet wasresuspended in high potassium buffer. Experiments were performedon the day of cell isolation and 4 to 8 myocytes were studied fromeach heart.
The methods and protocols used in the study were all in accor-dance with the University of Pittsburgh Institutional Animal Careand Use Committee and complied with the Guide for the Care andUse of Laboratory Animals as adopted by the NIH.
Data Acquisition and AnalysisL-type Ca2� currents were studied using the conventional whole-cellconfiguration of the patch clamp technique.30 Patch pipettes hadresistances of 1 to 2.5 m� when filled with (in mmol/L): 130 CsCl,20 tetraethylammonium chloride (TEA-Cl), 5 MgATP, 5 EGTA, 0.1Tris-GTP, and 5 HEPES (pH 7.2). Cells were bathed in K�-freesolution containing (in mmol/L): 140 NaCl, 5.4 CsCl, 2.5 CaCl2, 0.5MgCl2, 11 glucose, and 5.5 HEPES (pH 7.4). Currents recordedusing an Axopatch 200 amplifier were filtered at 5 kHz and sampledat 10 kHz using a Digidata 1200a interface and pCLAMP (version9.2) software (Axon Instruments). The magnitude of the peak inwardcurrent ICa,L was measured during 100-ms voltage-clamp steps to 0mV applied following a 50-ms prepulse to 30 mV from a holdingpotential of 80 mV every 6 seconds. All recordings were made 3to 5 minutes after gaining whole-cell access and after ICa,L hadstabilized.31 Series resistance was partially compensated to achievevalues of �3.0 m� to prevent large voltage errors when measuringlarger (1.5 nA) whole-cell ICa,L. Capacitance measurements wereobtained from membrane test parameters using Axon software.Capacitance of male and female prepubertal myocytes was 67.2�2.1and 69.9�3.7 pF, respectively. Adult male and female myocytecapacitance was 110.7�7.3 and 109.3�8.7 pF, respectively. ICa,L wasisolated by blocking K� channels with Cs� and TEA-Cl, inactivatingNa� channels with a voltage-clamp prepulse step to 30 mV andeliminating the driving force for Cl currents by measuring ICa,L closeto the predicted Cl equilibrium potential (0 mV).
The voltage dependence of Ca2� channel activation and inacti-vation was determined as described previously.32 Parameters forthe voltage dependence of activation were obtained from the leastsquares fit of data points to the equation:
g/gmax�1/{1�exp[(VTV0.5)/b]},
where g/gmax represents normalized Ca2� conductance, VT representstest potentials from 30 to 30 mV, V0.5 is the potential at half-maximal activation, and b is the slope. Parameters for the voltagedependence of inactivation were obtained from the equation:
I�Iir�(1Iir)/{1�exp[(VcV0.5)/b]},
where I is the normalized magnitude of the peak inward currentmeasured during a test pulse to 0 mV following a 5-secondconditioning pulse (Vc) to Vm between 90 and 30 mV, Iir is theinactivation-resistant current, V0.5 is the potential at which inactiva-tion was half maximal, and b is the slope. The current elicited duringthe test pulse was normalized to the magnitude of the currentrecorded during a pretest pulse to 0 mV, which preceded eachconditioning pulse. This corrected for changes in current magnitudeattributable to rundown.31 Time to half inactivation of ICa,L (t1⁄2) wasdetermined by fitting the inactivating component of the ICa,L trace(defined as the region between the peak Ca2� current and the end ofthe depolarizing pulse to 0 mV) to the biexponential curve fittingfunction of Clampfit (Axon Instruments). The larger of the 2exponential components (97% of the inactivation curve) was used tomeasure t1⁄2. Results are reported as the mean�SE of at least 3 ormore independent experiments. Statistical comparisons between 2groups of experimental data were performed using the Student’s2-tailed t test.
Action PotentialsAPs were recorded using the current-clamp mode as previouslydescribed33 with an internal solution containing (in mmol/L): 150KCl, 5 MgATP, 5 EGTA, 0.1 Tris-GTP, and 5 HEPES (pH 7.2).Cells were bathed in an extracellular solution containing (in mmol/L): 140 NaCl, 5.4 KCl, 2.5 CaCl2, 0.5 MgCl2, 11 glucose, and 5.5HEPES (pH 7.4), at 35°C. APs were elicited by injecting currentpulses (1 to 2 nA) of 5-ms duration through the patch pipette atfrequencies of 0.33 or 1 Hz. Once stable, AP recordings wereobtained, myocytes were exposed to 5 �mol/L E4031 to completelyblock IKr,33,34 and recordings were continued to monitor AP prolon-gation and the incidence of EADs. Fisher’s exact test was used to testthe null hypothesis of equal probability (P) of EADs between male
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and female myocytes. A probability of �2% (P�0.02) is consideredto be a rejection of the null hypothesis and indicates a statisticallysignificant difference in the likelihood of firing an EAD betweenmale and female myocytes.
Quantitative Assays of Protein and mRNATissues from the apex or base of rabbit hearts were dissected fromexactly the same regions as described for the isolation of ventricularmyocytes for voltage-clamp studies. The tissues were disruptedusing a PowerGen model 125 homogenizer (setting 5, 30 seconds) in1 mL enhanced lysis buffer (ELB) (250 mmol/L NaCl, 0.1% NP-40,50 mmol/L HEPES [pH 7.0], 5 mmol/L EDTA, 0.5 mmol/Ldithiothreitol) supplemented with a cocktail of protease and phos-phatase inhibitors. The extract was rocked for 15 minutes at 4°C,cellular debris was removed by centrifugation (12 000g, 5 minutes),and supernatant containing the protein extract was stored at 80°C.Protein concentrations were determined by the Bradford method(Bio-Rad protein assay). Cell lysates (50 �g protein/lane) werefractionated by SDS-PAGE, transferred to poly(vinylidene difluo-ride) membranes (Immobilon-P, Bedford, Mass), and incubatedovernight at 4°C with a rabbit antibody (Affinity Bioreagents,Golden, Colo) that was directed against Cav (1.2) (diluted 1:1000) ormouse �-actin (Sigma, St Louis, Mo; diluted 1:20 000), followed byhorseradish peroxidase–conjugated second antibody (Sigma). Theantigen–antibody complex was visualized with Millipore Immo-bilon Western Chemiluminescent horseradish peroxidase sub-strate. Digitized fluorograms were quantified by using NIH Image1.6 Software.
mRNA Extraction and Real-Time PCRLeft ventricle epicardial tissue from the apex or base of rabbit heartswas disrupted as described for protein extracts but in guanidineisothiocyanate buffer, and mRNA was purified by centrifugation
through cesium chloride.34a The resulting RNA (200 ng) wassubjected to reverse transcription in 100 �L of Geneamp PCR buffer(Applied Biosystems) containing 1 mmol/L dNTPs, 2.25 mmol/Lrandom hexamers, 7.5 mmol/L MgCl2 and Superscript II (Invitro-gen). The reaction was carried out in a thermocycler at 25°C for5 minutes, 48°C for 30 minutes, and 95°C for 5 minutes. Thereal-time PCR was performed using 2 �L of the reverse transcrip-tion reaction products in a total volume of 50 �L containing 25�L of ABsolute SYBR Green mix (Thermo Scientific) and 20nmol/L primers. The reaction was carried out in an AppliedBiosystems 7900HT thermocycler. Cav(1.2) expression was nor-malized to that of GAPDH. The primers used included thefollowing: Cav(1.2) forward, 5-CATTGGGAACATCGTGATTGTC-3; Cav(1.2) reverse, 5-CAGGCGAACATGAACTGCAG-3; GAPDHforward, 5-TCCTGGGCTACACCGAGG-3; and GAPDH reverse,5-TGGCACTGTTGAAGTCGCAG-3. Relative quantitation was car-ried out using the 2��Ct method.
Action Potential Modeling of Cardiac MyocytesLeft ventricular epicardial APs were simulated using the Luo–Rudy(LRd) model of the mammalian ventricular AP.35,36 The modelincorporates the transient outward K� current, Ito,37 and ioniccurrents are mathematically represented by the Hodgkin–Huxleyformulation. The computational model includes simulations for iontransporters and pumps that regulate Na�, K�, and Ca2� concentra-tions across the sarcolemmal and SR membranes. The model tracksthe dynamic changes in intracellular Ca2� by incorporating Ca2�
release and uptake from the SR network, a delay for Ca2� diffusionfrom the longitudinal to junctional SR, NCX, and Ca2� buffering bycalmodulin, troponin (in the cytoplasm), and calsequestrin (in theSR). Experimentally determined sex, age, and regional differences incurrent densities and voltage-dependent parameters for ICa,L (Tables1 and 2) were incorporated into the AP model by modifying the
Table 1. Parameters of Voltage-Dependent ICa,L Activation and Inactivation in Prepubertal Myocytes
Prepubertal
Activation Inactivation
Vr (mV) V0.5 (mV) Slope n V0.5,inact (mV) Slope Ir (%) n t1/2 (ms) n
Cumulative data were analyzed as in Materials and Methods and expressed as means�SE. Vr indicates reversal potential; Ir, inactivation resistant current. *Thevoltage of half-maximal inactivation (V0.5,inact) was significantly shifted to hyperpolarized potentials in female compared with male myocytes (P�0.03); †the slope factorin male† and female** apex myocytes was significantly different from base myocytes (P�0.001); ‡the time to half inactivation of ICa,L (t1/2) was significantly differentbetween male apex and base (P�0.0001); §male and female apex (P�0.05); n, number of cells in each group.
Table 2. Parameters of Voltage-Dependent ICa,L Activation and Inactivation in Adult Myocytes
Adult
Activation Inactivation
Vr (mV) V0.5 (mV) Slope n V0.5,inact (mV) Slope Ir (%) n t1/2 (ms) n
Cumulative data were analyzed as in Materials and Methods and expressed as means�SE. Vr indicates reversal potential; V0.5, the voltage that gave half-maximalactivation or inactivation (V0.5,inact); Ir, inactivation resistant current. *Time to half inactivation of ICa,L in adult myocytes was significantly different from correspondingsex and regions of prepubertal myocytes; n, number of cells in each group.
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equations representing ICa,L. The online data supplement providesdefinitions of the simulations parameters used to model APs from thebase of prepubertal and adult male and female myocytes. To mimicLQT2, the rapid component of the delayed rectifier K� current, IKr,was suppressed by either 50% or 100%.15 The last AP from a trainof 50 APs with a simulated cycle length of 1 second displayed wasused to evaluate the effects of altered ICa,L on APs with suppressedIKr. Simulations were repeated to examine the influence of the NCXand its current, INCX, on APD and EAD generation during 50% or100% IKr block with and without an increase of ICa,L. The LRd modelhas been extensively used in several studies during the past decadeto understand the mechanisms of arrhythmias arising from ionchannel mutations and/or drug block.38–40
Results
Sex and Age Differences in Arrhythmia PhenotypeAdult rabbit hearts exhibited the expected sex differences inarrhythmia phenotype as that reported for clinical drug-induced LQT2. As shown in Figure 1, perfusion of adult malehearts with E4031 produced a marked prolongation of APDs(�1 second) yet failed to develop TdP (1 of 8 hearts had anarrhythmia) (Figure 1a). In contrast, female hearts treatedwith the same concentration of E4031 consistently developedTdP in 7 of 8 hearts (Figure 1b). The arrhythmia phenotypewas opposite in hearts isolated from prepubertal rabbitswhere perfusion with E4031 elicited TdP in male hearts (7 of10 hearts, as in Figure 1c) but failed to elicit TdP inprepubertal female hearts (2 of 18 developed TdP, Figure 1d).Note the drug E4031 was effective in all cases and caused amarked APD prolongation in adult males and prepubertalfemale hearts yet no TdP. The highly reproducible sexdifferences of arrhythmia phenotype in adults and the reversein prepuberty did not correlate with sex differences in APDswhere prepubertal female hearts had longer APDs than theirmale counterparts.11
Sex and Regional Comparisons of Epicardial ICa,L
in Prepubertal Rabbit HeartsIn prepubertal hearts, peak whole-cell Ca2� currents (normal-ized to cell capacitance) were significantly higher in malecompared with female epicardial myocytes isolated from thebase of the left ventricles (Figure 2). ICa,L measured at 0 mVfrom the base of the heart was higher in absolute magnitudein male (6.4�0.5 pA/pF; cells [n]�26; hearts [H]�7)compared with female myocytes (5.0�0.2 pA/pF; n�17;H�4; P�0.03). Representative individual current traces weresuperimposed (Figure 2A) to demonstrate the differences inICa,L between the sexes. Current-to-voltage (I/V) relationshipswere plotted for test potentials between 30 and �60 mV(Figure 2B). I/V plots were bell-shaped for both sexes,reached a single maximum value at 10 mV, and had identicalreversal potentials (Vr) (see Table 1).
At the apex, no significant differences were found in peakICa,L between prepubertal male (5.0�0.3 pA/pF; n�30;H�9; P�0.02) and female (4.8�0.3 pA/pF; n�18; H�5)myocytes. The superposition of current traces from male andfemale myocytes (Figure 2C) demonstrated that at the apex,ICa,L was similar for both sexes. Averaged I/V relationships(Figure 2D) and cumulative data for ICa,L measured at 0 mV(Figure 2E) demonstrated that the normalized current magni-tudes were comparable in both sexes for myocytes isolatedfrom the apex of the hearts.
The apex–base distribution of ICa,L in prepubertal male andfemale ventricles were analyzed because previous reportsimplicated regional heterogeneities in current distribution ascontributors to dispersion of repolarization and arrhythmiavulnerability.11,20,33 Individual current traces and I/V plots(Figure 2A through 2E) showed that male epicardial cellsfrom the base had significantly higher peak ICa,L (6.4�0.5pA/pF) than those from the apex (5.0�0.3 pA/pF). In
MaleMale
a) Adult
c) Pre-puberty
+E-4031 +E-4031
+E-4031 +E-4031
FemaleFemale
b) Adult
d) Pre-puberty
Figure 1. Sex differences in arrhythmiaphenotype in rabbit hearts. a through deach illustrates a control AP measuredoptically using the voltage-sensitive dyedi4-ANEPPS before treatment with E4031.The arrhythmia phenotype of male (a andc) and female (b and d) on perfusion withE4031 (0.5 �mol/L) is illustrated for adults(a and b) and prepubertal rabbit hearts (cand d). E4031 elicited TdP in 5 to 10 min-utes in adult female hearts (b) and elicitedmarkedly prolonged APDs in adult malehearts without progressing to TdP, ven-tricular tachycardia, or ventricular fibrilla-tion (a). In contrast, E4031 elicited TdP inprepubertal male (c) but not female (d)hearts. Electric activity was recorded opti-cally for a minimum of 30 minutes afterperfusion with E4031 to verify that adultmale and prepubertal female hearts didnot develop EADs and TdP.
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female prepubertal rabbit hearts, apex–base differences inICa,L (4.8�0.3 pA/pF versus 5.0�0.2 pA/pF, respectively)were not statistically significant (Figure 2E).
Voltage Dependence of ICa,L in PrepubertalRabbit HeartsThe voltage dependence of ICa,L activation and inactivationwas measured to determine whether sex and regional differ-ences exist in these channel properties. ICa,L activation curvesfor male and female apex and base epicardial myocytes arepresented in Figure 3A. Although sex differences in ICa,L
activation were not observed, there were significant regionaldifferences in the slope factor for ICa,L activation in both sexes(Table 1). In males, the slope factor for current activation atapex and base was 7.9�0.84 and 7.1�0.67, respectively(P�0.001). The slope factor for current activation measuredin female apex and base myocytes was 8.0�0.67 and7.4�0.42, respectively (P�0.01). The higher slope factorimplied that during an AP the time course of Ca2� entry viavoltage-gated Ca2� channels may be faster at the apex thanthe base of the heart.
As shown in Figure 3B, ICa,L inactivation occurred at morenegative potentials in female compared with male myocytes. In
female apex and base epicardial myocytes, the voltage athalf-maximal inactivation (V0.5) was 33�0.6 and 32�0.6mV, respectively. The V0.5 of current inactivation in male apexand base cells occurred at significantly more positive potentialsof 30�0.5 and 29�0.6 mV, respectively (P�0.03).
Analysis of ICa,L inactivation kinetics revealed that the time tohalf-maximal inactivation, t1⁄2, was significantly longer in male(24.0�0.56 ms; n�30; H�9) compared with female(22.2�0.58 ms; n�18; H�5; P�0.05) myocytes at the apex. Nosex differences were observed at the base. In addition, there weremarked apex–base differences in t1⁄2 in male hearts; t1⁄2 wassignificantly longer at the apex than the base (24.0�0.56 versus20.6�0.45 ms; n�26; H�7; P�0.0001), whereas regionaldifferences in t1⁄2 were not observed in female hearts.
Sex and Regional Comparisons of ICa,L inAdult RabbitsIn contrast to the findings in prepubertal hearts, peak ICa,L
was significantly higher at the base of adult femalecompared with male myocytes. Representative currenttraces from myocytes with nearly identical membranecapacitance (Figure 4A), averaged I/V relationships (Fig-ure 4B) and cumulative data (Figure 4E) demonstrated the
A B C
D E
Figure 2. Sex differences in epicardial ICa,L in prepubertal rabbits. A, Representative ICa,L traces from male and female myocytes fromthe base of the left ventricle; the myocytes were chosen for their similar sizes or membrane capacitance (�1 pA/pF), the inward currentwas evoked by 100-ms depolarizing pulses to 0 mV. B, I/V relationships in male and female myocytes from the base of the left ventri-cle. C, As for A, representative traces from male and female myocytes from the apex. D, I/V relationships in male and female apexmyocytes. E, Cumulative peak ICa,L from male and female base and apex myocytes. At the apex, differences in peak ICa,L were not sig-nificantly different (n�18 to 30; P�0.70). At the base, cumulative differences in peak ICa,L between male and female myocytes were sta-tistically significant (*) (n�17 to 26; P�0.04).
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differences in ICa,L between the sexes. ICa,L at the base infemales was 9.5�0.7 pA/pF (n�11; H�5) compared with6.4�0.6 pA/pF, (n�11; H�5; P�0.002) for males. Nodifferences in ICa,L were found in adult apex myocytes. ICa,L
in female apex myocytes was 6.9�0.5 pA/pF (n�9; H�3)and in male myocytes from the same region was 7.3�0.4 pA/pF(n�7; H�4; P�0.5). The magnitude of representative currents(Figure 4C), I/V relationships (Figure 4D) and cumulative datafor ICa,L (Figure 4E), all demonstrated that ICa,L was similar at theapex for both sexes.
Regional comparisons revealed that peak ICa,L was 28%higher in female base (9.5�0.7 pA/pF; n�11; H�5) comparedwith apex (6.9�0.5 pA/pF; n�9; H�3; P�0.01) myocytes. Incontrast, there were no significant transepicardial differences inICa,L in adult male ventricular myocytes, with values of 6.4�0.5(n�11 H�5) and 7.3�0.4 pA/pF (n�7; H�4; P�0.3), respec-tively, for base and apex myocytes.
Voltage Dependence of ICa,L in AdultRabbit HeartsEvaluation of channel properties in adult ventricles revealedno significant sex or regional differences in ICa,L activation orinactivation (Figure 5A and 5B and Table 2). However,
significant differences in the rate of ICa,L inactivation wereobserved between adult and prepubertal hearts. The t1⁄2 for ICa,L atthe base of adult hearts was 17.3�0.64 and 17.0�0.87 ms formale and females, respectively. The t1⁄2 values in male and femaleapex myocytes of adult hearts were 17.2�0.50 and 18.8�1.1ms, respectively. These figures were statistically significant(P�0.01) for t1⁄2 values of prepubertal hearts (Tables 1 and 2).
Sex, Age, and Regional Distribution of Cav1.2�A reasonable explanation for sex differences in ICa,L is that sexsteroids modulate the expression levels of the Ca2� channelprotein Cav1.2�. Quantitative Western blot analysis of totalprotein supported the notion that differences in the currentdensity of ICa,L was attributable to differences in proteinlevels. Hearts from adult and prepubertal rabbits of each sex(n�4 per group) were rapidly isolated and flash-frozen inliquid nitrogen, and segments of tissue were dissected forprotein and mRNA analysis. The tissues were dissected fromexactly the same regions of epicardium as described for theisolation of myocytes for ICa,L. In adult rabbits, Cav1.2� wasexpressed at a statistically significant higher level at the baseof female hearts compared with at the apex and was higherthan at the base and apex of male hearts (Figure 6a and 6c).In prepubertal rabbits, Cav1.2� was higher at the base of malehearts compared with the apex and was higher than at the baseand apex of female hearts, but the differences were notstatistically significant (Figure 6b and 6d). Cav1.2� mRNAlevels exposed statistically significant higher levels at thebase than apex of adult female hearts but were otherwise notstatistically significant in either adult or prepubertal compar-isons of mRNA levels (Figure 6e and 6f).
Spatial Distribution of the Earliest EADsThe correlation between the arrhythmia phenotype and theenhanced ICa,L at the base of the hearts suggests that the earliestEADs that capture and progress to TdP should also occur at thebase of adult female and prepubertal male hearts. More pre-cisely, a higher Ca2� current density was measured from myo-cytes isolated from the top one-third of the base, just below theleft atrium, compared with the bottom one-third of the apex.
To measure the spatial distribution of the earliest EADs onthe epicardium, E4031 was added to the perfusate and mapsof optical APs were recorded to detect the earliest EADs thatappeared on the epicardium. As shown in Figure 7, thelocation of the earliest EADs clustered around the base of theheart in both adult female (Figure 7A; n�9/9 hearts) andprepubertal male hearts (Figure 7B; n�8/9 hearts). For thesemeasurements, only hearts that developed E4031-induced TdPwere considered in the analysis, and only 1 prepubertal maleheart had an early EAD that occurred below the midline (redhorizontal line, Figure 7A and 7B). A 1-tailed binomial test wasused to test the hypothesis that EADs occur with equal proba-bility at the base and the apex. For adult female and prepubertalmales, the probability values were 0.001953 and 0.017578,respectively. Thus, the clustering of EADs around the base of theheart was statistically significant, with P�0.002.
Sex Differences on the Incidence of EADs inIsolated MyocytesAPs were recorded from ventricular myocytes isolated fromthe base of prepubertal and adult male and female hearts;
A
B
Figure 3. Voltage dependence of ICa,L activation and inactivation inprepubertal rabbit myocytes. A, ICa,L activation curves from maleand female apex and base myocytes. B, Steady-state ICa,L inactiva-tion curves from male and female apex and base myocytes.
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then, treatment with E4031 revealed a sex-dependent propen-sity to fire EADs. As shown in Figure 8, prepubertal male(Figure 8A; n�4 cells; H�3 hearts) and adult female (Figure8D; n�6; H�4) myocytes fired EADs, whereas prepubertalfemale (Figure 8B; n�4; H�3) and adult male (Figure 8C;n�6; H�4) myocytes failed to fire EADs when treated withE4031. In all myocytes, E4031 produced a marked APDprolongation, but EADs could only be observed in prepuber-tal male and adult female myocytes. To observe EADs, theexternal Ca2� concentration had to be raised to 2.5 mmol/Land the myocytes had to be paced for 10 to 20 beats at 1 Hzfor prepubertal myocytes and at 0.33 Hz for adult myocytes.Fisher’s exact test was applied to test the null hypothesis thatboth males and females have equal likelihood of havingEADs. The null hypothesis is rejected because of its proba-
bility (P�0.0143 and 0.0076 for prepubertal and adult myo-cytes, respectively). With P�0.02, statistical significance isachieved for the greater incidence of EADs in prepubertalmales compared with female and for adult female comparedwith adult male myocytes.
AP Simulations: Influence of Elevated ICa,L onEAD InductionSimulations of the cardiac AP based on a modified version ofthe LRd model were used to evaluate the role of enhancedICa,L density as a predictor of the propensity to EADs indrug-induced LQT2. AP simulations for prepubertal and adultmyocytes from the base of the heart were generated byincorporating the experimentally determined current densitiesand voltage-dependent parameters for ICa,L (Figures 2 through5 and Tables 1 and 2) by modifying the equations represent-
A B
C D
EFigure 4. Sex differences in ICa,L are reversed inadult rabbits. A, Representative ICa,L traces fromadult female and male base myocytes with similarsizes or capacitance (�3 pA/pF). B, I/V relationshipsfrom adult female and male base myocytes. C, Rep-resentative traces from female and male base myo-cytes of similar capacitance (�3 pA/pF). D, I/V rela-tionships in female and male apex myocytes. E,Cumulative differences in peak ICa,L between femaleand male base myocytes. *The magnitude of peakICa,L between sexes was significantly different at thebase (n�9 to 11; P�0.01) but not at the apex. nsindicates not significantly different (n�7 to 9;P�0.50).
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ing ICa,L. To mimic LQT2, the rapid component of the delayedrectifier K� current, IKr, was suppressed by either 50% or100%. Figure 9a and 9b illustrates simulations of control APsin prepubertal male and female myocytes, respectively. Al-though experimental differences in ICa,L properties (see Table1) were incorporated in the simulations of control APs, therewere no discernable differences in the shape and time courseof prepubertal APs. However, the subsequent 50% block ofIKr resulted in the firing of EADs in the male (higher ICa,L) butnot the female model of a myocyte. In adults, female andmale myocytes were modeled according experimental differ-ences in their ICa,L properties (Table 2), and, again, there wereno discernible differences in control APs (Figure 9c and 9d).When a 50% inhibition of IKr was inserted, female myocytesfired EADs, whereas male myocytes did not (Figure 9c and9d). The theoretical analysis confirmed the experimentallyrecorded APs (Figure 8) using E4031 to suppress IKr andmimic the propensity to fire EADs, which, in turn, areconsistent with the arrhythmia phenotype recorded in intactperfused hearts. The simulations support the hypothesis that a25% to 30% increase of ICa,L was alone sufficient to promoteEADs in myocytes with reduced IKr.
Influence of INCXA reasonable concern is that the upregulation of ICa,L willincrease Ca2� influx on a beat-to-beat basis, which is likely tobe accompanied by an increase in INCX to increase Ca2� effluxand balance influx to efflux. Based on simulations of the AP,Figure 10 shows that in prepubertal male (Figure 10A) andadult female (Figure 10B) myocytes, an increase in INCX
(30%) had no discernible effect on APD and that during100% or 50% IKr block, the higher INCX does not inhibit thegeneration of EADs. Nevertheless, a 30% increase in INCX
decreased intracellular free Ca2� in the cytosol (Figure 10D)and the Ca2� concentration in the lumen of the SR (Figure10E). Moreover, during IKr blockade, increasing INCX alonedid not elicit EADs (Figure 10C), whereas increasing ICa,L
alone was sufficient to elicit EADs (Figure 9), which high-lights the importance of ICa,L as an important determinant ofEADs susceptibility and of arrhythmia phenotype.
DiscussionOur main findings are that ICa,L is elevated at the base of heartsthat are prone to EADs and TdP in E4031-induced LQT2 andthat the first EADs that elicit TdP originate from the base ofthe heart. Protein and mRNA levels suggest that these sexdifferences in ICa,L are predominantly attributable to sexdifferences in ion channel expression at the base but not theapex of the left ventricle. More precisely, we found that ICa,L
at the base is higher in prepubertal male than female myo-cytes and that this sex difference is unique to the base of theheart and not the apex, resulting in a gradient of ICa,L inprepubertal male but not in female hearts. Moreover, sexdifferences in ICa,L are reversed in adult rabbit hearts such thatthe adult female myocytes now have higher ICa,L at the basecompared with adult males. I/V relationships and the kineticsof ICa,L for all 4 groups of rabbits suggest that these sexdifferences in ICa,L are attributable to genomic changes in thedensity of functional L-type channels rather than alterationsin channel properties. Besides changes in peak ICa,L, differ-ences in the V0.5 suggest that inactivation of ICa,L occurs laterduring the AP of adult female myocytes, which would tend tocontribute to a slight increase in APD and higher Ca2� influxper AP. The other statistically different parameter was theslower inactivation kinetics of apical versus basal myocytesfrom prepubertal male hearts. The physiological conse-quences of slower inactivation remain unclear but suggestthat if all else remains unchanged, then the Ca2�-dependentinactivation of ICa,L is delayed at the apex, perhaps increasingCa2� influx during the AP plateau. Western blots of theCav1.2� subunit of Ca2� channel proteins revealed thatprotein levels are higher at the base of adult female heartscompared with the apex of female hearts and are higher thanprotein levels at the base and apex of adult male hearts. Inadult hearts, differences in Cav1.2� were statistically signif-icant and were consistent with functional measurements ofwhole-cell current densities. The mRNA coding for Cav1.2�was statistically (2.5�) higher at the base than the apex offemale hearts, but there were no other differences in mRNAbetween male and female hearts. Thus, the whole-cell current,protein, and mRNA analyses support the interpretation thatthere are sex differences in the expression of voltage-gated
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Figure 5. Voltage dependence of ICa,L activation and inactivationin adult rabbit myocytes. A, ICa,L activation curves from adultfemale and male apex and base myocytes. B, Steady-state ICa,L
inactivation curves from female and male apex and basemyocytes.
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L-type Ca2� channels at the base but not the apex of the heart.In prepubertal hearts, Cav1.2� protein levels were statisticallyhigher at the base of male hearts compared with the apex andhad a tendency to be greater than at the base and apex offemale hearts without reaching statistical significance. Simi-larly, message levels for Cav1.2� were not statistically dif-ferent in prepubertal hearts.
The correlation of the arrhythmia phenotype with (1)higher ICa,L, (2) higher protein levels, (3) the firing of EADs in
single cells and in simulations, and (4) a statistically higherincidence of EADs that originate first at the base of the heart,together, provide compelling evidence that ICa,L is an impor-tant determinant of the arrhythmia phenotype. A higher ICa,L
reduces the repolarization reserve and during IKr inhibitioncan promote EADs by 1 of 2 possible mechanisms: (1)spontaneous reactivation of ICa,L during the long AP plateau or(2) the reactivation of ICa,L triggered by an inward INCX, whichis, in turn, elicited by spontaneous SR Ca2� release. A 20% to
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Figure 6. Protein and message levels for Cav1.2� from the base and apex of adult and prepubertal female and male left ventricular tis-sues. Ventricular tissues from adult and prepubertal male and female hearts and female rabbit hearts were isolated, flash frozen, andprocessed to extract “total protein” or mRNA from the base and apex of each heart, as described in Materials and Methods. Proteinsamples from the base and apex of each heart were loaded on 16 lanes and run on the same gel to compare their concentrations ofCav1.2� compared with �-actin. A, Data from adult rabbits shows a Western blot for Cav1.2� obtained from the apex and base of 4adult male (hearts: 1 to 4) and 4 adult female hearts (hearts: 5 to 8) (a). Cumulative density histograms for Cav1.2� (normalized withrespect to �-actin) (c) and relative mRNA levels from real time PCR measurements (e) are summarized for 4 adult male and 4 adultfemale rabbit hearts. Protein and message levels were statistically higher at the base compared with the apex of adult female hearts(P�0.01). B, As for A but from prepubertal rabbits. Western blot for Cav1.2� obtained from the apex and base of 4 prepubertal female(hearts: 1 to 4) and 4 prepubertal male hearts (hearts: 5 to 8) (b). Cumulative density histograms for Cav1.2� (normalized with respect to�-actin) (d) and relative mRNA levels (f) are summarized for 4 prepubertal male and 4 prepubertal female rabbit hearts. Protein levelswere statistically higher at the base compared with the apex of prepubertal male hearts (P�0.01). There were no statistical differencesin message levels. For C through F, the ordinates represent are in an arbitrary scale derived from densitometry measurements. F indi-cates female; M, male; A, apex; B, base.
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30% increase in ICa,L is sufficiently large to enhance (1) Ca2�
influx per AP and intracellular Ca2� load, (2) luminal Ca2� inthe SR, (3) spontaneous SR Ca2� release and INCX during IKr
inhibition, and (4) thus initiate EADs that progress to TdP.Optical mapping of membrane potential changes showed thatin adult female hearts treated with E4031, EADs originatedpreferentially from the base. Similarly, AP recordings fromadult ventricular myocytes isolated from the base of the heartshowed that IKr blockade with E4031 elicited EADs in femalebut not in male myocytes. Thus, single-cell properties areconsistent with the arrhythmia phenotype of intact hearts. APsimulations confirmed that IKr inhibition prolonged APDswithout eliciting EADs and that an elevation of ICa,L wasnecessary and sufficient to elicit EADs. These data do notexclude sex differences in the expression of other Ca2�
channels and transporters, namely (1) INCX, (2) cardiac ryan-odine receptor 2, and/or (3) SERCA2, SR Ca2� pumps.
Sex Differences in ICa,L
Several studies have investigated sex differences in ICa,L invarious mammalian species, but the findings remain incon-clusive and no general consensus has thus far beenachieved. In 50- to 60-day-old rabbits, Pham et al reporteda transmural dispersion of ICa,L (higher on the epicardiumthan endocardium) at the base of female hearts that was
absent in male hearts.20 In contrast to rabbit hearts, a studyon mongrel dogs found uniformly higher levels of ICa,L infemale than male hearts across the left ventricular wall.41
In guinea pig hearts, the opposite result was obtained,where ICa,L was significantly higher in males than femaleseven when the female current density was measured atdifferent phases of the estrus cycle.42 Moreover, in mouseand rat hearts, no significant differences were found in ICa,L
between males and females.43,44 In human midmyocardialleft ventricular myocytes from patients with end-stageheart failure, ICa,L was found to be higher (�10%) in femalethan male hearts, but the difference did not reach statisticalsignificance.45 Nevertheless, simulations and experimentsshowed that at long cycle lengths, myocytes from womenwere prone to EADs, whereas myocytes from men rarelyfired an EAD.45 In the absence of similar studies in“healthy” human myocytes, Verkerk et al pointed out thatthe properties of myocytes from failing hearts were con-sistent with those obtained from healthy human hearts interms of rate-corrected QT, EAD susceptibility, and sexdifferences in APDs and, thus, proposed that the differ-ences in ICa,L between female and male hearts represent acharacteristic of normal human hearts.45
Previous studies were attentive to transmural differences inICa,L but neglected apex–base differences or differences in
Figure 7. Epicardial distribution of theearliest EADs in adult female and pre-pubertal male hearts with LQT2. A, Vm
was optically mapped from 256 sitesfrom the anterior surface of adult femalehearts (N�9). On perfusion with E4031(0.5 �mol/L), APs became prolongedand began to fire EADs. The earliestsite to fire an EAD was labeled with ared cross (X) to identify the regions ofmyocardium that are most susceptibleto fire an EAD. In 9 hearts, EADsclustered at the base and no EADsappeared first at the apex of heart. Thesuperposition of 2 APs recorded from apixel at the base and apex illustrate anexample of EADs that appeared first atthe base and did not propagate to theapex (Heart 1, middle) and EADs thatcaptured at the base and propagated tothe apex (Heart 2, right). The propensityof EADs to fire first at the base wasstatistically significant (P�0.02) basedon a 1-tailed binomial test. B, As for Aexcept that prepubertal male heartswere mapped to detect the regions ofthe heart that fired the earliest EADs.The earliest EADs occurred preferen-tially at the base (left; n�8/9) and thesuperposition of APs recorded from thebase and apex illustrate an example ofan EAD from the base that failed topropagate all the way to the apex (mid-dle) and an example of an EAD thatpropagated from the base to elicit asmaller, delayed depolarization at theapex (right).
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pre- versus postpuberty. The current findings of higher ICa,L
in adult female base myocytes are in agreement with previousstudies on rabbit20 and human45 hearts but extend the data toreveal ICa,L differences between apex and base and betweenpre- and postpuberty. It may be that once apex–base hetero-geneities are included in the analysis, ICa,L differences be-tween men and women will be statistically significant andwill expose larger ICa,L differences between the sexes. In ratand guinea pig hearts, sex differences in ICa,L were notdetected perhaps because the myocytes were isolated fromrandom regions of the left ventricle, and regional heteroge-neities of ICa,L may conceal differences of 25% to 30% incurrent density.44 In another study, no apex–base differencesin ICa,L were detected in mongrel dogs and human hearts, butthe study was not attentive to possible sex differences.46
More intriguing is the finding that higher ICa,L densities inadult and prepubertal rabbits correlate with the propensity toEADs at the base of the heart and the vulnerability to TdP inE4031-induced LQT2.11 Sex differences in arrhythmia phe-notype (as defined by E4031 in perfused hearts) arise fromthe properties of ventricular myocytes because E4031 elicitedEADs in freshly isolated adult female but not male myocytes
(Figure 8e and 8f). The significance of ICa,L differences inrabbit hearts is amplified by the remarkable similarity be-tween rabbit (Figure 8e and 8f) and human (Figure 1D45)recordings of male and female APs and the firing of EADs inhuman female but not male ventricular myocytes.
Regional Elevation of ICa,L As a Predictor ofArrhythmia PhenotypeNumerous studies showed that enhanced dispersion of repolar-ization is proarrhythmic and contributes to the initiation of TdPinduced by drugs that prolong the AP.11,17,20,33,46–49 In congen-ital, drug-induced LQT2 and in animal models of LQT2, APDprolongation does not automatically result in TdP and additionalfactors are needed to elicit EADs and TdP, with sex being amajor risk factor. Our findings of higher ICa,L at the base inprepubertal males and adult females correlate with sex- andage-related differences in arrhythmia phenotype are consistentwith the hypothesis that, in the setting of prolonged APDs, theseverity of Ca2� overload is a critical determinant of EADs andTdP. More precisely, the propensity to TdP in LQT2 is deter-mined by ICa,L: the higher the current, the greater the severity ofCa2� overload and the greater propensity that EADs originatefrom the base and progress to TdP.
Study LimitationsA comprehensive analysis of the cellular determinants ofLQT-mediated arrhythmias requires us to examine all chan-
A Pre-pubertal Male B Pre-pubertal Female
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Figure 8. EAD susceptibility in isolated ventricular myocytesfrom the base of the heart. Myocytes were isolated from thebase of rabbit hearts as described in Materials and Methodsand tested for their susceptibility to fire EADs once treated withE4031. In myocytes from prepubertal hearts, EADs occurredspontaneously in male (A) (n�4/4; H�3) but not in female (B)(n�0/4; H�3) ventricular cells. In myocytes isolated from adulthearts, there was a reversal of sex differences; EADs occurredin female (D) (n�5/6; H�4) but not in male (n�0/6; H�4) myo-cytes. Note that treatment with E4031 elicited a marked depo-larization or EAD (3) in adult female and prepubertal male myo-cytes compared with their adult male and prepubertal femalecounterpart. Fisher’s exact test rejects the null hypothesis ofequal probability of EADs between male and female myocyteswith P�0.02, such that statistical significance is reached topredict that EADs are more likely to occur with prepubertal malethan female myocytes and more likely to occur with adultfemale compared with male myocytes.
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Figure 9. LRd Simulations of APs from the base of adult andprepubertal hearts. Left graphs show APs from male base myo-cytes, and right graphs show APs from female base myocytes.The top row (a and b) illustrates LRd simulations of APs fromprepubertal myocytes; bottom row (c and d), AP simulations ofadult myocytes. The simulated AP shown in traces a through drepresent the 50th AP from a train of APs stimulated at a cyclelength of 1 second, either in the presence or absence of 100%or 50% IKr block (to mimic LQT2) in the simulated conditions. IKr
suppression leads to EAD development in the prepubertal malemyocytes (a) but not in prepubertal female myocyte (b). In con-trast, IKr suppression leads to EAD development in the adultfemale myocytes (d) but not in the adult male myocytes (c).The male vs female myocytes were simulated according to theexperimentally determined activation and inactivation param-eters shown in Tables 1 and 2 and experimental differencesin ICa,L.
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nels and transporters involved in Ca2� handling. One wouldreasonably expect that an increase in ICa,L would be matchedwith an increase in Ca2� pumps at the cellular membraneand/or NCX to balance the influx to the efflux of Ca2� duringan AP. Likewise, Ca2�-handling proteins like the Ca2� releasechannels (or ryanodine receptors) and Ca2�,Mg2�-ATPase(SERCA2) on the SR network may exhibit sex differencesand thereby contribute to the arrhythmia phenotype byaltering SR Ca2� overload, spontaneous SR Ca2� release, andthe initiation of EADs. In rat hearts, a recent study reportedhigher protein levels of Cav1.2, ryanodine receptor and NCXin females yet paradoxically Cav1.2 mRNA levels werehigher in males.21
In adult and prepubertal rabbit hearts, we found sexdifferences in NCX protein that were similar to Cav1.2� withhigher protein levels in adult female and prepubertal malesbased on Western blot analysis of prepubertal and adult rabbitleft ventricles (not shown). Similarly, the density of INCX washigher in prepubertal male compared with age-matched fe-male base myocytes (not shown). Further studies that probedifferences in Ca2� handling proteins are needed to obtain abetter understanding of all determinants of the arrhythmiaphenotype.
Optical mapping of membrane potential on the epicar-dium of rabbit hearts revealed a statistically significantpropensity of EADs to appear first at the base of the heart.The 2D nature of optical mapping does not resolve theexact origins of such EADs but does identify the break-
through sites that appear first on the epicardium. Until ahigh speed 3D technique is developed, one cannot com-pletely exclude the possibility that EADs originate fromdeeper layers at the base of the ventricles. However, it ishighly unlikely that EADs originate from deeper layersnear the apex, which then propagate inside the ventricularwall to breakthrough at the base before the apex.
The density of ICa,L depends on the number of functionalchannels and on the modulation of channel activity byregulatory peptides and multiple phosphorylation sitesthrough �-adrenergic activity. Sex-dependent regulation ofchannel activity presents another level of complexity that hasyet to be analyzed in a comprehensive fashion. The findingsraise important questions regarding genomic regulation of ionchannel expression by sex steroids. What mechanisms pro-duce sex differences in ion channel expression in prepubertybefore the surge of estrogen and testosterone? What cuesproduce regional differences in ion channel expression?
Nevertheless, the role of ICa,L as a determinant of arrhyth-mia phenotype in drug-induced LQT2 may be fundamental toour ability to evaluate the safety of new drugs that producesmall but measurable QT or APD prolongation. Female sex iswell known to be a risk factor to lethal TdP, but the currentdata support the more precise notion that sex differences inICa,L is a critical factor is the assessment of arrhythmogenicrisk. It is interesting to speculate that IKr inhibition may poseless of an arrhythmogenic risk if it is combined with ICa,L
and/or INCX inhibition. Similarly, congenital LQT2 may beasymptomatic well into adulthood; then, enhanced ICa,L and/or
A Adult B Pre-pubertal
200 ms200 ms 200 ms200 ms
49000 49200 49400 49600
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
49000 49200 49400 49600
0.0
0.5
1.0
1.5
2.0
2.5
[Ca2
+ ]i(m
M)
[Ca2
+ ]S
R(m
M)
30% ICa,L + 100% IKr Block
30% ICa,L + 30% I + 100%NCX IKr Block
[Ca2+]SR[Ca2+]i
C AdultICa,L±I ±NCX
200 ms
50 mV
50%IKr Block50%IKr Block + 30% INCX
100%IKr Block100%IKr Block + 30% INCX 30% ICa,L
30%INCX + 100% IKr Block
+ 100% IKr Block
D Intracellular Free Ca E SR Ca Content2+ 2+
Figure 10. Influence of INCX on EAD suscep-tibility. Simulated APs from the base of pre-pubertal male and adult female myocyteswere simulated as in Figure 9 but with a30% increase in INCX. A, APs from prepu-bertal male myocytes. B, APs from adultfemale base myocytes. A higher INCX hasno discernible effect on APD and does notinhibit the induction of EADs when ICa,L iselevated and IKr is inhibited. C, In adultmale and prepubertal female myocytes (ie,normal ICa,L), an increase in INCX alone didnot elicit EADs after imposing an IKr block.D and E, Changes in cytoplasmic free Ca2�
during an AP (D) and SR free Ca2� (E) with-out and with a 30% increase in ICa,L densityand 100% IKr block. Ca2� in control condi-tions (light traces), with higher INCX (boldtraces) and AP (dotted traces). A 30%increase in INCX caused a slight decrease infree Ca2� in both the cytosol and the SRlumen but did not inhibit EADs. The simu-lated APs shown represent the 50th APfrom a train of APs stimulated at a cyclelength of 1000 ms, either in the presence of100% or 50% IKr block.
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INCX through a genomic regulation can precipitate a shift inarrhythmia phenotype. Thus, sex steroids, heart failure, andcardiac hypertrophy may alter the LQT2 arrhythmia pheno-type through genomic regulation of Ca2� channels.
Sources of FundingThe study was supported by NIH grants HL57929 and HL 70722(to G.S.).
Female gender as a risk factor for torsades de pointes associated withcardiovascular drugs. JAMA. 1993;270:2590–2597.
2. Burke JH, Ehlert FA, Kruse JT, Parker MA, Goldberger JJ, Kadish AH.Gender-specific differences in the QT interval and the effect of autonomictone and menstrual cycle in healthy adults. Am J Cardiol. 1997;79:178–181.
3. Kuhlkamp V, Mermi J, Mewis C, Seipel L. Efficacy and proarrhythmiawith the use of d,l-sotalol for sustained ventricular tachyarrhythmias.J Cardiovasc Pharmacol. 1997;29:373–381.
4. Pratt CM, Camm AJ, Cooper W, Friedman PL, MacNeil DJ, MoultonKM, Pitt B, Schwartz PJ, Veltri EP, Waldo AL. Mortality in the SurvivalWith ORal D-sotalol (SWORD) trial: why did patients die? Am J Cardiol.1998;81:869–876.
5. Lehmann MH, Hardy S, Archibald D, MacNeil DJ. JTc prolongation withd,l-sotalol in women versus men. Am J Cardiol. 1999;83:354–359.
6. Drici MD, Burklow TR, Haridasse V, Glazer RI, Woosley RL. Sexhormones prolong the QT interval and downregulate potassium channelexpression in the rabbit heart. Circulation. 1996;94:1471–1474.
7. Drici MD, Clement N. Is gender a risk factor for adverse drug reactions?The example of drug-induced long QT syndrome. Drug Saf. 2001;24:575–585.
8. Roden DM. Long QT syndrome: reduced repolarization reserve and thegenetic link. J Intern Med. 2006;259:59–69.
9. Liu XK, Katchman A, Drici MD, Ebert SN, Ducic I, Morad M, WoosleyRL. Gender difference in the cycle length-dependent QT and potassiumcurrents in rabbits. J Pharmacol Exp Ther. 1998;285:672–679.
10. Zareba W, Moss AJ, Locati EH, Lehmann MH, Peterson DR, Hall WJ,Schwartz PJ, Vincent GM, Priori SG, Benhorin J, Towbin JA, RobinsonJL, Andrews ML, Napolitano C, Timothy K, Zhang L, Medina A. Mod-ulating effects of age and gender on the clinical course of long QTsyndrome by genotype. J Am Coll Cardiol. 2003;42:103–109.
11. Liu T, Choi BR, Drici MD, Salama G. Sex modulates the arrhythmogenicsubstrate in prepubertal rabbit hearts with Long QT 2. J CardiovascElectrophysiol. 2005;16:516–524.
12. Volders PG, Vos MA, Szabo B, Sipido KR, de Groot SH, Gorgels AP,Wellens HJ, Lazzara R. Progress in the understanding of cardiac earlyafterdepolarizations and torsades de pointes: time to revise currentconcepts. Cardiovasc Res. 2000;46:376–392.
13. January CT, Riddle JM. Early afterdepolarizations: mechanism ofinduction and block. A role for L-type Ca2� current. Circ Res. 1989;64:977–990.
14. Sipido KR, Callewaert G, Carmeliet E. Inhibition and rapid recovery ofCa2� current during Ca2� release from sarcoplasmic reticulum inguinea pig ventricular myocytes. Circ Res. 1995;76:102–109.
15. Viswanathan PC, Rudy Y. Cellular arrhythmogenic effects of con-genital and acquired long-QT syndrome in the heterogeneous myocar-dium. Circulation. 2000;101:1192–1198.
16. Volders PG, Kulcsar A, Vos MA, Sipido KR, Wellens HJ, Lazzara R,Szabo B. Similarities between early and delayed afterdepolarizationsinduced by isoproterenol in canine ventricular myocytes. Cardiovasc Res.1997;34:348–359.
17. Choi BR, Burton F, Salama G. Cytosolic Ca2� triggers early afterdepo-larizations and Torsade de Pointes in rabbit hearts with type 2 long QTsyndrome. J Physiol. 2002;543:615–631.
18. Choi BR, Salama G. Simultaneous maps of optical action potentials andcalcium transients in guinea-pig hearts: mechanisms underlying con-cordant alternans. J Physiol. 2000;529(pt 1):171–188.
19. Patterson E, Ma L, Szabo B, Robinson CP, Thadani U. Ovariectomy andestrogen-induced alterations in myocardial contractility in female rabbits:role of the L-type calcium channel. J Pharmacol Exp Ther. 1998;284:586–591.
20. Pham TV, Robinson RB, Danilo P Jr, Rosen MR. Effects of gonadalsteroids on gender-related differences in transmural dispersion of L-typecalcium current. Cardiovasc Res. 2002;53:752–762.
21. Chu SH, Sutherland K, Beck J, Kowalski J, Goldspink P, Schwertz D.Sex differences in expression of calcium-handling proteins and beta-adrenergic receptors in rat heart ventricle. Life Sci. 2005;76:2735–2749.
22. Bers D. Excitation-Contraction Coupling and Contractile Force. 2nd ed.Dordrecht, The Netherlands: Kluwer Academic Publishers; 2001.
23. Jost N, Virag L, Bitay M, Takacs J, Lengyel C, Biliczki P, Nagy Z,Bogats G, Lathrop DA, Papp JG, Varro A. Restricting excessive cardiacaction potential and QT prolongation: a vital role for IKs in humanventricular muscle. Circulation. 2005;112:1392–1399.
24. Eckardt L, Haverkamp W, Borggrefe M, Breithardt G. Experimentalmodels of torsade de pointes. Cardiovasc Res. 1998;39:178–193.
25. Eckardt L, Haverkamp W, Mertens H, Johna R, Clague JR, Borggrefe M,Breithardt G. Drug-related torsades de pointes in the isolated rabbit heart:comparison of clofilium, d,l-sotalol, and erythromycin. J CardiovascPharmacol. 1998;32:425–434.
26. Zabel M, Hohnloser SH, Behrens S, Li YG, Woosley RL, Franz MR.Electrophysiologic features of torsades de pointes: insights from a newisolated rabbit heart model. J Cardiovasc Electrophysiol. 1997;8:1148–1158.
27. Beyer C, McDonald P. Hormonal control of sexual behaviour in thefemale rabbit. Adv Reprod Physiol. 1973;6:185–219.
28. Salama G, Lombardi R, Elson J. Maps of optical action potentials andNADH fluorescence in intact working hearts. Am J Physiol. 1987;252:H384–H394.
29. Hool LC, Middleton LM, Harvey RD. Genistein increases the sensitivityof cardiac ion channels to beta-adrenergic receptor stimulation. Circ Res.1998;83:33–42.
30. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improvedpatch-clamp techniques for high-resolution current recording fromcells and cell-free membrane patches. Pflugers Arch. 1981;391:85–100.
31. Sims C, Harvey RD. Redox modulation of basal and beta-adrenergicallystimulated cardiac L-type Ca(2�) channel activity by phenylarsine oxide.Br J Pharmacol. 2004;142:797–807.
32. Belevych AE, Warrier S, Harvey RD. Genistein inhibits cardiac L-typeCa(2�) channel activity by a tyrosine kinase-independent mechanism.Mol Pharmacol. 2002;62:554–565.
33. Cheng J, Kamiya K, Liu W, Tsuji Y, Toyama J, Kodama I. Heteroge-neous distribution of the two components of delayed rectifier K� current:a potential mechanism of the proarrhythmic effects of methanesulfona-nilide class III agents. Cardiovasc Res. 1999;43:135–147.
34. Sanguinetti MC, Jurkiewicz NK. Two components of cardiac delayedrectifier K� current. Differential sensitivity to block by class III antiar-rhythmic agents. J Gen Physiol. 1990;96:195–215.
34a.Chomczynski P, Sacchi N. Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem.1987;162:156–159.
35. Luo CH, Rudy Y. A dynamic model of the cardiac ventricular actionpotential. I. Simulations of ionic currents and concentration changes. CircRes. 1994;74:1071–1096.
36. Faber GM, Rudy Y. Action potential and contractility changes in[Na(�)](i) overloaded cardiac myocytes: a simulation study. Biophys J.2000;78:2392–2404.
37. Dumaine R, Towbin JA, Brugada P, Vatta M, Nesterenko DV, Nest-erenko VV, Brugada J, Brugada R, Antzelevitch C. Ionic mechanismsresponsible for the electrocardiographic phenotype of the Brugadasyndrome are temperature dependent. Circ Res. 1999;85:803– 809.
38. Clancy CE, Rudy Y. Linking a genetic defect to its cellular phenotype ina cardiac arrhythmia. Nature. 1999;400:566–569.
39. Clancy CE, Rudy Y. Cellular consequences of HERG mutations in thelong QT syndrome: precursors to sudden cardiac death. Cardiovasc Res.2001;50:301–313.
40. Nakajima T, Hayashi K, Viswanathan PC, Kim MY, Anghelescu M,Barksdale KA, Shuai W, Balser JR, Kupershmidt S. HERG is protected
Sims et al Sex Differences in Rabbit Cardiac ICa,L e99
at University of Pittsburgh--HSLS on May 15, 2008 circres.ahajournals.orgDownloaded from
from pharmacological block by alpha-1,2-glucosyltransferase function.J Biol Chem. 2007;282:5506–5513.
41. Xiao L, Zhang L, Han W, Wang Z, Nattel S. Sex-based transmuraldifferences in cardiac repolarization and ionic-current properties in canineleft ventricles. Am J Physiol Heart Circ Physiol. 2006;291:H570–H580.
42. James AF, Arberry LA, Hancox JC. Gender-related differences in ven-tricular myocyte repolarization in the guinea pig. Basic Res Cardiol.2004;99:183–192.
43. Trepanier-Boulay V, St-Michel C, Tremblay A, Fiset C. Gender-baseddifferences in cardiac repolarization in mouse ventricle. Circ Res. 2001;89:437–444.
44. Leblanc N, Chartier D, Gosselin H, Rouleau JL. Age and gender dif-ferences in excitation-contraction coupling of the rat ventricle. J Physiol.1998;511(pt 2):533–548.
45. Verkerk AO, Wilders R, Veldkamp MW, de Geringel W, Kirkels JH, TanHL. Gender disparities in cardiac cellular electrophysiology and arrhyth-
mia susceptibility in human failing ventricular myocytes. Int Heart J.2005;46:1105–1118.
46. Szentadrassy N, Banyasz T, Biro T, Szabo G, Toth BI, Magyar J, LazarJ, Varro A, Kovacs L, Nanasi PP. Apico-basal inhomogeneity in distri-bution of ion channels in canine and human ventricular myocardium.Cardiovasc Res. 2005;65:851–860.
47. Kanai A, Salama G. Optical mapping reveals that repolarization spreadsanisotropically and is guided by fiber orientation in guinea pig hearts.Circ Res. 1995;77:784–802.
48. Efimov IR, Huang DT, Rendt JM, Salama G. Optical mapping of repo-larization and refractoriness from intact hearts. Circulation. 1994;90:1469–1480.
49. Liu DW, Gintant GA, Antzelevitch C. Ionic bases for electrophysiolog-ical distinctions among epicardial, midmyocardial, and endocardialmyocytes from the free wall of the canine left ventricle. Circ Res.1993;72:671–687.
e100 Circulation Research May 9, 2008
at University of Pittsburgh--HSLS on May 15, 2008 circres.ahajournals.orgDownloaded from
