The effects of intracellular Ca2+ on cardiac K+ channel expression and activity: novel insights from genetically altered mice
2 Division of Cardiovascular Medicine, University of California, Davis, Davis, CA
6 Department of Veterans Affairs, Northern California Health Care System, Mather, CA
5 Department of Physiology and Cell Biology, Ohio State University, Columbus, OH
3 Department of Internal Medicine
4 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, OH, USA
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1 Pharmacology Department, Hebei Medical University Shijiazhuang, China
Abstract
We tested the hypothesis that chronic changes in intracellular Ca2+ (Ca2+i) can result in changes in ion channel expression; this represents a novel mechanism of crosstalk between changes in Ca2+ cycling proteins and the cardiac action potential (AP) profile. We used a transgenic mouse with cardiac-specific overexpression of sarcoplasmic reticulum Ca2+ ATPase (SERCA) isoform 1a (SERCA1a OE) with a significant alteration of SERCA protein levels without cardiac hypertrophy or failure. Here, we report significant changes in the expression of a transient outward K+ current (Ito,f), a slowly inactivating K+ current (IK,slow) and the steady state current (ISS) in the transgenic mice with resultant prolongation in cardiac action potential duration (APD) compared with the wild-type littermates. In addition, there was a significant prolongation of the QT interval on surface electrocardiograms in SERCA1a OE mice. The electrophysiological changes, which correlated with changes in Ca2+i, were further corroborated by measuring the levels of ion channel protein expression. To recapitulate the in vivo experiments, the effects of changes in Ca2+i on ion channel expression were further tested in cultured adult and neonatal mouse cardiac myocytes. We conclude that a primary defect in Ca2+ handling proteins without cardiac hypertrophy or failure may produce profound changes in K+ channel expression and activity as well as cardiac AP.
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Introduction
Ion channels are transmembrane proteins that allow rapid transfer of ion fluxes across cell membranes. Previous evidence has suggested a critical role for intracellular Ca2+ (Ca2+i) in the control of gene expression (Bading et al. 1993; Hardingham et al. 1997). Several lines of evidence suggest that channel biosynthesis may be regulated by Ca2+i (Sherman et al. 1985; Klarsfeld et al. 1989; Chiamvimonvat et al. 1995). However, no studies to date have directly documented the roles of Ca2+i in ion channel expression in vivo.
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To directly test the role of Ca2+i in modulating ion channel expression and activity in vivo, we took advantage of a transgenic mouse model in which levels of the sarcoplasmic reticulum (SR) Ca2+ pump in the heart were altered. This is a transgenic model of cardiac-specific ectopic overexpression of SERCA1a. The SERCA pumps are a family of highly conserved transmembrane proteins of 110 kDa, encoded by three homologous genes: SERCA1, SERCA2 and SERCA3 (Arai et al. 1994). The SERCA2 gene encodes two isoforms, SERCA2a and SERCA2b. SERCA2a is the primary isoform expressed in the heart (Zarain-Herzberg et al. 1990). SERCA2b is expressed in all tissues and plays an essential housekeeping role (Gunteski-Hamblin et al. 1988). The SERCA1 gene encodes two isoforms, SERCA1a and SERCA1b, expressed in adult and neonatal fast-twitch skeletal muscle, respectively, but never in the heart (Brandl et al. 1986). SERCA3 is expressed primarily in epithelial, endothelial cells and cells of the immune system (Anger et al. 1994).
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Ectopic overexpression of the fast twitch isoform, SERCA1a, in the heart using the cardiac-specific -myosin heavy chain promoter, results in structural and functional substitution of this isoform with a 2.5-fold increase in the amount of total SERCA protein (Loukianov et al. 1998; Lalli et al. 2001). The level of endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin and tropomyosin, were unchanged. Analysis of cardiac function using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in the transgenic mouse hearts (Loukianov et al. 1998). The results from this transgenic mouse model clearly demonstrate that Ca2+ handling is significantly affected. These animals exhibit no abnormal cardiac phenotypes and are ideal models in which we can directly test the role of Ca2+i alterations in cardiac ion channel expression and activity.
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Methods
All animal care and procedures were approved by the University of California, Davis Institutional Animal Care and Use Committee. Transgenic (TG) mice that specifically overexpress SERCA1a in the heart using the cardiac-specific -myosin heavy chain promoter in FVB/N background have been generated (Loukianov et al. 1998; Lalli et al. 2001). The copy number of the transgene in the line used in our experiments (line no. 38) was nine copies (Loukianov et al. 1998). Transgene-negative littermates were used as wild-type controls.
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Electrocardiographic (ECG) recordings
ECG recordings were obtained using Bioamplifier (BMA 831, CWE, Incorporated, Ardmore, PA, USA) (Zhang et al. 2002). The animals were anaesthetized with 250 mg kg–1 of tribromoethanol (Avertin) I.P. and placed on a temperature-controlled warming blanket at 37°C. The temperature of the animals was monitored during the recordings using a rectal probe. Four consecutive 2 min epochs of ECG data were obtained from each animal. Signals were low-pass filtered at 0.2 kHz and digitized using Digidata 1200 (Axon Instrument, CA, USA) and a custom-written software. A total of 100 beats were analysed manually from each animal by two blinded observers to assess the interobserver variability. The QT interval was determined manually by placing cursors on the beginning of the QRS and the end of the T wave. The rate-corrected QT interval (QTc) was calculated using modified Bazett's formula as reported by Mitchell et al. whereby the RR interval was first expressed as a unitless ratio (RR in ms (100 ms)–1). The QTc interval was defined as the QT interval (in ms)/(RR/100)1/2 (Mitchell et al. 1998).
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Electrophysiological recordings from transgenic animals
Single mouse ventricular myocytes were isolated from transgenic and wild-type animals from the same littermates at 10–12 weeks of age as previously described (Ahmmed et al. 2001). Mice were anaesthetized with pentobarbital (I.P. 40 mg kg–1) and hearts were rapidly excised. Due to the known electrophysiological heterogeneity in various regions of the heart, we used only left ventricular free-wall cells for our electrophysiological recordings. APs were recorded at room temperature using the perforated-patch technique (Ahmmed et al. 2000). All other experiments were performed using the conventional whole-cell patch-clamp technique (Hamill et al. 1981) at room temperature.
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For AP recordings, the patch pipettes were backfilled with amphotericin (200 μg ml–1). The pipette solution contained (mM): potassium glutamate 120, KCl 25, MgCl2 1, CaCl2 1, Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid) 10, pH 7.4 with KOH. The external solution contained (mM): NaCl 138, KCl 4, MgCl2 1, CaCl2 2, NaH2PO4 0.33, glucose 10, Hepes 10, pH 7.4 with NaOH.
For K+ current recording, the external solution contained (mM): NMG 130, KCl 5, CaCl2 1, MgCl2 1, nimodipine 1 μM, glucose 10, Hepes 10, pH 7.4 with HCl. The pipette solution contained (mM): KCl 140, Mg-ATP 4, MgCl2 1, EGTA 10, Hepes 10, pH 7.4 with KOH. For whole-cell Ca2+ current measurement, the external solution contained (mM): N-methyl glucamine 140, CsCl 5, CaCl2 2, MgCl2 0.5 glucose 10, Hepes 10, pH 7.4 with HCl. The pipette solution contained (mM): CsCl 125, TEA 20, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; Molecular Probes) 0.05, Mg-ATP 4, Hepes 10, pH 7.3 with CsOH. All experiments were performed using 3 M KCl agar bridges. Cell capacitance was calculated as the ratio of total charge (the integrated area under the current transient) to the magnitude of the pulse (20 mV). Currents were normalized to cell capacity to obtain the current density. The series resistance was compensated electronically. In all experiments, a series resistance compensation of 90% was obtained. Currents were recorded using Axopatch 200B amplifier (Axon Instrument), filtered at 10 kHz using a 4-pole Bessel filter and digitized at sampling frequency of 50 kHz. Data analysis was carried out using custom-written software and commercially available PC-based spreadsheet and graphics software (MicroCal Origin version 6.0).
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Neonatal mouse cardiac myocytes in culture
Single cardiac myocytes were isolated from 5 day old neonatal mice (FVB/N) as previously described (Nuss & Marban, 1994; Chiamvimonvat et al. 1995). Mice were anaesthetized with pentobarbital (40 mg kg–1) intraperitoneally. Animals were killed by decapitation and hearts excised under sterile conditions into a filter-sterilized (0.2 μm) nominally Ca2+- and bicarbonate-free Hanks Hepes-buffered solution, pH 7.4 at room temperature. Atrial and great vessel tissues were carefully removed. The ventricles were washed and minced in this Ca2+-free buffered solution containing trypsin (Difco, 1.5 mg ml–1) and DNase (1 ml (100 ml)–1 of the enzyme solution). The tissues were then incubated with 2 ml of the enzyme solution and stirred continuously at 37°C. The supernatant was removed and the enzyme solution replaced at 5 min intervals. The supernatant obtained after the first 15 min was discarded. Thereafter, the supernatant was diluted 1: 1 in fetal bovine serum. The cells were centrifuged at 1200 g for 3 min and resuspended in MEM culture medium (Invitrogen) containing 5% fetal bovine serum (Invitrogen), penicillin G (sodium) and vitamin B12. The viability of the cells was assessed using the trypan blue dye exclusion test. Generally, we obtained a viability of 90–98%. The cells were plated in tissue culture dishes and maintained at 37°C in a 5% CO2 incubator and re-plated at a cell density of 1.5–2.5 x 105 ml–1 after 20 min into new tissue culture dishes containing glass cover-slips (pretreated with laminin in order to remove endothelial cells, which tend to attach to the tissue culture dishes during this 20 min interval). Finally, cells were maintained at 37°C in a 5% CO2 incubator and were used for electrophysiological recording within 24–72 h of cell isolation.
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Adult mouse ventricular myocyte culture
Tissue culture dishes were precoated for 1 h with 10 μg ml–1 mouse laminin (Invitrogen) in phosphate-buffered saline (PBS; Invitrogen) with 1% penicillin–streptomycin (PS; Invitrogen) at room temperature. Freshly isolated left ventricular free-wall cardiac myocytes were suspended in minimal essential medium (MEM; Sigma M1018) containing 1.2 mM Ca2+, 2.5% preselected fetal bovine serum (FBS; Invitrogen), and 1% PS (pH 7.35–7.45). After myocytes were pelleted by gravity for 10 min, the supernatant was aspirated and the myocytes were washed twice using the same protocol. The myocytes were then plated at 0.5–1 x 104 cells cm–2 in MEM containing 2.5% FBS and 1% PS. After 1 h of culture in a 5% CO2 incubator at 37°C, the medium was changed to FBS-free MEM and this was changed every 48 h during cell culture.
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Experimental protocols
Increase in intracellular Ca2+. The first group of cells were exposed to the culture medium containing 5 mM of external CaCl2 or 40 mM KCl for 24–72 h in an attempt to raise the intracellular Ca2+. This group of cells were compared with the control which was maintained in the same culture medium except for concentrations of external Ca2+ and K+, which were 1.8 and 5.0 mM, respectively.
Decrease in intracellular Ca2+. The second group of cells were exposed to 10 μM BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, a cell membrane permeant Ca2+ chelator, Molecular Probes) for 48–72 h. A fresh stock of 50 mM BAPTA-AM was made for each experiment using dimethyl sulfoxide (DMSO, Sigma). This group of cells was compared with a control, which was maintained in a culture medium containing 0.02% of DMSO. The external concentration of Ca2+ and K+ for both groups was kept constant at 1.8 and 5.0 mM, respectively.
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Whole-cell K+ currents were recorded from different groups of cells after 48–72 h in culture using external and internal solutions as described above for K+ currents.
Western blot analysis
Immunoblots were performed as previously described (Xu et al. 2003) using whole-tissue homogenates as well as membrane fraction. Mice were anaesthetized with pentobarbital (40 mg kg–1) intraperitoneally and hearts were rapidly excised. The membrane fraction prepared from LV free wall of transgenic animals and wild-type littermates was used. All procedures were performed at 4°C, the centrifuge rotor was precooled, and centrifugation was performed at 4°C. All solutions contained the following protease inhibitors: protein inhibitor cocktail, benzamidine (0.1 mg ml–1), calpain inhibitors (80 ng ml–1), pepstatinA (1 μg ml–1) and phenylmethyl sulfonyl fluoride (PMSF) (100 ng ml–1). All protease inhibitors were obtained from Sigma Chemical Co. Tissues were minced, diluted in lysis buffer (10 mM Hepes, 350 mM sucrose, 5 mM EDTA, 1 ml (100 mg of tissue)–1), and homogenized. Nuclei and debris were pelleted by centrifugation at 2000 g for 10 min. This procedure was repeated, and the supernatants from both low-speed spins were pooled and centrifuged at 100 000 g for 1 h. The pellet was solubilized in lysis buffer and 1% Triton X-100, sonicated and centrifuged at 100 000 g for 1 h. The supernatant was pooled and protein content of the solubilized membrane preparations was determined using a Bio-Rad protein assay kit. Solubilized membranes were aliquoted and stored at –20°C until used.
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The following primary antibodies were used: (1) anti-Kv4.2, (2) anti-Kv4.3, (3) anti-Kv1.5, (4) anti-Kv2.1 and (5) anti-Kv1.4 (Alomone Laboratories, Israel), (6) anti-KChIP2 (Kv4 channel interacting protein) (Santa Cruz) and (7) anti-GAPDH antibody (Sigma) was used as an internal loading control. Quantification of the signals was performed by densitometry (ImageQuant). Standard curves were generated for each antibody to ascertain that the measurements were in a linear range for both the control and the K+ channel antibody detection. The protein bands were normalized to the GAPDH band in each sample. The value was then averaged from all the different sets of experiments.
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Confocal Ca2+ imaging
Enzymatically isolated free-wall LV myocytes were loaded with the Ca2+ indicator fluo 3-AM. (Wang et al. 2001) Confocal line-scan imaging was performed using a Zeiss Pascal confocal microscope equipped with an argon laser (488 nm) and a 40 x, 1.3 NA oil immersion objective. Line-scan images were acquired at sampling rates of 0.7 ms per line and 0.07 μm per pixel, with radial and axial resolutions of 0.4 and 1.0 μm, respectively. Ca2+ transients were expressed as the normalized local fluorescence (F/Fo), where Fo refers to the fluorescence level before depolarization as described (Harkins et al. 1993).
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Where appropriate, pooled data are presented as means ± S.E.M. Significant differences between groups were tested using ANOVA. The null hypothesis was rejected when two-tailed P value < 0.05.
Results
QT intervals are prolonged in SERCA1a OE mice
Figure 1A shows significant prolongation of the QTc intervals in SERCA1a OE mice compared with wild-type littermates. However, heart rate (RR interval) and PR interval were unaffected (Fig. 1B).
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A, examples of ECG recordings from SERCA1a OE mice compared with their respective littermate controls. Note the evidence of QT prolongation in SERCA1a OE mice compared with wild-type littermates. B, summary data for RR, QTc and PR intervals. *P < 0.05, n = 20 animals for each group.
Action potentials are prolonged in SERCA1a OE mice
We recorded APs from single LV free-wall myocytes isolated from SERCA1a OE transgenic mice and compared with their littermate controls (Fig. 2A). APs were markedly prolonged in myocytes isolated from SERCA1a OE mice compared with those of their wild-type littermates. Summary data show significant prolongation of both APD50 and APD90 in SERCA1a OE animals (Fig. 2B).
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A, representative AP recordings from isolated cardiac myocytes from SERCA1a OE mice compared with their littermate controls. B, summary data for APD at 50 and 90% repolarization (APD50 and APD90; n = 10 animals for each group and 3–4 myocytes were analysed per animal, *P < 0.01). The stimulation frequency was 1 Hz.
The L-type Ca2+ current density is not altered in SERCA1a OE myocytes
We next examined the inward L-type Ca2+ and outward K+ currents from the two sets of animals. There were no significant differences in the Ca2+ current density between SERCA1a OE mice compared with the respective wild-type littermates (Fig. 3). The data in the SERCA1a OE mice are different from previously reported findings with this model; however, different recording conditions were used in that study (Lalli et al. 2001).
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A, examples of whole-cell L-type Ca2+ currents recorded from SERCA1a OE mice compared with their littermate controls. The holding potential used was –55 mV. B, summary of current density–voltage relations from SERCA1a OE myocytes compared with wild-type controls (P = NS, n refers to numbers of cells).
Outward K+ currents are down-regulated in SERCA1a OE myocytes
In order to further investigate the basis for the cardiac AP prolongation observed in the transgenic animals, we next examined whole-cell outward K+ currents from single free-wall LV myocytes from SERCA1a OE mice compared with the respective littermate controls. SERCA1a OE mice showed a significant down-regulation of the outward K+ currents (Fig. 4). A previous study documented four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes: a rapidly inactivating, transient outward K+ current, Ito,f; a slowly inactivating K+ current, IK,slow; a non-inactivating, steady state current, Iss; and a slowly inactivating transient outward current, Ito,s, which is observed mainly in septal cells (Xu et al. 1999b). The three components of the K+ currents in free-wall ventricular myocytes were distinguished using exponential fits to the decay phases as previously described (Xu et al. 1999a) (Fig. 4C). There were no significant changes in the fast or slow time constants of current decay (f and s). However, the SERCA1a OE mice showed a significant down-regulation of the amplitude of Ito,f, IK,slow and Iss. There were no significant differences in the cell capacitance obtained from the two different groups of animals.
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A, examples of whole-cell outward K+ currents from SERCA1a OE mice compared with wild-type littermates. The holding potential used was –80 mV. B, summary of current density–voltage relations from SERCA1a OE mice compared with wild-type littermates. n = 8 animals for each group, 3–4 myocytes were analysed from each animal (*P < 0.05). C, the decay phases of the transient outward currents (2.5 s) evoked during a depolarizing voltage step to +60 mV were fitted by two exponential decays using the following expressions: y(t) = A1 exp(–t/f) + A2 exp(–t/s) + Ass, where t is time, f and s are the fast and slow time constants, A1 and A2 are the amplitudes of the fast and slow inactivating current components, respectively, and Ass is the amplitude of the steady state, non-inactivating component. For all fits, time zero was set at the peak of the outward current. D and E, summary data for f, s and A1, A2 and Ass components (*P < 0.05).
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Quantitative immunoblot analysis
In order to define the possible mechanisms of changes in the K+ current density observed in the transgenic animals, we quantified the K+ channel protein levels using antibodies specific to Kv4.2 and Kv4.3, the molecular correlates of Ito,f (Guo et al. 1999; Wickenden et al. 1999) as well as antibodies specific to Kv1.5 and Kv2.1, the molecular correlates of IK,slow in mice (Xu et al. 1999a; Nerbonne, 2000; London et al. 2001). Previous studies have shown that the expression of Ito can be significantly modulated by the accessory subunit KChIP with KChIP2 as the main cardiac isoform (An et al. 2000; Kuo et al. 2001). Antibodies specific to KChIP2 were also used in our experiments. Figure 5 shows examples of immunoblots using antibodies directed against Kv4.2 (panels A and B), Kv4.3 (panel C) Kv1.4 (panel D) from the membrane fraction isolated from LV free-wall of transgenic compared with the wild-type littermates. Negative controls were performed with the antibody preincubated with the blocking peptide and labelled as ‘Neg control’ (panel B). Lower bands are GAPDH, which was used as an internal control. Summary data in panel E reveal a significant down-regulation of Kv4.2, Kv4.3, Kv1.5, Kv2.1 and KChIP2 protein expression in SERCA1a OE mice compared with wild-type littermates (*P < 0.05). In contrast, there were no significant differences in the protein expression level for Kv1.4 (panels D and E).
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A, examples of immunoblots with positive labelling of protein bands using an antibody specific to Kv4.2 comparing SERCA1a OE LV free-wall protein samples to the respective littermate control samples. Each lane was loaded with increasing amounts of protein (30, 60 and 120 μg of protein). The expected size of the Kv4.2 protein is 70 kDa. Lower bands in A, C and D are GAPDH (expected size of 27 kDa). B, examples of negative controls for Kv4.2 antibody using ventricular samples from SERCA1a OE mice. C, examples of immunoblots with positive labelling of protein bands using an antibody specific to Kv4.3 (expected size of 66 kDa) comparing SERCA1a OE and wild-type samples. Each lane was loaded with increasing amounts of protein (30, 60 and 120 μg). D, examples of immunoblots with positive labelling of protein bands using an antibody specific to Kv1.4 (expected size of 96 kDa) comparing SERCA1a OE and wild-type samples. Each lane was loaded with increasing amounts of protein (30, 60 and 120 μg) as in A and C. E, summary data comparing SERCA1a OE samples to the respective littermate controls (n = 7 animals for each group, *P = 0.049, 0.04, 0.04, 0.03 and 0.04 for KChIP2, Kv4.2, Kv4.3, Kv2.1 and Kv1.5, respectively, P = NS for Kv1.4).
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In order to further ascertain that the reduction in membrane protein levels of the Kv channels were due to the impairment of the membrane targeting or the reduced protein expression, we repeated the Western blot experiments using whole-tissue homogenates obtained from LV free wall and found a significant reduction in the expression levels of Kv1.5, Kv2.1, Kv4.2, Kv4.3 and KChIP2 in SERCA1a OE mice compared with wild-type littermates. The data obtained using the whole-tissue homogenates were similar to that using membrane fraction. These new findings suggest that the reductions were probably due to reduced protein expression.
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Line-scan images of cardiac myocytes from SERCA1a OE mice compared with wild-type littermates
We next obtained line-scan images from SERCA1a OE myocytes and compared these with the littermate controls to assess the [Ca2+]i transients (measured as F/Fo) as previously described (Gomez et al. 1997). Simultaneous APs were generated via perforated patch pipettes. SERCA1a OE myocytes showed a significant increase in the peak Ca2+i transients (measured as peak F/Fo ratio, n = 11 for both genotypes, P < 0.05, Fig. 6A). This was associated with a rapid decline in the [Ca2+]i transients, consistent with a faster time course of Ca2+ removal in SERCA1a OE myocytes. The decay of Ca2+i transients was fitted using a single exponential function. The time constants are summarized in Fig. 6B (*P < 0.05). The data confirm the previous finding of an increase in the beat-to-beat Ca2+i transient in the SERCA1a OE mice (Lalli et al. 2001).
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A, representative examples of line-scan images in response to stimulated AP showing kinetics of Ca2+i transients recorded from myocytes of SERCA1a OE mice compared with wild-type littermates. Sample records showing line-scan fluorescence image (top), and Ca2+i (measured as F/Fo) (bottom). The fluorescent signal (F) was normalized to the signal before depolarization (Fo). Scale bars represent 20 μm. B, summary data for the peak F/Fo (*P < 0.05). The Ca2+i transient decay was fitted using a single exponential function with time constants (in ms) summarized in the lower panel.
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Effects of changes in intracellular Ca2+ on K+ current in cultured neonatal and adult mouse cardiac myocytes
In order to directly establish the causal relations between changes in Ca2+i and K+ current densities, we recorded outward K+ currents from neonatal mouse cardiac myocytes which had been maintained for 48–72 h in culture media containing 5 mM of Ca2+ or 40 mM of K+ (to depolarize the membrane potential) or BAPTA-AM (10 μM to decrease Ca2+i) as compared with control cells which had been maintained in culture medium containing 1.8 mM Ca2+ and 5 mM K+. We have chosen this in vitro model since we have previously documented in this primary culture system that manipulation of the external Ca2+ concentration or addition of BAPTA-AM can lead to changes in Ca2+i using fura-2 as an indicator (Chiamvimonvat et al. 1995). The experiments were designed to test the effects of Ca2+i using two opposing conditions of increased versus decreased Ca2+ concentrations instead of testing for reversibility in order to try to avoid prolonged culture time which may induce as yet some undefined phenotypic drift. Current density was calculated from the current amplitude normalized by the cell capacitance. To further demonstrate that the results obtained using the neonatal cardiac myocytes were representative of the phenomenon observed in adult animals, we performed additional experiments using adult left ventricular free-wall myocytes in culture and manipulated the external K+ concentration (40 mM K+) for 48 h. The K+ current density recorded from the high [K+] group were then directly compared with that of the control cells kept in culture for 48 h.
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Figure 7 shows examples of the outward K+ current traces from the four different groups of neonatal cardiac myocytes at 48 h. Summary data of the current densities–voltage relations of the peak outward K+ current and the sustained components (labelled in the figure as Ito and ISS, respectively) are shown in Fig. 7B after 2 and 3 days in culture. There was a significant decrease in the peak outward K+ current densities in cells which had been maintained in high Ca2+ or high K+ media after 2 or 3 days in culture compared with control. In contrast, the peak outward K+ current was significantly increased in the BAPTA-AM group compared with control after 3 days in culture. Figure 7C shows comparable data obtained using adult left ventricular free-wall myocytes in culture. There was a significant decrease in the outward K+ current density in the cells maintained in a high external K+ concentration for 48 h compared with control. Of note, the current density shown for control cells in Fig. 7C was smaller than data obtained from wild-type animals shown in Fig. 4 since these cells had been maintained in culture for 48 h while data in Fig. 4 were obtained from freshly isolated ventricular myocytes.
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A, examples of outward K+ current traces recorded from neonatal mouse cardiac myocytes in culture media containing different Ca2+ concentrations; control, BAPTA-AM (10 μM), high Ca2+ (5 mM) or high K+ (40 mM) for 48 h. B, summary data of the peak outward and sustained components from the four different groups of cells (*P < 0.05 compared with control, n = 24–42 cells). C, similar data were obtained using adult left ventricular free-wall myocytes in culture. Upper panels show examples of outward K+ current traces recorded from adult mouse left free-wall ventricular myocytes in culture media containing high K+ (40 mM) compared with control conditions for 48 h. Bottom panel shows summary data of the peak outward and sustained components from the high K+ group compared with control cells in culture at 48 h (*P < 0.05 compared with control, n = 10 cells).
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Discussion
Previous studies have reported a reduction in SERCA2 mRNA and protein levels in failing hearts, resulting in a decrease in Ca2+ accumulation in the SR (Kiss et al. 1995; O'Rourke et al. 1999). We hypothesize that modulation of ion channel expression in cardiac hypertrophy and failure may be closely linked to the known alterations of Ca2+ homeostasis in these conditions. Here, we took advantage of a transgenic mouse model with known pertubations of one of the Ca2+ handling proteins. This allowed us to determine directly the effects of changes in Ca2+ homeostasis on ion channel expression in an in vivo model.
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We observed a significant down-regulation of the outward K+ currents including Ito,f, IK,slow and ISS in SERCA1a OE mice. This was associated with a marked AP prolongation and QT prolongation on surface ECG. We further established that changes in outward K+ current density resulted, at least in part, from alterations in the Kv4.2, Kv4.3, Kv2.1 and Kv1.5 as well as KChIP2 protein expression. Finally, we further documented a significant alteration in the beat-to-beat Ca2+i transient in the mouse model with genetic manipulation of the SERCA pumps. To recapitulate the in vivo experiments, the effects of changes in Ca2+i on ion channel expression were further tested in cultured neonatal and adult mouse cardiac myocytes. Indeed, an increase in [Ca2+]i in neonatal cardiac myocytes resulted in a decrease in both the transient and sustained components of the outward K+ currents after 2–3 days in culture.
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Roles of Ca2+i in modulation of ion channel activity and expression
Transcription of numerous genes has been shown to be regulated by Ca2+i(Carafoli et al. 1999). Ca2+ appears to act in conjunction with calmodulin, activating specific kinases that phosphorylate transcription factors in the nucleus or prior to their entry into the nucleus (Rao et al. 1997; Molkentin et al. 1998; Carafoli et al. 1999). The Ca2+-activated phosphatase calcineurin has been suggested to play a critical role in translating altered Ca2+ signalling into changes in the gene transcriptions linked to the development of cardiac hypertrophy and failure (Molkentin et al. 1998). Upon activation, calcineurin dephosphorylates the nuclear factor of activated T cells (NFAT). Recently, it was shown using NFATc3 knockout and NFAT reporter mice that the calcineurin/NFATc3 pathway plays an important role in mediating the down-regulation of K+ channels expression, including Kv1.5, Kv2.1, Kv4.2, and Kv4.3, associated with myocardial infarction or adrenergic stimulation (Rossow et al. 2004). It is possible that the calcineurin/NFAT pathway is activated by the increased intracellular Ca2+ and mediates the down-regulation of the K+ channel expression in SERCA1a OE mice. Additional signalling factors which may be involved include rennin–angiotensin, protein kinase A and C, MAP kinase, calmodulin-dependent (CAM) kinase, as well as adrenergic pathways (Packer, 1992; Colucci & Braunwald, 1997; Molkentin et al. 1998). In the SERCA1a OE model in particular, cardiac hypertrophy and failure was not observed suggesting that the down-regulation in the K+ channel expression was not secondary to the cardiac hypertrophy or failure but rather occur as a result of the signalling pathways which are activated by intracellular Ca2+. The
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change of Ca2+ signalling responsible for the down-regulation of K+ channels in ventricular myocytes in SERCA1a OE mice may involve an increase in the peak magnitude of the Ca2+ transient or the enhanced spontaneous Ca2+ sparks (or Ca2+ leak) due to SR Ca2+ overload. Future studies will be required to completely explain the signalling pathways involved in the down-regulation of K+ channel expression in the setting of increase intracellular Ca2+.
Indeed, cytosolic Ca2+ has also been shown to play a critical role in the regulation of biosynthesis of ionic channels, such as the nicotinic acetylcholine receptor (Shainberg et al. 1987; Klarsfeld et al. 1989), L-type Ca2+ channel expression (Renganathan et al. 1999), and Na+ channel biosynthesis (Sherman & Catterall, 1984; Sherman et al. 1985; Offord & Catterall, 1989; Taouis et al. 1991; Duff et al. 1992; Chiamvimonvat et al. 1995). Our data agree well with these previous studies that expression of ion channels in cardiac myocytes may be regulated by Ca2+i; however, further work is needed to address whether the significant changes in K+ channel expression in these mouse models could be correlated with either a change in peak, diastolic or total Ca2+i. Furthermore, in SERCA1a OE cardiac myocytes, the ATP consumption would be increased as a result of the enhanced ATPase activity, which might be involved in the down-regulation of K+ channels.
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Down-regulation of one or more of the underlying outward K+ currents has been documented in a variety of heart failure models including human heart failure (Beuckelmann et al. 1993; Nbauer et al. 1993; Tomita et al. 1994; Kb et al. 1996, 1998). The mechanistic basis of the down-regulation of Ito in human heart failure appears to be a decrease in the transcription of the Kv4.3 gene (Kb et al. 1998). In addition, significant electrical remodelling has been documented in animal models of atrial fibrillation as well as in humans (Gaspo et al. 1997; Van Wagoner et al. 1999; Yue et al. 1999). Several lines of evidence point to a role of intracellular Ca2+ overload (Nattel, 1999), e.g. histological changes compatible with Ca2+ overload-induced injury (Goette et al. 1996). In addition, Ca2+ channel blockers appeared to suppress the electrical remodelling in rapid-pacing-induced atrial fibrillation (Daoud et al. 1997). An increase in the frequency of depolarization may increase Ca2+ entry and hence an increase in the time-averaged Ca2+i level.
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The present report suggests the important finding that changes in K+ channel expression can follow alterations in calcium homeostasis, prior to cardiac hypertrophy and failure. On the other hand, other reports have suggested that inhibition of K+ currents can serve as a triggering event in cardiac hypertrophy as a result of the disturbances in calcium homeostasis following AP prolongation (Wickenden et al. 1999; Kassiri et al. 2002). However, this issue remains controversial since another mouse model with a prolonged cardiac AP did not show evidence of cardiac hypertrophy (Barry et al. 1998). Furthermore, no evidence of cardiac hypertrophy was documented in patients with long QT syndrome and prolonged cardiac repolarization (Nador et al. 1991).
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The SR Ca2+ overload, caused by the enhanced SERCA activity, and the prolongation of the QT interval, as observed in SERCA1a OE mice, is expected to increase the propensity to arrhythmia in SERCA1a OE mice. However, these mice do not exhibit an increased mortality compared with the littermate controls. It is likely that cardiac arrhythmias may only be uncovered during stress e.g. with programmed stimulation during electrophysiological studies or with adrenergic stimulation.
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Finally, recent studies using SERCA gene transfer both in vivo and in vitro have provided compelling evidence that SERCA gene transfer can be effectively used to enhance cardiac contractility and rescue the contractile function in failing myocardium (Miyamoto et al. 2000). Indeed, adenoviral-mediated gene therapy using the SERCA pump has been proposed as a therapeutic option for heart failure (Hajjar et al. 2000). Our present data show that a primary alteration in this critical Ca2+ handling protein, without cardiac hypertrophy and failure, can produce profound changes in K+ channel expression and cardiac AP profile. Taken together, our data suggest that a coexpression of the SERCA pump as well as K+ channels are likely to be necessary to rescue the full phenotype of cardiac failure (Ennis et al. 2002). This, in fact, will have important therapeutic implications.
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6 Department of Veterans Affairs, Northern California Health Care System, Mather, CA
5 Department of Physiology and Cell Biology, Ohio State University, Columbus, OH
3 Department of Internal Medicine
4 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, OH, USA
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1 Pharmacology Department, Hebei Medical University Shijiazhuang, China
Abstract
We tested the hypothesis that chronic changes in intracellular Ca2+ (Ca2+i) can result in changes in ion channel expression; this represents a novel mechanism of crosstalk between changes in Ca2+ cycling proteins and the cardiac action potential (AP) profile. We used a transgenic mouse with cardiac-specific overexpression of sarcoplasmic reticulum Ca2+ ATPase (SERCA) isoform 1a (SERCA1a OE) with a significant alteration of SERCA protein levels without cardiac hypertrophy or failure. Here, we report significant changes in the expression of a transient outward K+ current (Ito,f), a slowly inactivating K+ current (IK,slow) and the steady state current (ISS) in the transgenic mice with resultant prolongation in cardiac action potential duration (APD) compared with the wild-type littermates. In addition, there was a significant prolongation of the QT interval on surface electrocardiograms in SERCA1a OE mice. The electrophysiological changes, which correlated with changes in Ca2+i, were further corroborated by measuring the levels of ion channel protein expression. To recapitulate the in vivo experiments, the effects of changes in Ca2+i on ion channel expression were further tested in cultured adult and neonatal mouse cardiac myocytes. We conclude that a primary defect in Ca2+ handling proteins without cardiac hypertrophy or failure may produce profound changes in K+ channel expression and activity as well as cardiac AP.
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Introduction
Ion channels are transmembrane proteins that allow rapid transfer of ion fluxes across cell membranes. Previous evidence has suggested a critical role for intracellular Ca2+ (Ca2+i) in the control of gene expression (Bading et al. 1993; Hardingham et al. 1997). Several lines of evidence suggest that channel biosynthesis may be regulated by Ca2+i (Sherman et al. 1985; Klarsfeld et al. 1989; Chiamvimonvat et al. 1995). However, no studies to date have directly documented the roles of Ca2+i in ion channel expression in vivo.
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To directly test the role of Ca2+i in modulating ion channel expression and activity in vivo, we took advantage of a transgenic mouse model in which levels of the sarcoplasmic reticulum (SR) Ca2+ pump in the heart were altered. This is a transgenic model of cardiac-specific ectopic overexpression of SERCA1a. The SERCA pumps are a family of highly conserved transmembrane proteins of 110 kDa, encoded by three homologous genes: SERCA1, SERCA2 and SERCA3 (Arai et al. 1994). The SERCA2 gene encodes two isoforms, SERCA2a and SERCA2b. SERCA2a is the primary isoform expressed in the heart (Zarain-Herzberg et al. 1990). SERCA2b is expressed in all tissues and plays an essential housekeeping role (Gunteski-Hamblin et al. 1988). The SERCA1 gene encodes two isoforms, SERCA1a and SERCA1b, expressed in adult and neonatal fast-twitch skeletal muscle, respectively, but never in the heart (Brandl et al. 1986). SERCA3 is expressed primarily in epithelial, endothelial cells and cells of the immune system (Anger et al. 1994).
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Ectopic overexpression of the fast twitch isoform, SERCA1a, in the heart using the cardiac-specific -myosin heavy chain promoter, results in structural and functional substitution of this isoform with a 2.5-fold increase in the amount of total SERCA protein (Loukianov et al. 1998; Lalli et al. 2001). The level of endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin and tropomyosin, were unchanged. Analysis of cardiac function using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in the transgenic mouse hearts (Loukianov et al. 1998). The results from this transgenic mouse model clearly demonstrate that Ca2+ handling is significantly affected. These animals exhibit no abnormal cardiac phenotypes and are ideal models in which we can directly test the role of Ca2+i alterations in cardiac ion channel expression and activity.
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Methods
All animal care and procedures were approved by the University of California, Davis Institutional Animal Care and Use Committee. Transgenic (TG) mice that specifically overexpress SERCA1a in the heart using the cardiac-specific -myosin heavy chain promoter in FVB/N background have been generated (Loukianov et al. 1998; Lalli et al. 2001). The copy number of the transgene in the line used in our experiments (line no. 38) was nine copies (Loukianov et al. 1998). Transgene-negative littermates were used as wild-type controls.
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Electrocardiographic (ECG) recordings
ECG recordings were obtained using Bioamplifier (BMA 831, CWE, Incorporated, Ardmore, PA, USA) (Zhang et al. 2002). The animals were anaesthetized with 250 mg kg–1 of tribromoethanol (Avertin) I.P. and placed on a temperature-controlled warming blanket at 37°C. The temperature of the animals was monitored during the recordings using a rectal probe. Four consecutive 2 min epochs of ECG data were obtained from each animal. Signals were low-pass filtered at 0.2 kHz and digitized using Digidata 1200 (Axon Instrument, CA, USA) and a custom-written software. A total of 100 beats were analysed manually from each animal by two blinded observers to assess the interobserver variability. The QT interval was determined manually by placing cursors on the beginning of the QRS and the end of the T wave. The rate-corrected QT interval (QTc) was calculated using modified Bazett's formula as reported by Mitchell et al. whereby the RR interval was first expressed as a unitless ratio (RR in ms (100 ms)–1). The QTc interval was defined as the QT interval (in ms)/(RR/100)1/2 (Mitchell et al. 1998).
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Electrophysiological recordings from transgenic animals
Single mouse ventricular myocytes were isolated from transgenic and wild-type animals from the same littermates at 10–12 weeks of age as previously described (Ahmmed et al. 2001). Mice were anaesthetized with pentobarbital (I.P. 40 mg kg–1) and hearts were rapidly excised. Due to the known electrophysiological heterogeneity in various regions of the heart, we used only left ventricular free-wall cells for our electrophysiological recordings. APs were recorded at room temperature using the perforated-patch technique (Ahmmed et al. 2000). All other experiments were performed using the conventional whole-cell patch-clamp technique (Hamill et al. 1981) at room temperature.
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For AP recordings, the patch pipettes were backfilled with amphotericin (200 μg ml–1). The pipette solution contained (mM): potassium glutamate 120, KCl 25, MgCl2 1, CaCl2 1, Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid) 10, pH 7.4 with KOH. The external solution contained (mM): NaCl 138, KCl 4, MgCl2 1, CaCl2 2, NaH2PO4 0.33, glucose 10, Hepes 10, pH 7.4 with NaOH.
For K+ current recording, the external solution contained (mM): NMG 130, KCl 5, CaCl2 1, MgCl2 1, nimodipine 1 μM, glucose 10, Hepes 10, pH 7.4 with HCl. The pipette solution contained (mM): KCl 140, Mg-ATP 4, MgCl2 1, EGTA 10, Hepes 10, pH 7.4 with KOH. For whole-cell Ca2+ current measurement, the external solution contained (mM): N-methyl glucamine 140, CsCl 5, CaCl2 2, MgCl2 0.5 glucose 10, Hepes 10, pH 7.4 with HCl. The pipette solution contained (mM): CsCl 125, TEA 20, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; Molecular Probes) 0.05, Mg-ATP 4, Hepes 10, pH 7.3 with CsOH. All experiments were performed using 3 M KCl agar bridges. Cell capacitance was calculated as the ratio of total charge (the integrated area under the current transient) to the magnitude of the pulse (20 mV). Currents were normalized to cell capacity to obtain the current density. The series resistance was compensated electronically. In all experiments, a series resistance compensation of 90% was obtained. Currents were recorded using Axopatch 200B amplifier (Axon Instrument), filtered at 10 kHz using a 4-pole Bessel filter and digitized at sampling frequency of 50 kHz. Data analysis was carried out using custom-written software and commercially available PC-based spreadsheet and graphics software (MicroCal Origin version 6.0).
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Neonatal mouse cardiac myocytes in culture
Single cardiac myocytes were isolated from 5 day old neonatal mice (FVB/N) as previously described (Nuss & Marban, 1994; Chiamvimonvat et al. 1995). Mice were anaesthetized with pentobarbital (40 mg kg–1) intraperitoneally. Animals were killed by decapitation and hearts excised under sterile conditions into a filter-sterilized (0.2 μm) nominally Ca2+- and bicarbonate-free Hanks Hepes-buffered solution, pH 7.4 at room temperature. Atrial and great vessel tissues were carefully removed. The ventricles were washed and minced in this Ca2+-free buffered solution containing trypsin (Difco, 1.5 mg ml–1) and DNase (1 ml (100 ml)–1 of the enzyme solution). The tissues were then incubated with 2 ml of the enzyme solution and stirred continuously at 37°C. The supernatant was removed and the enzyme solution replaced at 5 min intervals. The supernatant obtained after the first 15 min was discarded. Thereafter, the supernatant was diluted 1: 1 in fetal bovine serum. The cells were centrifuged at 1200 g for 3 min and resuspended in MEM culture medium (Invitrogen) containing 5% fetal bovine serum (Invitrogen), penicillin G (sodium) and vitamin B12. The viability of the cells was assessed using the trypan blue dye exclusion test. Generally, we obtained a viability of 90–98%. The cells were plated in tissue culture dishes and maintained at 37°C in a 5% CO2 incubator and re-plated at a cell density of 1.5–2.5 x 105 ml–1 after 20 min into new tissue culture dishes containing glass cover-slips (pretreated with laminin in order to remove endothelial cells, which tend to attach to the tissue culture dishes during this 20 min interval). Finally, cells were maintained at 37°C in a 5% CO2 incubator and were used for electrophysiological recording within 24–72 h of cell isolation.
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Adult mouse ventricular myocyte culture
Tissue culture dishes were precoated for 1 h with 10 μg ml–1 mouse laminin (Invitrogen) in phosphate-buffered saline (PBS; Invitrogen) with 1% penicillin–streptomycin (PS; Invitrogen) at room temperature. Freshly isolated left ventricular free-wall cardiac myocytes were suspended in minimal essential medium (MEM; Sigma M1018) containing 1.2 mM Ca2+, 2.5% preselected fetal bovine serum (FBS; Invitrogen), and 1% PS (pH 7.35–7.45). After myocytes were pelleted by gravity for 10 min, the supernatant was aspirated and the myocytes were washed twice using the same protocol. The myocytes were then plated at 0.5–1 x 104 cells cm–2 in MEM containing 2.5% FBS and 1% PS. After 1 h of culture in a 5% CO2 incubator at 37°C, the medium was changed to FBS-free MEM and this was changed every 48 h during cell culture.
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Experimental protocols
Increase in intracellular Ca2+. The first group of cells were exposed to the culture medium containing 5 mM of external CaCl2 or 40 mM KCl for 24–72 h in an attempt to raise the intracellular Ca2+. This group of cells were compared with the control which was maintained in the same culture medium except for concentrations of external Ca2+ and K+, which were 1.8 and 5.0 mM, respectively.
Decrease in intracellular Ca2+. The second group of cells were exposed to 10 μM BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, a cell membrane permeant Ca2+ chelator, Molecular Probes) for 48–72 h. A fresh stock of 50 mM BAPTA-AM was made for each experiment using dimethyl sulfoxide (DMSO, Sigma). This group of cells was compared with a control, which was maintained in a culture medium containing 0.02% of DMSO. The external concentration of Ca2+ and K+ for both groups was kept constant at 1.8 and 5.0 mM, respectively.
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Whole-cell K+ currents were recorded from different groups of cells after 48–72 h in culture using external and internal solutions as described above for K+ currents.
Western blot analysis
Immunoblots were performed as previously described (Xu et al. 2003) using whole-tissue homogenates as well as membrane fraction. Mice were anaesthetized with pentobarbital (40 mg kg–1) intraperitoneally and hearts were rapidly excised. The membrane fraction prepared from LV free wall of transgenic animals and wild-type littermates was used. All procedures were performed at 4°C, the centrifuge rotor was precooled, and centrifugation was performed at 4°C. All solutions contained the following protease inhibitors: protein inhibitor cocktail, benzamidine (0.1 mg ml–1), calpain inhibitors (80 ng ml–1), pepstatinA (1 μg ml–1) and phenylmethyl sulfonyl fluoride (PMSF) (100 ng ml–1). All protease inhibitors were obtained from Sigma Chemical Co. Tissues were minced, diluted in lysis buffer (10 mM Hepes, 350 mM sucrose, 5 mM EDTA, 1 ml (100 mg of tissue)–1), and homogenized. Nuclei and debris were pelleted by centrifugation at 2000 g for 10 min. This procedure was repeated, and the supernatants from both low-speed spins were pooled and centrifuged at 100 000 g for 1 h. The pellet was solubilized in lysis buffer and 1% Triton X-100, sonicated and centrifuged at 100 000 g for 1 h. The supernatant was pooled and protein content of the solubilized membrane preparations was determined using a Bio-Rad protein assay kit. Solubilized membranes were aliquoted and stored at –20°C until used.
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The following primary antibodies were used: (1) anti-Kv4.2, (2) anti-Kv4.3, (3) anti-Kv1.5, (4) anti-Kv2.1 and (5) anti-Kv1.4 (Alomone Laboratories, Israel), (6) anti-KChIP2 (Kv4 channel interacting protein) (Santa Cruz) and (7) anti-GAPDH antibody (Sigma) was used as an internal loading control. Quantification of the signals was performed by densitometry (ImageQuant). Standard curves were generated for each antibody to ascertain that the measurements were in a linear range for both the control and the K+ channel antibody detection. The protein bands were normalized to the GAPDH band in each sample. The value was then averaged from all the different sets of experiments.
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Confocal Ca2+ imaging
Enzymatically isolated free-wall LV myocytes were loaded with the Ca2+ indicator fluo 3-AM. (Wang et al. 2001) Confocal line-scan imaging was performed using a Zeiss Pascal confocal microscope equipped with an argon laser (488 nm) and a 40 x, 1.3 NA oil immersion objective. Line-scan images were acquired at sampling rates of 0.7 ms per line and 0.07 μm per pixel, with radial and axial resolutions of 0.4 and 1.0 μm, respectively. Ca2+ transients were expressed as the normalized local fluorescence (F/Fo), where Fo refers to the fluorescence level before depolarization as described (Harkins et al. 1993).
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Where appropriate, pooled data are presented as means ± S.E.M. Significant differences between groups were tested using ANOVA. The null hypothesis was rejected when two-tailed P value < 0.05.
Results
QT intervals are prolonged in SERCA1a OE mice
Figure 1A shows significant prolongation of the QTc intervals in SERCA1a OE mice compared with wild-type littermates. However, heart rate (RR interval) and PR interval were unaffected (Fig. 1B).
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A, examples of ECG recordings from SERCA1a OE mice compared with their respective littermate controls. Note the evidence of QT prolongation in SERCA1a OE mice compared with wild-type littermates. B, summary data for RR, QTc and PR intervals. *P < 0.05, n = 20 animals for each group.
Action potentials are prolonged in SERCA1a OE mice
We recorded APs from single LV free-wall myocytes isolated from SERCA1a OE transgenic mice and compared with their littermate controls (Fig. 2A). APs were markedly prolonged in myocytes isolated from SERCA1a OE mice compared with those of their wild-type littermates. Summary data show significant prolongation of both APD50 and APD90 in SERCA1a OE animals (Fig. 2B).
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A, representative AP recordings from isolated cardiac myocytes from SERCA1a OE mice compared with their littermate controls. B, summary data for APD at 50 and 90% repolarization (APD50 and APD90; n = 10 animals for each group and 3–4 myocytes were analysed per animal, *P < 0.01). The stimulation frequency was 1 Hz.
The L-type Ca2+ current density is not altered in SERCA1a OE myocytes
We next examined the inward L-type Ca2+ and outward K+ currents from the two sets of animals. There were no significant differences in the Ca2+ current density between SERCA1a OE mice compared with the respective wild-type littermates (Fig. 3). The data in the SERCA1a OE mice are different from previously reported findings with this model; however, different recording conditions were used in that study (Lalli et al. 2001).
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A, examples of whole-cell L-type Ca2+ currents recorded from SERCA1a OE mice compared with their littermate controls. The holding potential used was –55 mV. B, summary of current density–voltage relations from SERCA1a OE myocytes compared with wild-type controls (P = NS, n refers to numbers of cells).
Outward K+ currents are down-regulated in SERCA1a OE myocytes
In order to further investigate the basis for the cardiac AP prolongation observed in the transgenic animals, we next examined whole-cell outward K+ currents from single free-wall LV myocytes from SERCA1a OE mice compared with the respective littermate controls. SERCA1a OE mice showed a significant down-regulation of the outward K+ currents (Fig. 4). A previous study documented four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes: a rapidly inactivating, transient outward K+ current, Ito,f; a slowly inactivating K+ current, IK,slow; a non-inactivating, steady state current, Iss; and a slowly inactivating transient outward current, Ito,s, which is observed mainly in septal cells (Xu et al. 1999b). The three components of the K+ currents in free-wall ventricular myocytes were distinguished using exponential fits to the decay phases as previously described (Xu et al. 1999a) (Fig. 4C). There were no significant changes in the fast or slow time constants of current decay (f and s). However, the SERCA1a OE mice showed a significant down-regulation of the amplitude of Ito,f, IK,slow and Iss. There were no significant differences in the cell capacitance obtained from the two different groups of animals.
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A, examples of whole-cell outward K+ currents from SERCA1a OE mice compared with wild-type littermates. The holding potential used was –80 mV. B, summary of current density–voltage relations from SERCA1a OE mice compared with wild-type littermates. n = 8 animals for each group, 3–4 myocytes were analysed from each animal (*P < 0.05). C, the decay phases of the transient outward currents (2.5 s) evoked during a depolarizing voltage step to +60 mV were fitted by two exponential decays using the following expressions: y(t) = A1 exp(–t/f) + A2 exp(–t/s) + Ass, where t is time, f and s are the fast and slow time constants, A1 and A2 are the amplitudes of the fast and slow inactivating current components, respectively, and Ass is the amplitude of the steady state, non-inactivating component. For all fits, time zero was set at the peak of the outward current. D and E, summary data for f, s and A1, A2 and Ass components (*P < 0.05).
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Quantitative immunoblot analysis
In order to define the possible mechanisms of changes in the K+ current density observed in the transgenic animals, we quantified the K+ channel protein levels using antibodies specific to Kv4.2 and Kv4.3, the molecular correlates of Ito,f (Guo et al. 1999; Wickenden et al. 1999) as well as antibodies specific to Kv1.5 and Kv2.1, the molecular correlates of IK,slow in mice (Xu et al. 1999a; Nerbonne, 2000; London et al. 2001). Previous studies have shown that the expression of Ito can be significantly modulated by the accessory subunit KChIP with KChIP2 as the main cardiac isoform (An et al. 2000; Kuo et al. 2001). Antibodies specific to KChIP2 were also used in our experiments. Figure 5 shows examples of immunoblots using antibodies directed against Kv4.2 (panels A and B), Kv4.3 (panel C) Kv1.4 (panel D) from the membrane fraction isolated from LV free-wall of transgenic compared with the wild-type littermates. Negative controls were performed with the antibody preincubated with the blocking peptide and labelled as ‘Neg control’ (panel B). Lower bands are GAPDH, which was used as an internal control. Summary data in panel E reveal a significant down-regulation of Kv4.2, Kv4.3, Kv1.5, Kv2.1 and KChIP2 protein expression in SERCA1a OE mice compared with wild-type littermates (*P < 0.05). In contrast, there were no significant differences in the protein expression level for Kv1.4 (panels D and E).
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A, examples of immunoblots with positive labelling of protein bands using an antibody specific to Kv4.2 comparing SERCA1a OE LV free-wall protein samples to the respective littermate control samples. Each lane was loaded with increasing amounts of protein (30, 60 and 120 μg of protein). The expected size of the Kv4.2 protein is 70 kDa. Lower bands in A, C and D are GAPDH (expected size of 27 kDa). B, examples of negative controls for Kv4.2 antibody using ventricular samples from SERCA1a OE mice. C, examples of immunoblots with positive labelling of protein bands using an antibody specific to Kv4.3 (expected size of 66 kDa) comparing SERCA1a OE and wild-type samples. Each lane was loaded with increasing amounts of protein (30, 60 and 120 μg). D, examples of immunoblots with positive labelling of protein bands using an antibody specific to Kv1.4 (expected size of 96 kDa) comparing SERCA1a OE and wild-type samples. Each lane was loaded with increasing amounts of protein (30, 60 and 120 μg) as in A and C. E, summary data comparing SERCA1a OE samples to the respective littermate controls (n = 7 animals for each group, *P = 0.049, 0.04, 0.04, 0.03 and 0.04 for KChIP2, Kv4.2, Kv4.3, Kv2.1 and Kv1.5, respectively, P = NS for Kv1.4).
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In order to further ascertain that the reduction in membrane protein levels of the Kv channels were due to the impairment of the membrane targeting or the reduced protein expression, we repeated the Western blot experiments using whole-tissue homogenates obtained from LV free wall and found a significant reduction in the expression levels of Kv1.5, Kv2.1, Kv4.2, Kv4.3 and KChIP2 in SERCA1a OE mice compared with wild-type littermates. The data obtained using the whole-tissue homogenates were similar to that using membrane fraction. These new findings suggest that the reductions were probably due to reduced protein expression.
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Line-scan images of cardiac myocytes from SERCA1a OE mice compared with wild-type littermates
We next obtained line-scan images from SERCA1a OE myocytes and compared these with the littermate controls to assess the [Ca2+]i transients (measured as F/Fo) as previously described (Gomez et al. 1997). Simultaneous APs were generated via perforated patch pipettes. SERCA1a OE myocytes showed a significant increase in the peak Ca2+i transients (measured as peak F/Fo ratio, n = 11 for both genotypes, P < 0.05, Fig. 6A). This was associated with a rapid decline in the [Ca2+]i transients, consistent with a faster time course of Ca2+ removal in SERCA1a OE myocytes. The decay of Ca2+i transients was fitted using a single exponential function. The time constants are summarized in Fig. 6B (*P < 0.05). The data confirm the previous finding of an increase in the beat-to-beat Ca2+i transient in the SERCA1a OE mice (Lalli et al. 2001).
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A, representative examples of line-scan images in response to stimulated AP showing kinetics of Ca2+i transients recorded from myocytes of SERCA1a OE mice compared with wild-type littermates. Sample records showing line-scan fluorescence image (top), and Ca2+i (measured as F/Fo) (bottom). The fluorescent signal (F) was normalized to the signal before depolarization (Fo). Scale bars represent 20 μm. B, summary data for the peak F/Fo (*P < 0.05). The Ca2+i transient decay was fitted using a single exponential function with time constants (in ms) summarized in the lower panel.
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Effects of changes in intracellular Ca2+ on K+ current in cultured neonatal and adult mouse cardiac myocytes
In order to directly establish the causal relations between changes in Ca2+i and K+ current densities, we recorded outward K+ currents from neonatal mouse cardiac myocytes which had been maintained for 48–72 h in culture media containing 5 mM of Ca2+ or 40 mM of K+ (to depolarize the membrane potential) or BAPTA-AM (10 μM to decrease Ca2+i) as compared with control cells which had been maintained in culture medium containing 1.8 mM Ca2+ and 5 mM K+. We have chosen this in vitro model since we have previously documented in this primary culture system that manipulation of the external Ca2+ concentration or addition of BAPTA-AM can lead to changes in Ca2+i using fura-2 as an indicator (Chiamvimonvat et al. 1995). The experiments were designed to test the effects of Ca2+i using two opposing conditions of increased versus decreased Ca2+ concentrations instead of testing for reversibility in order to try to avoid prolonged culture time which may induce as yet some undefined phenotypic drift. Current density was calculated from the current amplitude normalized by the cell capacitance. To further demonstrate that the results obtained using the neonatal cardiac myocytes were representative of the phenomenon observed in adult animals, we performed additional experiments using adult left ventricular free-wall myocytes in culture and manipulated the external K+ concentration (40 mM K+) for 48 h. The K+ current density recorded from the high [K+] group were then directly compared with that of the control cells kept in culture for 48 h.
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Figure 7 shows examples of the outward K+ current traces from the four different groups of neonatal cardiac myocytes at 48 h. Summary data of the current densities–voltage relations of the peak outward K+ current and the sustained components (labelled in the figure as Ito and ISS, respectively) are shown in Fig. 7B after 2 and 3 days in culture. There was a significant decrease in the peak outward K+ current densities in cells which had been maintained in high Ca2+ or high K+ media after 2 or 3 days in culture compared with control. In contrast, the peak outward K+ current was significantly increased in the BAPTA-AM group compared with control after 3 days in culture. Figure 7C shows comparable data obtained using adult left ventricular free-wall myocytes in culture. There was a significant decrease in the outward K+ current density in the cells maintained in a high external K+ concentration for 48 h compared with control. Of note, the current density shown for control cells in Fig. 7C was smaller than data obtained from wild-type animals shown in Fig. 4 since these cells had been maintained in culture for 48 h while data in Fig. 4 were obtained from freshly isolated ventricular myocytes.
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A, examples of outward K+ current traces recorded from neonatal mouse cardiac myocytes in culture media containing different Ca2+ concentrations; control, BAPTA-AM (10 μM), high Ca2+ (5 mM) or high K+ (40 mM) for 48 h. B, summary data of the peak outward and sustained components from the four different groups of cells (*P < 0.05 compared with control, n = 24–42 cells). C, similar data were obtained using adult left ventricular free-wall myocytes in culture. Upper panels show examples of outward K+ current traces recorded from adult mouse left free-wall ventricular myocytes in culture media containing high K+ (40 mM) compared with control conditions for 48 h. Bottom panel shows summary data of the peak outward and sustained components from the high K+ group compared with control cells in culture at 48 h (*P < 0.05 compared with control, n = 10 cells).
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Discussion
Previous studies have reported a reduction in SERCA2 mRNA and protein levels in failing hearts, resulting in a decrease in Ca2+ accumulation in the SR (Kiss et al. 1995; O'Rourke et al. 1999). We hypothesize that modulation of ion channel expression in cardiac hypertrophy and failure may be closely linked to the known alterations of Ca2+ homeostasis in these conditions. Here, we took advantage of a transgenic mouse model with known pertubations of one of the Ca2+ handling proteins. This allowed us to determine directly the effects of changes in Ca2+ homeostasis on ion channel expression in an in vivo model.
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We observed a significant down-regulation of the outward K+ currents including Ito,f, IK,slow and ISS in SERCA1a OE mice. This was associated with a marked AP prolongation and QT prolongation on surface ECG. We further established that changes in outward K+ current density resulted, at least in part, from alterations in the Kv4.2, Kv4.3, Kv2.1 and Kv1.5 as well as KChIP2 protein expression. Finally, we further documented a significant alteration in the beat-to-beat Ca2+i transient in the mouse model with genetic manipulation of the SERCA pumps. To recapitulate the in vivo experiments, the effects of changes in Ca2+i on ion channel expression were further tested in cultured neonatal and adult mouse cardiac myocytes. Indeed, an increase in [Ca2+]i in neonatal cardiac myocytes resulted in a decrease in both the transient and sustained components of the outward K+ currents after 2–3 days in culture.
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Roles of Ca2+i in modulation of ion channel activity and expression
Transcription of numerous genes has been shown to be regulated by Ca2+i(Carafoli et al. 1999). Ca2+ appears to act in conjunction with calmodulin, activating specific kinases that phosphorylate transcription factors in the nucleus or prior to their entry into the nucleus (Rao et al. 1997; Molkentin et al. 1998; Carafoli et al. 1999). The Ca2+-activated phosphatase calcineurin has been suggested to play a critical role in translating altered Ca2+ signalling into changes in the gene transcriptions linked to the development of cardiac hypertrophy and failure (Molkentin et al. 1998). Upon activation, calcineurin dephosphorylates the nuclear factor of activated T cells (NFAT). Recently, it was shown using NFATc3 knockout and NFAT reporter mice that the calcineurin/NFATc3 pathway plays an important role in mediating the down-regulation of K+ channels expression, including Kv1.5, Kv2.1, Kv4.2, and Kv4.3, associated with myocardial infarction or adrenergic stimulation (Rossow et al. 2004). It is possible that the calcineurin/NFAT pathway is activated by the increased intracellular Ca2+ and mediates the down-regulation of the K+ channel expression in SERCA1a OE mice. Additional signalling factors which may be involved include rennin–angiotensin, protein kinase A and C, MAP kinase, calmodulin-dependent (CAM) kinase, as well as adrenergic pathways (Packer, 1992; Colucci & Braunwald, 1997; Molkentin et al. 1998). In the SERCA1a OE model in particular, cardiac hypertrophy and failure was not observed suggesting that the down-regulation in the K+ channel expression was not secondary to the cardiac hypertrophy or failure but rather occur as a result of the signalling pathways which are activated by intracellular Ca2+. The
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change of Ca2+ signalling responsible for the down-regulation of K+ channels in ventricular myocytes in SERCA1a OE mice may involve an increase in the peak magnitude of the Ca2+ transient or the enhanced spontaneous Ca2+ sparks (or Ca2+ leak) due to SR Ca2+ overload. Future studies will be required to completely explain the signalling pathways involved in the down-regulation of K+ channel expression in the setting of increase intracellular Ca2+.
Indeed, cytosolic Ca2+ has also been shown to play a critical role in the regulation of biosynthesis of ionic channels, such as the nicotinic acetylcholine receptor (Shainberg et al. 1987; Klarsfeld et al. 1989), L-type Ca2+ channel expression (Renganathan et al. 1999), and Na+ channel biosynthesis (Sherman & Catterall, 1984; Sherman et al. 1985; Offord & Catterall, 1989; Taouis et al. 1991; Duff et al. 1992; Chiamvimonvat et al. 1995). Our data agree well with these previous studies that expression of ion channels in cardiac myocytes may be regulated by Ca2+i; however, further work is needed to address whether the significant changes in K+ channel expression in these mouse models could be correlated with either a change in peak, diastolic or total Ca2+i. Furthermore, in SERCA1a OE cardiac myocytes, the ATP consumption would be increased as a result of the enhanced ATPase activity, which might be involved in the down-regulation of K+ channels.
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Down-regulation of one or more of the underlying outward K+ currents has been documented in a variety of heart failure models including human heart failure (Beuckelmann et al. 1993; Nbauer et al. 1993; Tomita et al. 1994; Kb et al. 1996, 1998). The mechanistic basis of the down-regulation of Ito in human heart failure appears to be a decrease in the transcription of the Kv4.3 gene (Kb et al. 1998). In addition, significant electrical remodelling has been documented in animal models of atrial fibrillation as well as in humans (Gaspo et al. 1997; Van Wagoner et al. 1999; Yue et al. 1999). Several lines of evidence point to a role of intracellular Ca2+ overload (Nattel, 1999), e.g. histological changes compatible with Ca2+ overload-induced injury (Goette et al. 1996). In addition, Ca2+ channel blockers appeared to suppress the electrical remodelling in rapid-pacing-induced atrial fibrillation (Daoud et al. 1997). An increase in the frequency of depolarization may increase Ca2+ entry and hence an increase in the time-averaged Ca2+i level.
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The present report suggests the important finding that changes in K+ channel expression can follow alterations in calcium homeostasis, prior to cardiac hypertrophy and failure. On the other hand, other reports have suggested that inhibition of K+ currents can serve as a triggering event in cardiac hypertrophy as a result of the disturbances in calcium homeostasis following AP prolongation (Wickenden et al. 1999; Kassiri et al. 2002). However, this issue remains controversial since another mouse model with a prolonged cardiac AP did not show evidence of cardiac hypertrophy (Barry et al. 1998). Furthermore, no evidence of cardiac hypertrophy was documented in patients with long QT syndrome and prolonged cardiac repolarization (Nador et al. 1991).
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The SR Ca2+ overload, caused by the enhanced SERCA activity, and the prolongation of the QT interval, as observed in SERCA1a OE mice, is expected to increase the propensity to arrhythmia in SERCA1a OE mice. However, these mice do not exhibit an increased mortality compared with the littermate controls. It is likely that cardiac arrhythmias may only be uncovered during stress e.g. with programmed stimulation during electrophysiological studies or with adrenergic stimulation.
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Finally, recent studies using SERCA gene transfer both in vivo and in vitro have provided compelling evidence that SERCA gene transfer can be effectively used to enhance cardiac contractility and rescue the contractile function in failing myocardium (Miyamoto et al. 2000). Indeed, adenoviral-mediated gene therapy using the SERCA pump has been proposed as a therapeutic option for heart failure (Hajjar et al. 2000). Our present data show that a primary alteration in this critical Ca2+ handling protein, without cardiac hypertrophy and failure, can produce profound changes in K+ channel expression and cardiac AP profile. Taken together, our data suggest that a coexpression of the SERCA pump as well as K+ channels are likely to be necessary to rescue the full phenotype of cardiac failure (Ennis et al. 2002). This, in fact, will have important therapeutic implications.
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