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1Department of Cardiac Sciences, University of Calgary, Calgary, Alberta, Canada; and 2University of Washington, Seattle, Washington
Submitted 25 November 2005 ; accepted in final form 21 May 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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sodium current; transgenic mice; ryanodine receptor
Recent studies indicate that overexpression of calcineurin is associated with development of heart block, whereas overexpression of NF-AT3 is not, even though overexpression of NF-AT3 produced comparable extents of cardiac hypertrophy (14). These data suggest that nongenomic effects of calcineurin contribute to the phenotype. This possibility is in keeping with previous studies that found that calcineurin can activate a range of signal transduction pathways, including PKC (10). The mechanism of the heart block has not been elucidated. Although previous studies reported that overexpression of calcineurin causes an increase in the density of L-type calcium current (ICa-L) (36) and a decrease in the density of all components of transient outward potassium current (11), such ion channel defects could be compensatory to maintain excitability and contractility but do not easily explain heart block. Action potential (AP) recordings in calcineurin-overexpressing (CN) mouse hearts showed decreased maximum change of potential over time (dV/dtmax) of phase 0 of the AP, indicating the possibility of downregulation of the cardiac sodium channel. Moreover, profound downregulation of the sodium channel would be expected to produce a conduction defect. Accordingly, the density and function of the cardiac sodium channels were assessed at various stages of development in this transgenic mouse model. Parallel in vivo studies addressed the contribution of decreased sodium channel activity to the heart block phenotype.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Female wild-type (WT) mice and transgenic CN mice in the ICR background were compared (20). Electrophysiological studies were performed on only one mouse line since Molkentin et al. (20) have reported that all lines have identical cardiac phenotypes. All animals were housed in the animal care unit of the University of Calgary according to our animal care guidelines.
The genotypes from the F1 and F2 generations of the transgenic mice were determined by Southern blot (20), and all subsequent matings were determined by PCR on DNA from tail biopsy specimens. For PCR analysis, oligonucleotides for the human growth hormone sequence (5'-GTCTGACTAGGTGTCCTTCT-3'; 5'-CGTCCTCCTGCTGGTATAG) were used (18). The PCR conditions were as follows: denaturation at 95°C for 1 min, followed by 30 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C.
APs and sodium currents.
Voltage-dependent sodium currents (INa) and APs were measured in isolated ventricular cardiac myocytes with conventional patch-clamp methods in either voltage- or current-clamp configuration (11). A rapid solution changer (solution change <2 s) was used to expose the myocytes to a low-extracellular sodium concentration ([Na]o) solution (10 mM) just before application of the voltage-clamp pulses. All experiments were performed at room temperature. An Axopatch 200B (Axon Instruments) amplifier was used. Access resistance was <5 M
. Series resistance was routinely compensated to >80% (11). Capacitive transients were minimized by capacitance compensation and then digital subtraction. The current was filtered with a low-pass Bessel filter at 5 KHz and digitalized at a rate of 10 KHz. The peak transient current between its onset and up to 60 ms was measured as the amplitude of INa. The membrane potential was held at –120 mV. Test pulses of 60-ms duration were applied from –90 to +50 mV in 5-mV increments with 5 s between pulses. Voltage-dependent steady-state inactivation of INa was measured by applying paired pulses. The conditional pulse was 5 s in duration and varied from –130 to –25 mV, which was followed by a test pulse of –25 mV. The inactivation curves were fitted to the Boltzmann equation:
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Recovery from inactivation was measured with a standard two-pulse protocol. From a holding potential of –110 mV, a 50-ms conditional pulse to –25 mV was applied, followed by a test pulse with the same shape but at various intervals from 2 to 150 ms. The amplitude of the INa was normalized to the amplitude of the conditioning pulse. The protocols were repeated every 8 s. The time course of the recovery from inactivation was fitted to a two-exponential equation:
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f and s are the fast and slow time constants.
The strategy used to measure INa at physiological [Na]o was to measure INa conductance in proximity to its reversal potential. To have adequate voltage-clamp control under these physiological conditions, the pipette resistance was adjusted from 0.5 to 1 M. The access resistance was 1–2 M and was compensated to >90% (11). The test pulse potentials were applied from +20 to +70 mV for 60 ms from a holding potential of 120 mV. The capacitance currents were eliminated by electronic compensation and digital subtraction. The amplitudes of INa were <6 nA in all experiments.
Solutions.
The physiological [Na]o solution contained (mM) 145 NaCl, 1 CaCl2, 1.0 MgCl2, 5.0 HEPES, 0.1 CdCl2, and 5.5 glucose (pH 7.4, adjusted with NaOH). The pipette solution for the INa recording contained (mM) 120 CsCl, 20 tetraethylammonium chloride, 5 ATP-Mg, 0.5 GTP-Na2, 5 EGTA, 10 NaCl, 2 CaCl2, and 10 HEPES (pH 7.2, adjusted with CsOH). The calculated free Ca2+ concentration in the pipette solution was 
100 nM (7). The low-[Na]o extracellular solution contained (mM) 10 NaCl, 132 N-methyl-D-glucamine (NMG), 1 MgCl2, 5 CsCl, 5 HEPES, 5 CoCl2, 1 CaCl2, and glucose 5 (pH 7.4 with HCl). The pipette solution for the AP recording contained (mM) 110 K-aspartate, 10 KCl, 5 MgCl2, 2 5 ATP-Na, 10 EGTA, 10 HEPES, 1 CaCl2 (pH 7.2, adjusted with KOH). Tetrodotoxin (TTX) was obtained from Calbiochem. All other chemicals were acquired from Sigma.
Molecular mechanism of decrease in sodium channel expression by calcineurin. Neonatal myocytes were isolated and cultured from WT mice as described previously (35). For patch-clamp recordings, 2 x 105 myocytes were plated on laminin-coated coverslips. After 24 h in culture, the medium was replaced by serum-free medium, and the virus infection was delivered [control, adenovirus (Ad)-green fluorescent protein (GFP) at 5 tissue culture 50% infectious dose (TCID50)/myocyte or Ad-constitutively active calcineurin obtained from J. D. Molkentin (University of Cincinnati, Cincinnati, OH) at 5 TCID50/myocyte]. Typically 90% of the myocytes showed expression of GFP 24 h after infection. At 12 h after viral infection chronic pharmacological treatments [ryanodine (10 nM), BAPTA-AM (10 µM), and thapsigargin (1 µM)] were given. Patch-clamp measurements were performed 24 h after treatment.
Sodium channels were labeled with subtype-specific antibodies against voltage-dependent sodium channel (NaV)1.1, NaV1.3, and NaV1.5 subunits and examined under the confocal microscope. We viewed five to seven myocytes per experiment for each antibody used in this study, and the results were consistent for all slides examined. Antibodies were provided by W. A. Catterall and R. E. Westenbroek. Control cells included preincubation of the primary antibodies with their respective peptides or incubation with no primary antibody. These negative control studies showed no specific staining. Briefly, single ventricular myocytes were plated on laminin-coated glass coverslips and incubated (5% CO2, 37°C) for 6–12 h. Myocytes were fixed with 4% paraformaldehyde for 30 min, rinsed in PBS, 0.1 M Tris buffer (TB), and 0.1 M Tris-buffered saline (TBS), treated in 2% avidin-TBS and rinsed in TBS, treated in 2% biotin-TBS and rinsed in TBS, and blocked in 10% normal goat serum-TBS-0.1% Triton X-100. The myocytes were incubated with primary antibodies (diluted 1:20 in TBS containing 0.10% Triton X-100 and 10% normal goat serum) overnight at 4°C. Myocytes were then rinsed in TBS, incubated in biotinylated goat anti-rabbit IgG (diluted 1:300), rinsed in TBS, incubated in avidin D fluorescein (diluted 1:300), rinsed in TBS, rinsed in TB, and rinsed in distilled water briefly. Coverslips were mounted on slides with Vectashield mounting medium (Vector Laboratories). Cells were viewed with a Leica DM RXA2 confocal microscope. For control cells, either primary antibodies were preincubated with their antigenic peptide or no primary antibody was used. Apparent plasmalemmal expression was quantitated by subtracting the apparent intracellular staining from the putative surface staining.
Whole cell measurements of INa disclosed that total current was decreased; however, total measured current is the product of the number of functional channels (N), single-channel conductance, and open probability. Following the methods of Heinemann et al. (16), we performed nonstationary noise analysis, a validated method to estimate N (25). Currents were elicited from a holding potential of –80 mV with a prepulse of –120 mV for 500 ms before the test pulse of –20 mV for 25 ms. Myocytes were isolated from 5-day-old neonatal mice. The rationale for the choice of the 5-day-old neonates relates to the following: 1) the 
-myosin heavy chain promoter does not become robustly active until the first week after birth; 2) round and small cells allow optimal spatial clamp (<25 pF); and 3) our data showed that at this time the current density of INa was significantly reduced (P < 0.05) without a difference in capacitance between CN and WT mice.
Only for noise analysis, [Na]o was increased to 30 mM NaCl to increase unitary channel conductance. The concentration of NMG was reciprocally reduced to 112 mM. The pipette solution for the INa recording contained (mM) 120 CsCl, 20 tetraethylammonium chloride, 5 ATP-Mg, 0.5 GTP-Na2, 5 EGTA, 10 NaCl, 2 CaCl2, and 10 HEPES (pH 7.2, adjusted with CsOH). The pipette resistance was 1–3 M. No capacitance or series resistance compensation was applied. Analyses were performed with custom software developed in MATLAB (Coherent Therapeutics, Calgary, AB, Canada). Using 5 successive leak records (hyperpolarized to –100 mV from –80 mV) to correct 60 successive test records, we determined the mean current as a function of time. Using successive difference records, we estimated current variance as a function of time. Finally, using the effective variance least-squares fitting method of Orear (25) to analyze the plot of ensemble current variance as a function of mean current, we estimated N. A correlation time of 0.8 ms was estimated by autocovariance function centered at the time of peak current.
In vivo relevance of decreased activity of INa density to heart block phenotype. Surface ECGs were recorded in conscious, unsedated mice (34). Signal-averaged ECGs were obtained by aligning all normal sinus rhythm cardiac cycles to a common point in the ECG waveform. To do this, we created software in MATLAB designed to perform a wavelet transform of the ECG using the second derivative of the Gauss function as our wavelet [Gauss support (–5,5) and wavelet scale = 25 ms]. Each R wave produced a large-magnitude, local minimum in the wavelet transform space that could be easily localized with a threshold. The ECG for each cardiac cycle was defined as the voltages within a sampling window starting 35 ms before the first R wave and ending 35 ms before the next R wave. Through this process, the mean of the compiled cycles for each 60-s recording was calculated.
To assess the heart rhythm disturbances that caused the spontaneous SCDs, telemeters were chronically implanted and spontaneous SCD was observed in older free-roaming animals. The SCD occurred at least 1 wk (median 8 wk) after implantation of the telemeter, and the mice were not on any medications. To assess the morphological precursors of spontaneous heart block, repetitive episodes of the onset of heart block were superimposed and signal averaged with custom software in MATLAB (Coherence Therapeutics). The fiduciary point of the QRS for each sinus beat just prior to heart block was defined by a wavelet transform.
To further explore the pathophysiological role of profound downregulation of INa in the heart block phenotype in vivo, we assessed the incidence of heart block leading to SCD before and after encainide treatment (20 mg/kg ip) in mice at 28 days of age, a time at which spontaneous sudden deaths only rarely occur in this transgenic mouse model. The rationale for the use of encainide relates to the Cardiac Arrhythmia Suppression Trial (CAST) study, in which encainide induced sudden cardiac deaths in humans (1, 6, 15, 33). Moreover, 300–1,000 nM concentrations of encainide selectively block the cardiac sodium channel, with minimal effect on other cardiac currents (7, 8). Encainide does inhibit the delayed rectifier current (IKr) but only at high (micromolar) concentrations (12). In addition, we previously reported (11) that dofetilide, the selective IKr channel blocker, never caused sudden death in this transgenic model. Although the more specific sodium channel blocker TTX was used in vitro, it is not possible to use TTX in vivo, since TTX blocks the neuronal sodium channels at nanomolar concentrations but the cardiac sodium channels only at micromolar concentrations (12). Thus, if TTX had been given in vivo to mice, only neurological responses would have been seen. Moreover, TTX would have compromised neural regulation of breathing, which would have confounded any in vivo result.
The QRS duration and amplitude were measured in sinus rhythm just before the onset of spontaneous SCD in free-roaming CN mice with implanted telemeters. The QRS interval was measured as the time between its onset to the time of the onset of the J wave. The termination of the QRS is defined as its position just before the onset of the J wave.
Statistical analysis. Data are presented as means ± SE. One-way ANOVA with Dunnett's multiple-range test was used, with a P < 0.05 considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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25% of the CN mice died of SCD. No deaths occurred in the WT mice (n = 40) over this time course. To assess whether decreases in sodium current density underlie the decrease in dV/dtmax of phase 0 of the APs, INa was recorded. Representative examples of the families of sodium channel current records elicited by the protocol are shown in Fig. 2A: WT on the left and CN on the right. Figure 2B shows mean current-voltage relationships, comparing WT to CN at 28 days (n = 5 each). Peak current density is significantly reduced. To assess whether shifts in the current-voltage relationship exist, comparing WT to CN, currents were normalized to the peak current and the results are plotted in Fig. 2A (inset). The normalized current-voltage relationships are similar in both WT and CN mice, with the peak current developing at a voltage of –30 mV. Thus the character of the current-voltage relationship of the residual currents in CN mice is similar to that seen in WT mice. Figure 2C shows the progressive time-dependent changes in INa density at various times during development. At 42–50 days, INa was virtually abolished. These data indicate that decrease in INa is responsible for the decrease in dV/dtmax of the APs.
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60% of the sodium channels would be available for activation in CN mice, whereas 70% of the channels would be available in WT mice. These data indicate that shifts in steady-state inactivation could only contribute modestly to the measured downregulation of the peak density of INa. To assess whether changes in recovery from inactivation could contribute to the observed decrease in current density, recovery properties were evaluated. Recovery from inactivation was assessed with the protocol illustrated in Fig. 2E. The mean time course of recovery was compared in WT vs. CN mice (28 days). No significant change in the time course of recovery from inactivation was noted. Since only minimal changes in steady-state inactivation and recovery from inactivation of INa were observed and since INa is virtually abolished in 50-day-old CN mice, it is likely that changes in density of INa were related to changes in N.
Nonphysiological conditions (e.g., [Na]o of 10 mM) were required to biophysically estimate INa density in ventricular myocytes. To record INa under more physiological [Na]o conditions (145 mM), the density of the TTX-sensitive INa was measured at potentials in proximity to its reversal potential of +45 mV in the smaller atrial cells from WT and CN mice (Fig. 2, F and G). The slope of the current-voltage relationship should measure whole cell INa conductance. In WT myocytes at 28 days the conductance was 5.9 ± 0.06 nS/pF, whereas in CN myocytes it was 1.4 ± 0.14 nS/pF (n = 3 each). These data provide confirmation of the measurements of INa recorded in the traditional manner.
Molecular mechanism of decrease in sodium channel expression by calcineurin. Infection of neonatal cardiac myocytes with an adenovirus that overexpresses constitutively active calcineurin significantly decreased density of INa to a magnitude similar to that seen in adult cardiac myocytes freshly isolated from these transgenic mice (Fig. 3A). Since previous studies indicate that calcineurin can modulate ryanodine receptor function (2, 4), we assessed whether strategies designed to alter intracellular calcium homeostasis could rescue the downregulation of INa. Accordingly, we assessed the ability of chronic (24 h) in vitro treatment with ryanodine (10 nM), BAPTA-AM (10 µM), and thapsigargin (1 µM) to rescue the downregulation of INa. Figure 3A shows mean density of INa (pA/pF). All of these treatments rescued INa density to a value indistinguishable from that in WT cells. Importantly, these treatments had no effect on WT cells (Fig. 3B). Thus the effects of these drugs were specific to cells overexpressing calcineurin, but not to WT cells. To assess the physiological relevance of these changes in INa, the dV/dtmax of phase 0 was evaluated in spontaneous APs in separate cells (Fig. 3, C and D). As expected, the changes in INa were paralleled by changes in dV/dtmax. These data indicate that this rescue of the decreased density of INa has physiological relevance.
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Total cardiac sodium channel protein expression was evaluated with two methods: 1) Western blots on whole ventricle homogenates in 28-day-old WT and CN hearts and 2) immunocytochemistry in neonatal mouse cardiac myocytes infected with an adenovirus containing constitutively active calcineurin (provided by J. D. Molkentin) vs. a GFP adenoviral control (Fig. 6). The overall results were similar with both methods. Figure 6A shows sodium channel expression with an antibody against NaV1.5 in sib pair animals (lanes 1 and 2; lanes 3 and 5 are from sib pairs); lanes 5 and 6 represent the negative controls in which the antibodies were previously blocked with pure peptide. Western blot indicated that overexpression of calcineurin does not alter the overall NaV1.5 protein expression. To confirm these data, immunocytochemistry was performed with antibodies against NaV1.1, 1.3, and 1.5. (Fig. 6B). Negative control experiments involved incubation in the absence of primary antibody. Negative controls showed no specific staining. The mean data in arbitrary units of epifluorescence showed no significant change in expression in any of the isoforms examined. The arbitrary units of epifluorescence were for NaV1.1, 7.7 ± 1 for WT and 7.5 ± 1 for CN; for NaV1.3, 8.0 ± 1 for WT and 7.1 for CN; and for NaV1.5, 9.3 ± 0.3 for WT and 9.5 ± 0.3 for CN. Overexpression of calcineurin has no gross effects on overall sodium channel protein expression as assessed by these two techniques.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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APs and INa: part of the pathway to heart block. Decreased activity of INa was consistently manifest in three separate cell types: 1) adult cardiac myocytes from CN mice, 2) WT neonatal cardiac myocytes treated with adenoviral overexpression of calcineurin, and 3) day 5 neonatal myocytes from CN mice. Importantly, the decrease in INa activity in the day 5 neonatal myocytes from CN mice occurred without a significant difference in cell size (compared to WT) as assessed by capacitance values. Therefore, decrease in INa does not appear to be a nonspecific downstream target secondary to cardiac hypertrophy. In review, consistent results observed in three separate cell types confirms that decreased activity of INa is associated with calcineurin overexpression.
The evidence that decreased activity of INa contributes to the common final pathway to heart block includes the following. 1) There is a consistent time course of the decrease in activity of INa and the development of heart block and asystole in vivo. 2) Sodium channel current activity asymptotes to tiny values during aging in CN mice, and sodium channel activity is necessary for cardiac propagation. 3) Encainide, a selective sodium channel blocker, produces exaggerated block of INa in vitro and triggers heart block and asystole in vivo in young CN mice but not in WT mice. This ECG phenotype is identical to that which occurs during spontaneous SCD in older CN mice. 4) Adenoviral overexpression of calcineurin in isolated WT neonatal cardiac myocytes recapitulates the decrease in activity of INa. 5) The magnitude of the QRS (R3 beat, Fig. 10) just preceding heart block is significantly less (P < 0.0005) than in the precursor beats (R1 and R2 beats, Fig. 10), indicating that the defect predisposing to heart block exists in the QRS. 6) Our previous study (14) reported that the pacing cycle length that elicits heart block in CN mice is well correlated with left ventricular effective refractory period. Activity of INa is an important determinant of excitability and ventricular effective refractory period. 7) Previous studies have reported that in the absence of sodium current, "slow" action potentials can still be elicited with phase 0 being carried by ICa-L (3, 17, 19), but the magnitude of this current is critically dependent on catecholamines. Blockade of adrenergic maintenance of this "slow" action potential would be expected to precipitate heart block. In the present study, the beta-blocker propranolol consistently triggered heart block, asystole, and death in CN mice. 8) In humans, mutation in the cardiac sodium channel that decreases functional sodium channel activity creates the same phenotype—heart block (30). Probst et al. (26, 27) reported a splicing mutation that decreases SCN5A gene expression, leading to hereditary progressive cardiac conduction defect. 9) Haploinsufficiency of this SCN5A trait in combination with aging causes hereditary Lenegre heart block. These data, taken in concert, provide evidence that decrease in cardiac sodium channel activity contributes to the common final pathway to heart block in CN mice. However, it is clear that the effect on the sodium channel is indirect. The sodium channel is likely a downstream target for abnormalities in calcium homeostasis and PKC activation.
Molecular mechanism of downregulation of cardiac sodium channel. Chronic (24 h) in vitro treatment with ryanodine, thapsigargin, and BAPTA-AM rescues the downregulation of INa seen with overexpression of calcineurin. To further address whether altered calcium homeostasis could activate downstream signal transduction pathways, we assessed whether the PKC inhibitor bisindolylmaleimide I could rescue the downregulation of INa. Cross talk between signal transduction pathways could be mediated by alterations in calcium homeostasis. The involvement of the PKC pathway as a downstream signaling pathway for calcineurin is in keeping with studies of De Windt et al. (10).
The molecular mechanisms observed in neonatal ventricular myocytes treated with an adenovirus overexpressing constitutively active calcineurin are qualitatively similar to those observed in primary cultures of myocytes from 14-day-old CN mice treated with the same pharmacological treatments. Thus the insights into mechanism appear to apply to both kinds of cells.
In terms of calcium homeostasis, Chu et al. (9) reported that overexpression of calcineurin increased protein expression and activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a and increased expression of phospholamban and its phosphorylation. These biochemical findings were associated with an increase in the Ca2+ transient amplitude (1.5-fold) and the rate of Ca2+ signal decay (2-fold). Thus abnormalities of SERCA2a and phospholamban cannot explain the result observed here. Calcineurin has been reported to interact with the ryanodine receptor (4, 19) and inositol 1,4,5-trisphosphate receptor and with the FKBP12 complex (5, 13). However, the molecular mechanism by which calcineurin interacts with these proteins is uncertain, and whether PKC is an upstream mediator of this interaction is also unknown.
Previous studies by Qu et al. (28) reported that PKC activation decreases functional sodium channel expression. Activation of PKC by oleoyl-2-acetyl-sn-glycerol (10 µM) decreased the sodium current by 33% at a holding potential of –114 mV and 56% at –94 mV. This reduction in peak current was partially related to an 8- to 14-mV shift of steady-state inactivation in the hyperpolarized direction. In the present study, we found only a modest shift in the steady-state inactivation of INa. In the study by Qu et al. (28), single-channel recordings showed that the probability of channel opening was reduced by PKC activation but single-channel conductance was unaffected. Murray et al. (21) and Shin and Murray (32) also addressed the mechanism of the effects of PKC on decreased activity of the cardiac sodium channel. Their studies provide evidence that PKC modulates the human cardiac sodium channel by at least two mechanisms, one similar to that seen with rat brain channels at a conserved serine (residue 1503) involving a conserved putative PKC site and a second mechanism more specific to the cardiac isoform. Importantly, Murray et al. (32) also reported decreased functional sodium channel activity without a change in overall protein expression (Western blots). Our data are consistent with the previous molecular studies.
Potential clinical relevance. Previous studies have reported that calcineurin plays an important role in pathological cardiac hypertrophy (10, 11, 14, 20, 35). Cardiac hypertrophy can lead to calcium overload due to increases in calcium entry via ICa-L (35), abnormalities of calcium uptake into the sarcoplasmic reticulum (SR), calcium buffering within the SR, and release from the SR and/or removal from the cell by the sodium/calcium exchanger. Abnormalities of calcium homeostasis can activate a multitude of downstream targets. One of these targets is PKC. Activation of PKC decreases the activity of INa, contributing to the heart block phenotype in CN mice. Importantly, calcium homeostasis is dynamic. Interventions designed to enhance sequestering and/or buffering of calcium may have therapeutic activity.
In conclusion, decrease in activity of INa contributes to heart block and asystole in murine hearts overexpressing calcineurin.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and CN (
) mice. OS, overshoot potential; dV/dtmax, maximum change of potential over time; Threshold, excitability threshold.
