Modulation of Cardiac Na+ Current Phenotype by β1-Subunit Expression
Abstract—Na+ current (INa) is smaller, activates and inactivates more slowly, and displays less negative voltage dependence of inactivation in the neonatal rat than in the adult rat. We have observed very similar changes when INa is recorded as a function of time in culture in mouse atrial tumor (AT-1) cells. The differences between mature and immature INa are reminiscent of those observed when skeletal muscle Na+ channel α subunits are expressed alone (immature) or with the β1 subunit (mature). In the present experiments, we tested the hypothesis that suppression of β1-subunit expression by antisense oligonucleotides would prevent the development of a mature INa. The mouse β1 subunit was cloned from an AT-1 cDNA library and found to be identical to that in the rat at 216/218 amino acids. AT-1 cells exposed to anti-β1 antisense oligonucleotides displayed an immature INa at day 8 in culture, whereas untreated cells or cells exposed to sense oligonucleotides displayed a mature INa. This result was observed with 2 different oligonucleotides, and neither affected the rapidly activating component of the delayed rectifier K+ current, another current recorded in AT-1 cells. These findings indicate that in these cells, the gating of INa is modulated by β1 expression and that α-β1 coexpression is required for the development of a mature cardiac INa phenotype.
In adult cardiac myocytes, the fast inward Na+ current (INa) activates and inactivates rapidly. However, INa in neonatal rat myocytes displays different physiological properties: it activates and inactivates much more slowly than that in adults, and the midpoint for inactivation is less negative.1 In vitro studies have found that a “mature” phenotype can be restored to neonatal cells by long-term (>20-hour) exposure to membrane-permeant cAMP analogues, such as chlorophenylthio (CPT) cAMP, or by coculture with sympathetic neurons.1 2 However, short-term exposure to CPT cAMP did not restore the mature phenotype. These data have been interpreted as suggesting a “trophic” role of activation of intracellular signaling through cAMP-dependent pathways in maturation of the Na+ channel. However, the nature of this trophic effect has not been elucidated.
We have reported a very similar maturation process when INa in AT-1 cells is compared after 3 and after >7 days in culture.3 AT-1 cells were originally isolated from atrial tumors arising in mice carrying a transgene in which the atrial natriuretic factor promoter drove expression of the SV40 large-T antigen.4 As we and others have previously reported, AT-1 cells exhibit biochemical, histological, and electrophysiological properties very similar to those of mouse atrial myocytes.5 6 7 8 We found that INa in cells cultured for 3 days is smaller and activates and inactivates much more slowly than INa recorded in cells cultured for 10 days.3 The voltage dependence of activation was similar at the 2 time points, but the voltage dependence of inactivation was, as in the rat myocyte experiments, less negative in the 3-day cells. In cells cultured from days 0 to 3 in medium containing CPT cAMP, a mature INa phenotype was observed, with a greater amplitude, more rapid activation, and more negative voltage dependence of inactivation than in 3-day control cells.
The differences between INa in AT-1 cells at 3 and 10 days in culture or between that recorded in neonatal and in adult rat myocytes are reminiscent of the differences observed when Na+ channel α subunits are expressed with and without the ancillary β1 subunit: Isom et al9 reported that coexpression of β1 with rat brain α subunits in Xenopus oocytes resulted in INa that was larger, activated and inactivated more rapidly, and displayed voltage dependence of inactivation that was more negative than that observed when the α subunit alone was expressed. As discussed further below, the extent to which β1 modulates the function of expressed cardiac α subunits in Xenopus oocytes is controversial, and the extent to which the experiments in oocytes reflect information obtained in mammalian systems is uncertain. We hypothesized that the phenotype switch that we and others have observed in maturing INa was attributable to an α-β1 interaction in cardiac myocytes. In the present set of experiments, we tested this hypothesis with the use of antisense oligonucleotides targeting β1 expression in AT-1 cells. Portions of the present study have been reported previously in abstract form.10
Materials and Methods
Cloning and Sequencing the Mouse β1 Subunit
Plaques (5×105) of an AT-1 cDNA library in λgt10 (a gift of Dr Mike Tamkun, Colorado State University, Fort Collins, Colo) were screened under low-stringency conditions (20% formamide, 42°C) using the rat Na+ channel β1-subunit cDNA as a probe (a gift of Dr Al George, Vanderbilt University School of Medicine, Nashville, Tenn). Library screening was performed according to standard methods11 with 3 rounds of plaque purification. Probes were labeled using the Prime-It II kit (Stratagene) and [α-32P]dATP (NEN). DNA of positive phage clones was prepared using the Wizard Lambda Prep DNA purification system (Promega), and inserts were released via EcoRI digestion followed by subcloning into the EcoRI site of pBluescript KS− (Stratagene). Sequencing was performed partly by manual sequencing using Sequenase 2.0 (USB) and partly by using an automated sequencer (model 373A, Applied Biosystems). The mouse Na+ channel β1-subunit sequence has been deposited in GenBank under accession No. U85786.
A polymerase chain reaction (PCR) fragment consisting of nucleotides 1 to 360 of the mouse β1 subunit was subcloned into the PCR II vector (Invitrogen). In the present study, numbering starts at the initial adenine at the putative translation start codon. The orientation of the insert was established by sequencing. The plasmid was linearized by digestion at the 5′ end of the insert with XhoI. From this template, an RNase protection probe was generated by performing in vitro transcription using Sp6 RNA polymerase in a 10-μL reaction containing a final concentration of 1× the manufacturer’s transcription buffer, 500 μmol/L each of unlabeled ATP, CTP, and GTP, 10 μmol/L unlabeled UTP, and 40 μCi [α-32P]UTP. The reaction was allowed to proceed for 60 minutes at 37°C and was terminated by incubation for a further 20 minutes with RNase-free DNase I to remove the template DNA. The resulting radiolabeled RNA was purified of unincorporated nucleotides and protein by passage through a QIAquick PCR purification column (QIAGEN Inc). This radiolabeled probe RNA was then made up in hybridization buffer (80% formamide, 40 mmol/L PIPES, pH 6.4, 400 mmol/L NaCl, and 1 mmol/L EDTA) and retained for use in the hybridization reaction.
Total RNA was isolated from tissue or from AT-1 cells growing in culture using the acid phenol method.12 For the hybridization reaction, 10 μg of target RNA and 10 μg of yeast tRNA (as a neutral carrier) were mixed with ≈500 000 cpm of the radiolabeled probe RNA. The total volume of the hybridization was made up to 30 μL with hybridization buffer. The mixture was heated to 85°C and then slowly cooled to 45°C overnight. Control reactions with no target RNA and 20 μg of yeast tRNA were run to allow differentiation of the bands that result from hybridization of the probe RNA to the target RNA from those that result from secondary structures in the probe RNA. After the hybridization was complete, 350 μL of RNase digestion buffer (10 mmol/L Tris, pH 7.5, 300 mmol/L NaCl, and 5 mmol/L EDTA) containing RNase A (30 μg/mL) was added to each hybridization and incubated at 30°C for 45 minutes. Digestion was terminated by adding SDS to a final concentration of 0.5% (wt/vol) and proteinase K to a final concentration of 250 μg/mL and by incubation at 37°C for 45 minutes. The mixture was then phenol-extracted, ethanol-precipitated, and made up in loading buffer (46% formamide, 0.125% bromphenol blue, 0.125% xylene cyanole, and 0.25× TBE). The samples were denatured by heating to 85°C and then run on a 4% denaturing polyacrylamide gel. Dried gels were exposed to film, and quantitative analysis of the bands was accomplished using a PhosphorImager (Molecular Dynamics).
AT-1 Cell Preparation
As previously described,3 cells obtained from Loren Field (Krannert Institute) were injected subcutaneously into new syngeneic hosts ([C57BL/6J×DBA/2J]F1 female mice, Jackson Laboratories, Bar Harbor, Me). In 6 to 8 weeks, subcutaneous tumors became palpable, and cells were isolated 12 to 16 weeks after inoculation. With each cell isolation, further syngeneic hosts were inoculated to propagate the colony. To isolate cells, tumor-bearing mice were anesthetized with isoflurane (Abbott Laboratories) and placed in 70% ethanol. The tumor mass was excised, rinsed with PBS, minced finely, and placed for 1 hour at 37°C with gentle rocking in PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin (pen-strep, GIBCO) and 0.1% collagenase. The cell suspension was spun, washed with PBS, resuspended, and then plated at a density of 250 to 325×103 cells/mL in 100-mm Primaria dishes (Falcon). The media (PC1 [Ventrex Laboratories], which included pen-strep, 10% FBS, and 10 nmol/L dexamethasone) were changed every other day until used. Primary cultures grew to confluence and usually beat spontaneously at ≈1 week. It has been reported by others5 that isolated cells can also be repassaged or frozen for subsequent replating, but this approach was not used in these experiments. For electrophysiological studies, cells were removed from the culture dish by a 2-minute exposure to a trypsin-containing solution (0.125% in Ca2+- and Mg2+-free Hanks’ solution), decanted into sterile culture tubes, and held at room temperature. Parallel control experiments were performed to study the effects of interventions: in each case, cells from an individual tumor isolation were cultured in multiple dishes, and untreated and treated cells were isolated and studied on the same day.
Two different pairs of oligonucleotides, both targeting the region surrounding the 5′ ATG translation start site, were tested. The antisense oligonucleotides were complementary to nucleotides 21 to 5 (designated #1) and nucleotides 4 to −11 (designated #2). The antisense oligonucleotides and the corresponding sense oligonucleotides were synthesized by a core facility at our institution. To assess whether the antisense oligonucleotide effect was INa specific, we also determined the effect of anti-β1 oligonucleotides on another current we routinely observe in AT-1 cells, the rapidly activating component of the delayed rectifier K+ current (IKr).8 As described below (Results), cells were studied on day 8 (after exposure to oligonucleotide on days 0 to 8 in culture) and on day 14 (after exposure to oligonucleotide on days 10 to 14). With each culture medium change (every 48 hours), fresh oligonucleotide (1 μmol/L) was added to the culture medium. Data were then obtained by studying sense-exposed and antisense-exposed cells on the same day.
Whole-cell recordings were performed using an Axopatch-200A patch-clamp amplifier (Axon Instruments, Inc). Microelectrodes were pulled from borosilicate glass (Fisher Scientific) and heat-polished. The recording temperature was 18°C. The currents were low pass–filtered at 5 to 10 kHz (Bessel filter, −3 dB), sampled at 25 kHz, and stored on hard disk for subsequent analysis. Data acquisition and command potentials were controlled with a commercial software program (pClamp6.0, Axon Instruments). Microelectrode tip resistance was ≈1 MΩ (0.8±0.3 MΩ, n=30). Junction potential was zeroed with a reference electrode into the standard bathing solution. Microelectrodes were gently lowered onto the cell surface, and gigaohm seal formation was achieved by suction (range, 5 to 50 GΩ). After the whole-cell configuration was established, the capacitive transients elicited by symmetrical 10-mV voltage-clamp steps from −80 mV were recorded at 50 kHz (filtered at a bandwidth of 10 kHz, −3 dB) for calculation of capacitive surface area, time constant, and access resistance. Thereafter, capacitance and series resistance compensation were optimized; ≈80% compensation was usually obtained. All currents were normalized to cell size, which was determined by recording the capacitative current (after compensation) elicited by a voltage clamp step from −80 to −90 mV. Individual cell capacitance was then calculated as ΔQ/ΔV=∫Idt/ΔV=∫Idt/10, where Q is charge, V is voltage, and I is current.
To measure INa, the pipette-filling solution contained (mmol/L) NaF 10, CsF 110, CsCl 20, EGTA 10, and HEPES 10, and the pH of the solution was adjusted to 7.35 with CsOH. The external K+-free solution contained (mmol/L) NaCl 20, CsCl 110, MgCl2 1, CaCl2 0.1, tetraethylammonium chloride 5, HEPES 10, NiCl2 0.2 (to block T-type Ca2+ currents), nisoldipine (to block L-type Ca2+ currents) 1 μmol/L, and glucose 10, and the pH of the solution was adjusted to 7.35 with CsOH. A pulse duration of 60 milliseconds from a holding potential of −120 mV was used to assess INa activation, and variable holding potentials with a test pulse to −30 mV were used to assess inactivation; pulses were delivered every 5 seconds. Peak INa was measured within a moving time window beyond capacitive transients.
To record IKr, the extracellular solution was normal Tyrode’s solution containing (mmol/L) NaCl 130, KCl 4, CaCl2 1.8, MgCl2 1, HEPES 10, and glucose 10, with the pH adjusted to 7.35 with NaOH. The intracellular pipette-filling solution contained (mmol/L) KCl 110, K4BAPTA 5, K2ATP 5, MgCl2 1, and HEPES 10, and the solution was adjusted to pH 7.2 with KOH, yielding a final intracellular K+ concentration of ≈145 mmol/L. Pulses of 1-second duration to a range of depolarizing potentials (−30 to +50 mV) from a holding potential of −40 mV were used; tail currents were measured as the difference between current recorded immediately after a step back to −40 mV and holding current at −40 mV. Chemicals were obtained from Sigma Chemical Co. Nisoldipine was obtained from Miles Pharmaceutical, Inc.
The voltage dependence of channel opening and inactivation was determined by fitting a Boltzmann function, y=1/(1+exp[−(V−V1/2)/k]), to activation or inactivation curves, where k represents the slope factor, and V1/2 indicates the voltage at which 50% of the channels were activated or inactivated. Inactivation was fit with monoexponential or biexponential functions; the choice between the 2 functions was made using goodness-of-fit criteria as in previous work.13 With depolarizing pulses to −30 mV, the “mature” phenotype is characterized by large currents (>20 pA/pF at −30 mV), biexponential inactivation with a slow time constant (τslow, ≤15 milliseconds), and a time to peak of <5 milliseconds; in addition, the V1/2 for inactivation is negative to −80 mV.3 By contrast, the “immature” INa is smaller (<15 pA/pF), inactivates more slowly and in a monoexponential fashion (τslow, ≥14 milliseconds), and activates more slowly (time to peak, >8 milliseconds); the V1/2 for inactivation is generally positive to −80 mV.
Data are presented as mean±1 SE. For comparisons among means of several treatment groups, ANOVA was used, with post hoc pairwise comparisons by the Duncan test if significant differences among means were detected. If only 2 groups were being compared, the Student t test was used. A value of P<0.05 was considered significant.
The full-length mouse β1 Na+ channel subunit cDNA was 1616 nucleotides, with a coding region of 654 nucleotides (218 amino acids). The sequence of the coding region is identical to that in the rat at 637/654 positions, and only 2 of these differences are nonconservative at the amino acid level; ie, the inferred protein is identical to that in the rat at 216/218 positions.
Our previous studies had demonstrated that the INa phenotype switch in AT-1 cells occurred by day 7 in culture.3 Therefore, to determine whether anti-β1 antisense prevented this change in phenotype, we assessed the effect of exposing cells to sense or to antisense oligonucleotides from day 0 to day 8 in culture, and INa was studied on day 8. INa recorded from AT-1 cells exposed to sense or to antisense oligonucleotide is presented in Figure 1⇓. INa in antisense-treated cells activated and inactivated more slowly than did INa in untreated control or sense-treated cells. As shown in Figure 2⇓, this reduction in INa amplitude was observed with both antisense oligonucleotides used, but not with the corresponding sense oligonucleotides. The voltage dependence of activation was no different in antisense-treated cells compared with in sense-treated (or control untreated) cells (Figure 3A⇓), and the slope factors were similar (antisense, 5.1±0.4 mV; sense, 4.8±0.4 mV; and untreated, 5.5±0.6 mV). However, the voltage dependence of inactivation was more positive in the antisense-treated cells (Figure 3B⇓), although again the slope factors were similar (antisense, 8.3±0.6 mV; sense, 7.3±0.2 mV; and untreated, 6.6±0.2 mV). A summary of the electrophysiological characteristics of INa recorded at day 8 in sense-exposed and antisense-exposed cells is presented in the Table⇓, along with a summary of data we3 have previously reported on INa at day 3 (immature phenotype) and at day 14 (mature phenotype). Antisense-exposed cells demonstrated all the characteristics of immature INa at 3 days, including decreased current amplitude, prolonged time to peak, monoexponential and slow inactivation, and less negative voltage dependence of inactivation. In addition, as shown in Figure 4⇓, recovery from inactivation was slower in cells studied at day 3 versus day 10 in culture (left) and in antisense-exposed cells versus sense-exposed cells (right).
In our previous studies,3 we demonstrated that the mature INa phenotype could be reverted to the immature one by exposure of cells from days 10 to 14 to the cAMP inhibitor Rp-cAMPS. Therefore, we undertook experiments in which AT-1 cells at 10 days in culture were exposed to anti-β1 sense or antisense oligonucleotide from day 10 to day 14. As shown in the Table⇑, antisense exposure from day 10 to day 14 restored the immature INa phenotype.
To guard against the possibility of nonspecific antisense results, 2 different oligonucleotides were tested in these experiments (Figure 2⇑), and the results with both were similar. In addition, another current in AT-1 cells, IKr, was virtually indistinguishable in antisense-treated cells compared with sense-treated cells. The maximum tail current amplitudes were 3.5±0.1 (antisense-exposed) versus 3.4±0.1 pA/pF (sense-exposed) (n=6 each), the midpoints for IKr activation were −0.7±0.3 and 4.6±0.3 mV, respectively, and the slope factors were 10 mV−1 in both cases.
RNase protection was used to determine whether anti-β1 antisense exerted its effects by altering steady-state mRNA abundance. As shown in Figure 5⇓, mRNA abundance, normalized to rat cyclophilin, was unaltered in antisense-exposed cells compared with sense-exposed and with unexposed cells; this result was replicated 3 times with the use of both oligonucleotide pairs. As discussed further below, this result indicates that the effect of anti-β1 antisense was likely mediated via a decrease in β1 protein (or other posttranslational effects) rather than altered mRNA stability or decreased β1 gene transcription.
In the present study, we have demonstrated that the normal maturation of INa in AT-1 cells can be prevented by inhibition of expression of the β1 subunit. The maturation of INa in AT-1 cells has characteristics very similar to those observed in other cardiac myocytes, strongly suggesting that an α-β1 interaction plays a critical role in this maturation process.
Function of β1 Subunits
When brain or skeletal muscle INa α subunits are coexpressed with the β1 subunit in Xenopus oocytes, the physiological behavior of the resultant INa is markedly altered: activation and inactivation are more rapid, recovery from inactivation is accelerated, and the current is larger.9 Although Na+ channel β1 mRNA is detected in heart,14 15 it has been controversial whether β1 modulates the function of cardiac INa. In coexpression studies in Xenopus oocytes, one group has reported no change in phenotype,14 another group has reported an increase in current amplitude as the major effect,15 and a third group has reported effects of β1 on the human cardiac α subunit that are similar to those with the brain α subunit.16 More recently, a role for both β1 and β2 subunits in channel localization has been proposed on the basis of the inferred immunoglobulin-like extracellular domains of the proteins.17 In addition, Isom et al18 have shown that (in contrast to data obtained in ooyctes) expression of brain α subunits in Chinese hamster ovary cells results in a rapidly activating and inactivating current. One possible explanation for this result could be the presence of an endogenous β1 subunit in these cells, but such a subunit was not found, implying that normal gating could be observed with expression of the brain α subunit alone in a mammalian cell line. In the Chinese hamster ovary cell studies, the major effect of α+β1 coexpression was to increase INa amplitude. In addition, the voltage dependence of inactivation was more negative, reminiscent of the effect of the maturation that we and others have observed in myocytes in culture (Figure 3B⇑).
Our data strongly suggest that an α-β1 interaction occurs during development and is responsible for the maturation of the cardiac INa. Moreover, our data indicate that an immature phenotype can be restored to a mature INa, eg, by Rp-cAMPS or by anti-β1 antisense intervention. This further raises the possibility that in disease, INa function may become abnormal in part because of loss of association of an important β1 subunit. We speculate that the result of such a loss would include a smaller INa as well as a shift of inactivation to more positive potentials. With the exception of Lue and Boyden,19 who found a smaller INa but a negative shift in inactivation in cells that had been subjected to a recent ischemic insult, systematic studies of the effects of heart disease on INa have not been conducted. As noted above, Zhang et al1 reported that INa in neonatal rat cells was smaller and activated and inactivated more slowly than that in adult cells; in their studies, inactivation was also shifted more negatively in the adult cells, similar to our findings.
Antisense Inhibition of Ion Currents in Excitable Cells
We and others have previously shown that exposure of cultured excitable cells to oligonucleotides targeting ion channel subunits can reduce ion current. When cultured human atrial myocytes were exposed to anti-Kv1.5 antisense oligonucleotides,20 the amplitude of the ultrarapid delayed rectifier K+ current was reduced, lending very strong support to the idea that Kv1.5 encodes the major α subunit for this current. Similarly, anti-Kv1.5 oligonucleotides reduced the delayed rectifier current in cultured glial cells21 and in cultured pituitary cells.22 In the latter experiments, oligonucleotides targeting Kv1.4 were shown to reduce transient outward current, implicating Kv1.4 as a gene encoding a transient outward–like current in these cells. Likewise, oligonucleotides targeting the cystic fibrosis transmembrane conductance regulator were found to reduce cAMP-activated Cl− current in cultured epithelial cells.23 Our finding that anti-minK oligonucleotides reduced the amplitude of IKr in AT-1 cells24 suggested an interaction between minK and HERG, the gene encoding the major α subunit underlying IKr. This concept has been supported by recent work from others25 that coexpression of minK and HERG resulted in larger IKr than did expression of HERG alone in transfected mammalian cells; moreover, evidence for physical association between the 2 proteins was presented.
To ensure that the effect of antisense treatment is specific, most previous studies have, like the experiments reported in the present study, used controls (sense and/or random oligonucleotides) and have tested more than one antisense-sense pair. The mechanisms whereby antisense oligonucleotides inhibit protein function are not certain. One possibility is that binding of oligonucleotide to mRNA destabilizes the mRNA or that the oligonucleotide prevents gene transcription.26 27 Either case would result in a decrease in steady-state mRNA abundance, but not all previous studies have examined this question; among the studies that have, some23 but not all24 observed the effect. An alternate explanation for the inhibitory effect of antisense oligonucleotides is inhibition of translation by oligonucleotide binding to mRNA. In this case, mRNA abundance would be unaltered, but functional effects would nevertheless be observed. Demonstration of decreased protein abundance23 can be used to support this idea. We did obtain polyclonal anti-β1 antibodies for such experiments (courtesy of William Catterall and Brian Murphy at the University of Washington, Seattle) but were unable to demonstrate immunoreactive β1 protein in the cells; we assume that this represents low basal expression of the protein in these cells. Finally, it is possible that antisense oligonucleotides produce their effects through mechanisms that are nonspecific or at least unrelated to inhibition of gene transcription or mRNA translation, eg, by directly inhibiting ion permeation or α-β1 interactions. We consider this unlikely since the effect was seen with 2 sense-antisense pairs and did not affect the function of another ion current present in these cells.
Can the Phenotype Switch Be Explained by Expression of Multiple α Subunits?
In our previous experiments in AT-1 cells, we found that the sensitivity of INa to tetrodotoxin was similar (EC50, 100 to 200 nmol/L) at the 2 time points. Since this EC50 value is typical of neither neuronal (EC50, <10 nmol/L) nor cardiac (EC50, generally >1 μmol/L) α subunits, we raised the possibility that multiple α subunits were present. The present findings, by indicating that the INa phenotype is dependent on β1 expression, argue that variability in expression of different α subunits is not the mechanism underlying the change in INa phenotype. Also arguing against this possibility is the finding by Zhang et al1 that the unitary conductance of INa was similar at the 2 time points. In addition, the tetrodotoxin sensitivity was similar at the 2 time points (despite a difference in phenotype), arguing against multiple α subunits. When we performed Northern analysis using 14-day AT-1 cell mRNA and a probe derived from an isoform-specific region (the DII-III linker) of the rat cardiac Na+ channel gene, we found only a single band (data not shown). These findings collectively argue against the expression of multiple Na+ channel isoforms as the mechanism underlying the change in INa that we and others have reported here and previously.1 2 3 We recognize that we have not formally eliminated the possibility that these cells (or indeed cardiac tissue in general) express multiple Na+ channel isoforms. This will require cloning experiments that are far beyond the scope of the present experiments. However, the multiple lines of evidence cited above strongly support the idea that the change in phenotype we observe is unlikely to be attributable to a switch in expression of multiple Na+ channel α isoforms.
Intracellular Signaling and INa Maturation
Our own data and those of others1 2 3 make it clear that activation of an intracellular signaling event also promotes the development of a mature INa phenotype. However, the relationship between activation of intracellular signaling and the α-β1 interaction that we have now demonstrated is not clear. One obvious possibility is that activation of intracellular signaling promotes increased transcription of the β1 subunit. Indeed, we find that mRNA encoding the β1 subunit is near absent in the neonatal rat heart and increases dramatically postnatally. However, we have also found that exposure of AT-1 cells to CPT cAMP from days 0 to 3 in culture does not alter β1 mRNA abundance, indicating that increased β1 expression cannot be invoked as a major link between the phenotype switch and activation of intracellular signaling. Other possibilities that may explain the relationship between intracellular signaling and the α-β1 interaction include promotion of translation by a signaling event, expression of a translation inhibitor in the absence of activated intracellular signaling, promotion or hindrance of steric interactions between α and β1 subunits during assembly or at the cell membrane by the presence of phosphorylated residues, and promotion of stability of a α-β1 complex by an intracellular signaling event. The necessity for both β1 expression and an intracellular signaling event is further reinforced by the fact that the β-adrenergic signaling cascade in cardiac myocytes only matures around birth,28 and sympathetic innervation of the heart occurs in the immediate postnatal period.29 Thus, during development, a mature INa phenotype must reflect both cardiac sympathetic innervation, a mature β-adrenergic signaling cascade, and expression, translation, assembly, and transport to the cell surface of β1 (and α) subunits. Our data raise the possibility that disruption of the interaction among these steps may be a crucial event not only in the prevention of a mature INa phenotype during development but also in the development of abnormal INa in acquired cardiac disease.
This study was supported in part by a grant from the US Public Health Service (HL-49989). Dr Roden is the holder of the William Stokes Chair in Experimental Therapeutics, a gift from the Daiichi Corp. The technical assistance of Holly Waldrop and the secretarial assistance of Cynthia Tillman are gratefully acknowledged.
Previously presented in abstract form (Circulation. 1996;94[suppl I]:I-287).
Correspondence to Dan M. Roden, MD, Director, Division of Clinical Pharmacology, 532 Medical Research Building, Vanderbilt University School of Medicine, Nashville, TN 37232-6602.
- Received January 20, 1998.
- Accepted June 2, 1998.
- © 1998 American Heart Association, Inc.
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