Surface electrocardiogram and action potential in mice lacking urea transporter UT-B

Abstract

UT-B is a urea transporter protein expressed in the kidney and in many non-renal tissues including erythrocytes, brain, heart, bladder and the testis. The objective of this study was to determine the phenotype of UT-B deletion in the heart. UT-B expression in the heart was studied in wild-type mice vs UT-B null mice by utilizing RT-PCR and Western blot. A surface electrocardiogram (ECG) recording (lead II) was measured in wild-type mice and UT-B null mice at the ages of 6, 16 and 52 weeks. For the action potential recording, the ventricular myocytes of 16 w mice were isolated and recorded by floating microelectrode method. The sodium current was recorded by the patch clamp technique. RT-PCR and Western blot showed the UT-B expression in the heart of wild-type mice. No UT-B transcript and protein was found in UT-B null mice. The ECG recording showed that the P-R interval was significantly prolonged in UT-B null mice ((43.5 ± 4.2), (45.5 ± 6.9) and (43.8 ± 7.6) ms at ages of 6, 16 and 52 weeks) vs wild-type mice ((38.6 ± 2.9), (38.7 ± 5.6) and (38.2 ± 7.3) ms, P<0.05). The atrial ventricular heart block type II and III only appeared in the aging UT-B null mice (52 w old). The amplitude of action potential and V max decreased significantly in UT-B null mice ((92.17 ± 10.56) and (101.89 ± 9.54) mV/s) vs those in wild-type mice (vs (110.51 ± 10.38) and (109.53 ± 10.64) mV/s, P<0.05). The action potential duration at 50% and 90% (APD50 and APD90) was significantly prolonged in UT-B null mice ((123.83 ± 11.17) and (195.43 ± 16.41) ms) vs that in wild-type mice ((108.27 ± 10.85) and (171.00 ± 15.53) ms, P<0.05). The maximal sodium current decreased significantly in UT-B null mice (−8.80 ± 0.92) nA vs that in wild-type mice ((−5.98 ± 1.07) nA, P<0.05). These results provide the first evidence that UT-B deletion causes progressive heart block in mice.

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Citation: Meng Y, Zhao C Y, Zhang X X, et al. Surface electrocardiogram and action potential in mice lacking urea transporter UT-B. Sci China Ser C-Life Sci, 2009,
52(5): 474-478, doi: 10.1007/s11427-009-0047-y
Surface electrocardiogram and action potential in mice
lacking urea transporter UT-B
MENG Yan1, ZHAO ChunYan2, ZHANG XueXin2, ZHAO HuaShan1, GUO LiRong1, LÜ Bin1, ZHAO
XueJian1†& YANG BaoXue1,3†
1 Department of Pathophysiology, School of Basic Medicine, Jilin University, Changchun 130021, China;
2 Department of Physiology, School of Basic Medicine, Jilin University, Changchun 130021, China;
3 Department of Medicine, University of California, San Francisco CA 94143, USA
UT-B is a urea transporter protein expressed in the kidney and in many non-renal tissues including
erythrocytes, brain, heart, bladder and the testis. The objective of this study was to determine the
phenotype of UT-B deletion in the heart. UT-B expression in the heart was studied in wild-type mice vs
UT-B null mice by utilizing RT-PCR and Western blot. A surface electrocardiogram (ECG) recording
(lead II) was measured in wild-type mice and UT-B null mice at the ages of 6, 16 and 52 weeks. For the
action potential recording, the ventricular myocytes of 16 w mice were isolated and recorded by float-
ing microelectrode method. The sodium current was recorded by the patch clamp technique. RT-PCR
and Western blot showed the UT-B expression in the heart of wild-type mice. No UT-B transcript and
protein was found in UT-B null mice. The ECG recording showed that the P-R interval was significantly
prolonged in UT-B null mice ((43.5 ± 4.2), (45.5 ± 6.9) and (43.8 ± 7.6) ms at ages of 6, 16 and 52 weeks)
vs wild-type mice ((38.6 ± 2.9), (38.7 ± 5.6) and (38.2 ± 7.3) ms, P<0.05). The atrial ventricular heart block
type II and III only appeared in the aging UT-B null mice (52 w old). The amplitude of action potential and
Vmax decreased significantly in UT-B null mice ((92.17 ± 10.56) and (101.89 ± 9.54) mV/s) vs those in
wild-type mice (vs (110.51 ± 10.38) and (109.53 ± 10.64) mV/s, P<0.05). The action potential duration at
50% and 90% (APD50 and APD90) was significantly prolonged in UT-B null mice ((123.83 ± 11.17) and
(195.43 ± 16.41) ms) vs that in wild-type mice ((108.27 ± 10.85) and (171.00 ± 15.53) ms, P<0.05). The
maximal sodium current decreased significantly in UT-B null mice (− −8.80 ± 0.92) nA vs that in wild-type
mice ((− −5.98 ± 1.07) nA, P<0.05). These results provide the first evidence that UT-B deletion causes
progressive heart block in mice.
urea transporter, heart block, electrocardiogram
Urea transporters (UT) are a family of membrane chan-
nel proteins that facilitate urea movement across bio-
logical membranes along a concentration gradient.
Seven isoforms of urea transporters have been identified
(UT-A1-6, UT-B)[1,2]. UT-B is abundantly expressed in
the endothelium of the vasa recta throughout the renal
medulla, where it contributes to urinary concentration
ability[3]. UT-B was also found to be expressed in sev-
eral other organs, such as marrow, heart, spleen, brain
and testis. Little has been known about the physiological
function of UT-B in extrarenal tissues. UT-B null mice
provided the first animal model of a urea transporter
deletion[4]. The objective of the present study was to
determine the features of ECG and action potential in
UT-B null mice and evaluate the physiological function
of UT-B in the heart.

Received May 23, 2008; accepted July 29, 2008
doi: 10.1007/s11427-009-0047-y
†Corresponding author (email: pro_2@jlu.edu.cn; baoxue.yang@itsa.ucsf. edu)
Supported by the National Natural Science Foundation of China (Grant No.
30670852).

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MENG Yan et al. Sci China Ser C-Life Sci | May 2009 | vol. 52 | no. 5 | 474-478
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1 Materials and methods
1.1 Experimental animals
Transgenic knockout mice deficient in UT-B protein
were supplied by the University of California, San Fran-
cisco[4]. All of the wild-type and UT-B null mice used in
this study were from the C57 genetic background and
bred in our laboratory. The genotype was identified by
Genomic PCR[4].
1.2 RT-PCR
The total RNA from whole mouse hearts was isolated by
homogenization in TRIZOL reagent (Invitrogen, Carls-
bad, CA, USA). cDNA was reverse-transcribed from
total RNA. The PCR amplification system (25 μL) con-
tained 1 μL cDNA, 2.5 μL 10×PCR buffer, 2 μL 25
pmol/L primers, 1 μL 5 mmol/L dNTP and 2.5 U Taq
DNA polymerase. β-actin was used as the reference
gene. Primers were as follows: 5′-TTGTTACCAACTG
GGACG-3′ (sense) and 5′-GGCATAGAGGTCTTTAC
GG-3′ (antisense) for β-actin, 5′-CCACTCTGACAGC
CCTCTT-3′ (sense) and 5′-GATGACACCCAGCAAC
GA-3′ (antisense) for UT-B. Amplification condition:
94℃ for 2 min, 1 cycle; 94℃ for 30 s, 55℃ for 30 s,
72℃ for 45 s, 30 cycles; 72℃ extension for 5 min, 10
μL PCR product were electrophoresed on a 1% agarose
gel.
1.3 Western blot
100 mg of mouse heart tissue was homogenized in an
isolation buffer. The protein concentration was deter-
mined using the Bio-Rad protein assay kit (BioRad,
Richmond, CA, USA). Heat denatured samples were
separated on a 10% SDS-polyacrylamide gel (110 v, 90
s) and transferred to polyvinylidene difluoride mem-
branes. Anti-UT-B polyclonal antibodies (1︰1000) and
anti-β-actin monoclonal Ab (1︰2000; Research Dia-
gnostics Inc, Flanders, NJ, USA) were used for Western
blot analysis.
1.4 Surface Electrocardiogram (ECG)
6, 16 and 52 w old wild-type mice and UT-B null mice
were randomly selected. 20 mice were studied per group.
Mice were anesthetized with ethylcarbamate (2 g/kg) by
intraperitoneal injection and were analyzed by surface
lead II ECG (needle electrode was pricked subcutane-
ously into the 3 limb: lower left limb, right upper ex-
tremity and right lower extremity). ECG recordings
were obtained and analyzed on a BL-420S biological
function recording system (Taimeng technology limited
company, China).
1.5 Action potential recording
Hearts were isolated and quickly perfused with a Tyrode
solution in a Langendorff perfusion system. The perfu-
sion speed was set at 5 mL/min. The composition of the
normal Tyrode solution was: NaCl 140 mmol/L, KCl 5.4
mmol/L, CaCl2 2.0 mmol/L, MgCl2 1.0 mmol/L, HEPES
5.0 mmol/L, glucose 10.0 mmol/L with pH titrated to
7.4. Borosilicate glass micropipettes were made from a
horizontal pipette puller (Narishige). The tip resistance
of the pipette was 10–20 mΩ when filled with 3 mol/L
KCl pipette solution. The action potential was recorded
by the “floating microelectrode method”. All data were
stored and analyzed using the BL-420S biological func-
tion recording system.
1.6 Urea measurement
Blood samples were obtained by tail bleeding, and
plasma was isolated from blood by centrifugation. Heart
tissue homogenates were obtained by homogenizing
fresh heart tissue in ddH2O. Heart tissue homogenate
urea and plasma urea was measured by colorimetry us-
ing a commercial kit (Roche Diagnostic, Indianapolis,
IN, USA).
1.7 Statistics
All data are presented as x ± SD. Results obtained in
UT-B null mice were compared with those of wild-type
mice by Student’s t test. When three groups were com-
pared, two-way ANOVA was performed. P<0.05 was
considered as statistically significant.
2 Results
2.1 The expression of UT-B mRNA and protein in
mouse heart tissue
To determine UT-B expression in mouse hearts, mRNA
and protein were detected by RT-PCR analysis and
Western blot analysis. RT-PCR analysis showed a
β-actin band in both wild-type mice and UT-B null mice.
UT-B mRNA was only found in the heart tissue of
wild-type mice, but not in UT-B null mice (Figure 1).
Western blot analysis showed that 46 kD UT-B pro-
tein was only detected in heart tissue of wild-type mice,
but not in UT-B null mice (Figure 2), though -actin was
found in both wild-type and UT-B null mice. These re-
sults confirmed UT-B expression in mouse hearts.

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476 MENG Yan et al. Sci China Ser C-Life Sci | May 2009 | vol. 52 | no. 5 | 474-478


Figure 1 UT-B mRNA expression in mouse heart tissue. β-actin was
used as reference gene (top). M, DNA marker (DL2000); +/+, wild-type
mice; −/−, UT-B null mice.



Figure 2 UT-B protein expression in mouse heart tissue. β-actin was
used as reference gene (bottom). +/+, wild-type mice; −/−, UT-B null
mice.

2.2 Heart block in UT-B null mice
ECG (lead-II) recording showed that the P wave time
and QRS time were not significantly different between
wild-type and UT-B null mice. However, there was
greater variability of ECG data in UT-B null mice. The
P-R interval was significantly prolonged in UT-B null
mice ((43.5 ± 4.2), (45.5 ± 6.9) and (43.8 ± 7.6) ms at
ages of 6, 16 and 52 w) vs wild-type mice ((38.6 ± 2.9),
(38.7 ± 5.6) and (38.2 ± 7.3) ms, P<0.05) (table 1 and
Figure 3). The P-R interval prolongation did not change
with aging, but atrial ventricular heart block type II and
III only appeared in aging UT-B null mice (52 w old)
(Figure 4), which indicates that the UT-B caused
age-dependent progressive heart block.
2.3 Action potential in ventricular cardiocytes
Action potential amplitude (APA), maximal rate of de-
polarizing velocity (Vmax), action potential duration at
10% (APD10), 50% (APD50) and 90% (APD90) were also

Table 1 The surface II standard lead ECG in wild-type and UT-B null mice ( x ± SD)a)
recorded as shown in Table 2. APA and Vmax in 16 weeks
old UT-B null mice significantly decreased vs those in
wild-type mice (P<0.05). APD50 and APD90 were sig-
nificantly prolonged in UT-B null vs those in wild-type
mice (P<0.05). The representative action potential fig-
ures are shown in Figure 5.
2.4 Serum urea and heart tissue urea
Serum urea ((17.93 ± 1.38) mmol/L) and heart tissue
urea ((21.22 ± 1.13) mmol/kg tissue weight) in UT-B
null mice were significantly higher than that in wild-
type mice (serum urea (14.88 ± 1.11) mmol/L, heart tis-
sue urea (16.56 ± 0.43) mmol/kg tissue weight, P<0.05).
The results suggest that urea accumulates in theheart
tissue of UT-B null mice (Figure 6).
3 Discussion
UT-B is highly expressed in the membrane of erythro-
cytes, and also expressed in kidney and non-renal tis-
sues[3]. UT-B protein expressed in the human erythrocyte
is identified as the Jk antigen[5]. Jk(a-b-) (Jknull) humans,
who are very rare, have a mild urine-concentrating de-
fect[6,7]. It has not been reported that Jknull individuals are
associated with any other clinical syndrome. An unex-
pected finding that a Jknull individual suffered with com-
plete heart block (unpublished data) activated us to
study the physiological role of UT-B in heart. A UT-B
null mouse is an optimal animal model to determine the
effect of UT-B deficiency and urea accumulation in car-
diophysiology.
The UT-B null mice were generated by a targeted
gene deletion of exons 4–7 of UT-B gene[4]. UT-B null
mice showed severe urea concentrating defect[4] and
early maturity of the male reproductive system[8]. In this
Mouse N HR P-R interval (ms) QRS (ms) P-R > 45 (ms)II and III degree heart block
+/+
6 w 20 643.18±53.88 38.6±2.9 11.5±0.7 0 0
16 w 20 634.23±65.42 38.7±5.6 11.6±1.4 1 0
52 w 20 672.01±113.75 38.2±7.3 11.1±2.2 2 0
−/−

6 w 20 638.40±67.05 43.5±4.2* 11.0±0.9 6 0
16 w 20 631.62±52.78 45.5±6.9* 11.5±1.1 9 0
52 w 20 584.30±123.09# 43.8±7.6* 11.2±2.3 8 4
a) * P<0.05 vs wild-type mice; # P<0.05 vs wild-type mice and 6, 16 and 52 w UT-B null group.

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MENG Yan et al. Sci China Ser C-Life Sci | May 2009 | vol. 52 | no. 5 | 474-478
477


Figure 3 ECG recording in wild-type and UT-B null mice. A, The II
standard lead ECG of wild-type and UT-B null mice; B, The P-R interval
in wild-type and UT-B null mice ( x ± SD, N=20, * P<0.05 vs wild-type
mice).



Figure 4 Representative ECG in UT-B null mice with heart block. A, II
degree atrial ventricular block; B, III degree atrial ventricular block.

study, surface ECG analysis showed that the P-R inter-
vals (lead-II) were significantly prolonged in 6, 16 and
52 w old UT-B null mice compared with age matched

Table 2 The parameter of action potential of ventricular cardiocyte ( x ± SD, n=10)


Figure 5 Ventricular Action potential in cardiocytes.



Figure 6 Urea concentrations in serum and heart tissue. x ± SD, n=6,
*P<0.05 vs wild-type mice.

wild-type mice. The atrial ventricular heart block type II
and III only appeared in the aging UT-B null mice (52 w
old), which reveals that UT-B deletion may be relative to
the development of progressive heart block. The P wave
represents the time course of AP conduction within a-
trium cordis. QRS waves represent the time course of
AP within ventricles. The P-R interval delay observed in
the present study indicates that the impetus of atrium
cordis spread through the atrioventricular junction to
ventricles was delayed. Analysis of the AP of ventricular
cardiocytes revealed that the APA and Vmax were sig-
nificantly inhibited, and APD50 and APD90 were signify-
cantly prolonged, which suggests that the excitability
and conductivity was decreased in UT-B null cardiocytes.
This data indicated that the deletion of UT-B caused the
heart block. However it is still not clear if the abnormal
cardioelectrophysiological phenotype in UT-B null mice
mouse APA(mV) Vmax(mV/s) APD10 (ms) APD50 (ms) APD90 (ms)
16 w +/+ 110.51±10.38 109.53±10.64 10.13±2.32 108.27±10.85 171.00±15.53
16 w −/−
92.17±10.56* 101.89±9.54* 12.70±2.70 123.83±11.17* 195.43±16.41*
* P<0.05 vs wild-type mice.

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478 MENG Yan et al. Sci China Ser C-Life Sci | May 2009 | vol. 52 | no. 5 | 474-478
resulted from UT-B protein deficiency in cardiocytes or
from urea accumulation in heart tissue.
Urea is the major end product of protein metabolism
in mammals. Urea is not only synthesized by the liver, it
is also synthesized in tissue which can synthesizes poly-
amine, such as heart, testis and brain[1]. Urea is a polar
molecule. Without any urea transporter, urea trans-
membrane permeability is very low. UT-B deletion in
the plasma membrane of the kidney vasa recta blocked
the intrarenal urea recycling, decreasing the urinary
concentration ability and raising blood urea level[4]. The
present study showed that urea concentration in the heart
tissue of UT-B null mice was also higher than that of
wild-type mice, which suggests that urea accumulates in
the heart tissue when urea permeability is low in the
plasma membrane of cardiocytes. The clinical data
showed that most patients with chronic renal failure and
blood urea increase had electrocardiographic abnormali-
ties. Arrhythmia is a common complication and cause of
death in urinemia patients. Dialysis in patients with renal
failure significantly reduced the rate of heart block[9],
which suggests that high blood and tissue urea level in
urinemia patients may cause arrhythmia, influencing on
the physical function of the heart. Weisensee, et al found

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In summary, we confirmed that the urea transporter
UT-B is expressed in cardiocytes, and that UT-B deletion
caused the urea accumulation in heart tissue and abnor-
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