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Endocrinology Vol. 147, No. 11 5160-5169
Copyright © 2006 by The Endocrine Society

Long-Term and Immediate Effect of Testosterone on Single T-Type Calcium Channel in Neonatal Rat Cardiomyocytes

Guido Michels, Fikret Er, Michael Eicks, Stefan Herzig and Uta C. Hoppe

Department of Internal Medicine III (G.M., F.E., M.E., U.C.H.), University of Cologne, Institute of Pharmacology (S.H.), and Center for Molecular Medicine Cologne (S.H., U.C.H.), University of Cologne, 50937 Cologne, Germany

Address all correspondence and requests for reprints to: Uta C. Hoppe, M.D., Department of Internal Medicine III, University of Cologne, Kerpener Strasse 62, 50937 Cologne, Germany. E-mail: uta.hoppe{at}uni-koeln.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the cardiovascular system, T-type calcium channels play an important role for the intracellular calcium homeostasis and spontaneous pacemaker activity and are involved in the progression of structural heart diseases. Androgens influence the cardiovascular physiology and pathophysiology. However, their effect on native T-type calcium currents (ICa,T) remains unclear. To test the chronic effect of testosterone on the cardiac ICa,T, cultured neonatal rat ventricular cardiomyocytes were treated with testosterone (1 nM-10 µM) for 24–30 h. Current measurements were performed after testosterone washout to exclude any acute testosterone effects. Testosterone (100 nM) pretreatment significantly increased whole-cell ICa,T density from 1.26 ± 0.48 pA/pF (n = 8) to 5.06 ± 1.75 pA/pF (n = 7; P < 0.05) and accelerated beating rate. This was attributed to both increased expression levels of the pore-forming subunits Cav3.1 and Cav3.2 and increased T-type single-channel activity. On single-channel level, the increase of the ensemble average current by testosterone vs. time-matched controls was due to an increased availability (58.1 ± 4.2 vs. 21.5 ± 4.0%, P < 0.01) and open probability (2.78 ± 0.29 vs. 0.85 ± 0.23%, P < 0.01). Cotreatment with the selective testosterone receptor antagonist flutamide (10 µM) prevented these chronic testosterone-induced effects. Conversely, acute application of testosterone (10 µM) decreased T-type single-channel activity in testosterone pretreated cells by reducing the open probability (0.78 ± 0.13 vs. 2.91 ± 0.38%, P < 0.01), availability (23.6 ± 3.3 vs. 57.6 ± 4.5%, P < 0.01), and peak current (–20 ± 4 vs. –58 ± 4 fA, P < 0.01). Flutamide (10 µM) did not abolish the testosterone-induced acute block of T-type calcium channels. Our results indicate that long-term testosterone treatment increases, whereas acute testosterone decreases neonatal rat T-type calcium currents. These effects seem to be mediated by a genomic chronic stimulation and a nongenomic acute inhibitory action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LOW-THRESHOLD, VOLTAGE-gated T-type calcium channels appear as particularly interesting candidates for playing a substantial role in various pathologies (1, 2). Their cloning and molecular characterization revealed that these channels have evolved apart from L-type and other neuronal calcium channels, suggesting a specific functional role (3). One of the first physiological functions proposed for T-type calcium channels was a support for a pacemaker current because they activate at very negative voltages and resulting calcium entry leads to membrane depolarization. This role is well recognized in the heart, both for sinus node function and spontaneous activity of neonatal cardiomyocytes (1, 4). Changes in the expression levels of T-type channels have been observed during the development of various organs (5). Particularly, in most mammalian species, T-type calcium currents (ICa,T) are robustly present in embryonic heart and both atrial and ventricular myocytes but are absent or much reduced in postnatal ventricular myocytes (6, 7). However, in pathologic states such as hypertrophy and heart failure, it has been speculated that ICa,T might play a role in the pathogenesis of ventricular calcium overload and arrhythmias because a reinduction of T-type calcium channels has been obtained in hypertrophic and failing ventriculocytes mediated by angiotensin II and endothelin 1 (2, 8).

Androgens influence cardiovascular physiology and pathophysiology (9, 10). Anabolic androgenic steroids have been associated with myocardial ischemia, hypertrophy, sudden cardiac death, and hypertension in athletes, leading to the view that androgens are detrimental for the cardiovascular system (11). However, besides these genetic, mainly testosterone receptor-mediated pathways, recent experimental data indicated another, genomic-independent way of testosterone action on the cardiovascular system, i.e. short-term administration of testosterone acutely induces vasodilation in the systemic, coronary and pulmonary vascular beds (12, 13), and acutely protected cardiomyocytes against hypoxic injury by directly opening ATP-sensitive potassium channels of the inner mitochondrial membrane in isolated cells and mitoplasts (14). So far no detailed analysis of potential effects of androgens on cardiac T-type calcium channels is available. In the present study, we demonstrate distinct acute vs. chronic testosterone effects on single cardiac T-type calcium channels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication 85-23, revised 1996).

Myocyte isolation
Neonatal Sprague Dawley (1–2 d old) rats were killed by decapitation. The hearts were quickly removed via thoracotomy and transferred to an ice-cold Tyrode solution. Ventricles were cut off and stored in Tyrode solution. Myocytes were then isolated by further incubation in a shaker bath (37 C), with a solution containing collagenase (type II; Biochrom AG, Berlin, Germany) 200 IU/ml and trypsin 0.1% (Sigma, Munich, Germany) for 30 min. Dissociated cells were plated on gelatin-coated (Sigma) coverslips. For whole-cell patch-clamp experiments, 3- to 5-d-old monolayer cultures were dispersed by trypsin and replated at a low density to study electrically isolated cells within 2–8 h (15).

Electrophysiology
Patch-clamp recordings in the whole-cell configuration.
Whole-cell Ca2+ currents were recorded using standard microelectrode patch-clamp techniques (6, 16, 17). Currents were recorded and digitized with an Axopatch 200B amplifier and Digidata 1200 interface (Axon Instruments, Foster City, CA) by using custom software. The T-type calcium current (ICa,T) and L-type calcium current (ICa,L) were discriminated by changing the holding potential from –100 mV (T-type plus L-type calcium current) to –50 mV (L-type calcium current) for 200 msec before applying the test pulse from –50 to +10 mV. ICa,T was obtained by subtracting these current sets at the different holding potentials as previously described (2). The recording bath solution was composed of (millimoles): tetraethylammonium (TEA)-Cl 136, CaCl2 2, MgCl2 2, HEPES 25 and glucose 20 (pH was adjusted to 7.4 with TEA hydroxide). The corresponding pipette solution contained (millimoles): CsCl 130, EGTA 10, Mg-ATP 2, HEPES 25 (pH was adjusted to 7.2 with Cs-hydroxide). Data were not corrected for the liquid junction potential of 6.7 mV.

Patch-clamp recordings of single channels in the cell-attached configuration.
Single calcium channels were recorded in cell-attached configuration of the patch-clamp technique (depolarizing test pulses of 150 msec duration at 0.5 Hz). Single-channel measurements and analysis were done as previously reported (18). The external solution for single-channel experiments contained (millimoles): potassium glutamate 120, KCl 25, MgCl2 2, HEPES 10, EGTA 2, CaCl2 1, and dextrose 10 (pH was adjusted to 7.4 with KOH). Borosilicate pipettes (5–8 M{Omega}) were filled with (millimoles) BaCl2 110 and HEPES 10 (pH of 7.4 was adjusted with TEA hydroxide). An Axopatch 200B amplifier, Digidata 1200 interface (Axon Instruments), and custom software were used for pulse generation, data acquisition (10 kHz), and filtering (2 kHz, –3 dB, 4-pole Bessel filter). All experiments were performed at room temperature (21–23 C).

Data analysis
Linear leak and capacity currents were digitally subtracted using the average currents of nonactive sweeps. Openings and closures were identified by the half-height criterion. The open probability (defined as the relative occupancy of the open state during active sweeps, Po), the availability (fraction of sweeps containing at least one channel opening), and the peak ensemble average current (Ipeak, obtained visually) were calculated from single-channel and multichannel patches. In the latter case, they were corrected for n, the number of channels in the patch (n was defined as the maximum current amplitude observed, divided by the unitary current). Single-channel amplitudes were determined by direct measurements of fully resolved openings or as the maximum of Gaussian fits on amplitude histograms. Peak current (Ipeak) was corrected by division through n. The availability was corrected by the square root method: (1-availabilitycorrected) is the nth root of (1-availabilityuncorrected). The corrected open probability was calculated on the basis of the corrected number of active sweeps, i.e. total open time divided by (n x availabilitycorrected x number of test pulses x pulse length). Closed-time and first-latency analyses were carried out only in one-channel patches. Time constants of open- and closed-time histograms ({tau}open, {tau}closed) were obtained by maximum likelihood estimation (PStat software; Axon Instruments). The voltage dependence of activation and inactivation was analyzed using the Boltzmann function: Y = Ymax/{1 + e (V 0.5-V/k)}, where V0.5 is the voltage of half-maximal activation and k is the slope factor (18, 19). Time-dependent inactivation (percent) was determined by taking the peak maximum of the ensemble average current at the beginning of a pulse (Ipeak) and the remaining peak current at the end of a test pulse (I150): inactivation (percent) = 1-(I150/Ipeak).

Pooled data are presented as mean ± SEM. A two-tailed t test was used for statistical examinations, using unpaired format for comparison of different channels (chronic effect) and paired format for same experimental arrangements (acute effect). Probability values of P < 0.05 were deemed significant.

Preparation of membrane protein and Western blotting
Cardiomyocytes were collected from petri dishes and resuspended in buffer containing 1 mM phenylmethylsulfonyl fluoride, 10 mM each Tris/HCl (pH 7.5) and EDTA, 50 mM each NaCl and NaF, 20 µg/ml Aprotinin, 0.1% (vol/vol) each Triton X-100 and ß-mercaptoethanol. Preparation of total membrane protein was proceeded according to Zigrino et al. (20), with minor variations. The suspension was subjected to three subsequent freeze-thaw steps on dry ice-ethanol/37 C and afterward sheared by passing 20 times through a 20-gauge needle. After centrifugation at 300 x g, the supernatant was centrifuged at 16,000 x g to sediment the membrane fraction. Protein concentration was assayed using a commercial protein assay (BCA method, Pierce, Rockford, IL). After standard Laemmli SDS-PAGE (7.5%) and Western blotting (Tankblot system, nitrocellulose membrane; Amersham Pharmacia Biotech, Little Chalfont, UK), channel protein was detected using anticalcium channel Cav3.1 ({alpha}1G, Sigma) as first antibody and enhanced chemiluminescence staining (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Equal protein transfer among lanes was verified by reversible staining with Ponceau red and use of a commercial protein assay (BCA method; Pierce). Densitometrical analysis of protein induction was performed on a GS-800 calibrated densitometer (Bio-Rad Laboratories, Hercules, CA) with the Quantity One Alias analysis software (Bio-Rad).

RNA isolation and quantitative real-time PCR
Total RNA was extracted from neonatal cardiomyocytes using the RNeasy kit, and first-strand cDNA was synthesized by reverse transcription of 2 µg total RNA from different treatment groups using the Omniscript reverse transcription kit (QIAGEN, Hilden, Germany) according to the manufacture’s instructions. For identification of androgen receptor mRNA, PCR primers were based on the rat epididymal androgen receptor cDNA sequence (21): forward primer, 5'-CGA AGG CAG CAG CAG CGT GAG A-3'; backward primer, 5'-GCG AGC GGA AAG TTG TAG TAG T-3'. This responds to sequences of bp 1590–1611 and 2090–2069 of the cDNA and would be expected to produce a PCR product of 501 bp. PCR was performed with Taq polymerase for 35 cycles with an annealing temperature of 58 C (22). Quantitative real-time PCR was performed on a LightCycler 1.5 instrument using FastStart DNA MasterPlus SYBR Green I (Roche Molecular Biochemical, Mannheim, Germany). Gene-specific primers were the following: Cav3.1 ({alpha}1G) forward primer, 5'-CAG ACC TGC TGA CTG TGA GGA A-3'; backward primer, 5'-CGG CAC ATG TAG CTG TCA TTG-3' (GenBank accession no. Cav3.1 rat: AF027984); Cav3.2 ({alpha}1H) forward primer, 5'-GCG TGA CAC TGG GCA TGT T-3'; backward-primer, 5'-GGC GAA GAA GGC AAA GAT GA-3' (GenBank accession no. Cav3.2 rat, AF290213) (1, 23). Succinate dehydrogenase complex, subunit A (flavoprotein) expression of each sample was used as endogenous control: forward-primer, 5'-TGG GAA CAA GAG GGC ATC TG-3'; backward primer, 5'-CCA CCA CTG CAT CAA ATT CAT G-3' (GenBank accession no. NM_130428) (24). Quantitative real-time PCR was carried out under the following cycling conditions: stage 1, 95 C for 10 min [repeat one time (rep 1)]; stage 2, 95 C for 10 sec, 60 C for 5 sec, and 72 C for 10 sec with signal acquisition at 81 C for 1 sec (determined from melting curve analyses) (rep 40); stage 3, 65 C for 10 sec (rep 1). Analysis of the PCR curves was performed with the second derivate maximum method of the LightCycler software (25, 26). All sample measurements were repeated at least three times and results are given as mean ± SEM.

Drugs
Testosterone (4, 5{alpha}-dihydrotestosterone), S(-)-Bay K 8644, mibefradil, flutamide, and formestane (Sigma) were dissolved in an ethanol stock solution before they were added to the bath solution or culture medium, as indicated. The final concentration of ethanol was less than 0.01%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Chronic testosterone treatment increases cardiac T-type calcium channel density and beating frequency of neonate cardiocytes
To test the chronic effect of testosterone on the cardiac ICa,T, cultured neonatal rat ventricular cardiomyocytes were treated with testosterone 1, 10, and 100 nM for 24–30 h. Current measurements were performed in standard recording bath solution after testosterone washout for 10 min to exclude any acute testosterone effects. Testosterone pretreatment increased the whole-cell ICa,T current density, compared with time-matched control cells from 1.26 ± 0.48 pA/pF (at –30 mV; n = 8) to 2.78 ± 0.65 pA/pF (testosterone 1 nM; n = 10; P = ns), 3.21 ± 1.18 pA/pF (testosterone 10 nM; n = 8; P = ns), and 5.06 ± 1.75 pA/pF (testosterone 100 nM; n = 7; P < 0.05) (Fig. 1Go, A and B). Thus, increase of ICa,T current density could be observed at testosterone concentrations present during anabolic androgen application in men (27, 28). Because low voltage-activated, rapidly inactivating T-type calcium channels are known to modulate spontaneous beating frequency of neonatal cardiomyocytes, we evaluated the effect of chronic testosterone exposure on the electrical activity of neonate cell cultures. Untreated control cells contracted at a slow basal frequency, which was accelerated in a concentration-dependent manner by testosterone (EC50 108.3 ± 7.5 nM, n = 7; Fig. 1CGo). Testosterone application (24–30 h) increased the cell size (1 nM: 15.1 ± 1.8 pF; 10 nM: 22.2 ± 2.6 pF; 100 nM: 24.0 ± 4.6 pF; n = 12 each), compared with control cells (16.7 ± 1.6 pF, n = 12), although this did not reach statistical significance. Cotreatment with the selective testosterone receptor antagonist flutamide 100 nM prevented the testosterone-induced increment of ICa,T current density and beating rate, suggesting testosterone-induced modulation of ICa,T via the genomic androgen receptor-mediated pathway.


Figure 1
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  		FIG. 1. Chronic testosterone exposure increased whole-cell ICa,T density. A, Original representative patch-clamp current recordings of control vs. testosterone (Testo) 100 nM pretreated cardiomyocytes at variable test potentials from –50 to +10 mV using consecutively two different holding potentials (Hp) of –100 (left) and –50 mV (middle). Traces shown at right represent the difference current attributable to T-type Ca2+ current. B, Current densities of ICa,T at –30 mV in control vs. testosterone (1, 10, and 100 nM) pretreated cardiomyocytes. C, Concentration-dependent acceleration of the spontaneous beating frequency of neonatal cardiomyocytes cultures by chronic testosterone treatment (n = 7 each concentration). *, P < 0.05 vs. control

 
Chronic testosterone treatment increases cardiac T-type calcium channel expression
To confirm that neonatal cardiomyocytes express androgen receptors, RT-PCR was performed, demonstrating the expected PCR product of 501 bp (Fig. 2AGo). To test whether chronic testosterone exposure affected T-type calcium channel expression, membrane protein of Cav3.1 and mRNA levels of Cav3.1/Cav3.2 were determined from neonatal rat cardiomyocytes cultured under control conditions, in the presence of testosterone (100 nM) and testosterone (100 nM) + flutamide (100 nM). Testosterone pretreatment markedly increased Cav3.1/Cav3.2 expression, which could be prevented by flutamide (Fig. 2Go, B and C). Ethanol (0.01%) and flutamide (100 nM) had no effect on Cav3.1/Cav3.2 transcript levels.


Figure 2
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  		FIG. 2. Androgen receptor is expressed in neonatal cardiomyocytes, and chronic testosterone treatment increases expression of the T-type calcium channel in cardiomyocytes. A, RT-PCR confirmed expression of androgen receptors in neonatal cardiomyocytes (expected PCR product 501 bp). B, Detection of Cav3.1 in membrane protein fractions from neonatal rat cardiomyocytes cultured under control conditions in the presence of testosterone (100 nM) or testosterone + flutamide (100 nM). Loading was 35 µg of membrane protein per lane. Testosterone increased the expression of Cav3.1, whereas flutamide abolished the testosterone-induced effect. Densitometric values for Cav3.1 protein levels (n = 3). Co, Control; T, testosterone; Flu, flutamide. Values are mean ± SEM. *, P < 0.05 vs. control; **, P < 0.05 vs. testosterone. C, Cav3.1/Cav3.2 mRNA levels determined by quantitative real-time PCR (n = 5). Co, Control; EtOH, ethanol (0.01%); T, testosterone (100 nM); Flu, flutamide (100 nM). Values are mean ± SEM. *, P < 0.05 vs. control; **, P < 0.05 vs. testosterone.

 
Characteristics of single cardiac T-type calcium channels
Increase of whole-cell ICa,T current density might be due only to increased expression levels of the T-type calcium channel or additional changes of single-channel activity. Therefore, we determined single calcium channel characteristics of neonatal cardiomyocytes. Consistent with previous whole-cell and single-channel studies (6, 29, 30), we obtained two visually distinct types of single calcium channel activities in cell-attached patch-clamp recordings from cardiomyocytes of neonatal rats: the high-voltage activated L- and the low-voltage-activated T-type calcium channels. The two types of calcium channels differed in their biophysical and pharmacological properties (Figs. 3–5GoGoGo and Table 1Go).


Figure 3
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  		FIG. 3. Original traces form three experiments, illustrating the differences among the T-type (left), L-type (middle), and L-type calcium channel with S(-)-Bay K 8644 (right) in neonatal rat cardiomyocytes. Patches were depolarized from –80 mV (holding potential) to various test potentials (TP), as indicated. Two traces are shown for each indicated TP. Scale bars, 30 ms and 1 pA, respectively.

 

Figure 4
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  		FIG. 4. Biophysical properties of calcium channels in neonatal rat cardiomyocytes. A, Single-channel amplitude i of the T-type calcium channel (open symbols, n = 6) and L-type calcium channel under control conditions (closed symbols, n = 5) as a function of the test potential. Holding potential was –80 mV throughout. Slope conductances as depicted here were calculated based on pooled data (see text). B, Voltage dependence of single-channel activity of T-type calcium channels. Patches were depolarized from –80 mV to various test potentials (activation, right) or from various holding potentials (inactivation, left) to –20 mV. Data were averaged from n = 5 activation and n = 5 inactivation experiments. The curves shown here were fitted using the Boltzmann equation and the pooled data as displayed. Mean values indicated in the text were derived from Boltzmann fits of individual experiments.

 

Figure 5
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  		FIG. 5. Effect of mibefradil (10 µM) on single T-type calcium channels. The top illustrates the pulse protocol (holding potential –80 mV, test potential –20 mV). The middle represents consecutive sweeps: left, control currents; right, acute effect of mibefradil. The bottom trace depicts ensemble averages from 120 traces before (control) and 240 traces after mibefradil application. Scale bars, 30 ms and 1 pA (unitary current traces) or 16 fA (ensemble averages), respectively.

 

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  		TABLE 1. Single-channel mechanism of the T-type calcium channel blockers mibefradil (10 µM) and nickel (300 µM)

 
The L-type calcium channel activated at higher voltages, had a higher single-channel amplitude (conductance 15.7 ± 0.9 pS, n = 5; Fig. 4AGo), and openings were more evenly distributed, compared with T-type channels. Because calcium channel agonists resolve full-amplitude openings of L-type channels, especially at more negative test potentials, we performed additional recordings in the presence of S(-)-Bay K 8644 (1 µM), which increased conductance values to 20.2 ± 0.2 pS (n = 3). Thus, our conductance measurements were just within the expected range for L-type calcium channels (18, 31, 32).

The T-type calcium channel activated at lower voltages, had a lower single-channel amplitude (single-channel conductance 5.1 ± 0.8 pS, n = 6), and openings occurred in well-separated bursts at any given test potential. Detailed analysis of the voltage dependence of activation and inactivation (Fig. 4BGo) demonstrated the early activation behavior of the T-type calcium channel. Half-maximal activation was observed at –29.1 ± 2.6 mV with a slope of 3.0 ± 0.4 mV (n = 5). The T-type channel inactivated half-maximally at –59.0 ± 5.6 mV with a slope factor of –8.3 ± 2.0 mV (n = 5). The biophysical properties were well in accordance with previous single-channel studies from guinea pig cardiomyocytes (33). Cardiac T-type calcium channels in cardiomyocytes were blocked by 300 µM nickel and 10 µM mibefradil (Fig. 5Go and Table 1Go), as expected from previous studies on cardiovascular cells (18, 34, 35), whereas the dihydropyridine S(-)-Bay K 8644 (1 µM) had no effect on the T-type calcium channels. These results confirmed the typical behavior of native T-type calcium channels (31, 36, 37, 38). Moreover, our data showed that the biophysical and pharmacological properties of the T-type calcium channel were clearly distinguishable from those of L-type calcium channels.

Chronic testosterone treatment increases cardiac T-type calcium channel activity
In addition to an increase of Cav3.1/Cav3.2 expression, chronic testosterone application modulated single T-type calcium channel function. After testosterone pretreatment (10 µM for 24–30 h), current measurements were performed in standard recording bath solution after testosterone washout for 10 min to exclude any acute testosterone effects. Testosterone pretreatment markedly increased T-type calcium channel activity (Fig. 6Go). The increase of the ensemble average current by testosterone vs. time-matched controls (Table 2Go) was due to an increased availability (58.1 ± 4.2 vs. 21.5 ± 4.0%, P < 0.01) and an increased open probability (2.78 ± 0.29 vs. 0.85 ± 0.23%, P < 0.01). The higher open probability of testosterone pretreated cardiomyocytes was predominantly caused by the shorter first latency (Table 2Go). Cotreatment with the selective testosterone receptor antagonist flutamide 10 µM abolished the testosterone-induced functional modulation of the T-type calcium channel.


Figure 6
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  		FIG. 6. Effect of testosterone (10 µM) on single T-type calcium channels. The top illustrates the pulse protocol (holding potential –80 mV, test potential –20 mV). Middle: left, sequential unitary current records without (control); middle, after 24–30 h treatment with testosterone (pretreated); right, acute effect of testosterone on testosterone-pretreated cells (acute effect). Bottom: ensemble-average currents calculated from sweeps without testosterone (left: control 300 sweeps), after chronic testosterone exposure (middle: pretreated 180 sweeps), and after acute testosterone addition to pretreated cells (right: acute effect 360 sweeps). Scale bars, 30 ms and 1 pA (unitary current traces) or 18 fA (ensemble averages), respectively.

 

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  		TABLE 2. Single-channel mechanism of chronic testosterone modulation (10 µM) without and with flutamide (10 µM) on native cardiac T-type calcium channels (holding potential –80 mV, test potential –20 mV)

 
To further support that the observed chronic testosterone effect was mediated via the testosterone receptor rather than by estrogens catalyzed by local aromatase, we determined the testosterone effect in the presence and absence of the aromatase inhibitor formestane (4OH-androstendione; 10 nM). On the single-channel level, T-type channel activity was still markedly increased by testosterone in the presence of formestane with only slight, nonsignificant attenuation of the testosterone action by aromatase inhibition: formestane-testosterone (n = 8), compared with testosterone without formestane (n = 10) of same cell preparations: peak average current –31 ± 7 vs. –47 ± 4 fA (P = ns), channel availability 38.8 ± 6.31 vs. 51.4 ± 2.7% (P = ns), and open probability 2.04 ± 0.51 vs. 2.85 ± 0.32% (P = ns). These results further supported that at least the major effect of the observed chronic testosterone-induced activation on T-type calcium channels was mediated by testosterone receptors.

Acute testosterone exposure blocks T-type calcium channels
Given our observation of higher basal T-type calcium channel activity after chronic testosterone treatment, we decided to evaluate the acute effect of testosterone on T-type channels in testosterone-pretreated cells. Original current traces, mean data, time course of the open probability, and single-channel parameters demonstrated that testosterone 10 µM acutely decreased T-type channel activity on the single-channel level (Figs. 6Go and 7Go and Table 3Go). We observed a pronounced reduction of the open probability (0.78 ± 0.13 vs. 2.91 ± 0.38%, P < 0.01 vs. testosterone-pretreated cells, P = ns vs. controls), availability (23.6 ± 3.3 vs. 57.6 ± 4.5%, P < 0.01 vs. testosterone pretreated cells, P = ns vs. controls), and peak current (–20 ± 4 vs. –58 ± 4 fA, P < 0.01 vs. testosterone pretreated cells, P = ns vs. controls), compared with time-matched controls. This indicated an acute antagonistic effect of testosterone on T-type calcium channels, which reversed the chronic testosterone-mediated effects. Acute testosterone application of 100 nM also significantly reduced the open probability (0.59 ± 0.09 vs. 1.18 ± 0.13%, n = 4), availability (14.8 ± 2.06 vs. 27.1 ± 0.85%, n = 4), and peak current (–16 ± 3 vs. –32 ± 3 fA, n = 4) of nonpretreated cells (all P < 0.05 vs. control). Whereas testosterone changed the slow and fast gating properties (i.e. reduction of availability and open probability), it did not exert a very rapid block in the range of microseconds (Table 3Go), which would manifest as a reduced apparent single-channel current amplitude at our recording band width (18) common for small pore-blocking particles. Flutamide (10 µM) did not prevent the testosterone-induced block of T-type calcium channels indicating a receptor-independent mechanism (Table 3Go). Acute testosterone application dose-dependently reduced the spontaneous beating rate of neonatal cardiomyocytes (control: 85.6 ± 9.3, n = 14; 1 nM: 88.8 ± 10.4 bpm, n = 16, P = ns; 10 nM: 82.3 ± 5.8 bpm, n = 12, P = ns; 100 nM: 75.6 ± 3.6 bpm, n = 15, P = ns; 1 µM: 65.5 ± 5.3 bpm, n = 13, P = 0.08; 10 µM: 62.4 ± 6.9 bpm, n = 14, P = 0.06; 100 µM: 59.8 ± 4.2 bpm, n = 18, P = 0.01 vs. control). Ethanol 0.01% did not affect beating rate (84.3 ± 9.9 bpm, n = 16, P = ns vs. control).


Figure 7
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  		FIG. 7. Acute effect of testosterone (10 µM) on single T-type calcium channel. Open-probability (averaged over 2-min recording periods) of native T-type calcium channel vs. time. Channels were depolarized to –20 mV and exposed after 6 min to 10 µM testosterone (open circle, n = 8); control experiments (filled square, n = 4).

 

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  		TABLE 3. Single-channel mechanism of acute testosterone modulation (10 µM) without and with flutamide (10 µM) of native cardiac T-type calcium channels of testosterone pretreated cells (holding potential –80 mV, test potential –20 mV)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study examined the effect of testosterone on native T-type calcium channels. Our results show that testosterone exhibits distinct chronic vs. acute effects on the T-type channel: chronically the hormone increases single-channel activity, transcripts, and membrane protein levels of the pore-forming subunits and acutely antagonizing the T-type single-channel. Moreover, the underlying testosterone-induced mechanisms seem to differ, suggesting a nongenomic acute or rapid testosterone effect and a genomic chronic modulation of cardiac T-type calcium channels by testosterone.

Native T-type calcium currents of the cardiovascular system are believed to contribute to spontaneous pacemaker activity. Moreover, they are involved in maintenance of vascular tone, control of cell growth, modulation of atrial natriuretic peptide secretion, and cardiovascular remodeling (6, 31). In ventricular myocardium expression of T-type calcium channels peaks between postnatal d 4 and 8 and gradually declines or even disappears thereafter (6). In contrast to adult ventricular myocytes, robust T-type currents are observed in Purkinje cells, sinoatrial node cells, and latent pacemaker cells from a variety of species, supporting the notion that T-type channels are important in cardiac conduction and pacemaking (37, 39). Interestingly, studies indicate that T-type calcium channels may reappear in ventricular myocardium with left ventricular hypertrophy or cardiac failure and under neurohumoral stimulation (2, 40). In these pathological states, increased calcium channel expression may induce calcium overload, trigger arrhythmias, and activate calcium-dependent signaling pathways that mediate remodeling and cell apoptosis.

The cardiovascular system is a chronic and acute target for androgens (14, 41). Beginning in the third decade, aging is associated with a progressive decline in serum testosterone levels. The decrease in serum testosterone level during aging is characterized by high interindividual variability; thus, by far not all aging men will become hypogonadal to a clinical significant degree. Total testosterone levels less than 7 nM (normal 15–25 nM) confirms late-onset hypogonadism in the aging male and indicate that benefits might be derived from testosterone therapy. The long-term effect of testosterone therapy with supraphysiological testosterone levels on the cardiovascular system are unknown, especially those regarding the potential benefit of hormone replacement in elderly men who suffer from several concomitant diseases (42, 43). At 100 nM testosterone, we observed the first significant increase of T-type calcium current (Fig. 1BGo), a concentration mimicking serum levels of individuals undergoing a testosterone replacement therapy (>100 nM) (42, 44). Although no significant ICa,T has been recorded in atrial and ventricular cells isolated from human heart with various pathophysiological states (45), detection of Cav3.1 and Cav3.2 transcripts in the human heart suggest an involvement of these calcium channels (46). A reappearance of ICa,T under supraphysiological testosterone levels (sexual-steroid hormone treatment) might induce changes in the electrophysiological properties (calcium overload) of adult cardiomyocytes and increase the probability of spontaneous action potentials, thus increasing the likelihood for arrhythmias.

However, the role of these hormones in the regulation on cardiac T-type calcium channels is still unclear. Modulation of L-type calcium channels by different androgens has so far mainly been detected in various cell-types. Testosterone opens L-type calcium channels in osteosarcoma cells (47), decreases calcium uptake by coronary smooth muscles (12, 48) and ventriculocytes (49), and up-regulates the expression of the L-type calcium channel {alpha}-subunits in smooth muscle cells (10). Whereas several hormones and neurotransmitters have been reported to also affect T-type calcium currents (37, 50, 51), modulation by testosterone has not yet been analyzed.

Three T-type calcium channel subtypes (Cav3.1, Cav3.2, Cav3.3) have been identified by molecular cloning and electrophysiological studies (37). Whereas the two isoforms Cav3.1 and Cav3.2 can be detected in cardiomyocytes of neonatal rats, Cav3.1 is the predominant transcript in these cells (6). In the present study, we obtained higher protein concentrations of this pore-forming {alpha}1G subunit in the plasma membrane of neonate ventricular myocytes after testosterone pretreatment. Additionally, we obtained an increase of transcript levels of Cav3.1 and Cav3.2 on testosterone treatment. This elevated T-type channel expression together with the observed increase of channel activity on the single-channel level resulted in higher whole-cell T-type current densities associated with an accelerated beating rate of cardiomyocytes on chronic testosterone exposure. Both of these chronic testosterone effects could be abolished by flutamide, indicating a genomic pathway. The mechanism of the chronic functional modification of T-type single-channel activity by testosterone remains speculative. Possible factors include a changed coexpression of auxiliary subunits; an altered splice behavior; or an alteration of regulator proteins like protein kinases, protein tyrosine kinases, and G proteins (37, 38). These hypotheses should be addressed in further molecular biological and proteomic (trafficking) studies.

Previous studies also indicated an influence of estradiol on T-type calcium channels (50). Estrogens are in part being catalyzed by peripheral cyp450 aromatase, which metabolizes C19 steroids such as androstenedione and testosterone to estrone and 17ß-estradiol (52). To discriminate the contribution of aromatization, we performed experiments in the presence of the aromatase inhibitor formestane. Formestane slightly attenuated but, however, did not abolish the stimulating effect of testosterone on T-type channel activity. This further supported that the major effect observed by long-term testosterone was mediated via testosterone receptors with possibly a minor contribution of the estrogen pathway.

In addition to chronic hormone actions, evidence for rapid, nongenomic effects of androgens is increasing (12, 14). Nongenomic testosterone actions can be distinguished from genomic effects by a more rapid onset (seconds to minutes), insensitivity to inhibition of RNA and protein synthesis, and the fact that observed effects may not be blocked by classical testosterone receptor antagonists. In the present study, testosterone, given as a bolus, led to an acute inhibition of the T-type single calcium channel. The interaction of testosterone with the T-type calcium channel seemed to rely on two mechanisms: a rapid mechanism with a reduction of the open probability and the ensemble average current and a slow mechanism with a reduction of the channel availability, whereas no effect on current amplitude (very rapid mechanism) was detectable. This indicates that testosterone might interact at the internal site with the pore-forming Cav3.x subunit. The acute blocking effect of testosterone was not inhibited by the antiandrogen flutamide, excluding mediation by intracellular androgen receptors. Thus, acute testosterone blockade of T-type calcium channels occurs within minutes via a nongenomic mechanism.

Interestingly, in vitro the chronic testosterone effect on T-type single channels was antagonized by the acute testosterone action. This observation suggests that testosterone chronically might be involved in setting the basal activity of T-type single calcium channels, whereas in addition acute changes of hormone levels might rapidly modulate T-type calcium currents.


   Acknowledgments
 
We thank N. Henn, I. Berg, and M. Weber for skillful technical assistance.


   Footnotes
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ho 2146/3-1) and by the Marga und Walter Boll-Stiftung.

Disclosures: G.M., F.E., M.E., S.H., and U.C.H. have nothing to declare.

First Published Online July 27, 2006

Abbreviations: ICa,T, T-type calcium current; Ipeak, peak ensemble average current; TEA, tetraethylammonium; {tau}closed, time constant of closed-time histogram; {tau}open, time constant of open-time histogram.

Received February 14, 2006.

Accepted for publication July 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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