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Am J Physiol Heart Circ Physiol 279: H2133-H2142, 2000;
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Vol. 279, Issue 5, H2133-H2142, November 2000

Phenotypical features of long Q-T syndrome in transgenic mice expressing human Na-K-ATPase alpha 3-isoform in hearts

Sharon E. O'Brien1, Michael Apkon1, Charles I. Berul2, H. T. Patel2, Kurt Saupe3, Mathias Spindler3, Joanne S. Ingwall3, and Raphael Zahler1

1 Departments of Internal Medicine and Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510; 2 Department of Pediatrics, Tufts University School of Medicine, Boston 02111; and 3 Department of Medicine, Harvard University School of Medicine, Boston, Massachusetts 02118


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To understand why the adult human heart expresses three isoforms of the sodium pump, we generated transgenic mice (TGM) with 2.3- to 5.5-fold overexpression of the human alpha 3-isoform of Na-K-ATPase in the heart. Hearts from the TGM had increased maximal Na-K-ATPase activity and ouabain affinity compared with control hearts, even though the density of Na-K-ATPase pump sites (of all isoforms) was similar to that of control mice. In perfused hearts, contractility both at baseline and in the presence of ouabain tended to be greater in TGM than in controls. Surface electrocardiograms in anesthetized TGM had a steeper dependence of Q-T on sinus cycle length, and Q-T intervals measured during atrial pacing were significantly longer in TGM. Q-T dispersion during sinus rhythm also tended to be longer in TGM. Thus TGM overexpressing human alpha 3-isoform have several of the phenotypical features of human long Q-T syndrome, despite the absence of previously described mutations in Na+ or K+ channels.

sodium; myocytes; perfused heart; ouabain


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NA-K-ATPASE IS AN ENZYME that is critical to the normal mechanical and electrical function of the heart. Na-K-ATPase is comprised of two protein subunits, alpha  and beta , both of which have at least three isoforms. Moreover, there are important functional differences among the three alpha -isoforms that are found in heart. The alpha 1-isoform is ubiquitous and appears to be a stable housekeeping isoform. In the heart, alpha 2- and alpha 3-isoforms manifest differences in expression regionally, developmentally, and in response to certain physiological stresses (6, 7, 11, 17, 32, 36, 37). In the rat, the alpha 3-isoform is distinguished from the alpha 1-isoform by a decreased affinity for Na+, an apparently higher extrusion rate of Na+, and a 1,000-fold greater affinity for ouabain (5, 13, 22, 41).

We hypothesized that these differences in Na-K-ATPase function might be related to known isoform structural differences in the myocardium during development and at times of stress. For example, one might expect that contractile cells expressing increased amounts of the alpha 3-isoform would have higher intracellular Na+ concentrations ([Na+]i), as already shown in noncontractile HeLa cells (41). Additionally, one might anticipate that if contractile cells expressing increased amounts of the alpha 3-isoform were exposed to low concentrations of cardiac glycosides, the higher affinity alpha 3-pumps would be inhibited, increasing [Na+]i and thereby intracellular Ca2+ concentration ([Ca2+]i). There would thus be a significantly greater inotropic response in cells expressing increased amounts of the alpha 3-isoform than in control cells.

Furthermore, the sodium pump is electrogenic. It causes a net flux of ions out of the cell and thus contributes to a background hyperpolarizing current. The transmembrane gradient of Na+ maintained by the sodium pump also affects systems, such as the Na+-Ca2+ exchanger, which influence repolarization. For these reasons, increased expression levels of the alpha 3-isoform in cardiac myocytes could affect not only the contractile but also the electrophysiological properties of the myocardium (26).

We thus developed transgenic mouse (TGM) lines in which high levels of the alpha 3-isoform are expressed in the myocardium of adult animals. By utilizing both in vitro and in vivo techniques, in two separate lines of TGM, we were able to test the functional importance of isoform expression by comparing myocardial contractile and electrophysiological properties in control mice and TGM. We found that the TGM have several of the phenotypical features of human long Q-T syndrome.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Development of transgenic mouse lines. Previous authors have reported that the adult mouse myocardium lacks detectable alpha 3-mRNA and alpha 3-protein (2). We thus performed Western blot analysis on microsomes made from the control mouse myocardium using two different isoform-specific antibodies against rat alpha 3-protein. Even at long exposure times, only a faint band at the 97-kDa position was seen (Fig. 1). Although crossreactivity to other isoforms cannot be excluded, based on previous localization data in normal rat hearts (38), we believe that this signal represents alpha 3-protein located in conduction tissue or junctional complex. This is supported by immunohistochemical studies we performed on control mouse heart, in which we observed reactivity to alpha 3-protein only in conduction tissue (data not shown).


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  		Fig. 1.   A: Southern blot analysis of mouse tail DNA. We labeled a 3.6-kb fragment of the microinjected construct and used this to probe Eco RI/Kpn I-digested genomic DNA from mouse tails. Lane 1, positive control (plasmid DNA cut with Eco RI and Kpn I); lane 2, negative control (normal mouse tail DNA). Lanes 3-11, tail DNA from 9 offspring of oocyte injection showing signal from transgene DNA in lanes 6 and 11. Lane 11 animal was founder of line 52 (see text). B: evaluation of expression levels of alpha 3-protein in control (N) and transgenic mouse (TGM) heart. We performed a Western blot using microsomal protein from heart of a control male mouse and titrated amounts of microsomal protein as shown from heart of a male TGM of line 52. We stained the blot with F9G10 antibody (previously shown to be isoform-specific for alpha 3-protein; see Ref. 39). TGM heart expresses a 97-kDa protein that is immunoreactive with F9G10, consistent with alpha 3-isoform expression. Expression level of this protein is much greater in TGM heart than in control mouse heart (comparing lanes 1 and 4, both loaded with 32 µg protein). Level of alpha 3-isoform expression in multiple other control mouse hearts studied was at lower limits of detectability (see text). We obtained similar results using primary antibody CM, a polyclonal antibody also isoform-specific for alpha 3-protein (data not shown). In addition, similar quantitative Western blots performed on specimens from multiple other hearts showed on average 5.5-fold greater expression of alpha 3-protein in line 52 TGM hearts than in control hearts. C: Western blot performed as above on rat microsomal specimens but stained with McB2 antibody, which is isoform-specific for alpha 2-protein. Lane 1, rat skeletal muscle; lane 2, rat kidney; lanes 3-5, heart from control mouse; lanes 6-8, heart from line 52 TGM. Lanes 3 and 6 were loaded with 12.9 µg protein; lanes 4 and 7, 14.3 µg protein; and lanes 5 and 8, 16.5 µg protein. Tissue standards in lanes 1 and 2 confirm isoform-specificity under the conditions we used. Lanes 3-8 are representative of multiple experiments on hearts from different animals, which showed that expression level of alpha 2-protein was lower in TGM heart.

Thus to examine the functional effects of alpha 3-isoform expression, we developed lines of TGM that overexpress human alpha 3-protein (30) in the heart. First, we ligated a 640-bp fragment of rat alpha -myosin heavy chain 5'-upstream promoter region (19) to the cloned human alpha 3-cDNA. In an attempt to achieve higher levels of expression, we also made a construct in which the 3.3-kb Sph I segment of the 5.5-kb fragment of mouse alpha -myosin heavy chain promoter (29) drove expression of the same cDNA. In both cases, we inserted a fragment containing the SV40 small t-antigen splice and polyadenylation signal immediately 3' to the full-length human alpha 3-cDNA. We verified that the construct was correct by restriction mapping and partial sequencing. We then isolated and purified a restriction fragment of each plasmid, which contained the promoter, cDNA, and SV40 elements but excluded almost all the plasmid sequence, and injected it into mouse oocyte pronuclei. Animal experiments were performed under a protocol approved by the Yale Animal Care and Use Committee.

Western blotting. We evaluated expression of Na-K-ATPase isoform proteins by Western blotting of microsomal preparations using the isoform-specific antibodies 6H (anti-alpha 1), McB2 (anti-alpha 2), and F9G10 and CM (anti-alpha 3), generously supplied by M. Kashgarian, K. Sweadner, M. Caplan, and K. P. Campbell (24, 28, 31, 39). We verified isoform specificity using immunoblots of human brain, muscle, and kidney control specimens as previously described (38).

To prepare microsomes, we washed mouse hearts twice in ice-cold PBS with 0.315 M sucrose, 20 mM Tris, and 1 mM EDTA (pH 7.5) with leupeptin and aprotinin added, and then disrupted the tissue with a Dounce homogenizer. After homogenization, we performed successive centrifugations (1,000 g for 10 min and 9,000 g for 15 min). We then resuspended the pellet from a 12,000 g, 1-h centrifugation in 0.25 M sucrose with 10 mM HEPES and determined the protein concentration using a colorimetric protein assay (Bio-Rad, Hercules, CA). Microsomes were stored at -70°C until use. We made no attempt to exclude conduction tissue from the microsome preparations, but because conduction tissue is estimated to constitute <2% of cardiac mass (35), the results below are unlikely to have been affected by the inclusion of varying amounts of conduction tissue.

We loaded microsomal protein (10-30 µg) on 7% SDS-PAGE gels, performed electrophoresis, and transferred the protein electrically to Immobilon-P membranes (Millipore, Bedford, MA). We blocked the membranes by treating them with a solution of 20 mM Tris (pH 7.5), 150 mM NaCl, 5% nonfat dry milk, and 0.1% Tween 20 at room temperature for 1 h. We then incubated them with primary antibody for 1 h and then washed them. We used primary antibodies at the following dilutions: 6H at 1:5,000; McB2 at 1:20-1:40; F9G10 at 1:100-1:500; and CM at 1:2,000. We diluted secondary antibody (peroxidase-conjugated sheep anti-mouse IgG or donkey anti-rabbit IgG; Amersham, Little Chalfont, UK) to 1:5,000 and detected signals with the ECL kit (Amersham). We quantitated the results by including titrated amounts of microsomal protein on each blot (Fig. 1), measuring band intensity using densitometric scanning, and deriving from this a range in which band intensity was a linear function of protein (17, 41). Each numerical value of relative protein expression reported below is derived from at least four quantitative blots.

Ouabain binding. We studied ouabain binding to the microsomal membranes as previously described (15). Briefly, we made crude microsomal preparations from control and TGM tissue as above. A 40-µg aliquot of microsomal protein was added to 250 µl of a solution containing (in mM) 5 ATP, 10 MgCl2, and 100 Na+, with 2-40 nM [3H]ouabain and graded amounts of cold ouabain. We allowed ouabain binding to proceed during a 45-min incubation at 37°C and terminated the binding reaction by vacuum filtering onto HAWP membranes (Millipore). The amount of [3H]ouabain bound to the membrane was determined by liquid scintillation counting. We chose the 45-min incubation period because in preliminary experiments we verified that specific binding saturated by 30 min, remained stable for at least 90 min under the K+-free conditions employed, and increased linearly with membrane concentration.

Na-K-ATPase activity. We performed the basic assay as per Forbush (9) using the modifications detailed by Jewell and Lingrel (13). This assay measures Pi liberated from ATP colorimetrically. We defined Na-K-ATPase activity as the fraction of total ATPase activity, expressed as micromoles Pi per milligram of protein per hour, which is inhibited by 1 mM ouabain. We permeabilized microsomes (5-10 µg in 50 µl) prepared as above for 10 min at room temperature by adding 100 µl of 0.8 µg/ml SDS and 1% BSA in 25 mM imidazole (pH 7.4); preliminary experiments verified that these conditions yielded maximal ATPase activity. After this treatment, we added 600 µl of 0.3% BSA in 25 mM imidazole to lower the SDS concentration and transferred 100-µl aliquots to 15-ml polypropylene tubes. Next, we added 500 µl of assay buffer (containing in mM) 100 NaCl, 10 KCl, 40 choline chloride, 60 Tris, 1 EDTA, 4 MgCl2, and 4 Tris-ATP (pH 7.4) to each tube and incubated the samples for 90 min at 37°C. At this point we added 1 ml of molybdate mixture (0.5% ammonium molybdate, 3% SDS, and 3% ascorbic acid in 0.5 N HCl). After 10 min more on ice, we added 1.5 ml of arsenite solution (2% sodium m-arsenite, 2% sodium citrate, 2% acetic acid) and incubated the tubes for 10 min at 37°C. We then measured absorbance at 705 nm (A705) on a spectrophotometer. Pi standards (0 and 50 µM) were included with each trial. In preliminary experiments we verified that A705 was a linear function of Pi at concentrations ranging from 2 to 100 µM, and that Na-K-ATPase activity was a linear function of incubation time (through 90 min) and of membrane protein concentration.

Phosphorylation assay. Following the methods of Arguello et al. (1), we carried out Na+-activated phosphorylation at 4°C in a medium containing 100 mM NaCl, 2 mM MgCl2, 1.8 µM gamma -[32P]ATP, 0.2 mM EGTA, 40 mM HEPES (pH 7.2 at 4°C), ouabain, and 150 µg/ml of membrane protein. In control tubes, NaCl was substituted by 100 mM KCl. We initiated reactions by adding gamma -[32P]ATP and stopped the reactions after 30 s with nine volumes of an ice-cold solution containing 5% TCA, 5 mM Na2ATP, and 2.5 mM NaH2PO4. We then filtered the samples as described, washed them twice with 5 ml of the above ice-cold solution, and measured radioactivity using a liquid scintillation counter.

Isolation of cells. We prepared isolated cardiac myocytes by enzymatically digesting retrogradely perfused mouse hearts (21). For most experiments, solutions were based on Krebs-Henseleit buffer (containing in mM: 118 NaCl, 4 KCl, 1.2 NaH2PO4, 0.5 EDTA, and 2.5 NaHCO3), although in later experiments we used a similar buffer based on HEPES but not containing NaHCO3. We equilibrated bicarbonate-based solutions with a 5% CO2-95% O2 gas mixture, during which pH was 7.4. We sterile filtered all solutions.

To perform myocyte isolation, we first anesthetized adult male mice with methoxyflurane. We opened the chest and removed the heart and ascending aorta and transferred them to ice-cold solution A (Krebs-Henseleit with 10 µM Ca2+, 10 mM MgSO4, and 10 mM glucose). We then cannulated the ascending aorta and secured it to a 21-gauge blunted needle, which we then attached to a water-jacketed perfusion column warmed to 37.5°C. We perfused the hearts for 7 min with solution A and then switched to solution B [same as solution A but containing 1 mg/ml collagenase (type II, Worthington, lot F5P860), 1 mg/50 ml protease (type XIV, Sigma), and 220 µM Ca2+]. In some experiments, we added 1 mg/ml BSA (Sigma) to solution B. We continued the perfusion until the hearts were soft. After perfusion, we separated the ventricles from the atria, chopped them into 2- to 3-mm pieces, and triturated them in solution C (Krebs-Henseleit with 220 µM Ca2+, 1% BSA, and 10 mM glucose) to release single ventricular myocytes. We introduced the myocytes into physiological solutions at gradually increasing Ca2+ concentrations ([Ca2+]) to a final [Ca2+] of 1 mM.

Contractility studies in isolated myocytes. We placed cells in a chamber on the stage of an inverted microscope and superfused them with HEPES buffer alone or with 10 nM ouabain added. We stimulated the cells via a pair of platinum electrodes at a frequency of 0.5 Hz, using a 5-ms pulse width. We monitored the transilluminated image of the myocyte using a video camera (Pulnix T640, MOSFET) at 240 frames/s. We analyzed the images using a video edge-motion detector (Crescent Electronics, Sandy, UT), which generated an analog signal proportional to the cell length. We digitized the analog signals and stored them on a personal computer using an analog instrumentation interface (Labmaster Scientific Solutions) and acquisition software (pCLAMP, Axon Instruments).

We recorded data in sets of 16 twitches. We excluded those twitches that were not accurately followed by the edge detector (there was no systematic difference between control and TGM cells regarding unused twitch data) and averaged the remaining twitches from that set of 16 twitches. Usually no more than three twitches from each group had to be excluded but never more than eight. We determined the myocyte shortening fraction by calculating the change in length between systole and diastole and dividing the result by the maximum length in diastole. We performed this measurement at baseline as well as during the administration of ouabain.

Perfused heart experiments. These experiments were performed as described by Saupe et al. (27). Briefly, we perfused mouse hearts with phosphate-free Krebs-Henseleit buffer containing 2 mM Ca2+ and 10 mM glucose in a isovolumic Langendorff heart preparation in which volume (left atrial filling pressure) and aortic resistance could be modified. We monitored left ventricular and aortic pressures by means of indwelling catheters coupled to Statham P23 pressure transducers. We paced the hearts at 6.8 Hz (400 beats/min), holding the coronary perfusion pressure constant at 75 mmHg. We determined positive and negative changes in pressure over time (dP/dt) on-line.

Electrophysiology studies. We performed in vivo experiments to study cardiac electrophysiological characteristics of living control mice and TGM. We anesthetized the mice with ketamine and pentobarbital sodium (both 0.033 mg/gm ip). We acquired surface electrocardiograms (ECGs) and intracardiac electrogram signals by previously described methods (4). In brief, we recorded surface ECGs using a standard six-lead configuration, digitized the information, and stored it on a personal computer. The mean sinus cycle length (SCL, beat-to-beat heart rate), P wave duration (atrial conduction), P-R interval (composite of atrial and atrioventricular nodal conduction), QRS duration (ventricular depolarization), J-T interval (ventricular repolarization), and Q-T interval (surrogate of action potential duration) were measured with electronic calipers by two observers who were blinded to the identification of the mice. The P-R interval is marked from the beginning of the surface P wave to the beginning of the QRS complex. We calculated Q-T dispersion from the six limb leads as previously described (18), correcting for heart rate by dividing by SCL. We recorded intracardiac ECGs using a 1.7-Fr octapolar catheter (NuMed, Hopkinton, NY) introduced through a right jugular vein cut down and advanced into the heart up to the tricuspid annulus. In this position, we could pace the right atrium and ventricle and also record electrograms. For the data presented here, we studied 15 TGM and 13 control mice from line 52 (see RESULTS) and 10 TGM and 8 control mice from line 203. Data from the two lines were similar and were pooled in the presentation.

Statistical analysis. We present data as means ± SE. We performed statistical analysis using unpaired Student's t-test, simple linear regression with two-equation method to test for differences, and nonlinear regression (JMP, SAS Institute, Cary, NC). Ouabain response in perfused hearts was modeled by nonlinear fitting to an exponential function. We fitted data on ouabain inhibition of Na-K-ATPase activity to classical receptor-binding models of the form y = v1/ [1 + (x/k1)a1] + v2 [1 + (x/k2)a2], where y is the measured Na-K-ATPase activity, x is the ouabain concentration ([ouabain]), v is the component of maximal activity attributable to sites 1 and 2, k is the affinity of sites 1 and 2, and a is the Hill coefficient for sites 1 and 2.

A P value of <0.05 was considered statistically significant. For those comparisons whose P values were between 0.06 and 0.08, we found that the statistical power values ranged from 0.346 to 0.446.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vitro evaluation of TGM. Seven founder mice were generated from microinjections of the fragment that contained the 640-bp promoter, and four founder mice were obtained from the fragment that contained the 3.5-kb promoter. From each group, we chose a TGM line with the highest expression levels of immunoreactive alpha 3-protein in heart. This resulted in two lines of CD-1 mice hemizygous for the transgene, as confirmed by Southern blot analysis of genomic DNA obtained from tail specimens (Fig. 1A). The TGM used were all hemizygous, so nontransgenic littermates could be used as controls.

Grossly, these founders demonstrated no phenotypical changes. TGM hearts were morphologically normal at the gross and histological level, and the heart weight-to-body weight ratio of TGM did not differ from that of controls. The hearts of both lines of transgenic animals consistently expressed a 97-kDa protein that was immunoreactive with two isoform-specific anti-alpha 3-antibodies as confirmed by Western blotting, as well as by immunochemistry (data not shown). Quantitative Western blotting showed that TGM hearts of line 52 (which carried the fragment containing the 640-bp promoter) expressed high levels of immunoreactive alpha 3-protein (Fig. 1B); the level of alpha 3-isoform expression in the TGM heart was, on average, 5.5-fold higher than in control hearts. The barely detectable alpha 3-isoform signal in the control heart probably represents expression restricted to cardiac conduction system and junctional complexes (38). No alpha 3-isoform expression was detected in organs of TGM other than the heart and brain. The expression level of alpha 3-protein in hearts from TGM line 203 (which carried the fragment containing the 3.5-kb promoter) was 2.3-fold higher than in control hearts. The expression level of immunoreactive alpha 2-protein, however, was about one-third less in TGM hearts than in control hearts (Fig. 1C). The expression level of immunoreactive alpha 1-protein in the TGM heart was not significantly different from that in the control heart.

Several additional in vitro assays confirmed that greater numbers of alpha 3-isoform pumps were present and functional in TGM hearts. First, we performed quantitative competitive [3H]ouabain binding, which in rodent tissue measures only high-affinity Na-K-ATPase sites (alpha 2- or alpha 3-isoforms). Such experiments on heart microsomes indicated that the affinity measurement (KD) for the high-affinity component in TGM heart was 38.8 nM compared with 144 nM for control mouse hearts. In addition, the high-affinity ouabain KD measured in TGM hearts was similar to the KD of human alpha 3-protein measured independently in SY5Y human neuroblastoma cells (40). Furthermore, Scatchard analysis of the ouabain binding data suggested that the density of high-affinity pump sites in TGM hearts was 2.3-fold greater than in control hearts (control hearts 4 ± 1.8 pmol/µg protein, TGM line 52 hearts 10 ± 1.2 pmol/µg protein; P = 0.05, n = 4 for each group; 95% confidence interval for difference 0.1-10.9 pmol/µg protein). Finally, in assays of Na-K-ATPase activity, the fraction of Na-K-ATPase activity sensitive to 1 µM ouabain was 15% in TGM hearts compared with <5% in control hearts.

The density of Na-K-ATPase pump sites (of all isoforms) measured by quantitative phosphorylation assay was nonsignificantly greater in TGM hearts than in control hearts (control hearts 9.1 pmol/mg protein, TGM line 52 hearts 11.5 pmol/mg protein; P = 0.08, n = 8 control hearts and 9 TGM hearts; 95% confidence interval for difference -5.1 to 0.3 pmol/mg protein).1 Yet maximal Na-K-ATPase activity in control hearts (n = 5) was 8.83 ± 4.31 compared with 28.63 ± 6.22 µmol Pi · mg protein-1 · min-1 in TGM hearts, a threefold difference that was just short of statistical significance (P = 0.07, n = 7; 95% confidence interval for difference -1.7 to 35.1 µmol Pi · mg protein-1 · min-1). This discordance between modestly increased pump-site density and apparently markedly increased maximal activity in the TGM is actually consistent with a previous study (41), which suggested that the alpha 3-isoform has several-fold greater activity than the other isoforms. When we combine these data with results of earlier workers (2, 23) who described Na-K-ATPase isoform distribution in the normal rat and mouse heart, the results are consistent with a model in which alpha 3-pumps make up only 8% of total pumps in the normal adult mouse heart but 36% of total pumps in the TGM heart.

Contractility in isolated myocytes. Fig. 2 illustrates the effect of 10 nM ouabain on the shortening fraction of isolated cardiac myocytes from control and transgenic animals. Ouabain causes increased contractility by inhibiting sodium pumps; we chose the concentration of 10 nM because this level of ouabain would be expected to inhibit only alpha 2- and alpha 3-isoform pumps [<2% of alpha 1-pumps are inhibited at this ouabain concentration ([ouabain]); see Ref. 13]. The vertical axis of Fig. 2 plots the percent increase in shortening fraction of myocytes induced by exposure to 10 nM ouabain. In 15 myocytes from 7 TGM of line 52, ouabain caused the shortening fraction to increase 53% (±27%) compared with a 21% (±12%) increase in 15 myocytes from 6 control mice (P = not significant for the comparison between control and TGM myocytes). Although these data are not definitive, possibly because of the relatively small number of myocytes studied, an enhanced myocyte contractility response of TGM myocytes to low-dose ouabain would be consistent with our in vitro results, which suggest that a larger fraction of the pumps in TGM heart are sensitive to low levels of ouabain.


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  		Fig. 2.   Effect of ouabain on shortening fraction of isolated myocytes from control and TGM heart. We prepared cardiac myocytes as described in the text and stimulated them electrically, measuring changes in length with a video edge-motion detector. Shortening fraction was measured both in buffer alone and in the presence of 10 nM ouabain. Because basal shortening fraction (measured in buffer alone) varied considerably among myocytes, we used the following approach to compare the ouabain response in control and TGM myocytes: for each myocyte, we expressed the shortening fraction observed in the presence of 10 nM ouabain as a percent increase over shortening fraction measured in buffer alone. We averaged these values for the control and TGM myocytes, and plotted them on the vertical axis.

Perfused-heart experiments. We studied hearts from eight TGM and seven control mice. Peak positive dP/dt (a measure of contractility) increased by 59% and 77% in hearts from TGM and control mice, respectively, when treated with 100 µM ouabain (Fig. 3). Similarly, developed pressure, another index of contractility, increased by 35-40 mmHg (45-47%). Developed pressure at each of seven [ouabain] averaged 6-11% greater in TGM than control hearts, and peak positive dP/dt was 13-18% greater. At each individual value of [ouabain], the difference between control mice and TGM dP/dt did not reach statistical significance. When the control and TGM curves were each fitted to exponential functions, however, the difference between the intercepts of the two exponential curve fits was significant (P < 0.05; 95% confidence interval for difference -9.48 to -0.51). These data thus suggest modestly greater contractility in intact TGM hearts than in control hearts.


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  		Fig. 3.   Effect of ouabain concentration ([ouabain]) on peak positive change in pressure over time (dP/dt) in isolated perfused mouse hearts. We studied working perfused hearts from 8 control mice and 7 TGM. We perfused each heart first with buffer alone and subsequently with increasing [ouabain]. As shown in the figure, dP/dt in hearts from TGM was somewhat greater than in control hearts at all [ouabain] levels, although the positive inotropic effect of ouabain began to occur at similar concentrations in both groups of hearts. The difference in contractility in TGM hearts compared with control hearts was statistically significant (P < 0.05 for difference between the intercepts of the curve fits).

Surface ECGs. We obtained surface ECGs in 46 mice (Fig. 4). We measured SCL, P-R interval, QRS duration, and Q-T duration during sinus rhythm (Fig. 4, A and B) and during fixed-rate atrial pacing (Fig. 4, C and D). We attempted invasive electrophysiological studies in 25 of the mice and were successful in 21. 


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  		Fig. 4.   Surface electrocardiograms (ECGs) from control mice (wild-type, WT) (A and C) and TGM (TG) (B and D), recorded during sinus rhythm (A and B) and during right atrial pacing at a cycle length of 150 ms (C and D). The murine ECG is obtained from surface limb leads with band-pass filtering between 5 and 250 Hz, amplified at 0.1 mV/cm, and digitized for on-line analysis. The QRS complex has an R and R' deflection, both included in whole QRS measurement of ventricular depolarization. The QRS complexes have been aligned (solid vertical lines) at the Q wave for each pair of recordings to allow comparison of the Q-T intervals between control mice and TGM. The broken vertical line marks the terminations of the T waves in control mice: after 1 spontaneous QRS complex (A) and after the final complex at the termination of atrial pacing (C). The apparent alternation in QRS morphology in the paced ECGs C and D represents an artifact of pacing. The actual ventricular depolarizations have a cycle length of 150 ms.

There was no difference in average age or body weight between control mice and TGM. There were also no significant differences in P-R interval or QRS duration between TGM and control mice (Table 1). Average SCL was longer in control mice, although the difference was not statistically significant (P = 0.1). Because the SCL varied between the control and transgenic groups, and because the Q-T interval is known to depend on SCL, we used linear regression to determine whether the Q-T versus SCL relationship differed between control mice and TGM (Fig. 5). In both the control group and the TGM group there was a significant correlation between SCL and Q-T interval (P < 0.001). In addition, the slope of the regression line for the control mice was significantly greater than the slope of the regression line for the TGM, indicating a steeper dependence of Q-T on SCL in the TGM (slope for control group 0.186, slope for TGM 0.446; P = 0.016 for difference in slopes between control and TGM groups; 95% confidence interval for difference in slopes 0.11-0.41).

                              
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  		Table 1.   Results of surface ECGs obtained from control and alpha 3-isoform transgenic mice



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  		Fig. 5.   Dependence of Q-T interval on sinus cycle length (SCL) in control mice and TGM during normal sinus rhythm. We measured Q-T interval from 6-lead surface ECGs in 21 control mice (A) and 25 TGM (B). Persons performing the measurement were blinded to the identity of the mice. We analyzed the data using linear regression. Data from control mice and TGM are plotted on identically proportioned axes and the slope of the Q-T vs. SCL relationship is seen to be more than twice as steep in the TGM group. This difference was statistically significant (see text).

Inspection of Fig. 5 suggests that, at any given cycle length the Q-T interval was longer in the TGM. To confirm this and eliminate SCL variability as a factor, we studied 18 additional mice during atrial pacing at a fixed cycle length of 150 ms (Figs. 4, C and D, and 6). The Q-T interval measured during atrial pacing (Fig. 6) was significantly longer in the TGM than in the control group (102.6 ± 2.9 vs. 91.3 ± 2.3 ms; P = 0.017; 95% confidence interval for difference 3.3-19.2 ms). In addition, Q-T dispersion, measured during sinus rhythm in 9 controls and 15 TGM, tended to be greater in TGM than in controls (Q-T dispersion corrected for heart rate 0.257 ± 0.039 vs. 0.176 ± 0.030 ms; P = 0.06; 95% confidence interval for difference -0.18 to 0.02 ms).


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  		Fig. 6.   Comparison of Q-T intervals in surface ECGs of control mice and TGM measured during pacing at a cycle length of 150 ms. There were 9 mice in each group. Q-T intervals were measured as in Fig. 5.

Dysrhythmias. In addition to Q-T-interval prolongation and increased Q-T dispersion, ventricular arrhythmias are a manifestation of human long Q-T syndrome. We observed reproducible spontaneous ventricular tachycardia in 2 of 25 transgenic mice instrumented with intracardiac catheters compared with 0 of 21 control animals instrumented identically (Fig. 7). These tachycardias were verified to be ventricular tachycardia (VT) by intracardiac electrophysiological recording. In addition, we observed T wave alternans, another feature of human long Q-T syndrome, in 2 of 25 TGM compared with 0 of 21 control mice (Fig. 7). These were not the same two mice noted to have VT.


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  		Fig. 7.   Surface ECGs demonstrating electrophysiological abnormalities observed in 4 different transgenic mice. A: spontaneous nonsustained ventricular tachycardia (VT) and its termination. VT occurred during normal sinus rhythm in a mouse with an intracardiac pacing catheter in place across the tricuspid valve. B: nonsustained VT evoked by ventricular pacing at a cycle length of 100 ms. In both A and B, intracardiac electrophysiological recording confirmed that the rhythm was VT. C and D: 2 examples of T wave alternans during normal sinus rhythm. The T waves and their direction are indicated by arrows.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The electrocardiographical Q-T interval as a function of SCL was significantly longer in mice with cardiac overexpression of the alpha 3- isoform of Na-K-ATPase than in control mice. The TGM also tended to have increased Q-T dispersion. In addition, some of the TGM exhibited VT and T wave alternans, abnormalities that are also associated with human long Q-T syndrome. Because the electrocardiographical changes were present in two TGM lines, it is very unlikely that the phenotype results from positional effects of the transgene insertion site. Furthermore, there are several potential mechanisms by which upregulated alpha 3-isoform expression could lead to a prolonged Q-T. First, because extrusion of sodium by the Na-K-ATPase is electrogenic and produces a hyperpolarizing current, a decrease in the Na+ affinity of the pump, which is a property of the alpha 3-isoform, could lead to a decrease in the net electrogenic Na+ flux associated with the action potential. This would prolong the upstroke of the action potential, which can lengthen the Q-T interval (26). Second, it is possible that changes in Na-K- ATPase, by altering the [Na+]i or intracellular K+ concentration, might secondarily affect other ion channels or transporters, resulting in decreased ion fluxes and delayed repolarization. For example, increased [Na+]i, which as mentioned has been associated with alpha 3-isoform overexpression, is known to decrease Na-Ca exchange, leading to increased [Ca2+]i. This, in turn, has been associated with early afterdepolarizations and torsade de pointes, manifestations of clinical long Q-T syndrome (33). An alternative explanation, however, is that the lower expression level of the alpha 2-isoform found in our TGM may lead to increased [Ca2+]i (12). Thus increased expression of the human alpha 3-isoform (and/or decreased expression of the alpha 2-isoform) in the TGM heart may cause certain phenotypical features of human long Q-T syndrome.

Because only two of the TGM had VT and two other TGM had T wave alternans, these particular observations might have been the result of chance. However, spontaneous ventricular arrhythmias were not seen in any of the 21 control animals, nor have they ever been observed in any of the more than 100 control mice who have undergone invasive electrophysiological studies as part of a variety of different protocols (C. Berul, unpublished data, 1998). Thus our mice may in fact be prone to developing spontaneous tachyarrhythmias, as are humans with long Q-T syndrome.

Long Q-T syndrome has recently been shown to be genetically heterogeneous, linked in certain kindreds to mutations in genes coding for the SCN5A Na+ channel gene and the HERG and KVLQ-T1 K+ channel genes (14, 34). In many cases of human long Q-T syndrome, however, these genes are intact, suggesting the presence of as-yet-unrecognized mutations that lead to the phenotype. Thus alterations in Na-K-ATPase could represent a novel molecular mechanism for human long Q-T syndrome. Specifically, this syndrome could result either from mutations in Na-K-ATPase genes or from disturbances in the regulation of isoform expression within the heart, such as in our TGM.

The first published report of a TGM model of long Q-T syndrome has recently appeared (16). These authors expressed an NH2-terminal fragment of the rat delayed rectifier K+ channel in the mouse heart. The resulting TGM had Q-T prolongation and VT. The Q-T interval was measured manually from signal-averaged tracings, either from a chronically implanted one-lead system or with three leads during general anesthesia. The corrected Q-T interval (Q-Tc) from surface ECGs in three transgenic lines was 16%, 22%, and 67% longer than that of control mice; a statistically significant 11% increase in uncorrected Q-T was found using implanted telemetry. By comparison, our paced mice had a 12% increase in Q-T (measured using 6 or 12 leads). The slope of the Q-T versus SCL regression lines was greater in TGM than control mice in this previously published study, as it was in our animals. Similarly, the K-channel TGM had more premature ventricular contractions, couplets, and wide QRS tachycardia than controls. The tachycardias in the K-channel TGM, however, were diagnosed as VT on the basis of surface ECGs alone.

Because the Q-T interval must vary with heart rate, the Bazett formula is commonly used to correct Q-T interval for SCL in human subjects (3). The Bazett formula, however, is an empirical formula that corrects the Q-T interval to a SCL of 1 s, corresponding to a heart rate of 60 beats/min. There is thus no reason to expect that the same formula would be applicable to mice, whose heart rates are in the range of 400-600 beats/min (SCL 100-150 ms). This has been verified by Mitchell et al. (20). Because in our study the average SCL during sinus rhythm differed between the control and TGM groups, we needed to address the problem of heart rate dependence of the Q-T interval. We did this using two independent approaches. First, we analyzed the dependence of Q-T on cycle length during sinus rhythm by performing linear regression, because this approach makes no a priori assumptions about the biological basis of Q-T dependence on SCL. When we did the linear regressions, we found a statistically significant difference between the slopes of the regression lines in the two groups: there was a significantly steeper dependence of Q-T on SCL in the TGM compared with the control group. Second, in different groups of mice we measured the Q-T interval during atrial pacing at a set cycle length. When SCL was thus controlled by pacing, we again observed that the Q-T interval was significantly longer in TGM than in control animals.

We recognize that parallels between the abnormalities found in our mice and those in human long Q-T syndrome, although suggestive, are incomplete. For example, although humans with long Q-T syndrome are at increased risk of sudden death, we have not been able to observe a sufficient number of TGM and control mice for a long enough period to determine whether the lifespan of the TGM is significantly shorter. It is also true that autonomic influences are important in human long Q-T syndrome, whereas we did not measure or control for possible differences in autonomic tone in our mice. A number of methodological controversies persist regarding measurement of Q-T intervals and Q-T dispersion (18, 33); we followed procedures recommended by recent publications studying mouse electrophysiology (4, 20).

In addition to the electrophysiological manifestations of alpha 3-isoform overexpression, we expected to see a greater inotropic response to low-dose ouabain in TGM hearts than in control mouse hearts, because human alpha 3-protein has a 1,000-fold higher affinity for ouabain than mouse alpha 1-protein. The index of contractility dP/dt was indeed greater in perfused TGM hearts than in control hearts, but there was no difference between control mice and TGM in dose threshold for the response to ouabain. The reduced alpha 2-protein expression in TGM hearts could cause increased calcium transients (12), explaining the increased contractility. When we studied isolated myocytes, we did observe that the shortening-fraction response to low-dose ouabain was approximately twice as great in TGM cardiomyocytes as it was in control myocytes. The difference between the responses of TGM and control myocytes did not reach statistical significance, however, possibly due to the considerable variability in the data and the small number of cells studied thus far. Curiously, although the TGM myocytes appeared to respond more vigorously to low-dose ouabain than did control myocytes, there was no such difference in the ouabain response of perfused hearts, with neither normal nor transgenic whole perfused hearts responding to [ouabain] <10 µM. Myocytes may be a more sensitive indicator of Na-K-ATPase pump inhibition because they twitch under relatively unloaded conditions, as opposed to isovolumically perfused whole hearts, which contract with defined preload and afterload. Alternatively, the [ouabain] in the interstitium of the perfused hearts might differ from that in the perfusate, obscuring any difference between low and high ouabain doses.

In the rat, alpha 3-protein is expressed at relatively high levels in the heart during the perinatal period, but the alpha 3-isoform expression level decreases markedly after birth (17, 23). In contrast, sodium current in neonatal heart increases markedly just before birth and continues to increase from the neonatal period to adulthood (25). These and other developmental changes in cardiac ion channels may be related to the observation that human neonates have a slightly prolonged Q-T interval compared with older children and adults (8, 10). If human neonatal myocardium expresses alpha 3-protein in abundance, as the rat neonatal myocardium does, this developmental change in the Q-T interval could be explained by developmental changes in alpha 3-isoform expression.


    ACKNOWLEDGEMENTS

We thank Wei Sun, Rena Borukhovitch, and Mark Lufburrow for expert technical assistance.


    FOOTNOTES

We gratefully acknowledge support from the Donaghue Foundation, American Heart Association, Connecticut Affiliate, National Science Foundation, and National Heart, Lung, and Blood Institute Grant HL-57949.

Address for reprint requests and other correspondence: R. Zahler, Southern California Permanente Medical Group, 1526 N. Edgemont St., Los Angeles, CA 90027 (E-mail: raphael.x.zahler{at}kp.org).

1 The absolute values given for the density of Na-K-ATPase pump sites of all isoforms are not directly comparable to the values for density of high-affinity sites, because the former was derived from quantitative phosphorylation assay and the latter was obtained by analysis of ouabain binding data.

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.

Received 23 August 1999; accepted in final form 27 April 2000.


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