Skip Navigation

Cardiovascular Research 2003 57(1):28-36; doi:10.1016/S0008-6363(02)00673-9
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Liu, X.-K.
Right arrow Articles by Ebert, S. N
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, X.-K.
Right arrow Articles by Ebert, S. N
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

In vivo androgen treatment shortens the QT interval and increases the densities of inward and delayed rectifier potassium currents in orchiectomized male rabbits

Xiao-Ke Liua,b, Alexander Katchmana, Bernard H Whitfielda,c, Grace Wana, Einsley M Janowskia, Raymond L Woosleyd and Steven N Eberta,*

aDepartment of Pharmacology, Georgetown University Medical Center, Georgetown, Washington, DC 20007, USA
bMayo Clinic, Rochester, MI, USA
cNIH, Bethesda, MD, USA
dDepartment of Medicine, University of Arizona Health Sciences, Tucson, AZ, USA

eberts{at}georgetown.edu

* Corresponding author. Tel.: +1-202-687-1270; fax: +1-202-687-2585

Received 22 March 2002; accepted 17 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Women have longer rate-corrected QT intervals (QTc) and are at higher risk for developing life-threatening torsades de pointes ventricular arrhythmias than men, especially after taking medications that block cardiac human ether-a-go-go-related gene (HERG)-encoded K+ channels. The purpose of the present study was to determine if the male sex steroid hormone, dihydrotestosterone (DHT), influences QT intervals in orchiectomized (Orch) male rabbits. Methods: ECG and whole-cell patch-clamp analyses were employed to evaluate cardiac repolarization and K+ currents in hearts isolated from orchiectomized (Orch) male New Zealand White rabbits receiving subcutaneous sustained release pellets for either dihydrotestosterone (DHT) or placebo. The efficacy of the treatment paradigm was monitored by measuring plasma DHT concentrations before and after the treatment period (10–14 days). Results: The results show that rate- and drug-induced QT-lengthening is attenuated in hearts from DHT-treated rabbits relative to placebo-treated controls. No significant changes in QRS were observed in response to DHT, thereby indicating that DHT influences QT primarily through an effect on ventricular repolarization. In addition, hearts from DHT-treated rabbits displayed significantly less QT lengthening in response to quinidine challenge compared to placebo controls. Current densities for two important cardiac repolarizing K+ currents, IK1 and IKr, were found to be significantly increased in ventricular myocardium of DHT-treated rabbits. Further, the half-maximal voltage of activation (V1/2) for IKr was significantly shifted to more negative potentials in myocytes from DHT vs. placebo hearts (21.2±1.2 vs. 30.2±1.4 mV, respectively, n=12, P<0.001). Corresponding changes in rabbit ether-a-go-go-related gene (RERG) mRNA were not found when examined by Northern blot hybridization. Conclusions: These results suggest that the presence of male sex steroid hormones in male rabbits helps to suppress rate- and drug-induced delays in cardiac repolarization. DHT action produces increased current densities for IK1 and IKr and a left-shift in the V1/2 for IKr that could account, at least in part, for the observed QTc differences between males and females. Since little change was seen in ventricular RERG gene expression, DHT action in the heart may influence IKr via post-transcriptional and/or post-translational mechanisms.

KEYWORDS ECG; Gender; Gene expression; Hormones; K-channel; Repolarization

This article is referred to in the Editorial by A.F. James and J.C. Hancox (pages 1–4) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Female gender is a risk factor for drug-induced cardiac arrhythmias, with an approximately 2:1 preponderance of women:men developing torsades de pointes (TdP) arrhythmias in response to similar concentrations of a wide variety of drugs [1]. TdP is a potentially fatal form of polymorphic ventricular tachycardia that typically occurs in a setting of increased QT interval duration measured from electrocardiogram (ECG) recordings [2,3]. The generally longer baseline rate-corrected QT (QTc) in women compared to men has long been suspected as a contributory factor for why women are at higher risk than men for developing TdP arrhythmias [4,5].

Evidence from clinical studies suggests that the gender differences in QTc may be primarily due to the influence of male sex steroid hormones since prepubescent boys and girls have QTc values similar to those found in adult women, whereas postpubescent males generally have shorter QTc intervals [6,7]. In addition, a recent study that compared JTc intervals in castrated men to those found in intact men and women showed that the JTc intervals in the castrated male group were significantly longer than those in normal males and were similar to those found in normal females [8]. Moreover, testosterone replacement therapy in the castrated male group produced shortening of JTc similar to that observed in normal males. These results suggest that male sex steroid hormones can influence cardiac repolarization and may, therefore, be responsible for the shorter QTc values observed in men compared to those found in women.

We and others have shown that the rabbit heart is a good model to examine sex differences in cardiac repolarization because rabbits display sex differences in QT intervals and susceptibility to drug-induced TdP arrhythmias [9–13]. In the present study, we compare the effect of DHT versus placebo treatment in orchiectomized (Orch) male rabbits on baseline and drug-induced QT over a wide range of pacing rates using isolated perfused (Langendorff) heart preparations. In addition, current densities of two major repolarizing potassium currents, the rapid component of the delayed rectifier (IKr) and the inward rectifier (IK1), are evaluated in ventricular cardiomyocytes isolated from placebo- versus DHT-treated rabbits.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
New Zealand White rabbits (3–4 months old, weight 3–3.5 kg) were obtained from Covance Labs. (Denver, PA, USA), and orchiectomies were performed at Georgetown University by the veterinary staff. Briefly, rabbits were anesthetized with ketamine–xylizine (50 mg/kg and 10 mg/kg, i.m., respectively). Open castration was performed with hemostasis of tunica creemaster using a 3-clamp technique. Subcutaneous implantation of pellets slowly releasing DHT (200 mg each, Innovative Research of America, Sarasota, FL, USA) at 5 mg/day was performed at the time of orchiectomy as previously described [9]. The animals recovered in a heated environment and were given buprenorphine (0.02–0.05 mg/kg) for perioperative pain relief. Plasma DHT levels were measured from serum samples collected using a commercially available ELISA kit (ALPCO Labs., Windham, NH, USA) immediately prior to surgery and again at the time of sacrifice (10–14 days after surgery/pellet implantation). This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996), and all experiments were conducted in accordance with the guidelines of the Georgetown University Animal Care and Use Committee.

2.2 Langendorff preparation
Langendorff preparations were performed as previously described [10]. Briefly, rabbits were anesthetized with intravenous pentobarbital (35 mg/kg), anticoagulated with heparin injection (500 I.U., i.v.), and sacrificed with each heart being rapidly removed through a median sternotomy and immersed in an ice-cold oxygenated Tyrode's solution. After the removal of extraneous fat and connective tissue, hearts were perfused by the nonrecirculating Langendorff technique as previously described [10].

2.3 ECG recordings and QT measurements
Four silver–silver chloride electrode wires were positioned in a simulated Einthoven configuration with the reference and ‘foot’ electrodes fixed beneath the heart on the walls of a tissue bath with the approximate diameter of a rabbit thorax. The signals were amplified by an ECG amplifier (Colbourn Instruments, Lehigh Valley, PA, USA), allowing for the simultaneous recording of the three orthogonal signals designated as X, Y and Z. The ECG signals were filtered selectively at 60 Hz.

2.3.1 Baseline QT measurements
The stability of the electrophysiologic measurements in our Langendorff preparation has been previously reported [10,11]. After about 30 min of continuous pacing at a cycle length of 0.4 s, baseline QT was recorded. To evaluate the cycle length dependence of the baseline QT interval and the effect of a sex steroid hormone, the cycle length was switched to one of the following cycle lengths: 0.8, 1.2 and 2.3 s, and the QT interval was measured when stable (=5% change over a 5-min period).

2.4 Whole-cell patch-clamp recordings
Ventricular myocytes were isolated from rabbit hearts using a modification of the method previously described by Giles et al. [14]. For each treatment group (i.e. placebo and DHT), at least three cells from three different hearts were recorded for analysis of K+ currents. The whole-cell patch-clamp technique was used to record the membrane currents in single ventricular myocytes as we reported previously [10,15]. Pipettes with tip resistance of 1–4 M{Omega} were pulled from borosilicate glass (World Precision Instruments, Sarasota, FL, USA) and filled with an intracellular solution containing (mmol/l) KCl 125, NaCl 10, CaCl2 1, Mg-ATP 5, EGTA 14, HEPES 10, cAMP 0.1, adjusted with KOH to pH 7.2. A holding potential of –40 mV was used to inactivate fast sodium and T-type calcium currents. The external solution was Tyrode's solution containing (mmol/l) NaCl 137, KCl 5.4, HEPES 10.0, MgCl2 1.0, CaCl2 2.0, glucose 10.0, pH 7.4. Cd2+ (0.2 mmol/l) was used to block the L-type calcium channel and to shift the I–V relationship of Ito and IKr to more positive potentials [10].

Cardiac myocytes adhering to glass coverslips were placed in a small chamber mounted on the stage of an inverted microscope and superfused at 1.5 ml/min at room temperature (22–24 °C). Only rod-shaped single cells that were quiescent and exhibiting well-defined, cross-striations were studied. After establishment of the whole-cell configuration and measurement of cell capacitance, series resistance (≤10 M{Omega}) was compensated by ~70%. Junction potentials under these conditions were approximately –3 mV and not corrected. IKr currents were elicited from a holding potential of –40 mV by a series of 1.5-s test pulses from –10 to +50 mV in 10-mV increments. Membrane potential was then returned to the holding potential in order to observe IKr tail currents. The current–voltage (I–V) relationship for IKr was constructed by measuring the tail currents.

The inward-rectifier K+ currents (IK1) were elicited from a holding potential of –40 mV by a series of 250-ms test pulses ranging from –90 to –10 mV in 10-mV increments. The amplitude of IK1 at each voltage was determined by measuring the peak current relative to zero current.

2.5 Northern blot analysis
Total RNA was purified from equivalent regions (apex of ventricle, ~500 mg/sample) from castrated rabbits receiving either placebo or DHT (200 mg) sustained release pellets for a period of 14 days using TrizolTM (Invitrogen, Carlsbad, CA, USA), as previously described [16]. A 25-µg portion of RNA per sample was loaded and separated by agarose gel electrophoresis, transferred to BrightStar-PlusTM (Ambion, Austin, TX, USA) nylon membrane, and hybridized with a radiolabeled 3.5-kb HindIII–BamH1 HERG cDNA (GenBank accession no. U04720 [GenBank] ) fragment isolated from a pSP64 vector (kindly provided by Dr. Mark Curran) [17]. Please note that this HERG probe does not distinguish between the A and B isoforms of HERG since it contains sequences common to both [18,19]. After washing, the membrane was developed and quantitated using a Molecular Dynamics Phosphorimager (Model 445, Rockville, MD, USA). The membrane was then stripped, re-hybridized with a radiolabeled rat β-actin cDNA fragment [16], and reprocessed as above.

2.6 Data analysis and statistics
Data were recorded on a strip chart recorder (Gould Electronics, Cleveland, OH, USA) at a paper speed of 200 mm/s. Four representative beats from each set of XYZ recordings were chosen for QT measurement. The end of the T wave was defined as the latest point of the T wave as it merges with the electrical baseline in any lead [20].

QT intervals were interpreted in a blinded fashion by two investigators. Patch-clamp data were also acquired and analyzed in blinded fashion. All current measurements were normalized for total cell capacitance (i.e. pA/pF) to allow comparison between cells of various sizes. One-way analysis of variance (ANOVA) with Bonferroni posthoc testing for multiple comparisons was used to compare QT (and JT) measurements at specific pacing cycle rates and current densities at specified voltages. The Student's t-test was used to compare Northern blot data from placebo and DHT rabbits. Data are reported as mean±S.E.M., with differences between values being considered statistically significant when P≤0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
We previously showed that male rabbit hearts have shorter QT intervals and greater K+ current densities (IK1 and IKr) than female rabbit hearts [10]. To test the hypothesis that these differences were the result of male sex steroid hormone action(s), we performed a series of experiments on orchiectomized (Orch) male rabbits that were implanted with either placebo or dihydrotestosterone (DHT) sustained release pellets. The efficacy of the treatment paradigm was evaluated by measuring DHT in plasma samples obtained from the rabbits immediately before castration (time of pellet implantation) and again at the time of sacrifice. As shown in Table 1, Orch males receiving placebo pellets displayed plasma DHT concentrations similar to those found in intact female rabbits. In contrast, those receiving sustained-release DHT pellets had plasma DHT levels that were significantly elevated relative to the placebo-implanted controls (78±9 vs. 19±2 pg/ml, respectively, P<0.001, n=7–8/group). The Orch rabbits with DHT pellets had, on average, somewhat lower DHT concentrations than those observed prior to surgery (78±9 vs. 205±77 pg/ml, respectively), though these values were not found to represent statistically significant differences (P0.05, n=7–14/group). Thus, the treatment paradigm was effective at both significantly reducing DHT plasma concentrations in placebo-implanted Orch males and reconstituting DHT plasma levels in Orch males implanted with sustained-release DHT pellets.


View this table:
[in this window]
[in a new window]

  		Table 1 Serum DHT concentrations (pg/ml) in rabbits

 
Initially, we examined QT intervals in isolated perfused rabbit heart preparations (Langendorff) over a range of pacing rates. Sample ECG tracings recorded during these experiments are shown in Fig. 1. The presence of DHT helped to suppress the increases observed in QT interval produced by pacing the hearts at slower rates (increased cycle lengths) (Fig. 2). Normal male and female rabbit heart data [10] are shown for comparative purposes. Note that placebo-treated Orch males behaved like normal females while the DHT-treated Orch males behaved like normal males in this assay. Thus, the presence of DHT can lead to decreases in QT intervals in male rabbit hearts.


Figure 1
View larger version (52K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Sample ECG traces from Langendorff-perfused rabbit hearts isolated from Orch males implanted with either placebo or DHT sustained release pellets. The chart speed was 200 mm/s and each box in the grid represents 25 ms. The hearts were paced at a fixed cycle length of 0.4 s. The arrow indicates the position of the pacing spike, which we used to define the beginning of the QRS complex. The left-most dashed vertical line shown in each trace indicates the end of the QRS complex. The rightmost dashed vertical line shown in each trace depicts the end of the T-wave. JT=QT–QRS.

 

Figure 2
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effect of orchiectomy (Orch) and DHT treatment on baseline QT intervals at different pacing cycle lengths. ECGs were recorded from Langendorff-perfused hearts from placebo- or DHT-treated rabbits at the indicated pacing cycle lengths, and QT measurements were evaluated and compared to those found in normal male and female rabbit hearts in this experimental paradigm [10]. *, P<0.05 for comparison between DHT and placebo-treated Orch rabbit hearts (n=4 in each group).

 
Since QT intervals are a measure of the duration of ventricular depolarization as well as repolarization, we also compared JT intervals to obtain a more specific estimate for ventricular repolarization. We found similar results in these comparisons (Fig. 3). The most pronounced difference (P<0.05) occurred at a pacing cycle length of 1.2 s, with hearts from DHT-treated rabbits displaying JT intervals of 199±12 (n=4) while those from placebo-treated rabbits displaying JT intervals of 240±8 (n=4). In addition, no significant differences were observed in QRS (60±1 ms, n=4, vs. 61±3 ms, n=4, for Orch+placebo vs. Orch+DHT, respectively, pacing cycle length=2.3 s, P0.05). Thus, the QT differences observed in response to DHT primarily reflect changes in ventricular repolarization. These results demonstrate that DHT suppresses cycle length-dependent slowing of cardiac repolarization in male rabbit hearts.


Figure 3
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Comparison of JT intervals in Langendorff-perfused hearts from placebo and DHT-treated Orch rabbits. JT intervals, defined as QT–QRS (see Fig. 1) were compared from Langendorff-perfused hearts isolated from Orch male rabbits implanted with either placebo or DHT sustained release pellets (n=4/group); *, P<0.05.

 
To determine if the presence of DHT could also influence drug-induced QT prolongation, we evaluated QT responses to quinidine (1 µM) in the Langendorff preparation. As shown in Fig. 4, quinidine-induced QT-lengthening was attenuated in hearts isolated from DHT-treated Orch rabbits relative to the placebo controls. The biggest difference in degree of quinidine-induced QT prolongation ({Delta}QT) between placebo and DHT groups occurred at the slowest pacing rate (57±4 vs. 32±2%, respectively, pacing cycle length=2.3s, n=4, P<0.001). Thus, DHT appears to enhance the heart's ability to resist drug-induced QT prolongation.


Figure 4
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Comparison of quinidine-induced QT prolongation in isolated perfused rabbit hearts from placebo and DHT-treated Orch animals. Following baseline ECG recordings at the indicated pacing cycle lengths, quinidine (1 µM) was added to the perfusion buffer and QT measurements were obtained at the same pacing cycle lengths. The difference in QT ({Delta}QT) in the presence vs. the absence of quinidine was compared over this range of pacing cycle lengths as shown (n=4/group). *, P<0.05; **, P<0.01. ***, P<0.001.

 
To investigate potential mechanisms underlying the DHT-induced QT changes in male hearts, we examined repolarizing K+ currents in isolated ventricular cardiomyocytes using the whole-cell patch-clamp recording technique. We previously demonstrated that at least two important K+ currents, IK1 and IKr, were present at higher densities in male ventricular cardiomyocytes compared to those observed in females [10]. Thus, we chose to focus our initial efforts on these two currents.

The inward rectifier K+ current, IK1, was measured in ventricular cardiomyocytes isolated from Orch male rabbits that had received either placebo or DHT sustained-release pellets. The outward component of IK1 was significantly greater in ventricular cardiomyocytes from DHT rabbit hearts compared with those obtained from placebo hearts (e.g. 2.2±0.1 vs. 1.4±0.1 pA/pF at –60 mV, n=9–14/group, P<0.05, Fig. 5). No significant differences were observed in the inward portion of IK1 for these two groups. An example of a current tracing for IK1 is shown in the insert. We confirmed that this current was IK1 by using low concentrations of BaCl2 to block it (data not shown) [14]. Significant increases in IK1 densities were observed between test potentials of –70 and –20 mV in DHT ventricular cardiomyocytes, with the maximum at –60 mV (Fig. 5). These results show that IK1 in ventricular cardiomyocytes from DHT-treated rabbits was up to 50–60% greater than that found in ventricular cardiomyocytes from placebo-treated rabbits.


Figure 5
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Comparison of the inward rectifier K+ current, IK1, in ventricular cardiomyocytes isolated from placebo vs. DHT-treated Orch rabbits. The inward-rectifier K+ currents (IK1) were elicited from a holding potential of –40 mV by a series of 250 ms test pulses ranging from –90 to –10 mV in 10-mV increments. The amplitude of IK1 at each voltage was determined by measuring the peak current relative to zero current. Current densities (pA/pF) were plotted and compared as a function of test potential for ventricular myocytes isolated from Orch rabbits that had been treated with either placebo (n=14) or DHT (n=9) sustained-release pellets. *, P<0.05. Inset, example of an IK1 recording is depicted.

 
One of the most striking observations from this study came from our measurements of IKr. An example of IKr currents recorded from ventricular cardiomyocytes for these experiments is shown in Fig. 6 (inset). Substantially larger tail currents were recorded from the cells isolated from DHT-treated rabbits than those from the placebo group. To illustrate this point, we compared the mean current densities (pA/pF) over a range of activation voltages (mV) for each group and found significant increases in IKr between activation voltages of +20 and +50 mV in the DHT group relative to the placebo group (maximum at +50 mV: 0.64±0.05 vs. 0.45±0.03 pA/pF, respectively, n=12, P<0.001, Fig. 6). These results show that IKr densities are up to 40% greater in ventricular cardiomyocytes from DHT-treated versus placebo-treated Orch rabbits. Furthermore, comparison of the half-maximal voltage of activation (V1/2) for IKr in myocytes from DHT-treated rabbits versus placebo-treated rabbits reveals that the V1/2 was significantly shifted towards more negative potentials in the DHT group (21.2±1.2 vs. 30.2±1.4 mV, respectively, n=12, P<0.001). Thus, the presence of DHT facilitates activation of IKr at lower voltages and increases the density of this current in ventricular myocytes.


Figure 6
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Comparison IKr in ventricular cardiomyocytes isolated from placebo vs. DHT-treated Orch rabbits. IKr currents were elicited from a holding potential of –40 mV by a series of 1.5-s test pulses from –10 to +50 mV in 10-mV increments. Membrane potential was then returned to the holding potential in order to observe IKr tail currents. The current–voltage (I–V) relationship for IKr was constructed by measuring the tail currents and plotting the current density (pA/pF) versus test potential. ***, P<0.001, n=12. Inset, example of IKr recording.

 
To determine if the observed DHT-mediated increases in IKr were due to changes in RERG gene expression, we examined RERG mRNA levels in rabbit ventricle from placebo and DHT-treated Orch rabbits using Northern blots. RERG mRNA was chosen as the target for this experiment because it encodes the major channel subunit for IKr [21]. As shown in Fig. 7, no increase in RERG mRNA was detected in DHT-treated hearts relative to placebo-treated controls (P0.05, n=3/group). Although the probe used to detect RERG mRNA does not distinguish between the A and B isoforms, only one band of approximately 4 kb was detected, indicating that adult rabbit ventricle predominantly expresses only one isoform. This isoform is presumed to be the A isoform because cDNA sequence information (Genbank accession no. U87513 [GenBank] ) from rabbit ventricular RERG is highly homologous (92.2%) to the A isoform of HERG (Genbank accession no. U04720 [GenBank] ) [22]. The blot was stripped and reprobed for β-actin to ensure the integrity of the RNA samples. Interestingly, β-actin mRNA appeared to be slightly increased in the DHT group relative to placebo controls while RERG mRNA was similar in both groups. Consequently, relative RERG mRNA expression appears slightly decreased in the DHT samples (Fig. 7B). Therefore, the increased IKr densities observed in ventricular cardiomyocytes from DHT-treated rabbits are likely due to post-transcriptional and/or post-translational modifications of the RERG channel protein.


Figure 7
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Northern blot analysis of RERG mRNA expression in ventricular myocardium from placebo vs. DHT-treated Orch rabbits. (A) Phosphorimages of Northern blots: Lanes 1–3, RNA isolated from three different placebo hearts; Lanes 4–6, RNA isolated from three different DHT hearts. The blots were stripped and reprobed for β-actin mRNA as a control. (B) Signal intensities from the Northern blot data were analyzed using a phosphorimager, and the intensity of the RERG signal is plotted relative to the signal obtained for β-actin in each sample.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The principal finding from this study is that DHT enhances the ability of male rabbit hearts to suppress rate- and drug-induced delays in cardiac repolarization (i.e. DHT-induced shortening of QT–JT intervals). The conventional mechanism of action for DHT and other androgenic steroid hormones is to bind to androgen receptors, which, in turn, leads to changes in the expression of specific genes that are the targets for androgen receptor binding. It is now well-established that androgen receptors are expressed in ventricular tissue from several mammalian species, including humans [23–25] but there is very little information available about the identity of specific gene targets of androgen receptor activity in the heart. There is also evidence that sex steroid hormones such as testosterone and 17-β-estradiol can influence potassium currents through nongenomic mechanisms [26,27], so it remains to be determined which mode of action may be operative in the heart under physiological conditions. Another mitigating factor is the variability of DHT plasma concentration levels, which may not be a particularly good indicator of hormonal influence on cardiac repolarization parameters since their mode of action likely involves alteration in the expression patterns of specific target genes over a period of time.

Although the specific mechanism of DHT action in the heart is not known, its influence leads to increased current densities for at least two key repolarizing K+ currents, IK1 and IKr, that had previously been identified as being greater in ventricular cardiomyocytes isolated from male (compared to female) rabbit hearts [10]. It is clear, however, from this study and earlier work that the increased current density for IKr cannot be accounted for by a corresponding increase in RERG gene expression either in Orch males (this study) or in Ovx females [9] following DHT administration. Given the fact that the observed DHT-induced changes in IKr only produced about a 30% increase in current, it may be difficult to detect such relatively small changes in RERG gene expression (if present) in our heart samples. In addition, there may some heterogeneity of RERG gene expression [28] within the rabbit heart that could also influence the RNA results, and it is important to keep in mind that mRNA changes (or lack thereof) do not necessarily reflect alterations in RERG protein expression. These caveats notwithstanding, alternative explanations for how IKr is increased by DHT could include phosphorylation of existing RERG [29–31], changes in RERG protein trafficking to the membrane [32], and/or alterations in β-subunit association(s) [21,33,34]. This latter possibility is particularly intriguing since association between HERG and a putative β-subunit encoded by the KCNE1 gene (minK) has been shown to increase IKr density and cause an apparent leftward shift in V1/2 [33] similar to our findings with IKr in myocytes from DHT-treated rabbits. Irrespective of the specific mechanism, however, the greater densities of IKr and IK1 likely account, at least in part, for the enhanced ability of male hearts to buffer rate- and drug-induced prolongation of QT intervals.

Other cardiac ion currents also appear to contribute to the observed gender differences in cardiac repolarization [35], and evaluation of their activities in relation to DHT treatment will need to be performed to determine what role they may or may not play regarding gender differences in susceptibility to drug-induced cardiac arrhythmias. Our study does not, for example, address whether or not the slow component of the delayed rectifier K+ current, IKs, may also play a role. IKr was generally thought to be the predominant delayed rectifier current in adult rabbit ventricular myocardium [36–38]; however, several reports have shown that IKs is also present to a limited extent in rabbit ventricular myocytes [12,39,40]. Although it is possible that IKs could have been present in some of our IKr recordings since it would have been activated (if present) by the voltage protocol used for our IKr experiments, two key pieces of evidence strongly suggest that little, if any, IKs was present. First, we previously showed that the preparation and voltage-clamp methods employed for this study yield exclusively IKr [10,15]. This was shown by applying the IKr-selective inhibitor, E-4031 (5 µM), which totally abolished the tail currents in ventricular myocytes isolated from male and female rabbit hearts [10]. Despite this, one could argue that the DHT may have augmented dormant or otherwise previously quiescent IKs in the myocytes that we tested, but, if that were the case, then we should have expected to see a shift in the V1/2 for IKr towards more positive test potentials (right-shifted) since IKs, unlike IKr, continues to increase as the voltage of activation becomes more positive [39]. In fact, we observed exactly the opposite result (V1/2 for IKr was left-shifted in the presence of DHT), thereby providing the second key piece of evidence indicating that IKs was not likely present in the myocytes that we tested. Consequently, our results in no way imply that IKs and/or many other cardiac ion currents that were not evaluated in this study may or may not be influenced by DHT. Indeed, the results of a recent study [41] suggest that cardiac IKs can also be influenced by the sex of the patient.

The ability of DHT to influence the QT and JT intervals in Orch male rabbits is consistent with recent results reported by Pham et al. [12], which showed that Orch male rabbits displayed greater cardiac action potential duration in response to the IKr-selective blocker, dofetilide. In humans, QT intervals tend to become shorter in males near the time of puberty when testosterone and DHT serum levels rise dramatically [6,7]. Perhaps the strongest evidence in this regard comes from a recent study by Bidoggia et al. [8] where they compared JT intervals in normal men and women with those from castrated men and virilized women. Their results showed that castrated men had rate-corrected JT intervals (JTc) similar to those seen in control (normal) women and significantly longer than those found in control (normal) men. Moreover, testosterone administration in the castrated men produced shortening of JTc comparable to that observed in normal men. Our data suggest that similar influences of male sex steroid hormones may be operative in rabbits. These findings indicate that it is primarily the male sex steroid hormones that account for the observed differences in cardiac repolarization between males and females and that the isolated perfused rabbit heart preparation is a useful model for studying this phenomenon in the laboratory.

Time for primary review: 28 days.


   Acknowledgements
 
This work was supported by a grant (HL58743) from the NHLBI/NIH. The authors wish to thank Lisa Portnoy, DVM, Joseph Newsome, DVM, and Laura Rutter-Call, LVT, for their excellent veterinary assistance with this project.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Makkar R.R, Fromm B.S, Steinman R.T, Meissner M.D, Lehmann M.H. Female gender as a risk factor for Torsades de Pointes associated with cardiovascular drugs. J. Am. Med. Assoc. (1993) 270:2590–2597.[Abstract]
  2. Roden D.M, Spooner P.M. Inherited long QT syndromes: a paradigm for understanding arrhythmogenesis. J. Cardiovasc. Electrophysiol. (1999) 10:1664–1683.[ISI][Medline]
  3. Tamargo J. Drug-induced torsade de pointes: from molecular biology to bedside. Jpn. J. Pharmacol. (2000) 83:1–19.[CrossRef][Medline]
  4. Burke J.H, Ehlert F.A, Kruse J.T, et al. Gender-specific differences in the QT interval and the effect of autonomic tone and menstrual cycle in healthy adults. Am. J. Cardiol. (1997) 79:178–181.[CrossRef][ISI][Medline]
  5. Bazett H. An analysis of the time relationship of electrocardiograms. Heart (1920) 7:353–370.[ISI]
  6. Lehmann M.H, Timothy K.W, Frankovich D, et al. Age–gender influence on the rate-corrected QT interval and the QT-heart rate relation in families with genotypically characterized long QT syndrome. J. Am. Coll. Cardiol. (1997) 29:93–99.[Abstract]
  7. Rautaharju P.M, Zhou S.H, Wong S, et al. Sex differences in the evolution of the electrocardiographic QT interval with age. Can. J. Cardiol. (1992) 8:690–695.[ISI][Medline]
  8. Bidoggia H, Maciel J.P, Capalozza N, et al. Sex differences on the electrocardiographic pattern of cardiac repolarization: possible role of testosterone. Am. Heart J. (2000) 140:678–683.[CrossRef][ISI][Medline]
  9. Drici M.D, Burklow T.R, Haridasse V, Glazer R.I, Woosley R.L. Sex hormones prolong the QT interval and downregulate potassium channel expression in the rabbit heart. Circulation (1996) 94:1471–1474.[Abstract/Free Full Text]
  10. Liu X.-K, Katchman A, Drici M.-D, et al. Gender difference in the cycle length-dependent QT and potassium currents in rabbits. J. Pharmacol. Exp. Ther. (1998) 285:672–679.[Abstract/Free Full Text]
  11. Liu X.K, Wang W, Ebert S.N, et al. Female gender is a risk factor for torsades de pointes in an in vitro animal model. J. Cardiovasc. Pharmacol. (1999) 34:287–294.[CrossRef][ISI][Medline]
  12. Pham T.V, Sosunov E.A, Gainullin R.Z, Danilo P Jr., Rosen M.R. Impact of sex and gonadal steroids on prolongation of ventricular repolarization and arrhythmias induced by IK-blocking drugs. Circulation (2001) 103:2207–2212.[Abstract/Free Full Text]
  13. Hara M, Danilo P Jr., Rosen M.R. Effects of gonadal steroids on ventricular repolarization and on the response to E4031. J. Pharmacol. Exp. Ther. (1998) 285:1068–1072.[Abstract/Free Full Text]
  14. Giles W.R, Imaizumi Y. Comparison of potassium currents in rabbit atrial and ventricular cells. J. Physiol. (Lond.) (1988) 405:123–145.[Abstract/Free Full Text]
  15. Liu X.K, Katchman A, Ebert S.N, Woosley R.L. The antiestrogen tamoxifen blocks the delayed rectifier potassium current, IKr, in rabbit ventricular myocytes. J. Pharmacol. Exp. Ther. (1998) 287:877–883.[Abstract/Free Full Text]
  16. Ebert S.N, Thompson R.P. Embryonic epinephrine synthesis in the rat heart before innervation: association with pacemaking and conduction tissue development. Circ. Res. (2001) 88:117–124.[Abstract/Free Full Text]
  17. Sanguinetti M.C, Jiang C, Curran M.E, Keating M.T. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell (1995) 81:299–307.[CrossRef][ISI][Medline]
  18. London B, Trudeau M.C, Newton K.P, et al. Two isoforms of the mouse ether-a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current. Circ. Res. (1997) 81:870–878.[Abstract/Free Full Text]
  19. Lees-Miller J.P, Kondo C, Wang L, Duff H.J. Electrophysiological characterization of an alternatively processed ERG K+ channel in mouse and human hearts. Circ. Res. (1997) 81:719–726.[Abstract/Free Full Text]
  20. Browne K.F, Zipes D.P, Heger J.J, Prystowsky E.N. Influence of the autonomic nervous system on the Q–T interval in man. Am. J. Cardiol. (1982) 50:1099–1103.[CrossRef][ISI][Medline]
  21. Abbott G.W, Sesti F, Splawski I, et al. MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia. Cell (1999) 97:175–187.[CrossRef][ISI][Medline]
  22. Pearson W.R, Wood T, Zhang Z, Miller W. Comparison of DNA sequences with protein sequences. Genomics (1997) 46:24–36.[CrossRef][ISI][Medline]
  23. Marsh J.D, Lehmann M.H, Ritchie R.H, et al. Androgen receptors mediate hypertrophy in cardiac myocytes. Circulation (1998) 98:256–261.[Abstract/Free Full Text]
  24. McGill H.C Jr., Anselmo V.C, Buchanan J.M, Sheridan P.J. The heart is a target organ for androgen. Science (1980) 207:775–777.[Abstract/Free Full Text]
  25. Sheridan P.J, McGill H.C Jr., Aufdemorte T.B, Triplett R.G, Holt R.G. Heart contains receptors for dihydrotestosterone but not testosterone: possible role in the sex differential in coronary heart disease. Anat. Rec. (1989) 223:414–419.[CrossRef][Medline]
  26. Shuba Y.M, Degtiar V.E, Osipenko V.N, Naidenov V.G, Woosley R.L. Testosterone-mediated modulation of HERG blockade by proarrhythmic agents. Biochem. Pharmacol. (2001) 62:41–49.[CrossRef][ISI][Medline]
  27. Valverde M.A, Rojas P, Amigo J, et al. Acute activation of Maxi-K channels (hSlo) by estradiol binding to the β subunit. Science (1999) 285:1929–1931.[Abstract/Free Full Text]
  28. Brahmajothi M.V, Morales M.J, Reimer K.A, Strauss H.C. Regional localization of ERG, the channel protein responsible for the rapid component of the delayed rectifier, K+ current in the ferret heart. Circ. Res. (1997) 81:128–135.[Abstract/Free Full Text]
  29. Bruggemann A, Pardo L.A, Stuhmer W, Pongs O. Ether-a-go-go encodes a voltage-gated channel permeable to K+ and Ca2+ and modulated by cAMP. Nature (1993) 365:445–448.[CrossRef][Medline]
  30. Bian J, Cui J, McDonald T.V. HERG K(+) channel activity is regulated by changes in phosphatidyl inositol 4,5-bisphosphate. Circ. Res. (2001) 89:1168–1176.[Abstract/Free Full Text]
  31. Cui J, Melman Y, Palma E, Fishman G.I, McDonald T.V. Cyclic AMP regulates the HERG K(+) channel by dual pathways. Curr. Biol. (2000) 10:671–674.[CrossRef][ISI][Medline]
  32. Furutani M, Trudeau M.C, Hagiwara N, et al. Novel mechanism associated with an inherited cardiac arrhythmia: defective protein trafficking by the mutant HERG (G601S) potassium channel. Circulation (1999) 99:2290–2294.[Abstract/Free Full Text]
  33. McDonald T.V, Yu Z, Ming Z, et al. A minK-HERG complex regulates the cardiac potassium current I(Kr). Nature (1997) 388:289–292.[CrossRef][Medline]
  34. Mazhari R, Greenstein J.L, Winslow R.L, Marban E, Nuss H.B. Molecular interactions between two long-QT syndrome gene products, HERG and KCNE2, rationalized by in vitro and in silico analysis. Circ. Res. (2001) 89:33–38.[Abstract/Free Full Text]
  35. Pham T.V, Robinson R.B, Danilo P, Rosen M.R. Effects of gonadal steroids on gender-related differences in transmural dispersion of L-type calcium current. Cardiovasc. Res. (2002) 53:752–762.[Abstract/Free Full Text]
  36. Clay J.R, Ogbaghebriel A, Paquette T, Sasyniuk B.I, Shrier A. A quantitative description of the E-4031-sensitive repolarization current in rabbit ventricular myocytes. Biophys. J. (1995) 69:1830–1837.[Abstract/Free Full Text]
  37. Carmeliet E. Use-dependent block and use-dependent unblock of the delayed rectifier K+ current by almokalant in rabbit ventricular myocytes. Circ. Res. (1993) 73:857–868.[Abstract/Free Full Text]
  38. Carmeliet E. Use-dependent block of the delayed K+ current in rabbit ventricular myocytes. Cardiovasc. Drugs Ther. (1993) 7(Suppl. 3):599–604.[CrossRef][ISI][Medline]
  39. Salata J.J, Jurkiewicz N.K, Jow B, et al. IK of rabbit ventricle is composed of two currents: evidence for IKs. Am. J. Physiol. (1996) 271:H2477–H2489.[ISI][Medline]
  40. Lengyel C, Iost N, Virag L, et al. Pharmacological block of the slow component of the outward delayed rectifier current (IKs) fails to lengthen rabbit ventricular muscle QT(c) and action potential duration. Br. J. Pharmacol. (2001) 132:101–110.[CrossRef][ISI][Medline]
  41. Conrath C.E, Wilde A.A, Jongbloed R.J, et al. Gender differences in the long QT syndrome: effects of β-adrenoceptor blockade. Cardiovasc. Res. (2002) 53:770–776.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
A. F. James and J. C. Hancox
Sex, drugs and arrhythmia: are gender differences in risk of torsades de pointes simply a matter of testosterone?
Cardiovasc Res, January 1, 2003; 57(1): 1 - 4.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Liu, X.-K.
Right arrow Articles by Ebert, S. N
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, X.-K.
Right arrow Articles by Ebert, S. N
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?