| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Baylor College of Medicine, Houston, Texas 77030; and 2 University of Tokyo, Tokyo 113, Japan
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Skeletal muscle expresses at least two isoforms of the calcium release channel in the sarcoplasmic reticulum (RyR1 and RyR3). Whereas the function of RyR1 is well defined, the physiological significance of RyR3 is unclear. Some authors have suggested that RyR3 participates in excitation-contraction coupling and that RyR3 may specifically confer resistance to fatigue. To test this hypothesis, we measured contractile function of diaphragm strips from adult RyR3-deficient mice (exon 2-targeted mutation) and their heterozygous and wild-type littermates. In unfatigued diaphragm, there were no differences in isometric contractile properties (twitch characteristics, force-frequency relationships, maximal force) among the three groups. Our fatigue protocol (30 Hz, 0.25 duty cycle, 37°C) depressed force to 25% of the initial force; however, lack of RyR3 did not accelerate the decline in force production. The force-frequency relationship was shifted to higher frequencies and was depressed in fatigued diaphragm; lack of RyR3 did not exaggerate these changes. We therefore provide evidence that RyR3 deficiency does not alter contractile function of adult muscle before, during, or after fatigue.
ryanodine-sensitive calcium channel isoform 3; fatigue; excitation-contraction coupling
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
THERE ARE THREE KNOWN mammalian isoforms of the ryanodine-sensitive calcium channel (RyR): RyR1, primarily expressed in skeletal muscle; RyR2, the cardiac isoform; and RyR3, which is found at low levels in many tissues, including skeletal muscle, without any preferential tissue association (6, 14, 20). All three RyR isoforms function as release channels of intracellular calcium stores (6, 20); however, the precise function of RyR3 in calcium homeostasis in unclear (17, 23).
In the terminal cisternae of the sarcoplasmic reticulum, cell depolarization is detected by the voltage sensor, and calcium is released from the sarcoplasmic reticulum, leading to contraction in a process known as excitation-contraction coupling (6, 20). Several lines of evidence suggest involvement of RyR3 in this process. First, RyR3 has been localized to the terminal cisternae of the sarcoplasmic reticulum (5). Second, differences in calcium regulation between RyR1 and RyR3 suggest that RyR3 might participate in excitation-contraction coupling via calcium-induced calcium release. For example, the open probability of RyR3, when fully activated by calcium, is five times higher than that of RyR1 (2, 3). In vivo, this type of regulation could significantly impact calcium release and contractile function. On the basis of differences in calcium regulation, the most popularly theorized role of RyR3 in excitation-contraction coupling is that calcium released via RyR1 activates RyR3 to amplify the calcium signal (termed calcium-induced calcium release) (1, 17-19, 22, 23). The pervasiveness of this theory was a primary stimulus for the present study.
At the tissue level the distribution of RyR3 among skeletal muscles appears to correlate with fatigue resistance. Among skeletal muscles examined, diaphragm has the most RyR3 (5). Diaphragm, the primary muscle of inspiration and a muscle that resists fatigue (11), contains 50 times more RyR3 than extensor digitorum longus, an easily fatigued muscle (5). Additionally, neonatal muscle expresses more RyR3 (1, 23) and resists fatigue more effectively than adult muscle (10, 12, 13, 16, 25). Thus an attractive theory of the specific functional role of RyR3 exists: RyR3 contributes to fatigue resistance (1, 5).
Fatigue is the failure of muscle to maintain mechanical output during repetitive contractions (reviewed in Ref. 9). This can be measured as a loss of force production. The loss of force is accompanied by depression of tetanic calcium transients and metabolic alterations, including an increase in free radicals, a decline in pH, an increase in free magnesium, and an increase in resting calcium concentration (9, 24, 26-28). Each of these perturbations is known to depress RyR1 function (8).
Regulatory differences between RyR1 and RyR3 support the idea that RyR3 could supplement calcium release when RyR1 function is depressed. For example, magnesium may differentially modulate the two isoforms in vivo. The concentration of free magnesium in fatigued muscle is approximately twice that in rested muscle, likely as a result of ATP breakdown (28). RyR1 and RyR3 differ in sensitivity to magnesium. In general, agents that stimulate calcium release increase [3H]ryanodine binding, whereas the opposite is generally true of agents that depress calcium release; therefore, [3H]ryanodine binding is frequently used as a rapid screen of channel effectors. Magnesium depresses [3H]ryanodine binding of RyR3 by only ~25%, whereas it depresses [3H]ryanodine binding of RyR1 by 90% (18). These findings suggest the possibility that RyR3 remains active when exposed to levels of magnesium that inhibit RyR1.
We, therefore, combined the popular model of calcium-induced calcium release with known differences in channel regulation to develop a working model. If RyR3 were resistant to a fatigue-generated metabolite, which depresses RyR1 function, then RyR3 could effectively supplement calcium release and thereby maintain force production. We evaluated this model using diaphragm strips from adult RyR3-deficient mice and their littermates by testing two specific hypotheses: 1) during fatigue, lack of RyR3 accelerates the decline in force production and 2) in fatigued muscle, lack of RyR3 exaggerates the functional decrement. Our results led us to reject our hypotheses and the working model.
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Maintenance of knockout animals. The National Institutes of Health Guide for the Care and Use of Laboratory Animals dictated conditions of all procedures, which were approved in advance by the Institutional Review Board of Baylor College of Medicine. The mutant mice lacking RyR3 were established by the targeted gene disruption technique by using J1 embryonic stem cells, as described previously (21). The RyR3-deficient mice carry a mixed genetic background of C57BL and 129 strains and a mutation to exon 2 of the RyR3 gene. A total of 26 6- to 8-wk-old offspring of heterozygous parents were used in these experiments: 11 were RyR3 deficient, 6 were wild type, and 9 were heterozygous. Animal genotype was determined by PCR analysis after collection of contractile data.
Diaphragm preparation. Animals were anesthetized by methoxyflurane inhalation, then killed by cervical dislocation. The entire diaphragm was rapidly excised and immersed in room-temperature Krebs-Ringer solution containing (mM) 137 NaCl, 5 KCl, 1 NaH2PO4, 24 NaHCO3, 2 CaCl2, and 1 MgSO4. The solution was maintained at room temperature and aerated with 95% O2-5% CO2. A diaphragm strip was cut parallel to fiber orientation, with care taken to leave the rib and central tendon connected. The rib was fixed to a metal bar with silk suture (size 5-0), and the tendon was similarly tied to a force transducer (model BG 100, Kulite Instruments, Leonia, NJ) mounted on a micrometer. Diaphragm strips were stimulated by field stimulation with platinum electrodes. In pilot studies, maximal stimulation of diaphragm strips occurred at 16 V. For these experiments we utilized a supramaximal voltage of 25 V. We adjusted muscle length to produce optimum twitch force.
Experimental protocol. The solution was then replaced with 37°C Krebs solution, and the temperature was controlled by a digital water bath throughout the remainder of the experiment. After a 30-min thermoequilibration period, we recorded twitch characteristics, including twitch force, time to peak force, and one-half relaxation time. We then determined the force-frequency relationship. Tetanic contractions were stimulated at 2-min intervals (500-ms train duration); between each intermediate frequency (15, 30, 50, 80, 120, 160, 250 Hz), a maximum tetanic contraction (Po, 300 Hz) was elicited to serve as a reference for changes in force over time.
Five minutes after completion of the force-frequency protocol, we induced fatigue by stimulating the muscle at 30 Hz using a 1:4 duty cycle (0.5 trains/s, 500-ms train duration). For the second train and every 30 s thereafter, a 300-Hz stimulation replaced the 30-Hz stimulation. The final force-frequency data were collected after 5 min of fatiguing contractions by increasing the stimulus frequency (15, 30, 50, 80, 120, 160, 300 Hz) every 2 s. After a 3-min recovery period, a final train of stimulation of 300 Hz was applied. We measured the forces developed during stimulated contractions and the force sustained between trains of electrical stimulation (unstimulated force). Strips were measured for muscle length at optimum and then immediately trimmed of nonmuscle tissue and weighed. Force measurements were later normalized for functional cross section according to Close (4).PCR.
Sections of tail (1-3 cm) were collected at the time of
contractile studies and stored at 
20°C. DNA was harvested by
phenol-chloroform extraction. A solution containing PCR reagents (ExTaq
Polymerase Kit, Takara) and appropriate primers (Keystone Labs,
Camarillo, CA) was aliquoted into reaction tubes. Primers that annealed
to wild-type sequences were 5'-ATGAAGTTGTACTCCAGTGCATTG-3'
(P1) and 5'-TCCAGGAATCTCTGGTATACTAGGG-3' (P2). A separate
reaction combined P1 and P3, a primer that annealed within the exon 2 mutation: 5'-GCCACACGCGTCACCTTAATATGCG-3'. Genomic DNA was
added to a final concentration of 8 ng/µl. The reaction tubes were
then subjected to 35 temperature cycles (0.5 min at 94°C, 1 min at
60°C, 1 min at 72°C). Reaction products were electrophoresed in
a 1.5% agarose gel and detected by fluorescence of DNA-bound ethidium bromide.
Statistical analyses. Values are means ± SE. For differences in single measures among the three groups, we utilized a one-way ANOVA for parametric data or a Kruskal-Wallis ANOVA on ranks for nonparametric data. For recurrent measurements, we used a two-way repeated-measures ANOVA (15).
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Experimental model.
Consistent with previous reports, the apparent phenotype of the
RyR3-deficient mice was not grossly different from that of their
littermates (21). Genotype was determined by PCR, as detailed in
MATERIALS AND METHODS. Two sets of
primers annealing to alleles as diagramed in Fig.
1, top,
were used. Figure 1, bottom, shows representative PCR data from one animal of each genotype. The male-female distribution was similar among groups, with male
constituents as follows: 50% RyR3 deficient, 62.5% heterozygous, and
50% wild type. Body weight was not different among groups, averaging
20.5 ± 2.8 g. Average cross-sectional area of the diaphragm strips was 0.3 ± 0.1 mm2 and was not
different among groups.
|
Unfatigued contractile function.
The absolute forces developed by unfatigued muscle were similar among
groups. Table 1 shows the values obtained
for Po, twitch force, time to peak
force, and one-half relaxation time; there were no significant
differences. The average twitch-to-tetanus ratios were not
statistically different among groups: 0.22 ± 0.03 for
RyR3-deficient, 0.23 ± 0.04 for heterozygous, and 0.20 ± 0.03 for wild-type mice. The force-frequency relationships of the unfatigued muscles are illustrated in Fig. 2. These
relationships exhibit the characteristic sigmoidal shape. There were no
differences among groups. During the force-frequency protocol, muscles
from RyR3 knockout animals were as stable as those from wild-type
controls, as evidenced by the maintenance of
Po over time. Figure
3 shows the drop in maximum force over 30 min. In each group the total decrement averaged <10%.
|
|
|
Fatigue characteristics.
Diaphragm strips from the three groups responded similarly to the
fatigue protocol. Figure 4,
top, shows the change in developed force during 30-Hz stimulation. Force initially fell at a rapid rate,
then more gradually. This response was not different among groups.
Changes in maximal force (300-Hz stimulation) also followed a similar
pattern (Fig. 4, bottom).
|
|
Contractile function of fatigued muscle.
Fatigue produced a rightward shift and depression in the
force-frequency relationship. In Fig. 6 the
three-group average of the force-frequency relationship before fatigue
is shown, along with the fatigue-induced shift in each of the three
groups; there was no difference among groups. At the conclusion of the
contractile protocol, muscles were allowed to recover for 3 min.
Neither recovery of Po nor drop in
unstimulated force was different among groups. Po recovered to 56 ± 2% of
prefatigue values and unstimulated force dropped to 0.7 ± 0.2 N/cm2.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The diaphragm is peculiar among skeletal muscles. As the primary muscle of inspiration, it is crucial that the diaphragm be able to generate force sufficient for ventilation under a variety of conditions. Combined observations suggested a novel role for RyR3 in excitation-contraction coupling and fatigue resistance of the diaphragm (1).
We found no evidence that RyR3 is essential for normal contractile function. In unfatigued diaphragm from adult mice, contractile function was unchanged by a lack of RyR3. Our observations agree with those of Bertocchini et al. (1), whose data also indicated that RyR3 deficiency did not alter function in adult mouse diaphragm. However, this group did demonstrate a loss of function in diaphragm strips from neonatal, RyR3-deficient mice; the force-frequency relationship was shifted to higher frequencies than control diaphragm strips.
We also tested the postulate that RyR3 contributes to fatigue resistance. However, our studies do not support a role for RyR3 in resistance to fatigue. There was no divergence of developed or unstimulated force among groups. Bertocchini et al. (1) did not show data but mentioned that they observed no differences between normal and RyR3-deficient mice in developed force during repetitive contractions.
Conclusion. As evidenced by this study, mice without RyR3 develop and mature to an end point of normal contractile function. Before we can conclude that RyR3 is not essential in skeletal muscle, the plasticity of the tissue must be taken into account. With its ability to adapt to a variety of conditions (unloading, stretch, increased endurance or strength demands) taken into consideration, it is possible that skeletal muscle simply adapts to the loss of RyR3. For example, absence of RyR3 could result in modification of some other calcium release channel (RyR1) to serve the lost function of RyR3. Evidence of such adaptation would support the relevance of a functional role of an RyR3-type channel while securing the conclusion that the RyR3 isoform itself is nonessential.
Perspectives
With the absolute requirement of diaphragm contractile function and the findings of Bertocchini et al. (1) taken into consideration, RyR3 might be essential only under specific conditions where the protein is highly expressed (young diaphragm) and contractile demand is increased. For example, if the neonates were subjected to premature birth or inspiratory loading, would RyR3-deficient mice experience greater mortality? To our knowledge, no group has examined this possibility. However, given the mounting evidence against functional contribution of RyR3 to calcium transients and contractile function, this postulate seems unlikely. An alternative hypothesis is that RyR3 is involved in calcium signaling of some type other than excitation-contraction coupling. For example, microdomains around RyR3 channels could include calcium-sensitive components involved in gene regulation. Is it possible that RyR3-deficient muscles are slower than normal to adapt to changes in contractile demand? A final possibility is that RyR3 in adult skeletal muscle is simply vestigial, leftover from a developmental process in which it was important.| |
ACKNOWLEDGEMENTS |
|---|
The investigators thank Kathy Serysheva and Catherine Moore for assistance with the experiments; Laura Mackey, Dr. Barbara Williams, and Dr. Wienien Shou for advice on technical procedures; and Dr. Hakan Westerblad for advice on our fatigue protocol.
| |
FOOTNOTES |
|---|
This project was supported by National Institutes of Health Grants HL-45721 (M. B. Reid) and AR-41729 (S. L. Hamilton) and the National Space Biomedical Research Institute.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. B. Reid, Dept. of Medicine, Baylor College of Medicine, One Baylor Plaza, Rm. 520B, Houston, TX 77030-3498 (E-mail: reid{at}bcm.tmc.edu).
Received 3 February 1999; accepted in final form 25 May 1999.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Bertocchini, F.,
C. Ovitt,
A. Conti,
V. Barone,
H. Scholer,
R. Bottinelli,
C. Reggiani,
and
V. Sorrentino.
Requirement for the ryanodine receptor type 3 for efficient contraction in neonatal skeletal muscles.
EMBO J.
16:
6956-6963,
1997[Medline].
2.
Chen, S. R.,
P. Leong,
J. P. Imredy,
C. Bartlett,
L. Zhang,
and
D. H. MacLennen.
Single channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).
Biophys. J.
73:
1904-1912,
1998
3.
Chen, S. R.,
X. Li,
K. Ebisawa,
and
L. Zhang.
Functional characterization of the recombinant type 3 Ca2+ release channel (ryanodine receptor) expressed in HEK293 cells.
J. Biol. Chem.
272:
24234-24246,
1997
4.
Close, R. I.
Dynamic properties of mammalian skeletal muscle.
Physiol. Rev.
52:
129-197,
1972
5.
Conti, A.,
L. Garza,
and
V. Sorrentino.
Differential distribution of ryanodine receptor type 3 (RyR3) gene product in mammalian skeletal muscles.
Biochem. J.
316:
19-23,
1996.
6.
Coronado, R.,
J. Morrissette,
M. Sukhareva,
and
D. M. Vaughan.
Structure and function of ryanodine receptors.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1485-C1504,
1994
7.
Dietze, B.,
F. Bertocchini,
V. Barone,
A. Struk,
V. Sorrentino,
and
W. Melzer.
Voltage-controlled Ca2+ release in normal and ryanodine receptor type 3 (RyR3)-deficient mouse myotubes.
J. Physiol. (Lond.)
513:
3-9,
1998
8.
Ehrlich, B. E.,
and
A. R. Marks.
Regulation of the calcium release channel.
In: Ryanodine Receptors, edited by V. Sorrentino. New York: CRC, 1996, p. 17-30.
9.
Fitts, R.
Cellular mechanisms of muscle fatigue.
Physiol. Rev.
74:
49-94,
1994
10.
Franzinini-Armstrong, C.
Simultaneous maturation of transverse tubules and sarcoplasmic reticulum during muscle differentiation in the mouse.
Dev. Biol.
146:
353-363,
1991[Medline].
11.
Fratacci, M. D.,
M. Levame,
A. Rauss,
and
G. Atlan.
Rat diaphragm during postnatal development. II. Resistance to low- and high-frequency fatigue.
Reprod. Fertil. Dev.
8:
399-407,
1996[Medline].
12.
Fratacci, M. D.,
M. Levame,
A. Rauss,
H. Bousbaa,
and
G. Atlan.
Rat diaphragm during postnatal development. I. Changes in distribution of muscle fibre type and in oxidative potential.
Reprod. Fertil. Dev.
8:
391-398,
1996[Medline].
13.
Gosselin, L. E.,
B. D. Johnson,
and
G. C. Sieck.
Age-related changes in diaphragm muscle contractile properties and myosin heavy chain isoforms.
Am. J. Respir. Crit. Care Med.
150:
174-178,
1994[Abstract].
14.
Hakamata, Y.,
J. Nakai,
H. Takeshima,
and
K. Imoto.
Primary structure and distribution of a novel ryanodine receptor/calcium release channel from rabbit brain.
FEBS Lett.
312:
229-235,
1992[Medline].
15.
Johnson, R. A.,
and
D. W. Wichern.
Applied Multivariate Statistical Analysis. Englewood Cliffs, NJ: Prentice-Hall, 1982.
16.
Maxwell, L. C.,
R. J. McCarter,
T. J. Kuehl,
and
J. L. Robotham.
Development of histochemical and functional properties of baboon respiratory muscles.
J. Appl. Physiol.
54:
551-556,
1983
17.
Murayama, T.,
and
Y. Ogawa.
Properties of RyR3 ryanodine receptor isoform in mammalian brain.
J. Biol. Chem.
271:
5079-5084,
1996
18.
Murayama, T.,
and
Y. Ogawa.
Characterization of type 3 ryanodine receptor (RyR3) of sarcoplasmic reticulum from rabbit skeletal muscles.
J. Biol. Chem.
272:
24030-24037,
1997
19.
Sonnleitner, A.,
A. Conti,
F. Bertocchini,
H. Schindler,
and
V. Sorrentino.
Functional properties of the ryanodine receptor type 3 (RyR3) Ca2+ release channel.
EMBO J.
17:
2790-2798,
1998[Medline].
20.
Sutko, J.,
and
J. A. Airey.
Ryanodine receptor Ca2+ release channels: does diversity in form equal diversity in function?
Physiol. Rev.
76:
1027-1071,
1996
21.
Takeshima, H.,
T. Ikemoto,
M. Nishi,
N. Nishiyama,
M. Shimuta,
Y. Sugitani,
J. Kuno,
I. Saito,
H. Saito,
M. Endo,
M. Lino,
and
T. Noda.
Generation and characterization of mutant mice lacking ryanodine receptor type 3.
J. Biol. Chem.
271:
19649-19652,
1996
22.
Takeshima, H.,
M. Lino,
H. Takekura,
M. Nishi,
J. Kuno,
O. Minowa,
H. Takano,
and
T. Noda.
Excitation-contraction uncoupling and muscular degeneration in mice lacking functional skeletal muscle ryanodine receptor gene.
Nature
369:
556-559,
1994[Medline].
23.
Tarroni, P.,
D. Rossi,
A. Conti,
and
V. Sorrentino.
Expression of the ryanodine receptor type 3 calcium release channel during development and differentiation of mammalian skeletal muscle cells.
J. Biol. Chem.
272:
19808-19813,
1997
24.
Westerblad, H.,
and
D. G. Allen.
Myoplasmic free Mg2+ concentration during repetitive stimulation of single fibres from mouse skeletal muscle.
J. Physiol. (Lond.)
453:
413-434,
1992
25.
Westerblad, H.,
and
D. G. Allen.
The influence of intracellular pH on contraction, relaxation and [Ca2+]i in intact single fibres from mouse muscle.
J. Physiol. (Lond.)
466:
611-628,
1993
26.
Westerblad, H.,
and
D. G. Allen.
Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle.
Acta Physiol. Scand.
156:
407-416,
1996[Medline].
27.
Westerblad, H.,
S. Duty,
and
D. G. Allen.
Intracellular calcium concentration during low-frequency fatigue in isolated single fibers of mouse skeletal muscle.
J. Appl. Physiol.
75:
382-388,
1993
28.
Westerblad, H.,
J. A. Lee,
J. Lannergren,
and
D. G. Allen.
Cellular mechanisms of fatigue in skeletal muscle.
Am. J. Physiol.
261 (Cell Physiol. 30):
C195-C209,
1991
This article has been cited by other articles:
|
|
 |
|
  C. F. Perez, J. R. Lopez, and P. D. Allen Expression levels of RyR1 and RyR3 control resting free Ca2+ in skeletal muscle Am J Physiol Cell Physiol, March 1, 2005; 288(3): C640 - C649. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Krupnick, J. Zhu, T. Nguyen, D. Kreisel, K. R. Balsara, E. B. Lankford, C. C. Clark, S. Levine, H. H. Stedman, and J. B. Shrager Inspiratory loading does not accelerate dystrophy in mdx mouse diaphragm: implications for regenerative therapy J Appl Physiol, February 1, 2003; 94(2): 411 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Reid, J. Lannergren, and H. Westerblad Respiratory and Limb Muscle Weakness Induced by Tumor Necrosis Factor-{alpha}: Involvement of Muscle Myofilaments Am. J. Respir. Crit. Care Med., August 15, 2002; 166(4): 479 - 484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Rossi, R. Bottinelli, V. Sorrentino, and C. Reggiani Response to caffeine and ryanodine receptor isoforms in mouse skeletal muscles Am J Physiol Cell Physiol, August 1, 2001; 281(2): C585 - C594. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
),
heterozygous (
), and wild-type (
) mice before fatigue are shown.
Po, maximum tetanic contraction.
