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Cardiovascular Research 2007 74(1):85-95; doi:10.1016/j.cardiores.2007.01.001
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Copyright © 2007, European Society of Cardiology

Diminished Kv4.2/3 but not KChIP2 levels reduce the cardiac transient outward K+ current in spontaneously hypertensive rats

Diane Goltza,1, Jobst-Hendrik Schultza,1, Carolin Stuckea, Michael Wagnera,b, Peter Bassalaya, Alexander Peter Schwoerera, Heimo Ehmkea,* and Tilmann Volka,b,*

aInstitut für Vegetative Physiologie und Pathophysiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistraβe 52, 20246 Hamburg, Germany
bInstitut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstraβe 6, 91054 Erlangen, Germany

* Corresponding authors. T. Volk is to be contacted at Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstraβe 6, 91054 Erlangen, Germany. Tel.: +49 9131 85 24033; fax: +49 9131 85 22770. H. Ehmke, Tel.: +49 40 42803 3183; fax:+49 40 42803 4920. Email address: ehmke{at}uke.uni-hamburg.de tilmann.volk{at}physiologie2.med.uni-erlangen.de

Received 19 May 2006; revised 22 December 2006; accepted 2 January 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: A reduction of the Ca2+-independent transient outward potassium current (Ito) in epicardial but not in endocardial myocytes of the left ventricle has been observed in cardiac hypertrophy and is thought to contribute to the electrical vulnerability associated with this pathology.

Methods: In the present study we investigated the molecular mechanisms underlying regional alterations in Ito in hypertrophied hearts of spontaneously hypertensive rats (SHR) using the whole-cell patch-clamp technique, quantitative RT-PCR and heterologous expression of underlying ion channel subunits.

Results: Ito was significantly smaller in epicardial myocytes of SHR than in Wistar-Kyoto (WKY) controls (11.1±0.9 pA/pF, n=20 vs. 16.8±1.7 pA/pF, n=20, p<0.01), but not different in endocardial myocytes from both groups. Quantitative RT-PCR analysis of the genes encoding Ito revealed significantly lower levels of Kv4.2 and Kv4.3 mRNA in the epicardial region of SHR rats compared to WKY rats. In contrast, mRNA expression levels of all three splice variants of the β-subunit KChIP2 were significantly higher in both endo- and epicardial myocytes from SHR than from WKY rats. In parallel, inactivation of Ito, which is negatively modulated by KChIP2, was slowed down in SHR while recovery from inactivation remained unchanged. Heterologous co-expression of increasing amounts of KChIP2b together with a fixed amount of Kv4.2 in Xenopus laevis oocytes revealed a hyperbolic relation of recovery from inactivation and inactivation time constant, demonstrating that KChIP2 preferentially affects inactivation, if its expression level is high.

Conclusion: These results suggest that downregulation of Ito in the left ventricle of SHR is mediated by a reduced expression of Kv4.2 and Kv4.3 (but not of KChIP2), whereas the slower inactivation of Ito can be explained by increased expression levels of KChIP2 in SHR.

KEYWORDS Hypertrophy; Hypertension; K+ channel


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Hypertrophy of the heart is associated with several alterations in cardiac cellular electrophysiology, among which an increase in action potential duration (APD) is probably the most frequently observed phenomenon. A large fraction of the increase in APD has been attributed to a decrease in the Ca2+-independent transient outward potassium current (Ito) [1]. Ito, however, is not uniformly distributed among the left ventricle. For example, in epicardial and basal regions of the left ventricle, the magnitude of Ito is much larger than in endocardial and apical regions [2–4]. Hence, at baseline APD is shortest in epicardial and basal and longest in endocardial and apical regions. During cardiac hypertrophy, the reduction of Ito can decrease or even reverse the regional gradient in APD, which ultimately leads to an inversion of the T-wave in the surface ECG and may contribute to the increased susceptibility of hypertrophied hearts to ventricular arrhythmias [5–7].

The molecular mechanisms underlying the downregulation of Ito during cardiac hypertrophy are incompletely understood. In cardiac hypertrophy, several studies have found a decrease in expression levels of the potassium channel {alpha}-subunits Kv4.2 and Kv4.3 [8–12] which are thought to encode the major fraction of Ito in the myocardium of rodents (predominantly Kv4.2), dogs and humans (predominantly Kv4.3) [13,14]. One possible mechanism for the decrease of Ito in cardiac hypertrophy could therefore be a downregulation of Kv4 gene expression. In addition, however, β-subunits encoded by the KChIP gene family were identified which associate with the Kv4 {alpha} subunits and modify current sizes and kinetic properties to become more similar to the native Ito [15]. Accordingly, there is good reason to believe that KChIP2, the major cardiac KChIP isoform, is a key player in modulating Ito magnitude and indeed, recent data suggest that a downregulation of KChIP2 may contribute to the decrease of Ito in cardiac hypertrophy in mice [16] and rats [17].

In the present study we therefore investigated Ito current properties together with the mRNA expression levels of Kv4.2, Kv4.3 and KChIP2 in myocytes from the endo- and epicardial region isolated from spontaneously hypertensive rats (SHR), a genetic model of primary hypertension with secondary cardiac hypertrophy, and Wistar Kyoto (WKY) control animals. Furthermore, we co-expressed KChIP2 and Kv4.2 at different ratios in Xenopus laevis oocytes to investigate a potential influence of different KChIP2/Kv4.2 ratios on the kinetics of Kv4.2 mediated currents.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals, hemodynamic measurements and isolation of myocytes
SHR and WKY animals aged 10–14 months of either sex (weight male: ~380 g, female: ~240 g) were used in the present study. The animals were a generous gift from Lilly Deutschland GmbH, Bad Homburg. The investigation conforms with 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).

Rats were anesthetized by i.p. injection of Trapanal® (thiopenthal-Na+), 100 mg/kg body mass. A femoral artery catheter was inserted to measure peripheral blood pressure (BP). Then, the heart was quickly excised and myocytes were isolated from the endo- and epicardial region of the left ventricular free wall as described previously [4]. To obtain tissue for RT-PCR amplifications, endo- and epicardial tissue of the left ventricular free wall was separated and quick frozen in liquid nitrogen. This tissue was obtained from separate groups of animals not subjected to perfusion and myocyte isolation.

2.2 Quantitative PCR analysis
Total RNA was isolated using TRIzol Reagent (Invitrogen, Karlsbad, CA, USA) followed by chloroform extraction and isopropanol precipitation. RNA was quantified by spectrophotometric absorbency at 260 nm, purity confirmed by A260/A280 ratio. RNA samples were stored at –80 °C.

Single-stranded cDNA synthesis was performed using 200 ng of total RNA (for total volume of 100 µl) and TaqMan reverse transcription assay (Applied Biosystems, Branchburg, NJ, USA) with oligo (dT) as the primer according to the manufacturer's instructions. The cDNA was diluted 1:2.5 for PCR in the presence of double-stranded DNA binding dye (SYBR green). Quantitative real time PCR was carried out on a LightCycler 1.2 (Roche Diagnostics, Mannheim, Germany) using LightCycler FastStart DNA Master Plus SYBR Green I Kit. Primers were selected to amplify 70–285 bp fragments across exon–exon boundaries. The following pairs were used: Kv 4.2 forward: 5'-CCGAATCCCAAATGCCAATGTG-3', Kv 4.2 reverse: 5'-CCTGACGATGTTTCCTCCCGAATA-3' [18], Kv 4.3 forward 5'-AACATCTGCTCATCAATAAACTCGTG-3', Kv 4.3 reverse 5'-GCCTTGCCAGAATCCGTGTG-3', KChIP2 forward 5'-ACAGACCAAGTTCACACGCA-3', KChIP2 reverse 5'-TCGTTCTTGAAGCCTCGGT-3' (within Exon 4, amplification of genomic DNA was controlled by RT-probe). β-actin forward 5'-CGGGATCCCCGCCCTAGGCACCAGGGTG-3' and β-actin reverse 5'-GGAATTCGGCTGGGGTGTTGAAGGTCTCAAA-3'.

Since SYBR Green binds to any double stranded DNA, PCR-products were also run on 1% agarose gels with ethidium bromide staining to ensure that only one product was amplified. Dissociation curve plots (melting temperature analysis) confirmed specificity revealing only one peak for each product. To determine PCR efficiency, a standard curve with a decimal dilution series (3 times) of cDNA obtained from 1 µg RNA in reverse transcriptase step was run in duplicate for each experiment. Quantitative data of target mRNA levels were normalized to β-actin mRNA. β-actin mRNA levels were neither significantly different between endo- and epicardial regions nor between WKY animals and SHR. Results are expressed as the target/reference ratio of each sample with efficiency correction for individual PCR conditions.

2.3 Detection of KChIP2 splice variants
The primers and fluorescent probes listed below were chosen for detection of KChIP2 splice variants (KChIP2a–c). All sequences were checked with the program Oligo 5.0 for the absence of false priming sites, formation of primer dimers and primer/probe hybrids. KChIP2 splice variant 2b lacks exon 3, KChIP2c lacks both exon 2 and exon 3, respectively. Probes were designed to bind to exon–exon spanning sequences that are specific for a single splice variant. For KChIP2a and 2b, amplification primers bind exon 2 and 4, resulting in two amplicons differing in 64 bp of length, only one of which can bind the fluorescent probe. For KChIP2c amplification primers bind exon 1 and 4, resulting in three amplicons, respectively. Quantitative real time PCR was carried out on a LightCycler 1.2 (Roche Diagnostics, Mannheim) using LightCycler TaqMan Master Kit. Efficiency of fluorescent target amplification was proved constant in individual samples (n=12) using the standard curve and linear regression methods. PCR-products were run on 1% agarose gel with ethidium bromide staining to verify correct amplification. Results are expressed as the target/β-actin ratio of each sample with efficiency correction for individual PCR conditions. KChIP2a: Primer up, 5'-CTCAAGCTGCTGCCGTGC (in Exon 2); Primer lo, 5'-CTGCGTGTGAACTTGGTCTGT (in Exon 4); Flou probe, 5'-TGGACCCAGACAGCGTAGAGGATGAGT (binds Exon 3 and 4). KChIP2b: Primer up, 5'-CTCAAGCTGCTGCCGTGC (in Exon 2); Primer lo, 5'-ACACCGTGGATAATTCAAACTCA (in Exon 4); Flou probe, 5'-TGCCCTCAGTCAGTGAAAACAGCGTAGA (binds Exon 2 and 4). KChIP2c: Primer up, 5'-AGAGTTTGTCCGAATCCCGAGA (in Exon 1); Primer lo, 5'-ACACCGTGGATAATTCAAACTCA (in Exon 4); Fluo probe, 5'-CTCTACGCTGTCCGGTAAGCTGGTCATA (binds Exon 1 and 4). β-actin: Primer up, 5'-ACCTTCAACACCCCAGCCA; Primer lo, 5'-CAGTGGTACACCAGAGGCA; Fluo probe, 5'-ACGTAGCCATCCAGGCTGTGTTGTCC. Fluorogenic probes were labelled with the 5'-reporter 6-FAM and the 3'-quencher TAMRA.

2.4 Solutions and chemicals
Cardioplegic solution contained (in mM) NaCl 15, KCl 9, MgCl2 4, NaH2PO4 0.33, CaCl2 0.015, glucose 10, mannitol 238, titrated to pH=7.40 with NaOH. Giga-ohm seals were obtained in modified Tyrode's solution (control solution): NaCl 138, KCl 4, MgCl2 1, NaH2PO4 0.33, CaCl2 2, glucose 10, HEPES 10, titrated to pH=7.30 with NaOH. To inhibit Ca2+ currents, 0.3 mM CdCl2 was added to the solutions. The pipette solution contained glutamic acid 120, KCl 10, MgCl2 2, EGTA 10, Na2-ATP 2, HEPES 10, titrated to pH=7.20 with KOH. Oocytes were maintained in sterile modified Barth' saline (MBS) containing NaCl 88, KCl 1, NaHCO3 2.4, MgSO4 0.82, Ca(NO3)2 0.3, CaCl2 0.41, TRIS 15, titrated to pH=7.60 with HCl and supplemented with Penicillin/Streptomycin (10 mg/l each). For two-electrode voltage-clamp experiments, oocytes were bathed in NaCl-95 solution: NaCl 95, KCl 2, MgCl2 1, CaCl2 1, HEPES 10, titrated to pH=7,40 with TRIS.

2.5 Patch-clamp technique
The ruptured patch whole-cell configuration was used as described previously [6,19]. Membrane capacitance (Cm) and series resistance (Rs) were calculated using the automated capacitance compensation procedure of the EPC-9 (HEKA Elektronik, Lambrecht, Germany) amplifier. When filled with K-glutamate, pipette resistance averaged 3.2±0.1 M{Omega} (n=64). Rs averaged 5.7±0.2 M{Omega} (n=64) and was compensated by 85% leading to an average effective Rs of 0.9 M{Omega}. Pipette potentials (VPip) were corrected for liquid junction potentials (13 mV for pipette solution vs. control solution). Whole-cell currents were low-pass filtered at 1 kHz, sampled at 5 kHz and analyzed using the PulseFit software (HEKA Elektronik) and IGOR (WaveMetrics, Lake Oswego, OR, USA). All experiments were performed at room temperature (21-23 °C). Data are given as means±SEM. Statistical significance was calculated using an initial one-way ANOVA followed by a Bonferroni posthoc test using the software PRISM (Graph-Pad Inc., San Diego, CA, USA). Differences with p<0.05 were considered statistically significant.

2.6 Isolation, injection and maintenance of Xenopus oocytes
Female X. laevis were anesthetised by immersion in 0.2% MS-222 (titrated to pH=7.0 with NaHCO3) for 10 min. Ovarian lobes were surgically removed and oocytes were isolated by enzymatic digestion using collagenase A 0.8 U/ml MBS (SERVA, Heidelberg, Germany) for ~20 h. After digestion, oocytes were rinsed and incubated for 5 min in Ca2+-free NaCl-95 solution to remove debris and to stop collagenase-activity and subsequently transferred to MBS. The full-length cDNA encoding rat Kv4.2 was in Bluescript SK–, rat KChIP2b in pGEMT. Linearized plasmids were used as templates for cRNA synthesis using T7 (Kv4.2) or SP6 (KChIP2b) RNA polymerases (Promega GmbH, Mannheim, Germany). Defolliculated stage V-VI oocytes were injected with a 1:1 mixture of Kv4.2 (2.5 ngnl–1) and KChIP2b cRNA (0, 0.25, 0.5, 1.0, 2.5, 5, 10 or 20 ngnl–1) or with a 1:1 mixture of Kv4.2 (0.2, 2 or 20 ngnl–1) and KChIP2b cRNA (1.0 ngnl–1). cRNAs were dissolved in RNAse-free water and the total volume injected was ~50 nl per oocyte. After injection, oocytes were maintained in MBS and were studied after 24 to 48 h.

2.7 Two-electrode voltage-clamp experiments
Oocytes were transferred to a perfusion chamber (2.5 mmx20 mm), superfused with NaCl-95 solution and impaled with electrodes (0.3–1.5 M{Omega}) filled with KCl 3 M. Whole-cell currents were measured at room temperature (21–23 °C) with the two-electrode voltage-clamp technique using a Turbo Tec 05 amplifier (npi electronic, Germany) controlled by the Pulse-software (HEKA Elektronik) via an ITC-16 interface (Instrutech Corp., Long Island, N.Y., USA). An Ag–AgCl pellet placed directly in the bath solution served as reference electrode for the current injection circuit while an additional Ag–AgCl pellet located close to the oocyte was used to sense the bath potential in order to avoid series resistance problems. Pulsed current data were filtered at 1 kHz and were directly written to hard disc at a sample rate of 5 kHz. Whole-cell data were analyzed using the PulseFit software (HEKA Elektronik) and IGOR (WaveMetrics).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Hemodynamics of SHR and WKY controls
WKY controls (n=5) displayed a normal peripheral systolic and diastolic blood pressure of 122±5 and 80±6 mmHg, respectively. In SHR (n=10), both systolic and diastolic blood pressure were significantly increased and averaged 211±7 (p<0.0001) and 158±6 mmHg (p<0.0001), respectively. Heart rates were not significantly different and averaged 242±30 min–1 in WKY and 283±13 min–1 in SHR. The relative left ventricular weight was significantly larger in SHR (3.69±0.08 mg/g, n=10 vs. 1.97±0.05 mg/g, n=5, p<0.0001), indicating pronounced cardiac hypertrophy. Hypertrophy was also detected at the cellular level, as the cell capacitance, a marker of cell size, was significantly larger in myocytes isolated from SHR (284±23 pF, n=34 vs. 224±13 pF, n=30, p<0.05). We did neither observe pleural nor peritoneal effusions indicating that the hearts from SHR were at least not grossly insufficient.

3.2 Transient outward K+ current
Fig. 1 A+B show representative current traces recorded from myocytes isolated from endo-, and epicardial regions of WKY (A) and SHR (B). At depolarizing voltage steps exceeding VPip=–20 mV, a rapidly activating and inactivating Ito was observed in all cardiac myocytes. Ito was much larger in epicardial than in endocardial myocytes in both WKY and SHR. Epicardial myocytes isolated from SHR, however, displayed a significantly smaller Ito than those isolated from the corresponding region in WKY rats. In endocardial myocytes Ito magnitudes were similar in both groups.


Figure 1
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  		Fig. 1 Selective reduction of Ito in epicardial myocytes of SHR. Representative outward currents recorded from endo- and epicardial myocytes of WKY (A) and SHR (B). Currents were activated at a rate of 0.3 Hz by rectangular voltage pulses from a holding potential of VPip=–90 mV to values ranging from +60 mV to –60 mV in steps of –20 mV. Each voltage pulse was preceded by a depolarization to VPip=–50 mV for 20 ms to inactivate Na+ currents. The bath solution contained 0.3 mM Cd2+ to inhibit Ca2+ currents. Currents were normalized to Cm to correct for different cell sizes and are thus given in pApF–1. C, average current–voltage relations of Ito recorded from endocardial (WKY, n=9; SHR, n=10) and epicardial (WKY, n=20; SHR, n=20) myocytes of WKY (filled symbols) and SHR (open symbols). Ito was quantified by subtracting the current at the end of the voltage pulse (600 ms) from the peak current. **p<0.01; WKY vs. SHR.

 
Fig. 1C shows average current–voltage (I–V) relations from all myocytes investigated. In all myocytes Ito activated at pulse potentials positive to VPip=–20 mV and the I–V relation was linear from this potential up to VPip=60 mV. Ito was 34% smaller in epicardial myocytes from SHR (11.1±0.9 pA/pF, n=20 vs. 16.8±1.7 pA/pF, n=20; p<0.01, VPip=40 mV). In endocardial myocytes, Ito magnitudes were similar in both groups (2.5±0.7 pA/pF, n=9 vs. 2.8±0.6 pA/pF, n=10; n.s.).

In contrast to Ito, the steady-state current at the end of the voltage pulse was similar in endo- (6.7±0.7 pA/pF, n=8 vs. 5.2±0.8 pA/pF, n=10; n.s., VPip=40 mV) and epicardial myocytes (6.6±0.4 pA/pF, n=20 vs. 5.4±0.5 pA/pF, n=20; n.s., VPip=40 mV) of SHR and WKY animals, respectively. This indicates that Ito is specifically downregulated in epicardial myocytes of SHR.

3.3 Kinetic properties of Ito
Since altered channel kinetics may contribute to a smaller current magnitude of Ito, we analyzed its kinetic properties. Fig. 2 summarizes inactivation (A), steady-state inactivation (B), and recovery from inactivation (C) of Ito recorded from endo- and epicardial myocytes of WKY and SHR. Detailed characteristics of Ito kinetics are summarized in Table 1.


Figure 2
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  		Fig. 2 Kinetic properties of Ito in normal and hypertrophied hearts. Inactivation (A), steady-state inactivation (B), and recovery kinetics (C) of Ito recorded from endo- and epicardial myocytes of WKY (filled symbols) and SHR (open symbols). A, inactivation time constants ({tau}) of Ito were estimated at holding potentials ranging from VPip=0 mV to VPip=+60 mV by biexponential fitting of the current decay, yielding a short time constant of below 100 ms and a long time constant in the range of ~1 s. Only the short time constant is displayed. Biexponential fitting was chosen to exclude a potential contribution of slowly inactivating current components [31] to the inactivation time constant of Ito. B, steady-state inactivation was determined by a two-step pulse protocol: a conditioning pulse of 600 ms duration ranging from –80 mV to +10 mV in steps of 10 mV was followed by a step to +60 mV for 600 ms. The magnitude of Ito detected at +60 mV after the conditioning pulse was normalized to Ito recorded at conditioning potential of –90 mV in each individual experiment and is given as a function of the conditioning pulse potential. Data were fitted assuming a Boltzmann-kinetic of steady-state inactivation. C, recovery from inactivation was determined by two consecutive pulses to +60 mV, each of 600 ms. During the interval between the two depolarizations, VPip was returned to –90 mV. The interval between the voltage pulses ranged from 10 to 7500 ms and increased exponentially with 1.5 being the exponent. Pulses were delivered at 0.1 Hz. The magnitude of Ito recorded during the second voltage pulse was normalized to the magnitude of the first, and plotted versus the duration of the interval between the voltage pulses. Recovery curves were fitted using a bi-exponential function. *p<0.05, **p<0.01, ***p<0.001, SHR vs. WKY.

 

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  		Table 1 Kinetic properties of Ito

 
Fig. 2A shows inactivation time constants plotted versus the voltage pulse potential. In endocardial myocytes, the inactivation time constant tended to be larger in myocytes isolated from SHR, while it was significantly larger in epicardial myocytes from SHR than from WKY at voltages between VPip=20 and VPip=60 mV (60.3±2.8 ms (n=19) and 40.5±2.3 ms at VPip=40 mV; n=19; p<0.01).

Fig. 2B displays average steady-state inactivation curves of Ito. In both, WKY and SHR, the membrane potential at which half-maximal inactivation occurred (V1/2) was in the range of –40 mV, and it was similar in endo- and epicardial myocytes (see Table 1).

Recovery from inactivation (Fig. 2C) followed a bi-exponential characteristic resulting in a short time constant in the range of 30–60 ms and a long time constant in the range of 2500–5000 ms, and did not differ between WKY and SHR. The short time constant was larger in endocardial myocytes and contributed to a significantly less amount of total recovery from inactivation than in epicardial myocytes from both groups (Table 1).

3.4 Expression of Kv4.2, Kv4.3 and KChIP2 mRNA in WKY and SHR
Fig. 3 summarizes the expression levels of Kv4.2 (A), Kv4.3 (B) and KChIP2 (C). KChIP2 primers were designed to amplify the three splice variants known to be expressed in rat myocardium KChIP2 [20]. In WKY animals, expression of Kv4.2, Kv4.3 and KChIP2 mRNA was significantly lower in the endo- than in the epicardial region, suggesting that the difference in gene expression of both Kv {alpha}-subunits as well as the β-subunit KChIP2 contribute to the epicardial-to-endocardial gradient in Ito magnitude. Compared to WKY animals, Kv4.2 and Kv4.3 expression were significantly lower in epicardial myocytes from SHR, whereas in endocardial myocytes a significantly lower expression of Kv4.2 but not of Kv4.3 was observed. In contrast, KChIP2 expression was larger in both endo- and epicardial tissue of SHR, but to a lesser extent in epicardial regions.


Figure 3
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  		Fig. 3 Expression of Kv4.2, Kv4.3 and KChIP2 mRNA in hearts of WKY and SHR. Quantitative RT-PCR analysis of Kv4.2 mRNA (A), Kv4.3 mRNA (B) and KChIP2 mRNA (C) expression in epicardial and endocardial myocytes of WKY (n=8) and SHR (n=8). Values are means±SEM. *p<0.05, **p<0.01, ***p<0.001.

 
To address the question whether individual splice variants of KChIP2 are selectively regulated, experiments using fluorescent TaqMan probes designed to identify the three individual slice variants were performed (see Methods for details). Fig. 4 depicts expression levels of KChIP2a, KChIP2b, and KChIP2c. With some minor differences, the three splice variants display a similar expression pattern with epicardial expression being larger than endocardial in WKY animals. In SHR, expression of the three splice variants was larger in both endocardial and epicardial myocytes. These results demonstrate that KChIP2a, KChIP2b, and KChIP2c are affected in a similar manner by cardiac hypertrophy.


Figure 4
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  		Fig. 4 Expression of KChIP2a, KChIP2b and KChIP2c mRNA in hearts of WKY and SHR. Quantitative RT-PCR analysis of KChIP2a mRNA (A), KChIP2b mRNA (B) and KChIP2c mRNA (C) expression in epicardial and endocardial myocytes of WKY (n=8) and SHR (n=8). Values are means±SEM. *p<0.05, **p<0.01, ***p<0.001.

 
3.5 Effect of KChIP2 on inactivation and recovery of Kv4.2 currents in Xenopus oocytes
In epicardial myocytes from SHR the inactivation time constant of Ito was significantly larger than in epicardial myocytes from WKY rats. This could be explained by the increased ratio of KChIP2/Kv4.2 expression observed in SHR animals, since it has been shown that co-expression of Kv4.2 with KChIP2 increases the inactivation time constant of the corresponding current [20]. However, we did not observe any changes in the recovery from inactivation, which has also been shown to be affected by co-expression of KChIP2 in heterologous expression systems [20]. To investigate, whether alterations in the relation between Kv4.2 and KChIP2 expression levels can alter inactivation time constant and recovery from inactivation independently from each other, we co-expressed rat Kv4.2 and rat KChIP2 in X. laevis oocytes at different ratios. Although the expression of all three splice variants of KChIP2 was altered in SHR, KChIP2c expression revealed the largest difference between SHR and WKY. Consequently, it would have been the best suited target for the heterologous coexpression analysis. However, for technical reasons, we have not succeeded in cloning and expressing of KChIP2c and therefore chose to use KChIP2b which may be equally suitable for the analysis of the interaction of KChIP2 and Kv4.2: KChIP2a, KChIP2b and KChIP2c have been found to identically affect kinetic properties (current inactivation, steady-state inactivation and recovery from inactivation) of Kv4.2 mediated currents [20].

In Fig. 5 inactivation time constant (A) and recovery from inactivation (B) are plotted vs. different ratios of Kv4.2 and KChIP2b cRNA. With decreasing ratios of Kv4.2/KChIP2b cRNA, the time constant of inactivation significantly increased while the recovery from inactivation significantly decreased. To exclude the possibility that the observed kinetic changes were related to the increased amounts of total RNA injected, rather than to the altered ratio of Kv4.2/KChIP2b expression, control experiments were performed in which the concentration of KChIP2b was kept constant while the concentration of Kv4.2 was increased. Fig. 6 displays inactivation (A) and recovery from inactivation (B) under these conditions: in contrast to Fig. 5B, increased inactivation time constants and accelerated recovery from inactivation were observed at low amounts of total RNA injected. This indicates that the amount of total RNA injected is unlikely to contribute to the kinetic alterations observed. Fig. 5C depicts the relation of recovery from inactivation and inactivation time constant for all individual 498 oocytes shown in Fig. 5A and B. The time constant of recovery from inactivation and the inactivation time constant were inversely related in a hyperbolic fashion. Depending on the relation of KChIP2 and Kv4.2 expression levels, changes in inactivation time constant may be associated with large (when KChIP2 expression is low) or small (when KChIP2 expression is high) changes in the time constant of recovery from inactivation. The increase in inactivation time constant from ~40 ms in to ~50 ms in SHR compared to WKY animals in the present study may therefore well be associated with an undetectable small increase in the time constant of recovery from inactivation.


Figure 5
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  		Fig. 5 Effect of KChIP2 on inactivation and recovery of Kv4.2 currents in Xenopus laevis oocytes. A, Inactivation time constant plotted vs. different ratios of concentrations of KChIP2 and Kv4.2. Inactivation time constant was obtained by monoexponentially fitting the current decay after a voltage step to Vm=+60 mV. 54<n<68 for each column. ***p<0.001 vs. 0 ngnl–1 KChIP2b. The upper panel depicts representative current traces activated by a voltage step to Vm=+60 mV obtained from an oocyte expressing Kv4.2 only and an oocyte expressing Kv4.2 and KChIP2b (10 ngnl–1) (+KChIP). Current traces were normalized to their peak values and capacitive artefacts were removed for means of clarity. B, time constant of recovery from inactivation plotted vs. different ratios concentrations of KChIP2 and Kv4.2. Recovery from inactivation was determined by two consecutive pulses to Vm=+60 mV, each of 600 ms. During the interval between the two depolarizations, Vm was returned to –90 mV. The interval between the voltage pulses ranged from 10 to 7500 ms and increased exponentially with 1.5 being the exponent. Pulses were delivered at 0.1 Hz. The magnitude of the current recorded during the second voltage pulse was normalized to the magnitude of the first, and plotted versus the duration of the interval between the voltage pulses. Recovery curves were fitted using a monoexponential function. 54<n<68 for each column. ***p<0.001 vs. 0  ngnl–1 KChIP2b. The upper panel depicts representative current traces activated by the voltage protocol described above obtained from an oocyte expressing Kv4.2 only and an oocyte expressing Kv4.2 and KChIP2b (10 ngnl–1) (+KChIP). Current traces were normalized to their peak values and capacitive artefacts were removed for means of clarity. C, recovery time constant of each individual oocyte plotted vs. its inactivation time constant for all 498 oocytes investigated.

 

Figure 6
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  		Fig. 6 Effect of KChIP2 on inactivation and recovery of Kv4.2 currents in Xenopus laevis oocytes: variation of Kv4.2. A, Inactivation time constant plotted vs. different ratios of concentrations of KChIP2 and Kv4.2. Inactivation time constant was determined as described in the legend of Fig. 5A. 12<n<8 for each column. B, time constant of recovery from inactivation plotted vs. different ratios concentrations of KChIP2 and Kv4.2. Recovery from inactivation was determined as described in the legend of Fig. 5B. 8<n<12 for each column. *p<0.05, ***p<0.001.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The results of the present study show that cardiac hypertrophy in SHR is associated with a smaller Ito in epicardial but not in endocardial myocytes of the left ventricular free wall. The lower amplitude of Ito in myocytes of the epicardial region is associated with a lower mRNA expression level of both K+ channel {alpha}-subunits Kv4.2 and Kv4.3. In contrast, the expression level of the three splice variants of KChIP2 (KChIP2a–c) was larger in myocytes from both, endo- and epicardial regions of SHR animals. These changes at the level of gene expression were associated with a prolongation of the inactivation time constant. Heterologous expression of Kv4.2 and KChIP2b at different rations in X. laevis oocytes revealed that at low Kv4.2/KChIP2b ratios, changes in the ratio preferentially affect the inactivation time constant rather than the recovery from inactivation.

4.1 Regional distribution and kinetics of Ito in WKY and SHR
A smaller magnitude of Ito in SHR with long-standing hypertension compared to WKY control animals has been described earlier by Cerbai and coworkers [21]. The present study shows that the gradient in Ito density among the left ventricular free wall is affected in SHR in a similar way as in other models of cardiac hypertrophy [6,22,23]. This suggests that a regional reduction of Ito is a general response in cardiac hypertrophy and is independent of the underlying pathology. In contrast to other models of cardiac hypertrophy, which show no alterations of the kinetics of Ito [6,22,23], the inactivation time constant of Ito was significantly prolonged in epicardial myocytes from SHR. A similar trend was also observed in endocardial myocytes of SHR animals. This prolongation may be explained by the substantially increased ratio of KChIP2 to Kv4.2 expression in SHR, as our data obtained by heterologous expression of different ratios of Kv4.2/KChIP2 in X. laevis oocytes showed that at high levels of expression KChIP2 preferentially slows inactivation without affecting other kinetic properties of Ito. These results also suggest that changes in the expression level of KChIP2 and/or Kv4.2 may lead to functional channel proteins composed of different stoichiometries than the proposed composition of 4 {alpha}- and 4 β-subunits observed when Kv4.2 and KChIP2 were overexpressed in tissue culture [24].

4.2 Regional distribution of Kv4.2, Kv4.3 and KChIP2 expression in WKY rats
In the present study, expression levels of Kv4.2, Kv4.3 and KChIP2 were determined at the mRNA level, hence raising the question, whether similar expression levels are also present for the corresponding proteins. Although a correlation of mRNA and protein levels has been questioned for KChIP2 in the heart [25], a recent study using more specific antibodies against KChIP2 confirmed that KChIP2 mRNA and protein levels correlate very closely in the dog ventricle [26]. Furthermore, in the rat ventricle as well as in neonatal rat cardiac myocytes in culture, KChIP2 mRNA and protein expression are identically regulated [17]. Similarly, Kv4.2 mRNA and protein expression have been found to correlate very well in the rat ventricle [9]. Therefore, the differences detected in the present study at the mRNA level are likely to be also present at the protein level.

Our results confirm the presence of a steep gradient in Kv4.2 mRNA expression in the rat left ventricle [27,28]. Furthermore, we could show, that Kv4.3 expression is larger in the epicardial than in the endocardial region and is hence likely to contribute to the gradient in Ito. However, in contrast to previous studies [28], we also detected a gradient in the expression of KChIP2 mRNA. A potential reason underlying these discrepant findings may be that the dissection of endo- and epicardial tissue in small rodents, such as the rat, differs from laboratory to laboratory. In the present study, the left ventricular free wall was dissected in an endo-, a mid- and an epimyocardial layer, each of approximately equal thickness. Hence, endo- and epicardial tissue was limited to the very outer or inner ~0.5 mm of the left ventricular free wall and a potential contamination with tissue originating from the midmyocardial region was avoided. The endo-epicardial difference in KChIP2 mRNA expression was substantially lower than that observed in the dog or human left ventricular free wall [28], but we cannot exclude that it might have been larger, if we had restricted the isolation of the tissue even more to the respective site of origin. Accordingly, although the endo- to epicardial differences in KChIP2 expression in the rat left ventricular free wall are less pronounced than in the dog or human ventricle [28], regional differences in KChIP2 expression might contribute to the gradient in Ito current magnitude in the rat left ventricle.

4.3 Differences in Kv4.2, Kv4.3 and KChIP2 expression between WKY and SHR
In epicardial myocytes of SHR expression of Kv4.2 and Kv4.3 was significantly lower than in epicardial myocytes of WKY animals. A decrease in Kv4.2 expression has been suggested to underlie the decrease in Ito in other forms of cardiac hypertrophy such as renovascular hypertension [12] or after myocardial infarction [23]. Kv4.3 expression was also found to be decreased in both forms of cardiac hypertrophy [12,29]. The reduction of both Kv channel {alpha}-subunits is in agreement with the hypothesis that Ito in rodents is composed of heteromultimers of Kv4.2 and Kv4.3 [14]. This would also explain why Ito current magnitudes in endocardial myocytes from WKY and SHR were similar, although Kv4.2 expression was smaller in SHR than in WKY. In endocardial myocytes from SHR, Kv4.2 expression was only moderately decreased while Kv4.3 expression was unaltered. Such a small decrease (compared to the decrease observed in epicardial myocytes) might not be sufficient to manifest itself in detectable changes in current magnitude as Ito was very small (less than 3 pApF–1).

Surprisingly, we found a higher expression of the three splice variants of KChIP2 in both endo- and epicardial myocytes of SHR, thus making it unlikely that alterations in KChIP2 expression contribute to the observed smaller magnitude of Ito. This is in agreement with recent results that were obtained from human and canine failing left ventricle, in which a decrease in Ito was not accompanied by any changes in KChIP2 expression [30]. In the mouse, left ventricular hypertrophy induced by thoracic aortic banding was associated with a decrease in KChIP2 expression [16], but failed to affect Ito [30]. A very recent study found that abdominal aortic constriction in the rat was associated with a decrease in Ito and KChIP2 mRNA and protein [17]. These conflicting results may be caused by the different stimuli used to induce cardiac hypertrophy. Taken together, however, there appears to be increasing evidence that the decrease in Ito observed in cardiac hypertrophy and cardiac failure is a consequence of an altered expression of Kv {alpha}-subunits rather than that of accessory KChIP2 subunits.

In conclusion, the results of the present study suggest that the expression levels of the ion channel {alpha} subunits Kv4.2 and Kv4.3, and not those of the β subunit KChIP2, are the limiting factor of the modulation of the magnitude of Ito in rats. A reduction of Kv4.2 and Kv4.3 gene expression may be the molecular mechanism underlying the reduction of Ito in secondary cardiac hypertrophy.


   Acknowledgement
 
We are most grateful to Telse Kock for expert technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft.


   Notes
 
1 Both authors contributed equally to that work. Back

Time for primary review 42 days


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

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