HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 562/3/745    most recent jphysiol.2004.076216v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xu, Y. Articles by Chiamvimonvat, N. Search for Related Content PubMed PubMed Citation Articles by Xu, Y. Articles by Chiamvimonvat, N.

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
¹ Pharmacology Department, Hebei Medical University Shijiazhuang, China

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
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 (I_to,f), a slowly inactivating K⁺ current (I_K,slow) and the steady state current (I_SS) 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.

(Received 28 September 2004; accepted after revision 19 November 2004; first published online 25 November 2004)
Corresponding author N. Chiamvimonvat: Division of Cardiovascular Medicine, University of California, Davis, Genome and Biomedical Sciences Facility, Rm 6315, 451 East Health Sciences Drive, Davis, CA 95616, USA. Email: nchiamvimonvat{at}ucdavis.edu

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
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.

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).

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.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
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.

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 (QT_c) 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 QT_c interval was defined as the QT interval (in ms)/(RR/100)^1/2 (Mitchell et al. 1998).

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.

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).

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.

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.

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.

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.

The following primary antibodies were used: (1) anti-K_v4.2, (2) anti-K_v4.3, (3) anti-K_v1.5, (4) anti-K_v2.1 and (5) anti-K_v1.4 (Alomone Laboratories, Israel), (6) anti-KChIP2 (K_v4 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.

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/F_o), where F_o refers to the fluorescence level before depolarization as described (Harkins et al. 1993).

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

Top Abstract Introduction Methods Results Discussion References

 
QT intervals are prolonged in SERCA1a OE mice

Figure 1A shows significant prolongation of the QT_c intervals in SERCA1a OE mice compared with wild-type littermates. However, heart rate (RR interval) and PR interval were unaffected (Fig. 1B).

View larger version (17K): [in this window] [in a new window]   Figure 1.  QT intervals are prolonged in SERCA1a OE mice 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.

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).

View larger version (18K): [in this window] [in a new window]   Figure 2.  Action potentials are prolonged in SERCA1a OE mice 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.

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).

View larger version (14K): [in this window] [in a new window]   Figure 3.  L-type Ca2+ currents are not altered in SERCA1a OE mice 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).

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, I_to,f; a slowly inactivating K⁺ current, I_K,slow; a non-inactivating, steady state current, I_ss; and a slowly inactivating transient outward current, I_to,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 I_to,f, I_K,slow and I_ss. There were no significant differences in the cell capacitance obtained from the two different groups of animals.

View larger version (21K): [in this window] [in a new window]   Figure 4.  Down-regulation of outward K+ current in SERCA1a mice 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).

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 K_v4.2 and K_v4.3, the molecular correlates of I_to,f (Guo et al. 1999; Wickenden et al. 1999) as well as antibodies specific to K_v1.5 and K_v2.1, the molecular correlates of I_K,slow in mice (Xu et al. 1999a; Nerbonne, 2000; London et al. 2001). Previous studies have shown that the expression of I_to 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 K_v4.2 (panels A and B), K_v4.3 (panel C) K_v1.4 (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 K_v4.2, K_v4.3, K_v1.5, K_v2.1 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 K_v1.4 (panels D and E).

View larger version (30K): [in this window] [in a new window]   Figure 5.  Quantitative immunoblot analysis 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).

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/F_o) 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/F_o 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).

View larger version (29K): [in this window] [in a new window]   Figure 6.  Line-scan images of cardiac myocytes obtained from SERCA1a OE mice compared with wild-type littermates 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.

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.

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 I_to and I_SS, 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.

View larger version (52K): [in this window] [in a new window]   Figure 7.  Effects of changes in [Ca2+]i on outward K+ currents in neonatal mouse cardiac myocytes in culture 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).

	Discussion

Top Abstract Introduction Methods Results Discussion References

We observed a significant down-regulation of the outward K⁺ currents including I_to,f, I_K,slow and I_SS 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 K_v4.2, K_v4.3, K_v2.1 and K_v1.5 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.

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 K_v1.5, K_v2.1, K_v4.2, and K_v4.3, associated with myocardial infarction or ß-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

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.

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; Näbauer et al. 1993; Tomita et al. 1994; Kääb et al. 1996, 1998). The mechanistic basis of the down-regulation of I_to in human heart failure appears to be a decrease in the transcription of the K_v4.3 gene (Kääb 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.

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).

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 ß-adrenergic stimulation.

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.

	References

Top Abstract Introduction Methods Results Discussion References

 
Ahmmed GU, Dong PH, Song G, Ball NA, Xu Y, Walsh RA & Chiamvimonvat N (2000). Changes in Ca²⁺ cycling proteins underlie cardiac action potential prolongation in a pressure-overloaded guinea pig model with cardiac hypertrophy and failure. Circ Res 86, 558–570.[Abstract/Free Full Text]

Ahmmed GU, Xu Y, Hong Dong P, Zhang Z, Eiserich J & Chiamvimonvat N (2001). Nitric oxide modulates cardiac Na⁺ channel via protein kinase A and protein kinase G. Circ Res 89, 1005–1013.[Abstract/Free Full Text]

An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS & Rhodes KJ (2000). Modulation of A-type potassium channels by a family of calcium sensors. Nature 403, 553–556.[CrossRef][Medline]

Anger M, Samuel JL, Marotte F, Wuytack F, Rappaport L & Lompre AM (1994). In situ mRNA distribution of sarco (endo) plasmic reticulum Ca²⁺-ATPase isoforms during ontogeny in the rat. J Mol Cell Cardiol 26, 539–550.[CrossRef][Medline]

Arai M, Matsui H & Periasamy M (1994). Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ Res 74, 555–564.[Free Full Text]

Bading H, Ginty DD & Greenberg ME (1993). Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science 260, 181–186.[Abstract/Free Full Text]

Barry DM, Xu H, Schuessler RB & Nerbonne JM (1998). Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative K_v4 alpha subunit. Circ Res 83, 560–567.[Abstract/Free Full Text]

Beuckelmann D, Näbauer M & Erdmann E (1993). Alterations of K⁺ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ Res 73, 379–385.[Abstract/Free Full Text]

Brandl CJ, Green NM, Korczak B & MacLennan DH (1986). Two Ca²⁺ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell 44, 597–607.[CrossRef][Medline]

Carafoli E, Genazzani A & Guerini D (1999). Calcium controls the transcription of its own transporters and channels in developing neurons. Biochem Biophys Res Commun 266, 624–632.[CrossRef][Medline]

Chiamvimonvat N, Kargacin M, Clark R & Duff H (1995). Effects of intracellular calcium on sodium current density in cultured neonatal rat cardiac myocytes. J Physiol 483, 307–318.[Medline]

Colucci WS & Braunwald E (1997). Pathophysiology of heart failure. In Heart Disease, a Textbook of Cardiovascular Medicine, 5th edn, ed. Braunwald E, pp. 394–420. W.B. Saunders, Philadelphia, PA.

Daoud EG, Knight BP, Weiss R, Bahu M, Paladino W, Goyal R, Man KC, Strickberger SA & Morady F (1997). Effect of verapamil and procainamide on atrial fibrillation-induced electrical remodeling in humans. Circulation 96, 1542–1550.[Abstract/Free Full Text]

Duff HJ, Offord J, West J & Catterall WA (1992). Class I and IV antiarrhythmic drugs and cytosolic calcium regulate mRNA encoding the sodium channel alpha subunit in rat cardiac muscle. Mol Pharmacol 42, 570–574.[Abstract]

Ennis IL, Li RA, Murphy AM, Marban E & Nuss HB (2002). Dual gene therapy with SERCA1 and Kir2.1 abbreviates excitation without suppressing contractility. J Clin Invest 109, 393–400.[CrossRef][Medline]

Gaspo R, Bosch RF, Bou-Abboud E & Nattel S (1997). Tachycardia-induced changes in Na⁺ current in a chronic dog model of atrial fibrillation. Circ Res 81, 1045–1052.[Abstract/Free Full Text]

Goette A, Honeycutt C & Langberg JJ (1996). Electrical remodeling in atrial fibrillation. Time course and mechanisms. Circulation 94, 2968–2974.[Abstract/Free Full Text]

Gomez AM, Valdivia HH, Cheng H, Lederer MR, Santana LF, Cannell MB, McCune SA, Altschuld RA & Lederer WJ (1997). Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure. Science 276, 800–806.[Abstract/Free Full Text]

Gunteski-Hamblin AM, Greeb J & Shull GE (1988). A novel Ca²⁺ pump expressed in brain, kidney, and stomach is encoded by an alternative transcript of the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase gene. Identification of cDNAs encoding Ca²⁺ and other cation-transporting ATPases using an oligonucleotide probe derived from the ATP-binding site. J Biol Chem 263, 15032–15040.[Abstract/Free Full Text]

Guo W, Xu H, London B & Nerbonne JM (1999). Molecular basis of transient outward K⁺ current diversity in mouse ventricular myocytes. J Physiol 521, 587–599.[Abstract/Free Full Text]

Hajjar RJ, del Monte F, Matsui T & Rosenzweig A (2000). Prospects for gene therapy for heart failure. Circ Res 86, 616–621.[Abstract/Free Full Text]

Hamill O, Marty A, Neher E, Sakmann B & Sigworth F (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391, 85–100.[CrossRef][Medline]

Hardingham GE, Chawla S, Johnson CM & Bading H (1997). Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression. Nature 385, 260–265.[CrossRef][Medline]

Harkins AB, Kurebayashi N & Baylor SM (1993). Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3. Biophys J 65, 865–881.[Abstract/Free Full Text]

Kääb S, Dixon J, Duc J, Ashen D, Nabauer M, Beuckelmann DJ, Steinbeck G, McKinnon D & Tomaselli GF (1998). Molecular basis of transient outward potassium current downregulation in human heart failure: a decrease in K_v3 mRNA correlates with a reduction in current density. Circulation 98, 1383–1393.[Abstract/Free Full Text]

Kääb S, Nuss H, Chiamvimonvat N, O'Rourke B, Pak P, Kass D, Marban E & Tomaselli G (1996). Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ Res 78, 262–273.[Abstract/Free Full Text]

Kassiri Z, Zobel C, Nguyen TT, Molkentin JD & Backx PH (2002). Reduction of I_to causes hypertrophy in neonatal rat ventricular myocytes. Circ Res 90, 578–585.[Abstract/Free Full Text]

Kiss E, Ball N, Kranias E & Walsh R (1995). Differential changes in cardiac phospholamban and sarcoplasmic reticular Ca²⁺-ATPase protein levels: effects on Ca²⁺ transport and mechanics in compensated pressure overload hypertrophy and congestive heart failure. Circ Res 77, 759–764.[Abstract/Free Full Text]

Klarsfeld A, Laufer R, Fontaine B, Devillers-Thiery A, Dubreuil C & Changeux JP (1989). Regulation of muscle AChR alpha subunit gene expression by electrical activity: involvement of protein kinase C and Ca²⁺. Neuron 2, 1229–1236.[CrossRef][Medline]

Kuo HC, Cheng CF, Clark RB, Lin JJ, Lin JL, Hoshijima M, Nguyen-Tran VT, Gu Y, Ikeda Y, Chu PH, Ross J, Giles WR & Chien KR (2001). A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I_to and confers susceptibility to ventricular tachycardia. Cell 107, 801–813.[CrossRef][Medline]

Lalli MJ, Yong J, Prasad V, Hashimoto K, Plank D, Babu GJ, Kirkpatrick D, Walsh RA, Sussman M, Yatani A, Marban E & Periasamy M (2001). Sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca²⁺ handling capacity. Circ Res 89, 160–167.[Abstract/Free Full Text]

London B, Guo W, Pan X, Lee JS, Shusterman V, Rocco CJ, Logothetis DA, Nerbonne JM & Hill JA (2001). Targeted replacement of K_V5 in the mouse leads to loss of the 4-aminopyridine-sensitive component of I_K,slow and resistance to drug-induced qt prolongation. Circ Res 88, 940–946.[Abstract/Free Full Text]

Loukianov E, Ji Y, Grupp IL, Kirkpatrick DL, Baker DL, Loukianova T, Grupp G, Lytton J, Walsh RA & Periasamy M (1998). Enhanced myocardial contractility and increased Ca²⁺ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca²⁺-ATPase. Circ Res 83, 889–897.[Abstract/Free Full Text]

Mitchell GF, Jeron A & Koren G (1998). Measurement of heart rate and Q-T interval in the conscious mouse. Am J Physiol 274, H747–751.[Medline]

Miyamoto MI, del Monte F, Schmidt U, DiSalvo TS, Kang ZB, Matsui T, Guerrero JL, Gwathmey JK, Rosenzweig A & Hajjar RJ (2000). Adenoviral gene transfer of SERCA2a improves left-ventricular function in aortic-banded rats in transition to heart failure. Proc Natl Acad Sci U S A 97, 793–798.[Abstract/Free Full Text]

Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR & Olson EN (1998). A calcineurin dependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215–228.[CrossRef][Medline]

Näbauer M, Beuckelmann D & Erdmann E (1993). Characteristics of transient outward current in human ventricular myocytes from patients with terminal heart failure. Circ Res 73, 386–394.[Abstract/Free Full Text]

Nador F, Beria G, De Ferrari GM, Stramba-Badiale M, Locati EH, Lotto A & Schwartz PJ (1991). Unsuspected echocardiographic abnormality in the long QT syndrome. Diagnostic, prognostic, and pathogenetic implications. Circulation 84, 1530–1542.[Abstract/Free Full Text]

Nattel S (1999). Electrophysiologic remodeling: are ion channels static players or dynamic movers? J Cardiovasc Electrophysiol 10, 1553–1556.[Medline]

Nerbonne JM (2000). Molecular basis of functional voltage-gated K⁺ channel diversity in the mammalian myocardium. J Physiol 525, 285–298.[Abstract/Free Full Text]

Nuss HB & Marban E (1994). Electrophysiological properties of neonatal mouse cardiac myocytes in primary culture. J Physiol 479, 265–279.[Medline]

Offord J & Catterall WA (1989). Electrical activity, cAMP, and cytosolic calcium regulate mRNA encoding sodium channel alpha subunits in rat muscle cells. Neuron 2, 1447–1452.[CrossRef][Medline]

O'Rourke B, Kass DA, Tomaselli GF, Kaab S, Tunin R & Marban E (1999). Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies. Circ Res 84, 562–570.[Abstract/Free Full Text]

Packer M (1992). The neurohormonal hypothesis: a theory to explain the mechanism of disease progression in heart failure. J Am Coll Cardiol 20, 248–254.[Abstract]

Rao A, Luo C & Hogan PG (1997). Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15, 707–747.[CrossRef][Medline]

Renganathan M, Wang ZM, Messi ML & Delbono O (1999). Calcium regulates L-type Ca²⁺ channel expression in rat skeletal muscle cells. Pflugers Arch 438, 649–655.[CrossRef][Medline]

Rossow CF, Minami E, Chase EG, Murry CE & Santana LF (2004). NFATc3-induced reductions in voltage-gated K⁺ currents after myocardial infarction. Circ Res 94, 1340–1350.[Abstract/Free Full Text]

Shainberg A, Freud-Silverberg M & Brik H (1987). Changes in the levels of acetylcholine receptors mediated by calcium concentration in the sarcoplasmic reticulum. Prog Clin Biol Res 253, 303–314.[Medline]

Sherman SJ & Catterall WA (1984). Electrical activity and cytosolic calcium regulate levels of tetrodotoxin-sensitive sodium channels in cultured rat muscle cells. Proc Natl Acad Sci U S A 81, 262–266.[Abstract/Free Full Text]

Sherman SJ, Chrivia J & Catterall WA (1985). Cyclic adenosine 3':5'-monophosphate and cytosolic calcium exert opposing effects on biosynthesis of tetrodotoxin-sensitive sodium channels in rat muscle cells. J Neurosci 5, 1570–1576.[Abstract]

Taouis M, Sheldon RS & Duff HJ (1991). Upregulation of the rat cardiac sodium channel by in vivo treatment with a class I antiarrhythmic drug. J Clin Invest 88, 375–378.[Medline]

Tomita F, Bassett A, Myerburg R & Kimura S (1994). Diminished transient outward currents in rat hypertrophied ventricular myocytes. Circ Res 75, 296–303.[Abstract/Free Full Text]

Van Wagoner DR, Pond AL, Lamorgese M, Rossie SS, McCarthy PM & Nerbonne JM (1999). Atrial L-type Ca²⁺ currents and human atrial fibrillation. Circ Res 85, 428–436.[Abstract/Free Full Text]

Wang SQ, Song LS, Lakatta EG & Cheng H (2001). Ca²⁺ signalling between single L-type Ca²⁺ channels and ryanodine receptors in heart cells. Nature 410, 592–596.[CrossRef][Medline]

Wickenden AD, Lee P, Sah R, Huang Q, Fishman GI & Backx PH (1999). Targeted expression of a dominant-negative K_v2 K⁺ channel subunit in the mouse heart. Circ Res 85, 1067–1076.[Abstract/Free Full Text]

Xu H, Barry DM, Li H, Brunet S, Guo W & Nerbonne JM (1999a). Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative K_v2 alpha subunit. Circ Res 85, 623–633.[Abstract/Free Full Text]

Xu H, Guo W & Nerbonne JM (1999b). Four kinetically distinct depolarization-activated K⁺ currents in adult mouse ventricular myocytes. J General Physiol 113, 661–678.[Abstract/Free Full Text]

Xu Y, Tuteja D, Zhang Z, Xu D, Zhang Y, Rodriguez J, Nie L, Tuxson HR, Young JN, Glatter KA, Vazquez AE, Yamoah EN & Chiamvimonvat N (2003). Molecular identification and functional roles of a Ca²⁺-activated K⁺ channel in human and mouse hearts. J Biol Chem 278, 49085–49094.[Abstract/Free Full Text]

Yue L, Melnyk P, Gaspo R, Wang Z & Nattel S (1999). Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation. Circ Res 84, 776–784.[Abstract/Free Full Text]

Zarain-Herzberg A, MacLannan DH & Periasamy M (1990). Characterization of rabbit cardiac sarco (endo) plasmic reticulum Ca²⁺ ATPase gene. J Biol Chem 265, 4670–4677.[Abstract/Free Full Text]

Zhang Z, Xu Y, Song H, Rodriguez J, Tuteja D, Namkung Y, Shin HS & Chiamvimonvat N (2002). Functional roles of Ca_v3 (alpha_1D) calcium channel in sinoatrial nodes: insight gained using gene-targeted null mutant mice. Circ Res 90, 981–987.[Abstract/Free Full Text]

	Acknowledgements

 
The authors thank Dr Ebenezer N. Yamoah for helpful discussions and the UC Davis Health System Confocal Microscopy Facility. This work was supported by the Department of Veteran Affairs Merit Review Grant and the National Institutes of Health Grants (HL68507, HL67737) to N.C.

This article has been cited by other articles:

				G. J. Babu, P. Bhupathy, V. Timofeyev, N. N. Petrashevskaya, P. J. Reiser, N. Chiamvimonvat, and M. Periasamy Ablation of sarcolipin enhances sarcoplasmic reticulum calcium transport and atrial contractility PNAS, November 6, 2007; 104(45): 17867 - 17872. [Abstract] [Full Text] [PDF]

				N. Hiranandani, S. Raman, A. Kalyanasundaram, M. Periasamy, and P. M. L. Janssen Frequency-dependent contractile strength in mice over- and underexpressing the sarco(endo)plasmic reticulum calcium-ATPase Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R30 - R36. [Abstract] [Full Text] [PDF]

				W. P. Dirksen, V. A. Lacombe, M. Chi, A. Kalyanasundaram, S. Viatchenko-Karpinski, D. Terentyev, Z. Zhou, S. Vedamoorthyrao, N. Li, N. Chiamvimonvat, et al. A mutation in calsequestrin, CASQ2D307H, impairs Sarcoplasmic Reticulum Ca2+ handling and causes complex ventricular arrhythmias in mice Cardiovasc Res, July 1, 2007; 75(1): 69 - 78. [Abstract] [Full Text] [PDF]

				C. Pott, X. Ren, D. X. Tran, M.-J. Yang, S. Henderson, M. C. Jordan, K. P. Roos, A. Garfinkel, K. D. Philipson, and J. I. Goldhaber Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice Am J Physiol Cell Physiol, February 1, 2007; 292(2): C968 - C973. [Abstract] [Full Text] [PDF]