Calcium influx through long-lasting (“L-type”) Ca2+ channels (CaV) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca2+ overload, and interventions that reduce expression of the pore-forming α1 subunit may alleviate cytosolic Ca2+ excess. As a molecular approach to disrupt the assembly of CaV1.2 (α1C) channels at the cell membrane, we targeted the Ca2+ channel β2 subunit, an intracellular chaperone that interacts with α1C via its β interaction domain (BID) to promote CaV1.2 channel expression. Recombinant adenovirus expressing either the full β2 subunit (Full-β2) or truncated β2 subunit constructs lacking either the C terminus, N terminus, or both (N-BID, C-BID, and BID, respectively) fused to green fluorescent protein were developed as potential decoys and overexpressed in HL-1 cells. Fluorescence microscopy revealed that the localization of Full-β2 at the surface membrane was associated with increased Ca2+ current mainly attributed to CaV1.2 channels. In contrast, truncated N-BID and C-BID constructs showed punctate intracellular expression, and BID showed a diffuse cytosolic distribution. Total expression of the α1C protein of CaV1.2 channels was similar between groups, but HL-1 cells overexpressing C-BID and BID exhibited reduced Ca2+ current. C-BID and BID also attenuated Ca2+ current associated with another L-type Ca2+ channel, CaV1.3, but they did not reduce transient Ca2+ currents attributed to CaV3 channels. These results suggest that β2 subunit mutants lacking the N terminus may preferentially disrupt the proper localization of L-type Ca2+ channels in the cell membrane. Cardiac-specific delivery of these decoy molecules in vivo may represent a gene-based treatment for pathologies involving Ca2+ overload.
The predominant voltage-gated Ca2+ channels (CaV) in cardiac cells are the dihydropyridine-sensitive CaV1.2 (α1C) channels that mediate long-lasting (“L-type”) Ca2+ current as a critical step in excitation-contraction coupling (Bers, 2002). The CaV1.2 channels are multiprotein complexes composed of a large, pore-forming α1C subunit and accessory β and α2-δ subunits. The expression level of functional CaV1.2 channels is regulated by the β subunits (β1, β2, β3, and β4), which are cytoplasmic proteins encoded by four different genes, including multiple splice variants (Foell et al., 2004). The β2 subunit is predominantly expressed in rat ventricular myocytes, although other gene families have been reported. It is noteworthy that β2 acts as a molecular chaperone that escorts α1C to the sarcolemma to localize functional CaV1.2 channels at the surface membrane (Pragnell et al., 1994; De Waard et al., 1996; Opatowsky et al., 2003). Indeed, we recently reported that overexpression of β2 in isolated feline ventricular myocytes significantly increases Ca2+ influx and cardiac contractility (Chen et al., 2005). Although mutations in the β interaction domain (BID) of β2 were found to abolish α1C-β interaction (De Waard et al., 1994, 1996), two additional protein-interaction domains (SH3 and GK) were recently identified that also may interact with α1C subunit (Chen et al., 2004; Opatowsky et al., 2004; Van Petegem et al., 2004). Thus, it has been suggested that BID may not bind directly to α1C, but instead it is fundamentally essential for the structural integrity and bridging of the SH3 and GK domains.
Although physiologically essential to normal cardiac function, CaV1.2 channels also have been implicated in pathological processes involving Ca2+ overload, including cardiac ischemia and myocardial stunning. In these conditions, an elevated cytosolic Ca2+ ([Ca2+]cyt) in the myocardial cells is associated with a loss of myofilament sensitivity to Ca2+ (Bolli and Marban, 1999). Moreover, Ca2+ overload also plays an important role in cardiac apoptosis and necrosis (Aon et al., 2003). Even under conditions in which [Ca2+]cyt is within a normal range, it may be desirable to modestly depress [Ca2+]cyt as a therapeutic intervention to slow atrioventricular conduction in atrial fibrillation or to modestly depress contractility in hypertrophic cardiomyopathy. Currently, calcium channel blockers represent the drug therapies of choice for these conditions, but several features of their properties, including their short duration of action and vascular dilator effects, limit their usefulness.
In this respect, a gene therapy approach designed to reduce the availability of β2 subunits required for the expression of CaV1.2 channels may provide for the highly targeted and long-term relief of calcium overload. Accordingly, we engineered recombinant adenoviral constructs expressing truncated β2 to provide proof of concept that mutated β2 could potentially act as a dominant-negative construct to reduce CaV1.2 channels at the surface membrane. In the present study, we expressed the recombinant adenovirus constructs in HL-1 cells, a cell line derived from AT-1 mouse atrial tumor lineage that displays many normal markers of cardiac biology. HL-1 cells spontaneously depolarize and express ion channels required for generating action potentials (White et al., 2004), and they retain the signaling pathways to respond appropriately to inotropic and chronotropic agonists (Claycomb et al., 1998). The activity of the truncated β2 constructs was compared with the effect of full-length β2 (Full-β2), which we have shown enhances the expression of functional CaV1.2 channels in feline cardiac myocytes (Chen et al., 2005). The goal was to retain the ability of the β2 decoys to bind to α1C but to eliminate the amino and/or carboxyl termini of β2 that may be required for the expression of functional α1C subunits in the plasma membrane, thereby “trapping” CaV1.2 channels inside the cell. We also evaluated the effect of the β2 subunit mutants on Ca2+ current attributed to two other types of Ca2+ channels important in pacemaker activity or contractile function. The CaV1.3 channels (α1D) mediate L-type current at mid-voltages, and they are approximately 10-fold less sensitive to 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (nifedipine)-induced block than CaV1.2 channels at negative membrane potentials (Koschak et al., 2001). HL-1 cells also express CaV3 channels that activate at much lower voltages than L-type channels, and they mediate transient (“T-type”) current associated with normal pacemaker activity in cardiac cells (Xia et al., 2004; Zhang et al., 2005).
Materials and Methods
Culture of HL-1 Cells. HL-1 cells (kindly provided by Dr. W. C. Claycomb, Louisiana State University, Baton Rouge, LA) were grown and maintained in Claycomb medium (SAFC Biosciences, Lenexa, KS) supplemented with penicillin-streptomycin (1×) and 2 mM l-glutamine (Invitrogen, Carlsbad, CA). After trypsinization, dissociated cells were either plated in standard 60-mm tissue culture dishes (0.8–1.0 × 106 cells/ml) for mRNA and/or protein expression assays or in glass culture dishes (MatTek, Ashland, MA) for electrophysiology (60–80 × 103 cells/ml). Cells were then infected for 24 to 48 h with one of four different GFP-fused β2 subunit constructs, including Full-β2, or truncated β2 lacking either the N terminus (C-BID), the C terminus (N-BID), or both termini (BID only) or with the control GFP construct. The optimal dose of crude viral stock (multiplicity of infection, 50–100) needed to optimize protein expression without visible cell death was determined by fluorescent imaging and immunoblot techniques. Experimental conditions were developed that produced >95% infection of HL-1 cells for each of the constructs studied, as described previously (Fan et al., 2003; Telemaque-Potts et al., 2003; Chen et al., 2005). All studies on HL-1 cells were performed between 24 and 48 h after transfection.
Detection of CaL Channel Subunit Gene Expression in HL-1 Cells. To identify the endogenous β subunits expressed in HL-1 cells, total RNA was isolated from untreated HL-1 cells using a TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). Primers for PCR reactions were designed to detect CaV1.2 (α1C) and different β subunit isoforms (β1, β2a, β2b, β3, and β4). Sense and antisense primers were as follows: α1C:5′-CCGCCCACTACCAAGATCAAC-3′, 5′-TCGTGTCATTGACAATGCGG-3′, covering cDNA bases 2536 to 2791 (256 bp; GenBank accession number NM_012517.1 [rat]); β1:5′-TCCAGAAGAGCGGCATGTCCC-3′, 5′-AGGACGTACTCCCGTCTGAC-3′, covering cDNA bases 5 to 140 (136 bp; GenBank accession number NM_031173.2 [mouse]); β2a: 5′-CGAGTACGGGTGTCCTATG, 5′-CGTCCTATCCACCAGTCAT-3′, covering cDNA bases 31 to 320 (290 bp; GenBank accession number NM_053851.1 [rat]); β2b: 5′-AGGCAGTTGGTGTCTTCTC-3′, 5′-CGTCCTATCCACCAGTCAT-3′, covering cDNA bases 10 to 323 (314 bp; GenBank accession number AF423193 [rat]); β3:5′-CATCCCTGGACTTCAGAAC-3′, 5′-TGGTAGGCATCTGCATAGTC-3′, covering cDNA bases 1125 to 1301 (177 bp; GenBank accession number NM_007581.2 [mouse]); and β4:5′-CTCACCATATCCCACAGCAA-3′, 5′-TCCGGGTAATCTTCTTCCACCA-3′, covering cDNA bases 1266 to 1478 (213 bp; GenBank accession number NM_001037099.1 [mouse]).
Reactions contained 2 μl of cDNA, 10 pmol of each primer, 0.3 mM dNTPs, 1.0 mM MgSO4, and 0.5 units of Pfx DNA polymerase (Invitrogen) mixed with 1× enhancer buffer in a total volume of 25 μl. Amplification steps included initial denaturation at 94°C for 3 min, followed by 30 cycles (denaturation for 30 s at 94°C; annealing 30 s at 53°C; extension 30 s at 68°C). A final step for product extension was performed at 68°C for 5 min. The PCR products (5 μl) were separated using a 1.2% agarose gel stained with ethidium bromide for visualization. All oligonucleotide sequences were directed toward the mouse/rat sequences of the Ca2+ channel subunits.
Generation of β2 Subunit Constructs. A plasmid encoding the rat heart/brain β2a subunit cDNA, a generous gift from Dr. E. Perez-Reyes (Perez-Reyes et al., 1992), was used as a template for generating three PCR-based mutations of the β2a subunit. In brief, cDNA encoding the N-BID fragment (bp 376-1177), BID fragment (bp 867–1177), and C-BID fragment (bp 867-2427) were amplified, digested, and directionally cloned into pEGFP-C1 vector (Clontech, Mountain View, CA), an expression vector driven by a cytomegalovirus (CMV) promoter. All β2 subunit constructs were cloned in frame with an enhanced GFP reporter gene located at the amino-terminal end of the construct. A schematic representation of the various β2 subunit adenoviral constructs is illustrated in Fig. 1.
Preparation of Recombinant Adenovirus Construct. Recombinant adenovirus constructs were prepared based on the methods described by He et al. (1998) and made commercially available (AdEASY) by Stratagene (La Jolla, CA). The CMV-GFP-β subunit fragments were gel-purified and subcloned into the pShuttle vector. Then, it was linearized and cotransformed into Escherichia coli BJ5183 cells with pAdEasy-1, the adenoviral backbone. A CMV-GFP lacking any β subunit construct also was subcloned into a pShuttle vector to construct a control virus (Fan et al., 2003). Recombinants were then linearized and transfected into human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Manassas, VA) using FuGENE 6 reagent (Roche Molecular Systems, Alameda, CA). Virus-containing medium from HEK 293-infected cells was collected 3 to 6 days after infection, and proper expression of GFP-fused β subunits was confirmed by PCR and immunodetection. High-titer viral stock was generated by several rounds of amplification in HEK 293 cells and by purification by cesium chloride gradient ultracentrifugation (Niranjan et al., 1996; Telemaque-Potts et al., 2003). Final viral stock titer was determined by plaque assay (1–3 × 108 plaque-forming unit/μl).
Immunodetection. Forty-eight hours after infection, cells were washed twice with ice-cold phosphate-buffered saline (PBS), and then they were lysed in Triton lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 1% Triton X-100, 1% glycerol, 1× Sigma phosphatase inhibitor cocktail, and 1× Sigma protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Cell lysates were incubated on ice for 15 to 20 min and centrifuged at 4°C for 15 min at 14,000g. The supernatants were then collected. Protein concentration in supernatants was measured by the Bradford method (Bio-Rad, Hercules, CA). Equal protein samples (40 μg of total protein) were loaded on 3 to 8% NuPAGE Tris acetate gels (Invitrogen), and they were run and transferred to a polyvinylidene fluoride membrane. After blocking the membrane (Tris-buffered saline solution containing 10% nonfat dry milk and 0.1% Tween 20, immunoblotting was performed using mouse α1C monoclonal antibody (1:200; NeuroMabs, Davis, CA), mouse β-actin monoclonal antibody (1:5000; Sigma-Aldrich), and mouse GFP monoclonal antibody (1:2000; Invitrogen). Corresponding secondary horseradish peroxidase-conjugated antibodies were used (1:10,000–1:20,000 dilutions; Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Additional Western blots were probed with polyclonal antibodies using methods described previously (Pesic et al., 2004). These antibodies were obtained commercially (Alomone Labs, Jerusalem, Israel) and directed against CaV1.2 (α1C), CaV1.3 (α1D), CaV3.1 (α1G), and CaV3.2 (α1H). Protein bands were visualized by enhanced chemiluminescence, and the blots were exposed to Hyperfilm (GE Healthcare, Chicago, IL). Band densities were quantified by densitometry (Gel-Doc System; Bio-Rad), analyzed with Bio-Rad image analysis software, and they were normalized to β-actin expression.
Fluorescent Localization of Recombinant Adenovirus Proteins. Infected cells grown on glass-bottomed tissue culture dishes for 24 h after infection were washed, fixed in 4% paraformaldehyde, and kept in the dark at 4°C until fluorescent imaging. The images were acquired using a spinning disk confocal CARV II unit (BD Biosciences, San Jose, CA) on a Zeiss 200M microscope (Carl Zeiss, Thornwood, NY) fitted with a 100×/1.4 numerical aperture objective and a Retiga EXi camera (QImaging, Surrey, BC, Canada).
Whole-Cell Patch Clamp. Functional expression of Ca2+ channels was evaluated using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). After 24 h of infection with the different β2 constructs, culture dishes were mounted on the stage of an inverted microscope (Nikon, Tokyo, Japan), and HL-1 cells expressing GFP were identified by epifluorescence microscopy and selected for electrophysiological recordings. Cells were initially bathed in a solution containing 145 mM NaCl, 5.4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 5.5 mM dextrose. After formation of a conventional gigaohm seal, the patched membrane was ruptured to obtain the whole-cell configuration. Cells were then bathed in a solution that consisted of 20 mM BaCl2, 115 mM N-methyl-d-glucamine, 10 mM HEPES, 1 mM MgCl2, and 5.5 mM dextrose (pH 7.4; 300 mOsm). The pipette solution consisted of 120 mM cesium aspartate, 30 mM cesium chloride, 0.5 mM GTP, 5 mM MgATP, 5 mM creatine phosphate, and 5 mM sodium pyruvate (pH 7.2; 300 mOsm). Data acquisition and pulse generation were performed using pClamp software (Molecular Devices, Sunnyvale, CA) and an Axopatch 200A amplifier interfaced with a Digidata-1320 acquisition system (Molecular Devices). The data were sampled at 2 to 10 kHz, and they were filtered at 1 to 5 kHz. Capacitance and series resistance compensations were used, and leak current was linearly subtracted using a P/4 protocol, with a subtracting holding potential of –100 mV. Currents were normalized to cell size by dividing by cell capacitance (in pA/pF). Data analysis was performed using Clampfit (Molecular Devices) and Origin (OriginLab Corp., Northampton, MA), as described previously (Stimers et al., 2003). Activation curves were fit by the Boltzmann equation: I/Imax = A2 + (A1 – A2)/(1 + exp((V – V1/2)/s), where V is the command voltage, V1/2 is the potential of half-maximal activation, s is the slope factor, A1 is the maximum value of the ratio I/Imax (fixed at 1), and A2 is the minimum value of the ratio I/Imax. Steady-state inactivation curves also were fit according to the Boltzmann function I/Imax = A2 + (A1 – A2)/(1 + exp((V – V1/2)/s), to obtain half-maximal inactivation values.
Chemicals. Unless otherwise stated, all standard chemical reagents were purchased from Sigma-Aldrich.
Statistical Analysis. Data are reported as mean ± S.E.M. Statistical comparisons were performed using one-way analysis of variance followed by post hoc Student-Newman-Keuls test. Values of P less than 0.05 were considered significant.
Characterization of Ca2+Channel Subunits in HL-1 Cells. Before introducing β subunit decoys, we determined the native profile of β subunits in HL-1 cells. Transcripts for the CaV1.2 (α1C) subunit and for multiple β subunits, including β1, β2, β3, and β4, were detected (Fig. 2A). Subsequently, we confirmed the presence of nifedipine-sensitive, high-threshold Ca2+ channel current (ICa) attributed to CaV1.2 channels in these cells. Exposure of HL-1 cells to 10 μM nifedipine (n = 4) resulted in a marked inhibition of ICa elicited by stepwise 10-mV depolarizing pulses between –40 to +50 mV (Fig. 2B). In these recordings, the ICa resistant to block by nifedipine was attributed to the presence of Ca2+ channel types other than CaV1.2 (Xia et al., 2003; Hansen et al., 2004; Zhang et al., 2005).
Adenovirus Infection of HL-1 Cells. Initial experiments assessed the potential for cytotoxicity of the adenoviral treatment. The multiplicity of infection was adjusted to achieve >95% cell transduction by the adenovirus (determined by GFP fluorescence) at 48 h after viral treatment, with minimal (<10%) cytopathic effects visible as cell detachment, rounding, or blebbing. There was no evident difference in cytopathic effect between HL-1 cells treated with a control adenovirus (without construct) and the wild-type or mutant constructs. To confirm that adenoviral infection with GFP-fused constructs containing β2 subunits resulted in expression of the appropriate protein, immunoblots were performed on cell lysates collected from HL-1 cells 48 h after adenoviral infection. The use of an antibody directed against GFP confirmed that all GFP-fused β2 constructs were successfully expressed in the infected HL-1 cells (Fig. 3, lanes 2–6), whereas no immunoreactivity was detected in untreated HL-1 cells (Fig. 3, lane 1). Full-β2 was detected as an ∼106-kDa protein (lane 2), whereas truncation of the amino terminus resulted in detection of C-BID at ∼88 kDa (lane 3). Likewise, N-BID with a truncated carboxyl terminus was detected at ∼66 kDa (lane 4), and the isolated BID was detected at ∼42 kDa (lane 5). Control infection with GFP resulted in the expected ∼30-kDa protein (lane 6). Immunoblots were also performed to assess the expression level of CaV1.2 (α1C) in untreated HL-1 cells compared with HL-1 cells infected with GFP alone or GFP-fused β2 constructs (Fig. 3B). The averaged data from six independent blots indicated that the protein expression level of α1C was similar between untreated cells (lane 1), cells infected with GFP-fused Full-β2, C-BID, N-BID, or BID constructs (lanes 2 to 5), or cells infected with GFP alone (lane 6), suggesting that overexpression of the β2 constructs did not alter the total number of CaV1.2 channels in the HL-1 cells. Likewise, overexpression of the β2 constructs did not change the total number of CaV1.3 channels in the HL-1 cells (data not shown).
Cellular Localization of Various β2 Subunit Adenoviral Constructs. Cells were examined by fluorescence microscopy 24 h after treatment with adenoviral vectors. Preliminary experiments demonstrated that for adenoviral infection with either the Full-β2 construct or various mutated constructs, gene expression determined by GFP fluorescence was already present 24 h after viral infection. Maximal in vivo fluorescence was observed by 48 h, which was confirmed by immunoblots. Although the duration of gene expression was not formally investigated in this study, strong fluorescence was maintained for at least 5 days under similar experimental conditions. We postulated that overexpression of Full-β2 subunit would promote the normal trafficking of CaV1.2 channels to the plasma membrane. However, we anticipated that truncated β2 subunits would retain the ability to bind to the α1C subunit but display abnormal protein trafficking, and thereby not localize CaV1.2 channels at the surface membrane. Compared with untreated HL-1 cells (Fig. 4A), confocal images of HL-1 cells infected with GFP revealed a diffuse pattern of fluorescence in the cytosol (Fig. 4B), as reported previously in isolated cardiomyocytes (Telemaque-Potts et al., 2003; Chen et al., 2005). In contrast, overexpression of Full-β2 produced sarcolemmal localization (Fig. 4C, arrows), whereas C-BID and N-BID fusion proteins seemed primarily localized to intracellular vesicles (Fig. 4, D and E, respectively). Overexpression of BID produced a diffuse reticular pattern suggestive of cytosolic localization (Fig. 4F). Thus, the truncated β2 subunits showed punctate or diffuse intracellular localization that was distinctly different from the sarcolemmal localization of full-length β2. We also attempted to localize endogenous α1C subunit by immunostaining, using two different commercially available antibodies against CaV1.2 channels, but we were unable to detect a specific signal at the cell membrane (Supplemental Figs. A and B).
Effect of Overexpression of β2 Adenoviral Constructs on ICa. Subsequently, we compared the impact of overexpressing the β2 adenoviral constructs (24 h after infection) on whole-cell ICa elicited by 10-mV depolarizing steps from a holding potential of –40 mV to potentials between –40 and +50 mV (Fig. 5A). Initial studies suggested that this pulse protocol primarily activated high-threshold, nifedipine-sensitive CaV1.2 channels (Fig. 2B). Compared with cells infected with GFP alone (Fig. 5A), overexpression of Full-β2 markedly increased the membrane density of ICa (Fig. 5B) as reflected in the enhanced amplitude of the current-voltage (I-V) curve (Fig. 5F). Current densities were ∼2.5-fold higher at the peak of the I-V curve in Full-β2-overexpressing cells compared with the cells overexpressing GFP alone (–17.9 ± 3.0 and –7.2 ± 0.6 pA/pF, respectively; n = 10–12). To test the hypothesis that truncated β2 subunits serve as dominant-negative mutants, cells were infected with adenoviral constructs expressing N-BID, C-BID, or BID. Recordings of whole-cell ICa in these cells are depicted in Fig. 5 (C–E, respectively), and average I-V curves are plotted in Fig. 5F (n = 7–10). Cells infected with N-BID (Fig. 5C) showed ICa densities similar to GFP-infected cells (Fig. 5A), as verified by similar I-V curves. However, ICa was markedly reduced in C-BID- and BID-overexpressing cells (Fig. 5, D and E, respectively) compared with GFP alone (Fig. 5A). Overexpression of truncated C-BID and BID significantly decreased ICa density at the peak of the I-V curve to –2.7 ± 0.33 and –2.5 ± 0.7 pA/pF, respectively, compared with –7.2 ± 0.6 pA/pF in cells infected with GFP alone. Although Ca2+ channel β subunits can influence Ca2+ current kinetics, we did not observe consistent changes in the average inactivation rate constant (599 ± 50 ms) determined for the peak Ca2+ current elicited at +20 mV between HL-1 cells overexpressing the GFP, C-BID, and BID constructs.
Effect of Overexpression of C-BID and BID on Voltage-Dependent Activation and Inactivation of ICa. Although we hypothesized that C-BID and BID reduced ICa in HL-1 cells by retaining CaV1.2 channels intracellularly, we investigated the possibility that voltage-dependent gating also was altered. The voltage dependence of activation was evaluated by holding cells at –55 mV and imposing 10-mV depolarizing steps between –50 and +20 mV. Peak current at each voltage was plotted as a ratio of maximal current elicited at +20 mV, and the data were fit by a Boltzmann equation to obtain V1/2. The resulting activation curves in Fig. 6A revealed no significant difference (n = 4–5) in V1/2 values between cells overexpressing GFP (–14 ± 1 mV), C-BID (–12 ± 1 mV), or BID (–13 ± 1 mV). Inactivation was assessed by subjecting cells to 1400-ms prepulse voltages (10-mV steps) between –50 and 30 mV from a holding potential of –55 mV. Cells were briefly returned to –55 mV for 20 ms, and then they were pulsed to +20 mV to assess the effect of inactivating prepulses on channel availability. Peak current at each prepulse voltage was plotted as the ratio of maximal current elicited from a holding potential of –55 mV. Figure 6B shows data fit with the Boltzmann function, and it reveals that V1/2 values were not significantly different (n = 4–5) for HL-1 cells overexpressing GFP (–7 ± 2 mV), C-BID (–3 ± 2 mV), and BID (–5 ± 1 mV).
Effect of Overexpression of C-BID and BID on Ca2+Channels Other than CaV1.2. The finding that C-BID and BID suppressed functional CaV1.2 channels raised the important question of whether this dominant-negative effect was CaV1.2-specific, or whether it extended to other types of Ca2+ channels. In particular, L-type Ca2+ currents contributed by CaV1.3 channels (α1D) have been reported in HL-1 cells (Xia et al., 2004). The CaV1.3 channels can be distinguished from high-threshold CaV1.2 channels by their activation at mid-voltages and their lower sensitivity to nifedipine at negative membrane potentials (Koschak et al., 2001; Xu and Lipscombe, 2001; Xia et al., 2004; Zhang et al., 2005; Striessnig et al., 2006). Low-threshold T-type Ca2+ currents ascribed to CaV3 (α1G and α1H) channels also have been described in HL-1 atrial cells (Xia et al., 2003; Hansen et al., 2004; Yang et al., 2005). To collectively activate L-type and T-type channels to obtain “total current”, we imposed progressive 10-mV depolarizing steps over a wide voltage range from –80 to +50 mV. Total current was suppressed in HL-1 cells overexpressing C-BID and BID compared with those expressing only the GFP construct (Fig. 7A). Subsequently, the addition of 10 μM nifedipine revealed two components of total ICa. The nifedipine-resistant, residual ICa showed peak activation at +10 mV, revealing lower threshold Ca2+ current (Fig. 7B). Digital subtraction of this ICa component from total current revealed the nifedipine-sensitive ICa attributed to CaV1.2 channels that maximally activated at +30 mV (Fig. 7C). It is noteworthy that the I-V curve for the low-threshold, nifedipine-resistant ICa was significantly suppressed by C-BID and BID (Fig. 7B), suggesting that the dominant-negative effect of these β2 subunit mutants extends to Ca2+ channels other than CaV1.2.
Based on these findings, we questioned whether C-BID and BID act nonspecifically to suppress both T-type and L-type Ca2+ currents. It is noteworthy that the surface localization of L-type channels, including CaV1.2 and CaV1.3, is thought to rely at least partly on β subunits (Catterall et al., 2005). In contrast, T-type channels are missing the known motifs required for β subunit interaction (Perez-Reyes, 1998, 2003; Catterall et al., 2005). To confirm the expression of both CaV1.3 and CaV3 channels in the HL-1 cells, and thus a possible contribution to nifedipine-resistant ICa, we probed Western blots with α1 subtype-specific antibodies using mouse brain as a positive control in each of the left lanes (Fig. 8A). In addition to detecting CaV1.2, α1 subunits corresponding to CaV1.3, CaV3.1 and CaV3.2 were evident. Subsequently, we applied a depolarizing pulse between –80 and –10 mV to cells overexpressing GFP, C-BID, or BID to maximally activate low-threshold, T-type (CaV3) channels but avoid activation of mid- (CaV1.3) and high (CaV1.2)-threshold L-type channels (Fig. 8B, top traces). To further isolate T-type current, (1S,2S)-2-[2[[3-(2-benzimidazolylpropyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl methoxyacetate dihydrochloride hydrate (mibefradil) (7 μM), a preferential blocker of T-type channels (Martin et al., 2000; Xia et al., 2003) was applied and peak mibefradil-sensitive current elicited at –10 mV was calculated by digital subtraction (Fig. 8B, bottom traces). This transient current was fully intact after BID and C-BID overexpression, suggesting that the decoy effect of the β2 subunit mutants does not extend to T-type channels (Fig. 8). Rather, the dominant-negative effect of C-BID and BID on nifedipine-resistant current apparently reflects an action on CaV1.3 channels or other Ca2+ channel types.
The results of this study provide initial evidence that gene transfer of truncated β2 subunits into HL-1 cells prevents the proper localization of CaV1.2 channels in the surface membrane. We took advantage of the chaperone function of the β2 subunit to alter functional CaL channel expression using overexpressed truncated β2 subunit mutants in HL-1 cells. These mutants were designed to bind the pore-forming α1C subunits but prevent their proper insertion at the surface membrane form functional CaL channels. We have demonstrated that two engineered β2 subunit mutants, C-BID and BID, can serve a dominant-negative function in HL-1 cells, disrupt sarcolemmal localization of CaV1.2 channels, and specifically decrease ICa. Thus, the findings establish the principle that engineered β2 subunits represent a viable molecular approach for reducing the number of functional CaV1.2 channels in cardiomyocytes. In our experiments, the adenoviral delivery system seems to be highly effective in delivering the β2 subunit constructs to the HL-1 cells, and the cognate proteins were readily detected. We previously used adenoviral delivery to introduce the full-length β2 subunit into isolated feline ventricular myocytes to confirm the important role of this regulatory subunit in promoting CaV1.2 channel expression (Chen et al., 2005). In this earlier study performed in adult feline cardiomyocytes, we established that adenoviral-mediated gene delivery can indeed alter CaV1.2 channel expression and function within 24 to 48 h, a time course similar to that for HL-1 cells.
In the present study, we also examined the dominant-negative effects of C-BID and BID on additional Ca2+ channels coexpressed in HL-1 cells with CaV1.2. In our cells, mibefradil-sensitive, T-type current was not attenuated by either β2 subunit mutant, consistent with reports that CaV3 channels do not have binding motifs to permit β subunit interaction (Perez-Reyes, 1998, 2003; Catterall et al., 2005). However, other components of nifedipine-resistant current were suppressed by gene delivery of C-BID and BID. One target probably was the CaV1.3 channel that provides L-type current at mid-voltages in HL-1 cells, and it is 10-fold less sensitive to nifedipine than CaV1.2 channels. Although precise identification of CaV1.3 current is difficult due to the lack of a specific antagonist, members of the CaV1 gene family, including CaV1.3, share an α-interactive domain that enables α1-β interaction (Catterall et al., 2005). Thus, a shared feature of the CaV1 gene family may be susceptibility to dominant-negative β2 mutants. In this regard, one potential therapeutic benefit of our mutants may include the treatment of supraventricular tachyarrhythmias, because atrial cells seem to densely express CaV1.3 channels, which contribute to normal rhythmicity (Zhang et al., 2005). In contrast, CaV1.3 channels are only sparsely expressed in ventricular tissues, and CaV1.2 channels seem to be the primary pathway for voltage-gated Ca2+ influx. Thus, disruption of expression of CaV1.2 channels by our β2 subunit mutants may be beneficial in pathologies involving Ca2+ overload as a strategy to depress [Ca2+]cyt and restore normal contractility.
Our findings also provide initial insight into the interactions between the α1C subunit and truncated β2 subunit decoys that may be required to interrupt the trafficking of functional CaL channels to the surface membrane. In contrast to the observation that overexpression of the full-length β2 subunit at the surface membrane was associated with increased ICa, the highly distinctive vesicular patterns for N-BID and C-BID localization and the reticular pattern for BID suggest disruption of proper localization of CaL channel subunits. Indeed, each of the truncated β2 subunits was designed to retain the central BID sequence necessary for α1C-β interaction, but at least one of the two additional protein-interaction domains (SH3 and GK) recently reported to be required for channel assembly was eliminated (Chen et al., 2004; Opatowsky et al., 2004; Van Petegem et al., 2004). Thus, the SH3 domain was absent in C-BID, a significant portion of the GK domain was missing in N-BID, and the SH3 domain and a significant portion of the GK domain were missing in BID. In each case, the localization of β2 subunits was abnormal, supporting previous reports that the β subunits rely on at least three domains in the molecule to confer structural integrity.
Although all three truncated β2 subunits resulted in altered cellular localization, only C-BID and BID acted as dominant-negative subunits by markedly depressing ICa in the HL-1 cells. It seems logical to assume that the overexpression of the two successful β2 mutants, C-BID and BID, overwhelmed interactions between the endogenous α1C and β subunits to sequester α1C subunit. However, the abnormal vesicular localization of N-BID did not confer a decoy function, suggesting that it did not successfully compete for the α1C subunit although it shared the BID binding site with the C-BID and BID negative constructs. This lack of dominant-negative effect of N-BID is consistent with previous observations that a short N-terminal isoform of β1 subunit can modulate the gating of cardiac CaV1.2 channels, without promoting membrane trafficking of the channel complex (Cohen et al., 2005). Interestingly, a recent report demonstrated a link between the CaV1.2 activity and the cell endocytic machinery. In that study, a reduction in the number of channels present at the plasma membrane was observed when the SH3 domain of the β subunit (also located in the N terminus) was expressed in oocytes (Gonzalez-Gutierrez et al., 2007). The removal of the channels from the membrane by this β2-SH3 construct is not consistent with our observation that N-BID had no effect on Ca2+ currents. This suggests that additional portions of the N terminus of the β subunit may be involved in the regulation of the turnover of the L-type Ca2+ channels. Alternatively, it is possible that the spatial cellular localization of N-BID in these vesicle-like compartments may prevent binding interaction with the endogenous α1C protein.
Our study has several limitations. First, the HL-1 cells represent an atrial cell line that expresses robust Ca2+ currents and other markers of cardiac function, suggesting that it is a useful model in which to study Ca2+ channel regulation (Claycomb et al., 1998; White et al., 2004; Xia et al., 2004). However, the effectiveness of our dominant-negative β2 constructs clearly may differ between HL-1 cells and native cardiac cells that may express different levels of endogenous β subunits or even process the mutant β subunits differently. Indeed, the success of our β2 subunit mutants to reduce Ca2+ current in cardiac myocytes will depend on the rate of L-type channel turnover in the surface membrane in vivo. The precise turnover rate is unknown, but the fact that changes in Ca2+ current in response to norepinephrine or thyroid hormone are evident within 24 to 48 h suggests that the membrane turnover of L-type Ca2+ channels is a highly dynamic process (Kim et al., 1987; Maki et al., 1996). Second, we did not attempt to pinpoint the cellular sites involved in the aberrant localization of the C-BID, N-BID, and BID mutants. Further detailed colocalization studies will be needed to identify the cellular compartments or organelles that account for these expression patterns.
Although our initial attempt to detect by immunostaining the endogenous α1C subunit in the HL-1 cells was not successful (Supplemental Figs. A and B), we believe that colocalization of the endogenous α1C subunit with the GFP-tagged β2 subunit constructs would provide important insight into the mechanism by which these β2 subunits affect Ca2+ channel function. Finally, structure-function studies are needed to optimize the design of dominant-negative β2 constructs to obtain high-affinity molecules that serve as effective decoys for the L-type Ca2+ channels as a prelude to expressing it specifically in heart.
It is noteworthy that new molecular approaches designed to down-regulate L-type Ca2+ channel subunits in cardiac myocytes represent alternatives to the traditional use of organic Ca2+ channel blocking drugs to attenuate [Ca2+]cyt. The concept of directly titrating the number of Ca2+ channel proteins rather than altering the open-state probability of an existing Ca2+ channel population may have advantages, particularly if coupled to a cardiac-specific delivery system to provide durability of effect. For example, work by others shows that in vitro gene knockdown of the β2 subunit using RNA interference technology significantly decreased calcium transients in neonatal rat cardiomyocytes, prevented an increase in relative cell size, and abrogated phenylephrine-induced protein synthesis in a cellular model of hypertrophy (Cingolani et al., 2007). Furthermore, in an aortic-banded rat model of left ventricular hypertrophy, the same authors showed that knockdown of the β2 subunit was capable of attenuating cardiac hypertrophy without compromising systolic performance, suggesting that the targeted suppression of pathologies involving Ca2+ overload may be possible (Cingolani et al., 2007). The adenoviral vectors used in our study are primarily useful in vitro for proof-of-concept, because they are not regarded as ideal clinical vectors due to their immunogenic nature. However, other vectors including adeno-associated viruses containing cardiac-specific promoters may permit targeted knockdown of L-type channels in the heart.
In summary, the data presented in the current study do support the concept that adenovirus-mediated gene transfer of dominant-negative β2 subunit mutants can effectively and specifically modulate L-type calcium channel expression and function in the HL-1 cell model system of cardiomyocytes.
We thank Drs. Zhen Wang and Larisa Buzdugan for excellent technical support, and GibAnn Berryhill for assistance in preparing the manuscript.
This work was supported in part by the University of Arkansas for Medical Sciences (UAMS) Department of Internal Medicine Departmental Research Endowments (to S.T. and J.D.M.), UAMS Department of Pharmacology and Toxicology Research Funds (to J.R.S.), the Department of Veterans Affairs in the form of a VA Merit Award (to J.D.M.), and United States Public Health Service Grants R01 HL-68406 and R01 HL-050818 (to N.J.R.) from the National Institutes of Health (NIH). The University of California Davis/National Institute of Neurological Disorders and Stroke (NINDS)/National Institute of Mental Health (NIMH) NeuroMab Facility (mouse α1C subunit antibody) is supported by NIH Grant U24 NS-050606. The mouse α1C subunit monoclonal antibody (clone L57/46) used in the present study was developed by and/or obtained from the University of California Davis/NINDS/NIMH NeuroMab Facility and maintained by the Department of Pharmacology, School of Medicine, University of California, Davis, CA.
S.T. and S.S. contributed equally to this work.
A preliminary version of some of these results has been presented in abstract form at the following conference: Sonkusare S, Stimers JR, Grain T, Marsh JD, and Télémaque S (2006) Expression of β2 subunit mutants alters calcium currents in HL-1 cells (FASEB J20:A1114); Experimental Biology Meeting 2006; 2006 Apr 1–5; San Francisco, CA. American Society for Pharmacology and Experimental Therapeutics, Bethesda, MD.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: Cav, Ca2+ channel; BID, β interaction domain; SH3, Src homology 3; GK, guanylate kinase; GFP, green fluorescence protein; Full-β2, full β2 subunit; CaL, L-type Ca2+ channel; PCR, polymerase chain reaction; bp, base pair(s); CMV, cytomegalovirus; HEK, human embryonic kidney; ICa, Ca2+ channel current; I-V, current-voltage; V1/2, half-maximal activation.
- The American Society for Pharmacology and Experimental Therapeutics