Published online before print November 13, 2003, doi: 10.1161/01.RES.0000106134.69300.B7
© 2003 American Heart Association, Inc.
Cellular Biology |
Angiotensin II Signaling Pathways Mediate Expression of Cardiac T-Type Calcium Channels
From the Remodelage Tissulaire et Fonctionnel, Hôpital Marie Lannelongue, Le Plessis Robinson, France.
Correspondence to Laurent Ferron, CNRS UMR 8078, Remodelage Tissulaire et Fonctionnel, Hôpital Marie Lannelongue, 133 avenue de la Résistance, 92350 Le Plessis Robinson, France. E-mail laurent.ferron{at}ccml.u-psud.fr
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Abstract |
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Recent studies indicate that cardiac T-type Ca2+ current (ICaT) reappears in hypertrophied ventricular cells. The aim of this study was to investigate the role of angiotensin II (Ang II), a major inducer of cardiac hypertrophy, in the reexpression of T-type channel in left ventricular hypertrophied myocytes. We induced cardiac hypertrophy in rats by abdominal aorta stenosis for 12 weeks and thereafter animals were treated for 2 weeks with losartan (12 mg/kg per day), an antagonist of type 1 Ang II receptors (AT1). In hypertrophied myocytes, we showed that the reexpressed ICaT is generated by the CaV3.1 and CaV3.2 subunits. After losartan treatment, ICaT density decreased from 0.40±0.05 pA/pF (n=26) to 0.20±0.03 pA/pF (n=27, P<0.01), affecting CaV3.1- and CaV3.2-related currents. The amount of CaV3.1 mRNA increased during hypertrophy and retrieved its nonhypertrophic level after losartan treatment, whereas the amount of CaV3.2 mRNA was unaffected by stenosis. In cultured newborn ventricular cells, chronic Ang II application (0.1 µmol/L) also increased ICaT density and CaV3.1 mRNA amount. UO126, a mitogen-activated protein kinase kinase-1/2 (MEK1/2) inhibitor, reduced Ang II–increased ICaT density and CaV3.1 mRNA amount. Bosentan, an endothelin (ET) receptor antagonist, reduced Ang II–increased ICaT density without affecting the amount of CaV3.1 mRNA. Finally, cotreatment with bosentan and UO126 abolished the Ang II–increased ICaT density. Our results show that AT1-activated MEK pathway and autocrine ET-activated independent MEK pathway upregulate T-type channel expression. Ang II–increased of ICaT density observed in hypertrophied myocytes may play a role in the pathogenesis of Ca2+ overload and arrhythmias seen in cardiac pathology.
Key Words: angiotensin II • mitogen-activated protein kinase • T-type Ca2+ channel • cardiac hypertrophy • gene expression
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Introduction |
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The electrophysiological remodeling that occurs during cardiac pathologies, such as hypertrophy, contributes to myocardial remodeling and plays an important role in the development of arrhythmia, increasing the risk of morbidity and mortality.1 Reappearance of the T-type Ca2+ current (ICaT) has been observed in hypertrophied ventricular cells, in association with pressure overload, postinfarction, and cardiomyopathy.2–4 Little is known about the pathophysiological role of ICaT, but its activation at low voltage suggests that ICaT may be related to Ca2+ overload and arrhythmias.4 The relationship between ICaT and cardiac damage is highlighted by the therapeutic benefits of blocking ICaT.5,6 For example, the use of a selective ICaT antagonist, mibefradil, has been shown to improve survival in a rat model of chronic heart failure.7
Previous studies showed that ICaT is developmentally regulated in rodent ventricular cells.8,9 In the fetus, ICaT is generated by both CaV3.1 (
1G) and CaV3.2 (1H) pore-forming channels whereas in newborn rat ventricles, only CaV3.1-related current can be recorded. Furthermore, ICaT is no more detectable in adult rat ventricles. The cardiac pattern of expression of T-type channels, together with previous reports describing the relationship between ICaT and cell proliferation, is consistent with Ca2+ uptake through ICaT being dedicated to specific functions related to the cell cycle.10–12 ICaT density increases after growth hormone stimulation,13 and thus ICaT may also be associated with growth processes in nonproliferating adult hypertrophied myocytes.
Few studies have been reported concerning the effectors/mechanisms involved in regulation of the expression of cardiac T-type channels.14 Among the factors stimulated during cardiac hypertrophy, it has been shown that acute application of endothelin (ET)-1 to cultured newborn rat ventricular cells increases ICaT density due to the activation of protein kinase C.15 Conflicting results have been obtained concerning adrenergic stimulation, which has been reported to have no effect or to increase the density of ICaT in the ventricle.16,17 To our knowledge, the only study describing the effects of angiotensin II (Ang II) stimulation in cardiac tissue was performed in frog atrial cells and reported an increase in ICaT density in response to acute Ang II application.18
In the heart, Ang II is known to play a critical role in ventricular hypertrophy and several aspects of signal transduction in response to Ang II stimulation resemble features of the signal transduction induced by growth factors.19 Ang II exerts its hypertrophic effect by activating G-protein–coupled type 1 Ang II receptors (AT1) and initiating a number of well-defined intracellular signaling pathways.20 Considerable efforts are currently being made to identify the signaling molecules involved in hypertrophic responses, such as those involved in the mitogen-activated protein kinase (MAPK) cascade.21 Recent data have demonstrated that a MAPK cascade component, the extracellular signal-regulated kinase 1/2 (ERK1/2) activated by the MAPK kinase-1/2 (MEK1/2), may be an important component connecting several signaling pathways involved in hypertrophy.22
In this study, we demonstrate for the first time that Ca2+ entry through ICaT is increased by the Ang II–activated AT1 cascade in left ventricular hypertrophied myocytes. We also show that CaV3.1 and CaV3.2 subunits participated to the reexpressed ICaT and these subunits are differently regulated. Finally, Ang II–activated MEK pathway and ET-activated independent MEK pathway are shown to regulate the expression of ICaT-related subunits.
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Materials and Methods |
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Hypertrophic Model
Animals were cared for and used in accordance with the European convention for the protection of vertebrate animals used for experimental purposes, and institutional guidelines n° 86/609/CEE November 24, 1986. Adult male Wistar rats (IFFA Credo, France) weighing 150 to 170 g were subjected to abdominal aortic stenosis during 6 or 12 weeks as previously described.23 Stenosed rats (AS) were paired with sham-operated rats (CTL) of the same weight and age. Twelve-week rats were treated with losartan (AS/L) for 2 weeks (12 mg/kg per day) via Alzet miniosmotic pumps (model 2002) inserted into the intraperitoneal cavity.24 Losartan was generously provided by MERCK.
Isolation of Adult Cardiomyocytes and Culture of Newborn Cardiomyocytes
Left ventricular myocytes were isolated enzymatically from adult rat hearts by retrograde perfusion, as described elsewhere.25 Cultures of newborn myocytes were prepared from the ventricles of 1- to 2-day-old Wistar rats, as described by Renaud et al.26 Twenty-four hours after plating, myocytes were incubated for 48 hours with Ang II (0.1 µmol/L, Sigma) or Ang II plus antagonists/inhibitors in fetal calf serum-free medium supplemented with 10% heat-inactivated horse serum. The antagonists were AT receptor antagonists (losartan, 1 µmol/L and PD123319, 10 µmol/L; Sigma) and ET receptor antagonists (bosentan, 0.1 µmol/L, generously provided by Actelion, BQ123, 10 µmol/L, and BQ788, 10 µmol/L; Sigma). For MEK1/2 inhibition, we used UO126, 10 µmol/L, and PD98059, 10 µmol/L (Cell Signaling Technology).
Electrophysiological Recordings
Ca2+ currents were recorded by the whole-cell patch-clamp technique at room temperature (22°C to 24°C). The fire-polished pipettes used had a resistance of 1 to 3 M
when filled with pipette solution (in mmol/L: CsCl 10, Cs-aspartate 120, MgCl2 3, HEPES 10, EGTA 15, CaCl2 1.8, glucose 10, creatine phosphate 3.6, MgATP 5, Tris-GTP 0.2, pH 7.2 adjusted with CsOH). Ca2+ currents were recorded in an external solution containing 135 mmol/L TEA Cl, 1 mmol/L MgCl2, 10 mmol/L CaCl2, 10 mmol/L HEPES, 10 mmol/L glucose, 3 mmol/L 4-aminopyridine, 20 mmol/L CsCl, 0.03 mmol/L tetrodotoxin, pH 7.4 adjusted with CsOH. Additional details of recording and data analysis can be found elsewhere.9
Quantitative RT-PCR
Total RNA was prepared from ventricular tissues using Trizol (GIBCO-BRL). Reverse transcription (RT) and polymerase chain reaction (PCR) conditions, and the procedures used for quantification have been described elsewhere.9 Briefly, quantitative RT-PCR was performed with normalized RNA aliquots (1 µg) and a known amount of cRNA internal control (0.3x106 molecules for both transcript species, for tissues, and 2.5x106 molecules for cultures). The internal control differed from the target counterpart by only one restriction site. PCR was performed with a trace amount of 32P-labeled 5' primer. The number of copies per µg of total RNA was calculated using the following equation: [Internal control weight/(size in nucleotidesx330)]x6.02x1023, and from the relationship [molecules cRNA control/cpm cRNA control]xcpm target=target molecules per normalized RNA aliquot.
Statistical Analysis
Data are expressed as mean±SE. N and n correspond to the number of animals and the number of cells, respectively. Statistical significance was estimated by one-way ANOVA followed by Dunnett’s test to identify differences between groups. Differences were considered significant if P<0.05.
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Results |
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Ang II Is Involved in the Stenosis-Induced Increase in ICaT Density
We have used a model of stenosed rat treated with losartan to examine the putative effect of Ang II in the reexpression of ICaT. After 6 weeks of stenosis, although left ventricular cells were hypertrophied by 52% compared with control cells (234±6 pF, n=10 versus 154±29 pF, n=10; P<0.01), no ICaT was detected at this time. In long-term stenosis AS group (12 weeks), the cell membrane capacitance increased by 1.5-fold as in 6-week-stenosed group (Table 1). Two weeks of losartan treatment decreased heart weight/body weight ratio without affecting cell size (AS/L versus AS). As it has been shown that losartan treatment prevents increase in the interstitial collagen fraction in rat heart after aortic stenosis,24 the decrease in hypertrophy in AS/L rats was probably due to a decrease in interstitial fibrosis.
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After 12 weeks of stenosis, ICaT was recorded in AS cells (Figures 1A and 1B). After losartan treatment, ICaT is still expressed (Figure 1C) but with a significantly reduced density (0.20±0.03 pA/pF, n=27 in AS/L cells and 0.40±0.05 pA/pF, n=26 in AS cells; P<0.01) (Figure 2A). We have checked that in sham-operated losartan group, neither the procedure nor the 2 additional weeks have any effect on ICaT (0.41±0.04 pA/pF, n=9). We have also tested that ICaT density is insensitive to acute application of losartan (2 µmol/L) in freshly isolated hypertrophied myocytes (0.44±0.06 pA/pF, n=6). Then the reduction of ICaT density in AS/L results from AT1 blockade by losartan. As neither activation nor steady-state inactivation parameters of ICaT were modified after losartan treatment (Figure 2B and Table 2), it can be postulated that the blockade of the AT1 pathway induced the decrease in functional channel density, leading the decrease in ICaT density.
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The impact of cardiac hypertrophy on L-type Ca2+ current (ICaL) density remains unclear as previous studies have reported either a decrease or an increase of ICaL density during pathology (see review27). We found that neither stenosis-induced pressure overload nor losartan treatment altered ICaL density in hypertrophied myocytes (9.9±0.5 pA/pF, n=22, 10.9±1.0 pA/pF, n=26 and 10.0±0.7 pA/pF, n=27 for CTL, AS, and AS/L cells, respectively). The activation and steady-state inactivation voltage relationships of ICaL were also unaffected (Table 2).
Characteristics and Regulation of the Functional ICaT Pore-Forming 1 Subunits
We tested the Ni2+ sensitivity of ICaT in order to determine the 1 subunit–related currents induced during cardiac hypertrophy (Figure 3A). The Ni2+ dose-response relation indicated that ICaT was blocked in a biphasic manner with IC50 of 1.6 and 240 µmol/L. According to previous reports, 1.6 and 240 µmol/L Ni2+ sensitivity corresponds to sensitivity described for CaV3.2- and CaV3.1-related current, respectively.28,29 Also, the relative contribution of CaV-related current in ICaT was assessed from Ni2+ dose-response relation. We found that CaV3.1 and CaV3.2 contribute to 80% and 20%, respectively.
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Interestingly, because there was no difference between Ni2+ dose-response curves in AS and AS/L, it appeared that the relative contributions of CaV3.1 and CaV3.2 subunits to ICaT were not modified by losartan treatment (Figure 3A). These data indicated that the decrease in ICaT density after AT1 blockade was due to a proportional decrease in the density of functional CaV3.1 and CaV3.2 subunits. The Ang II signaling pathway then mediates upregulation of the expression of CaV3.1 and CaV3.2 subunits during stenosis-induced cardiac hypertrophy.
We investigated the regulation of CaV3.1 and CaV3.2 expression by quantifying transcript amount (Figure 3B). Neither aortic stenosis nor losartan treatment affected the amount of CaV3.2 mRNA (0.53±0.07, 0.52±0.06, and 0.51±0.10x106 molecules per µg of total RNA in CTL, AS and AS/L groups, respectively). As losartan treatment decreased the density of functional CaV3.2 subunits, Ang II probably regulated CaV3.2 channel expression at the posttranscriptional level. Conversely, the amount of CaV3.1 transcripts increased by almost 3-fold in hypertrophied group (0.70±0.04 versus 0.25±0.09x106 molecules in AS versus CTL cells; P<0.001), and this increase was abolished by losartan treatment (0.29±0.03x106 molecules). Therefore, during stenosis-induced hypertrophy, regulation of the amount of CaV3.1 mRNA is entirely dependent on Ang II signaling pathway.
Ang II Stimulates ICaT Channel Expression in Cultured Newborn Cells via the MEK1/2 Pathway
We investigated the Ang II intracellular signaling events involved in the expression of the T-type channels in ex vivo model. Previous experiments have indicated that chronic Ang II stimulation failed to reexpress ICaT in cultured adult ventricular cells (data not shown). Therefore, we have investigated the effect of Ang II in newborn ventricular cells that are known to exhibit basal ICaT.
Cultured newborn cells exhibited a typical ICaT, the density of which was increased under Ang II stimulation (8.1±0.8 pA/pF, n=10, for Ang II and 3.6±0.3 pA/pF, n=8, for CTL; P<0.001) (Figures 4A and 4B). Treatment of myocytes with losartan had no effect on basal ICaT density (3.5±0.8 pA/pF, n=8) and completely blocked the Ang II–induced increase of ICaT density (3.8±0.4 pA/pF, n=10; P<0.001) (Figure 4B). Because an AT2 receptor antagonist, PD123319, had no effect on Ang II–induced increase of ICaT density (6.8±0.9 pA/pF, n=5), then Ang II upregulates ICaT density via AT1 receptor.
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The increase in ICaT density was not associated with changes in the activation and steady-state inactivation parameters (Figure 4C). Thus, the Ang II–induced increase in ICaT density resulted from an increase in functional channel density in vitro. Interestingly, as in untreated cells, Ni2+ blocked ICaT in a monophasic manner with an IC50 of 260 µmol/L in Ang II–treated cells (Figure 4D). Thus, Ang II–stimulated cells exhibited CaV3.1-related current but are not potent to reexpressed functional CaV3.2 subunits.
We investigated whether ERK1/2 activation plays a role in the Ang II–induced increase in ICaT density using MEK1/2 inhibitors (UO126 and PD98059). Peak ICaT density with UO126 (5.0±0.2 pA/pF, n=6) did not differ significantly from that with PD98059 (5.2±0.5 pA/pF, n=6), and both reduced the Ang II–induced ICaT density by 65% (Figures 5A and 5B). The reduction of ICaT density was due to the inhibition of Ang II effect because ICaT was not significantly different in cultures treated with UO126 alone or untreated (3.0±0.7 pA/pF, n=6) (Figure 5B). Because no change in the activation and steady-state inactivation characteristics of ICaT was observed after MEK1/2 inhibition, we can propose that the regulation of functional CaV3.1 channel density is mediated by Ang II–activated MEK1/2-dependent pathway. Use of a higher concentration of MEK1/2 inhibitor (75 µmol/L) did not further inhibit the Ang II–induced increase in ICaT density (5.5±0.5 pA/pF, n=5). Then the remaining ICaT density is related to an Ang II–activated MEK1/2-independent pathway.
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We found that the Ang II–activated signaling pathway increased the number of CaV3.1 transcripts (1.93±0.02 and 3.16±0.16x106 molecules per µg of total RNA in untreated and Ang II–treated cells, respectively; P<0.001) (Figure 5C). The increase in CaV3.1 mRNA amount was partially prevented by applying PD98059 or UO126 (2.53±0.08 and 2.36±0.07x106 molecules, respectively, n=3; P<0.05). Although our data clearly reveal the implication of an activated MEK1/2-dependent pathway in the regulation of CaV3.1 transcript amount, an additional Ang II–activated pathway also seems to be involved in this process.
Autocrine Effect of ET on ICaT Channel Expression After Ang II Stimulation in Cultured Newborn Cells
It has been reported that in cultured cardiomyocytes, Ang II increases the synthesis of ET-1, which may act as an autocrine/paracrine factor.30 We tested the putative autocrine effect of ET on the expression of T-type channels using multiple ET receptor antagonists: a dual ETA and ETB (bosentan), specific ETA (BQ123), and specific ETB (BQ788) (Figure 6). We found that the Ang II–induced ICaT density was significantly reduced by bosentan (5.4±0.3 pA/pF, n=11; P<0.05) and BQ123 (4.8±0.4 pA/pF, n=6; P<0.05), whereas BQ788 had no effect (8.2±0.8 pA/pF, n=8). To rule out the possibility that bosentan decreased basal ICaT density instead of the Ang II–increased ICaT density, we verified that ICaT density remained stable in cells treated by bosentan alone (3.9±0.3 pA/pF, n=5). These results showed that ETA stimulation mediated an autocrine ET response that participated to upregulation of ICaT. In accordance to that, 48 hour application of ET (0.1µmol/L) increased ICaT density (5.4±0.8 pA/pF, n=7 versus 3.6±0.3 pA/pF, n=8 for CTL; P<0.05) and this increase was abolished by BQ123 (3.2±0.3 pA/pF, n=6).
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The increase in ICaT density observed in cultured ventricular cells stimulated with Ang II reflects an additive effect of the Ang II- and ET-activated signaling pathways. Because cotreatment with bosentan and UO126 abolished the Ang II–induced increase in ICaT density (3.0±0.6 pA/pF, n=6) (Figure 6), we can postulate that a MEK1/2 independent ET-signaling pathway is involved in the regulation of CaV3.1. The quantification of the amount of CaV3.1 mRNA indicated that bosentan treatment did not alter the Ang II–induced increase of CaV3.1 mRNA (3.13±0.06 versus 3.16±0.16x106 molecules per µg of total RNA, respectively). As expected, in culture newborn myocytes, chronic ET-1 application had no effect on basal CaV3.1 mRNA amount (1.9x106 molecules). Therefore, we can conclude that MEK1/2 independent ET-signaling pathway is involved in the translational/posttranslational regulation of CaV3.1 subunit.
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Discussion |
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This study provides data on the regulation of molecular and functional expression of T-type channels by Ang II in hypertrophied myocytes. We demonstrate that (1) Ang II via AT1 increased CaV3.1- and CaV3.2-related current density in stenosis hypertrophied myocytes, (2) CaV3.1 and CaV3.2 were differently regulated because Ang II via AT1 increased the amount of CaV3.1 mRNA but not those of CaV3.2, (3) in cultured newborn ventricular myocytes Ang II via AT1 increased ICaT density and the amount of CaV3.1 mRNA through MEK1/2 pathway, and (4) increased ICaT density also resulted of the autocrine action of ET released by Ang II.
Long-term aortic stenosis displayed expression of an ICaT with kinetic properties and CaV3.1 and CaV3.2 pore-forming units related to those described for fetal ventricular ICaT.9 However, compared with fetal ICaT, kinetic characteristics of ICaT in hypertrophied myocytes revealed a hyperpolarized shift in the voltage-dependent activation (-39.5±0.8 mV, n=7, versus -22.4±0.6 mV, n=9, for AS and 18-day-old fetal ventricular cells, respectively),9 and this hyperpolarized shift was insensitive to Ang II. Because Ca2+ calmodulin–dependent protein kinase II (CaMKII) shifts the activation of ICaT to more negative potentials in adrenal glomerulosa cells,31 and that CaMKII is upregulated in hypertrophied ventricles from rats,32 then CaMKII might be responsible for the hyperpolarized shift in ICaT activation in AS cells.
Interestingly, after 6 weeks of stenosis, although left ventricular cells were hypertrophied, no ICaT was yet reexpressed. We can note that between 6 and 12 weeks, cell membrane capacitance increased in the same way in AS than in control indicating that hypertrophy was compensated. Therefore, whereas a hypertrophic background is necessary to reexpress ICaT, long-term compensate phase of hypertrophy is required for efficient expression of T-type channels, which is likely due to the activation of a time-dependent regulator factor. Supporting that, we have observed that Ang II stimulation was inefficient to reexpress ICaT in cultured adult ventricular myocytes. Therefore, further ex vivo investigation of the regulation of ICaT had required myocytes potent to express ICaT such as newborn myocytes.
Ang II increased the expression of T-type channel subunits via AT1-stimulated signaling pathway. Ni2+ sensitivity revealed that functional CaV3.1 and CaV3.2 channels participate to Ang II–induced increase in ICaT density in stenosed rats. The amount of CaV3.2 mRNA remaining stable during stenosis-induced hypertrophy indicates that no hypertrophy-associated factor appears efficient to increase neither CaV3.2 promoter activity nor mRNA stability. It appears that the stenosis-induced increase in functional CaV3.2 proceeds through translational and/or posttranslational regulation. In newborn myocytes, CaV3.2 is also mainly regulated by translational and/or posttranslational mechanism because there is no more functional subunit while mRNA regulation persists.9 The blockade of functional CaV3.2 subunit is maintained in cultured newborn myocyte even after Ang II treatment. Therefore, we can propose that Ang II in addition to an unidentified stenosis-induced factor is crucial to ensure the increase of functional CaV3.2 subunit.
In contrast to CaV3.2 regulation, the amount of CaV3.1 mRNA increased during stenosis-induced hypertrophy and this increase entirely depends on the activation of AT1 signaling pathway. As a residual CaV3.1 related current persisted after AT1 blockade, we can assure that in addition to the transcriptional regulation, CaV3.1 expression is regulated through translational and/or posttranslational mechanisms.
The stenosis-associated changes in ICaT density and CaV3.1 mRNA amount can be reproduced by application of Ang II to newborn rat myocytes and thereafter blocked by losartan. Thus, Ang II–treated cultures of myocytes appear to be one relevant model for investigating the AT1 signaling pathways involved in the regulation of CaV3.1 expression. We show in this study that Ang II treatment leads to an increase in the amount of CaV3.1 mRNA, partly through ERK1/2-dependent activation. These data are consistent with a large body of study implicating the MAPK cascade in hypertrophic responses.19 It should be noted that ERK1/2 is activated by stenosis-induced cardiac pressure overload33 and that the MEK/ERK pathways have been shown to be involved in the reexpression of fetal genes in hypertrophic background.34 It seems reasonable to postulate that the stenosis-induced regulation of the amount of CaV3.1 mRNA is mediated by Ang II activated MEK/ERK pathway. Further studies are required to evaluate the role of Ang II–activated ERK1/2 in the regulation of T-type channel expression in pathological situations.
It was previously shown that some of the cardiovascular hypertrophic effects of Ang II result from the autocrine/paracrine release of ET.30,35 Consistent with this, we demonstrated that the effect of Ang II stimulation on ICaT density is decreased by ETA receptor blockade in cultured myocytes. Both ET-1 and Ang II activate similar G-protein–coupled receptor signaling pathways, including those activating ERK1/2.36 Another important finding is that the blockade of ET signaling pathways did not affect the transcriptional regulation of CaV3.1, neither in Ang II–induced ET autocrine effect nor in chronic application of ET. Therefore, ET signaling pathway regulates functional CaV3.1 protein density through a translational and/or posttranslational mechanism. Finally, coblockade of ERK1/2 and ETA decrease more strongly ICaT density than ERK1/2 blockade alone suggesting that ET regulates CaV3.1 through an independent MEK/ERK pathway. This indicates that Ang II and ET use different signaling mechanisms for ICaT expression in cultured myocytes.
It is not surprising that several hypertrophic factors regulated T-type channels because the blockade of Ang II effect did no wholly abolished ICaT in stenosed rats. As no more transcriptional upregulation of channel subunits occurs after losartan treatment, the residual ICaT likely results from translational and/or posttranslational mechanism. Cultured newborn cell data lead us to propose that this latter mechanism can be ensured by ET signaling pathways activation.
This study provides new evidence that Ang II is closely related to electrophysiological remodeling process that occurs during cardiac hypertrophy. In addition to ICaT, another inward current, If, has been shown to be upregulated by Ang II during cardiac hypertrophy.37,38 As ICaT and If are both considered to ensure pacemaker activity,39,40 alterations in ICaT and If density likely contribute to the increased propensity of the hypertrophied heart to develop arrhythmia.27 In addition to its involvement in abnormal electrical properties, ICaT has been shown to contribute to Ca2+-dependent hormone secretion.41,42 It would therefore seems to be important to consider the biological role of ICaT with respect to pathogenesis of Ca2+ overload effects4 such as the activation of Ca2+-dependent transduction pathways required after long-term compensate cardiac hypertrophy.
In conclusion, we found that Ang II through AT1-activated MEK-dependent pathway is responsible for the regulation of CaV3.1 transcription (promoter activity and/or mRNA stability), whereas ET through ETA-activated MEK-independent pathway is implicated in CaV3.1 posttranscriptional regulation (translational and/or posttranslational mechanism). Unfortunately, CaV3.2 is not expressed in cultured myocytes that avoid detailed description of mechanisms involved in its regulation. However, we show that CaV3.2 is only regulated at posttranscriptional level and this regulation is also mediated by Ang II. Altogether these regulations of the channel subunits lead to ICaT expression that contributes to cardiac electrophysiological remodeling. Future studies will be needed to precisely explain the relationship between ICaT expression and pathophysiological events.
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Acknowledgments |
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This work was supported by Centre National de la Recherche Scientifique and the Association Marie Lannelongue. We would like to thank Prof D. Feuvray for critical reading and helpful discussion. We would like to thank Dr M. Clozel of Actelion Pharmaceuticals Ltd, for providing us with bosentan and Merck Sharp & Dohme, for providing us with losartan.
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Footnotes |
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Original received April 21, 2003; resubmission received September 25, 2003; revised resubmission received October 30, 2003; accepted October 30, 2003.
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References |
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