Vol. 281, Issue 3, R917-R925, September 2001
The
1- and
2-isoforms of
Na-K-ATPase play different roles in skeletal muscle
contractility
Suiwen
He1,*,
Daniel A.
Shelly2,*,
Amy E.
Moseley1,
Paul F.
James1,
J. Howard
James3,
Richard J.
Paul2, and
Jerry B.
Lingrel1
1 Department of Molecular Genetics, Biochemistry, and
Microbiology and 2 Department of Molecular and Cellular
Physiology and 3 Department of Surgery, University of
Cincinnati, College of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
The Na-K-ATPase, which maintains the Na+ and
K+ gradients across the plasma membrane, can play a major
role in modulation of skeletal muscle contractility. Although both
1- and
2-isoforms of the Na-K-ATPase are
expressed in skeletal muscle, the physiological significance of these
isoforms in contractility is not known. Evaluation of the contractile
parameters of mouse extensor digitorum longus (EDL) was carried out
using gene-targeted mice lacking one copy of either the
1- or
2-isoform gene of the Na-K-ATPase. The EDL muscles from heterozygous mice contain approximately one-half of the
1- or
2-isoform, respectively,
which permits differentiation of the functional roles of these
isoforms. EDL from the
1+/
mouse shows
lower force compared with wild type, whereas that from the
2+/
mouse shows greater force. The
different functional roles of these two isoforms are further
demonstrated because inhibition of the
2-isoform with
ouabain increases contractility of
1+/
EDL. These results demonstrate that the Na-K-ATPase
1-
and
2-isoforms may play different roles in skeletal
muscle contraction.
sodium-potassium-adenosinetriphosphatase; extensor digitorum longus
muscle; ouabain; muscle fatigue
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INTRODUCTION |
THE NA-K-ATPASE is
an integral membrane protein that uses the energy from ATP hydrolysis
for the countertransport of three Na+ and two
K+ across the plasma membrane. This enzyme is critical for
maintaining the resting membrane potential and osmotic balance of cells
central to the excitability of muscle and nerve tissues. The
Na+ gradient is also the driving force for numerous
Na+-dependent secondary processes, including transport of
amino acids, Ca2+, and other nutrients.
The Na-K-ATPase is a heteromeric enzyme composed of two polypeptide
subunits: a 112-kDa
-subunit and a 40- to 60-kDa glycosylated
-subunit (reviewed in Ref. 28). The
-subunit
contains the cation- and ATP-binding sites to perform the catalytic and
transport activity of the enzyme. The
-subunit is involved in the
modulation of the Na+ and K+ affinity of the
enzyme (10, 11). There are distinct genes encoding
multiple isoforms of both
- and
-subunits in various species.
Four
-subunits (
1,
2,
3, and
4) and three
-subunits (
1,
2, and
3) have been
found (16, 29, 37-39, 41). The
1-isoform is expressed in nearly every tissue,
suggesting a possible housekeeping role for this isoform. In contrast,
expression of
2-,
3-, and
4-isoforms is restricted to several tissues:
2 in skeletal muscle, smooth muscle, heart, and brain
(glial cells);
3 in brain (neurons) and ovary; and
4 in sperm. The unique pattern of expression suggests
specific functional roles for these individual isoforms.
Among the four
-isoforms of the Na-K-ATPase, differences in cation
affinity, ATP affinity, and other kinetic parameters of the enzymatic
activity have been noted. Discrepancies in these parameters have been
reported, depending on the various tissues and species (Refs.
43 and 45; reviewed in Ref. 4). The major
biochemical difference among the four isoforms is their affinities to
cardiac glycosides such as ouabain. In rodents, the affinity of the
2- and
3-isoforms for ouabain is
1,000-fold greater than the
1-isoform (12,
42). Because multiple
-isoforms of the Na-K-ATPase display a
tissue-specific expression pattern that is generally consistent across
various species, the functional relevance and physiological
significance of such expression patterns have been investigated in
certain tissues such as heart and sperm (23, 47). However,
little work has been done in skeletal muscle.
In skeletal muscle, excitability and contractility depend on the
membrane potential and the Na+ and K+ gradients
across the plasma membrane. During action potentials, Na+
enters the cell in the depolarization phase and K+ leaves
the cell in the repolarization phase. The alteration of Na+
and K+ gradients across the plasma membrane is restored by
the Na-K-ATPase. If the rate of passive leakage of Na+ and
K+ through ion channels exceeds the rate of active ion
transport by the Na-K-ATPase, the excitability of the skeletal muscle
fibers will decrease, contributing to contractile fatigue. Alteration of skeletal muscle Na-K-ATPase activity has been observed in various physiological and pathophysiological states, such as thyroid hormone excess (reviewed in Ref. 6) or deficiency
(1), training and inactivity (33, 36, reviewed in Ref.
6), K+ deficiency (31), and
diabetes (26). In many studies, inhibition of the
Na-K-ATPase results in perturbation of Na+ and
K+ concentration both intracellularly and extracellularly.
Accumulation of K+ in the extracellular space, especially
around the T-tubule system, depolarizes the plasma membrane and makes
the muscle cells unexcitable (7). The relationship of
individual
-isoforms to muscle contractility has yet to be understood.
Using gene targeting to alter the expression levels of
1- and
2-isoforms of Na-K-ATPase in mice,
we have previously demonstrated that these two isoforms have distinct
physiological functions in heart (23). Hearts from mice
lacking one copy of the
2-isoform (
2+/
) are hypercontractile. In contrast,
genetic reduction of one copy of the
1-isoform
(
1+/
) depresses cardiac contractility. To
further define specific functional roles for the Na-K-ATPase
1- and
2-isoforms, we have analyzed
skeletal muscle contractility in mice heterozygous for the
1- and
2-isoforms. We demonstrated here
that under supramaximal stimulation conditions, the extensor digitorum
longus (EDL) muscle from
2+/
mice is
capable of producing more force than wild type, whereas muscles from
1+/
mice produce less force than wild
type. These results, along with those from our previous work
(23), clearly demonstrate unique roles for the Na-K-ATPase
1- and
2-isoforms in muscle contractility.
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MATERIALS AND METHODS |
Animals and genotypes.
Male mice, 12-15 wk old, were used for all studies described.
Heterozygous mice containing one copy of either the
1-
or
2-isoform gene of the Na-K-ATPase were generated
using gene-targeting methods described previously (23).
Genomic DNA was extracted from mouse tails using Puregene DNA isolation
kit (Gentra Systems, Minneapolis, MN). To identify genotypes,
isoform-specific probes for the respective
1- and
2-isoform gene were used in Southern blot analysis of genomic DNA (23). Mice were euthanized by CO2
inhalation followed by cervical dislocation, and EDL muscles from both
legs were dissected. For preparation of crude microsomes, EDL muscles
were frozen in liquid nitrogen for later use.
Intracellular Na+ and
K+ contents.
Measurement of intracellular Na+ and K+ was
performed as described (22). Briefly, ions from the
extracellular space were washed out by incubation in ice-cold
Na+- and K+-free buffer. Muscles were
homogenized in trichloroacetic acid followed by centrifugation to
remove precipitated proteins. Na+ and K+
concentrations in the supernatant were measured with a Shimadzu AA-6200
atomic absorption spectrophotometer (Shimadzu Scientific Instruments,
Columbia, MD). Ion concentration was expressed as micromoles per gram
wet weight.
Preparation of crude microsomes.
For each microsomal preparation, 20-30 EDL muscles from each
genotype (wild type,
1+/
, and
2+/
) were homogenized on ice with two 30-s
bursts of a Polytron homogenizer in 5 ml homogenization buffer [250 mM
sucrose, 30 mM imidazole (pH 7.5), and 1 mM EDTA]. The homogenates
were centrifuged at 3,000 g for 15 min at 4°C to remove
cellular debris. The supernatants were centrifuged at 115,000 g for 60 min at 4°C. The pellets were resuspended in 30 mM
imidazole buffer (pH 7.5) and 1 mM EDTA and centrifuged again at
115,000 g for 60 min. The pellets were finally resuspended
into 250 µl of imidazole and EDTA buffer. Protein concentrations were
measured using the bicinchoninic acid protein assay kit (Pierce,
Rockford, IL). Aliquots of microsomal preparations were stored at
80°C for Western blot analysis.
Quantitation of Na-K-ATPase isoform levels.
Western blot analysis was used to quantitate protein levels of the
Na-K-ATPase
-isoforms in EDL. Microsomal proteins were separated
using SDS-PAGE as described (27). To facilitate detection of the glycosylated
-subunits, sugar residues were removed with recombinant N-glycanase kit following manufacturer's
direction (Glyko, Novato, CA). Samples were incubated for 30 min at
37°C in sample buffer containing 50 mM Tris (pH 6.9), 5% SDS, 1%
-mercaptoethanol, and 10% glycerol and electrophoresed through 10%
polyacrylamide gels. The separated proteins were transferred to
polyvinylidene difluoride membranes (Amersham Life Science, Piscataway,
NJ) overnight at 4°C. Membranes were blocked with 2% BSA in TBST
solution [5 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% Tween 20] for
1 h at room temperature and then incubated in TBST containing
individual primary antibodies overnight at 4°C as follows:
1-isoform-specific monoclonal
6f, 1:1,000 (University
of Iowa Developmental Hybridoma Bank, Iowa City, IA) (44);
2-isoform-specific monoclonal McB2, 1:500 (generously
provided by Dr. Kathleen Sweadner) (46);
3-monoclonal antibody, 1:1,000 (Affinity Bioreagents,
Golden, CO); polyclonal
-KETYY, 1:10,000 (generously provided by Dr.
Jack Kyte);
1-polyclonal SpETB1, 1:1,500
(17);
2-polyclonal SpETB2, 1:1,500
(17); and
3-polyclonal RNTB3, 1:2,000
(generously provided by Dr. Kathleen Sweadner). The membranes were
incubated overnight at 4°C in TBST containing 1:20,000
peroxidase-conjugated secondary antibodies (Calbiochem, La Jolla, CA).
The membranes were processed using enhanced chemiluminescence system
(Amersham Life Science) and visualized with Kodak BioMax MR X-ray film.
Signal intensities were quantified by densitometry using ImageQuant
software. Two microsomal preparations were analyzed for EDL muscles of
each genotype. Samples containing various amounts of microsomal protein were analyzed in a single blot, and multiple exposures of the blot were
obtained to ensure linearity of signal intensity.
Measurement of EDL contractility.
After the resting length of each muscle was measured in situ,
dissected muscles were mounted vertically in a sealed cylindrical chamber fitted with a magnetic stirrer. By means of a small plastic ring tied to each tendon of EDL with surgical silk, muscles were mounted at one end to a fixed stainless steel post. The other end was
fixed to the lever of an isometric force transducer (Kistler Morse,
Redmond, WA), and the muscle length was adjusted to produce a passive
tension of 5 mN. Muscles were incubated in sterile Krebs solution (in
mM: 118 NaCl, 4.7 KCl, 25 NaHCO3, 2.5 CaCl2,
1.2 MgSO4, 1.2 NaH2PO4, 0.026 EDTA,
11 glucose equilibrated with 95% CO2-5% O2)
at room temperature (~22°C). Muscles were electrically stimulated
via two platinum electrodes positioned along either side of the muscle.
Supramaximal voltage was determined empirically using a series of short
(1.5 s) tetani and subsequently increasing the voltage for each
tetani. Similarly, supramaximal frequency of stimulation was
determined. Supramaximal electrical stimuli employed capacitor discharges of equal but alternating polarity (66 Hz at 15 V, 15-ms duration, circuit rate constant, 5 ms). Experiments consisted of a set
(~5) of twitches and a set (2-4) of tetani with
duration of 3-7 s. Digital recordings of force were obtained with
the BioPac data-acquisition system (BioPac System, Goleta, CA).
Measurements of force were normalized to muscle cross-sectional area
estimated as muscle weight divided by muscle length for comparison
among genotypes (32). Twitch and tetanus parameters were
analyzed by ANOVA or Student's t-test using the SigmaStat
statistical software package (Jandel Software, San Rafael, CA).
 |
RESULTS |
Quantitative analysis of
1- and
2-protein levels in EDL.
EDL, a fast-twitch skeletal muscle, was selected because of ease of
dissection and its small size such that it is not diffusion limited for
oxygen. The lack of either the
1- or
2-isoform gene in
1+/
or
2+/
animals was expected to decrease the
amount of the respective isoform by 50%. SDS-PAGE and quantitative
Western blot analysis were used to quantitate levels of
1- and
2-proteins in EDL (Fig. 1A). The protein level of the
1-isoform decreased by 48% in
1+/
EDL (Fig. 1B) and that of
the
2-isoform decreased by 46% in
2+/
EDL (Fig. 1C), close to the
expected 50%. Interestingly, the
1-protein level
increased by 39% in
2+/
EDL (Fig.
1B), while the
2-protein level was not
altered in
1+/
EDL (Fig. 1C).

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Fig. 1.
Western blot analysis of the -isoforms of Na-K-ATPase in
extensor digitorum longus (EDL). A: microsomal proteins were
separated using SDS-PAGE, and the gels were transferred to
polyvinylidene difluoride (PVDF) membranes for immunoblotting. Primary
antibodies of 6F (1:1,000), Mcb2 (1:500), and -KETYY (1:10,000)
were used to detect the 1-, 2-, and both
-isoforms, respectively. Different amounts of EDL samples were
loaded (150, 75, and 37.5 µg, from left to right,
for each genotype). For NIH/3T3 cells, 2.5, 5, and 10 µg of
proteins were loaded (left to right). WT, wild
type. B: signal intensities from A were
quantitated by densitometry. The amount of 1- and
2-protein in 1+/ EDL was
normalized to that of the WT and expressed as a percentage.
C: amount of 1- and 2-protein
in 2+/ EDL was quantitated as in
B. D: signal intensities from immunoblots using
6F and -KETYY in A were used to calculate the
percentages of 1- and 2-isoforms in WT,
2+/ , and 1+/
EDL. Values are means ± SE (n = 8).
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Besides quantitative analysis of individual
-isoforms in EDL from
1+/
and
2+/
mice, Western blot analysis was also used to quantitate the relative amounts of the
1- and
2-isoforms in EDL
by the method developed by our laboratory (24). Because
monoclonal antibodies used above exhibit different binding affinities
to the
1- and
2-isoforms, the relative
amounts of these two isoform proteins in EDL could not be directly
assessed. To resolve this, we used a polyclonal antibody (
-KETYY)
that recognizes an epitope common to all mouse
-isoforms, in
conjunction with a standard that contains only the
1-isoform and the isoform-specific monoclonal antibodies to characterize the relative amounts of each
-isoform protein in any
sample. In this study, NIH/3T3 cells, a murine cell line that contains
only the
1-isoform, were used as a reference to calculate the relative amounts of
1- and
2-isoforms (Fig. 1A). In wild-type EDL, the
2-isoform was predominant, accounting for 87% of total
-isoform of Na-K-ATPase, whereas the
1-isoform accounted for only 13% (Fig. 1D). In
2+/
EDL, the total amount of Na-K-ATPase
-isoforms was 66% of wild type, comprising 48%
2-
and 18%
1-isoform (Fig. 1D). In
1+/
EDL, the total amount of Na-K-ATPase
-isoforms was 93% of wild type, comprising 86%
2-isoform and 7%
1-isoform (Fig.
1D).
Western blot analysis was used to examine other Na-K-ATPase subunits in
EDL. The protein levels of
1,
2, and
3 are similar among wild type,
2+/
, and
1+/
EDL (Fig. 2). The
3-isoform, which is normally expressed in brain and
ovary, is not expressed in either
2+/
or
1+/
EDL (Fig. 2). The results show that
the total pool of other Na-K-ATPase subunits in the microsomal protein
preparation was not altered because of decreased protein levels of
1- or
2-Na-K-ATPase.

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Fig. 2.
Western blot analysis of the - and
3-subunits of Na-K-ATPase in EDL. Microsomal proteins
were separated using SDS-PAGE, and the gels were transferred to PVDF
membranes for immunoblotting. Primary antibodies of SpETB1 (1:1,500),
SpETB2 (1:1,500), RNTB3 (1:2,000), and 3 (1:1,000) were
used to detect the 1-, 2-,
3-, and 3-subunits, respectively.
Different amounts of EDL samples were loaded (10, 20, and 40 µg, from
left to right). For brain tissue as a positive
control, 5 µg of protein was loaded.
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EDL from
1+/
and
2+/
mice has distinct
alterations in muscle contractility.
The gross anatomy of EDL muscles from the wild-type,
2+/
, and
1+/
animals was examined for any alteration that might influence muscle
contractility (Table 1). The weight and
length of EDL from all three genotypes were similar. Cross-sectional
area of muscle fiber, an important parameter used to normalize the
contractile force, did not differ. Hypertrophy was not observed as
assessed by the ratio of EDL weight to animal body weight. Because the Na-K-ATPase is important for controlling ionic levels in tissues, the
concentrations of Na+ and K+ in EDL muscles
were also measured, but no significant alteration was found.
Isometric force during stimulation was measured to assess the
consequences of a reduction of individual
-isoform levels on EDL
contractility. The relationship of force and voltage, determined empirically, is shown in Fig. 3. The
2+/
produces more force for almost any
given voltage and has higher force at supramaximal voltage than wild
type. Figure 4A shows representative force traces for wild-type,
2+/
, and
1+/
EDL muscles. With supramaximal stimulation, the force quickly reached a
peak value and declined slightly during the process of stimulation. At
the end of stimulation, tension returned to the resting level. The peak
value of tetanic force was 384.4 ± 13.2 mN/mm2 for
2+/
EDL, i.e., 29% higher than EDL from
wild-type mice shown in Fig. 4B (297.0 ± 13.6 mN/mm2, P < 0.01). On the other hand, the
peak value for EDL from
1+/
mice was only
238.9 ± 17.1 mN/mm2, representing a 20% reduction
from the wild type (Fig. 4B; P < 0.05).
Twitch force, another independent contractile parameter for EDL, was
also measured. EDL from
2+/
mice was able
to produce 34% more twitch force (41.3 ± 1.8 mN/mm2,
P < 0.01) than wild-type EDL (30.8 ± 2.6 mN/mm2). However, the twitch force of
1+/
(29.2 ± 2.2 mN/mm2)
was not significantly different from wild type (Fig. 4C).
The greater force found in
2+/
EDL and
less force in
1+/
EDL compared with the
wild type suggest that the Na-K-ATPase
1- and
2-isoforms may play distinct roles in skeletal muscle contractility.

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Fig. 3.
Force-voltage relationship. A series of 1.5-s tetani was
elicited from WT and 2+/ EDL over an
increasing voltage range to empirically determine the supramaximal
stimulation voltage. Values are means ± SE (n = 5). Statistical analysis utilized unpaired Student's
t-test. * Significant difference between WT and
2+/ (P < 0.05).
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Fig. 4.
Tetanic and twitch contractions in EDL. A: EDL
muscles from WT, 2+/ , and
1+/ mice were used for contractility
measurement. Nonnormalized representative force recordings are shown.
B: peak values of tetanic contractions are shown for WT,
2+/ , and 1+/
EDL. Force was normalized to the cross-sectional area of EDL muscle,
which was estimated by the ratio of EDL weight (mg) and length (mm).
C: twitch forces were plotted and normalized to the
cross-sectional area of the muscle. Values are means ± SE
(n = 8). Statistical analysis utilized ANOVA, and
intergroup comparison used Scheffé's test. * Significant
difference between WT and 2+/
(P < 0.01). ** Significant difference between WT
and 1+/ (P < 0.05).
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To confirm that the contractile phase, but not the relaxation phase, of
skeletal muscle contraction is altered, parameters of contraction and
relaxation in EDL were further analyzed (Fig. 5). The rate of force development in the
tetanic contraction was increased in
2+/
mice (750.8 ± 29.9 mN · mm
2 · s
1) compared
with wild type (571.9 ± 28.7 mN · mm
2 · s
1,
P < 0.01), whereas the rate of tetanic contraction in
1+/
muscle was lower (521.9 ± 28.1 mN · mm
2 · s
1,
P > 0.05), but not statistically significant (Fig.
5A). For the relaxation phase of tetanic contractions,
half-relaxation time was not altered in either
2+/
(0.189 s) or
1+/
(0.194 s) compared with wild type
(0.192 s) (Fig. 5B). Our studies further indicate that the
relaxation phase, primarily controlled by sarco/endoplasmic
reticulum Ca2+-ATPase (SERCA), was not altered in
either heterozygous animal.

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Fig. 5.
Rate of tetanic contraction (A) and
half-relaxation time (B). A: rate of isometric
force development (dF/dt) was obtained by
dividing the normalized peak value of tetanus (mN/mm2) by
the time (s) needed to reach the peak value. The force trace is nearly
linear from initiation to the peak of the contraction. B:
half-relaxation time (T1/2) represents time (s)
required for force to decrease to one-half after termination of the
stimulus. Values are means ± SE (n = 8).
Statistical analysis utilized ANOVA, and intergroup comparison used
Scheffé's test. * Significant difference between WT and
2+/ (P < 0.01).
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Cardiac glycoside inhibition of the Na-K-ATPase
2-isoform increases contractility of
1+/
EDL.
The loss of one-half of the
1-isoform in
1+/
EDL results in less force. Because the
protein level of the
1-isoform increased by 39% in
2+/
EDL (Fig. 1C), it is
possible that the greater force observed in
2+/
EDL resulted from secondary
upregulation of the
1-isoform. To establish that the
greater force resulted specifically from a reduction in the
2-isoform and not a compensatory upregulation of the
1-isoform, ouabain (10
6 M) was applied to
the
1+/
EDL to specifically inhibit the
Na-K-ATPase while EDL contractility was measured. This concentration of
ouabain inhibits the
2-isoform but is not sufficient to
inhibit the
1-isoform because this isoform has a much
lower affinity for cardiac glycosides (12, 45). In these
experiments, EDL muscles from one leg were treated with ouabain, and
EDL muscles from the other leg were used as control. The EDL was
stimulated to produce several tetanic contractions and then incubated
in ouabain for 30 min (Fig.
6A). Stimulation was then
reapplied, and force was measured. A 17% increase of peak value of
tetanus (P < 0.05) was induced by ouabain treatment in
1+/
EDL (Fig. 6B). These data
indicate that inhibition of the
2-isoform in the
1+/
EDL by low concentrations of ouabain
elicits increased contractile force and further support our hypothesis
that greater force observed in
2+/
EDL
results from reduction of the
2-isoform but not from a
compensatory upregulation of the
1-isoform in
2+/
EDL.

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Fig. 6.
Effect of ouabain on tetanic contraction in
1+/ EDL. A: nonnormalized
representative force recordings before and after 10 6 M
ouabain treatment from 1+/ EDL.
B: peak value of tetanus contraction in
1+/ EDL. Peak value of isometric force
before ouabain treatment was normalized to 100%. Values are means ± SE (n = 7). Statistical analysis utilized paired
Student's t-test. * Significant difference
(P < 0.05).
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Fatigue study.
In many pathological states, downregulation of the expression of
Na-K-ATPase
-isoforms is correlated with skeletal muscle fatigue
(1, 26). Strenuous muscle activity can also cause a
significant flux of Na+ and K+ across the
plasma membrane that exceeds the capacity of the Na-K-ATPase activity
to restore the ionic gradients (8). We thus tested whether
muscle fatigue in the
1+/
and
2+/
EDL was altered. EDL was first
stimulated to produce a tetanus and then allowed to rest for 30 min.
Stimuli were repetitively applied for 15 min (1 stimulus/min) to induce
fatigue (Fig. 7A). At the
beginning of the train of stimulation, peak values of isometric tetani
were greater for
2+/
EDL and lower for
1+/
EDL than the wild-type EDL (Fig.
7B). However, at the end of the stimulation train, peak
values for all three genotypes were similar: wild type, 54.8 ± 7.7 mN/mm2;
2+/
, 76.9 ± 26.1 mN/mm2; and
1+/
,
59.9 ± 7.2 mN/mm2 (Fig. 7B). EDL from all
three genotypes exhibited similar time constants for fatigue measured
as the time to half the maximal tetanus peak value (wild type, 5.5 ± 0.5 min;
2+/
, 5.3 ± 0.6 min;
1+/
, 6.6 ± 0.6 min).

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Fig. 7.
Muscle fatigue in EDL. A: electrical
stimulation for 5 s was applied every minute for 15 min to examine
fatigue in EDL muscles from WT, 2+/ , and
1+/ mice. A representative trace for a WT
EDL is presented. B: peak value of tetanus contraction in
WT, 2+/ , and
1+/ mice. Peak values of isometric force
for each stimulation in A were plotted for the WT,
2+/ , and 1+/
EDL muscle. Values are means ± SE (n = 8 for the
WT, and n = 7 for both the
1+/ and 2+/ ).
Statistical analysis utilized ANOVA, and intergroup comparison used
Scheffé's test. * Significant difference between WT and
2+/ (P < 0.01).
** Significant difference between WT and
1+/ (P < 0.05).
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 |
DISCUSSION |
Isoform-specific roles of Na-K-ATPase.
Two observations from this study indicate that the Na-K-ATPase
1- and
2-isoforms may play different
roles in skeletal muscle contractility. First, distinct alterations in
tetanic force between
1+/
and
2+/
EDL were observed. Reduction of the
2-isoform results in an increased isometric force. This
is consistent with previous findings that the hearts from
2+/
mice are hypercontractile
(23). It is possible that the increased tetanic force in
2+/
EDL results from an increase of
sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER) Ca2+
content via inhibition of the Na+/Ca2+
exchanger activity. Interestingly, reduction of the
1-isoform results in a decreased isometric force in
1+/
EDL that is also similar to the
phenotypic change described in
1+/
hearts.
The mechanism for reduced force in
1+/
mice is presently not clearly understood.
Second, the relative amounts of the
1- and
2-isoforms in skeletal muscle are strikingly different
from those in cardiac muscle, further suggesting the
1-
and
2-isoforms play different roles in muscle
contractility. In EDL, the
2-isoform makes up 87% of
the total
-isoform, whereas the
1-isoform only
accounts for 13%. Interestingly, these percentages are almost reversed in cardiac muscle, where 70% of the total is
1-isoform
(unpublished observation). Skeletal muscle has a more developed
T-tubule system than cardiac muscle (15). The percentage
of T-tubule membrane area is up to 85% of the total sarcolemmal
membrane area in mammalian skeletal muscle (9). The unique
percentages of the two
-isoforms in these two types of striated
muscle may become more understandable when the isoform-specific roles
of the two
-isoforms are considered. Because skeletal muscle has
more sarcolemmal membrane area in the T-tubule system, more Na-K-ATPase
2-isoform may be required to modulate the
Na+/Ca2+ exchanger activity to exert the
regulation of SR/ER Ca2+ content.
Two recent findings strongly suggest that the
1- and
2-isoforms play different functional roles at the
cellular level. First, different locations for the
1-
and
2-isoforms in the plasma membrane of rat astrocytes
and arterial myocytes have been observed (25). Although
the
1-isoform is uniformly distributed within the plasma
membrane, the
2-isoform is concentrated in restricted regions of the plasma membrane (microdomains) where the
Na+/Ca2+ exchanger is also localized. These
microdomains are in close proximity to SR/ER and serve as a functional
compartment in which Na+ and Ca2+
concentrations can be regulated in a locally restricted cytosolic area
without altering global Na+ and Ca2+
concentrations in the cytosol.
Second, this laboratory has demonstrated that
1- and
2-isoforms of the Na-K-ATPase play different roles in
cardiac muscle contractility in mice (23). By reducing the
expression levels of either
-isoform by gene targeting, it was found
that with
2+/
animals, the heart is
hypercontractile, whereas hearts from
1+/
are hypocontractile even though reduction of the
1-isoform Na-K-ATPase in
1+/
hearts does not alter Ca2+
transients (23). In hearts from
2+/
animals, reduction of the
2-Na-K-ATPase may result in an increase of intracellular
Na+ concentration that lessens the Na+ gradient
across the plasma membrane. Because the Na+ gradient is the
driving force of the Na+/Ca2+ exchanger to
extrude intracellular Ca2+, inhibition of Na-K-ATPase
ultimately causes an increase of intracellular Ca2+ that is
transported into SR/ER by SERCA. In subsequent contractions, more
Ca2+ is released from SR/ER to produce higher
Ca2+ transients that result in the hypercontractile state
in the
2+/
hearts.
Na-K-ATPase and skeletal muscle contractility.
During the determination of the supramaximal stimulation parameters,
the
2+/
muscles generated more force at
any given stimulation voltage. Thus
2+/
EDL shows both an increased sensitivity to submaximal voltages and
greater forces at supramaximal voltages. This is consistent with the
traditional view that whole skeletal muscle regulates total force via
increased recruitment of fibers. It is possible that the increased
sensitivity of the
2+/
muscle is due to an
excitation/contraction coupling phenomenon. The loss of the
2-isoform may cause a slight depolarization. Under such
conditions, a muscle may be more easily stimulated, resulting in
greater recruitment of muscle fibers, leading to greater force at any
given voltage. However, this phenomenon cannot explain the greater
force in the
2+/
muscle at supramaximal
voltage where all fibers would be activated.
In the present study, we also found that 10
6 M ouabain
increased tetanic force in
1+/
EDL. Thus
inhibition of the
2-isoform resulted in more contractile force, possibly through increasing Ca2+ content in SR/ER.
This may be due to the putative functional association of the
2-Na-K-ATPase and the Na+/Ca2+
exchanger, whose functional role has recently been elucidated in
skeletal muscle (2). A study by Clausen and Everts
(7) reported that 10
6 M ouabain produces a
slight loss of contractility in the presence of 12.5 mM K+.
In our study, a physiological concentration of 4.7 mM KCl was used that
may contribute to the different observations of ouabain effect on
muscle contractility between the two studies. Additionally, this
discrepancy may be due to the different protocol used in our study. To
facilitate intracellular Ca2+ loading, EDL muscles in our
experiments were not stimulated during the preincubation with
10
6 M ouabain. The tetanic contractions were elicited
after the incubation period and compared with untreated EDL, whereas
the study by Clausen and Everts (7) applied several
conditional stimulations of short duration (0.5-2 s) during the
incubation period with ouabain. It is highly possible that any increase
of SR/ER Ca2+ content may be dampened by these conditional
stimulations. Therefore, increased force was never observed.
One hypothesis to explain our observations is that for any given
stimulus, the
2+/
EDL releases more
Ca2+, resulting in greater force. This applies equally to
the increased force during both the submaximal and supramaximal stages
of the force-voltage curve. However, Zhao et al. (48)
showed that in a single twitch, free Ca2+ concentration
([Ca2+]free) increases to ~16 µM in frog
fast-twitch muscle (48). Hollingworth et al.
(20) measured [Ca2+]free in
fiber bundles isolated from mouse fast-twitch muscles and showed that
Ca2+ release is slightly temperature dependent, increasing
[Ca2+]free to 17 and 22 µM at 16 and
28°C, respectively (20). It has been shown that in frog
fast-twitch skeletal muscle, the spatially averaged
[Ca2+]free can increase as high as ~18
µM, which is sufficient to saturate troponin C (3, 19).
Thus the actomyosin contractile apparatus would not be expected to be
Ca2+ limited during a contractile event in frog muscle.
Importantly, however, these authors also determined that the half-time
of the [Ca2+]free transient in mice is
remarkably rapid (~4 ms), approximately one-half that of the frog
(20). Given the relationship of the
2-isoform with the Na+/Ca2+
exchanger, it is possible that the [Ca2+]free
transient is either directly prolonged in EDL from
2+/
animals or that the release of
Ca2+ is much larger than normal such that it requires more
time to resequester effectively. In either case, this could lead to
higher tetanic steady-state [Ca2+]free,
resulting in potentiation of force.
Results of the fatigue study also support our hypothesis of higher
[Ca2+]free in
2+/
EDL. While the
2+/
EDL loses more force over the entire
time course of the fatigue protocol, these muscles reach the same basal
level of force as the wild-type and
1+/
EDL, and the time to half-maximal tetanus peak value is similar for all
three genotypes. This suggests that there are no intrinsic differences
in muscle ultrastructure or biochemistry but, rather, that
Ca2+ handling has been normalized by the frequent stimulations.
It is possible, given the phenotypic plasticity of muscle tissue, that
an increase in fast-twitch myosin isoforms in the
2+/
animal could contribute to the
increased force. Myosin isoform distribution can be altered by
electrical stimulation (34), thyroid hormone status
(21, 30), exercise (5, 18, 40), and disease
(13, 14, 35). Furthermore, it has been shown that in
rodent models of type I diabetes, there is often a change in
Na-K-ATPase activity (36) that is concurrent with changes in myosin isoforms.
Perspectives
Cardiac glycoside treatment of congestive heart failure also
inhibits the Na-K-ATPase activity in skeletal muscle and may result in
loss of muscle contractility. We present for the first time that this
inhibition can be therapeutically beneficial. Our study indicates that
a loss of one-half of the
2-isoform results in more
force in skeletal muscle. Increased force may be helpful for venous
return from peripheral veins to improve circulation or for preventing
respiratory muscle failure. Furthermore, it needs to be noted that this
study is based on mouse models whose
2-isoform is at
least 1,000-fold more sensitive to cardiac glycosides than the
1-isoform. However, in most mammals, including human, the difference in affinities for cardiac glycosides between the
1- and
2-isoforms is small. Cardiac
glycosides will influence the activity of both
2- and
1-isoforms in human skeletal muscle. The data here
demonstrate that inhibition of the
1-isoform results in
decreased muscle contractility, contributing to possible toxic side
effects of cardiac glycosides, whereas inhibition of the
2-isoform results in stronger contraction. Combining the
finding from skeletal muscle with the results from the heart
(23), it is reasonable to speculate that identification or
design of compounds that specifically inhibit the
2-isoform, but not the
1-isoform, may
reduce the toxicity without altering the therapeutic potency of
Na-K-ATPase inhibition. Specific inhibition of the
2-isoform by potential compounds may be beneficial for
both cardiac and skeletal muscle.
 |
ACKNOWLEDGEMENTS |
We thank Dr. M. H. Cougnon for carefully reading and discussing the manuscript.
 |
FOOTNOTES |
*
S. He and D. A. Shelly contributed equally to this paper.
Present address of P. F. James: Dept. of Zoology, Miami
University, Oxford, OH 45056.
Address for reprint requests and other correspondence: J. B. Lingrel, Dept. of Molecular Genetics, Biochemistry, and Microbiology, Univ. of Cincinnati, College of Medicine, Cincinnati, OH 45267 (E-mail:
jerry.lingrel{at}uc.edu).
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 11 November 2000; accepted in final form 16 May 2001.
 |
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