Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction.
ABSTRACT Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca(2+)/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fast-twitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca(2+)/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.
- SourceAvailable from: Manuel A Riquelme[Show abstract] [Hide abstract]
ABSTRACT: Normal myotubes and adult innervated skeletal myofibers express the glycoprotein pannexin1 (Panx1). Six of them form a "gap junction hemichannel-like" structure that connects the cytoplasm with the extracellular space; here they will be called Panx1 channels. These are poorly selective channels permeable to ions, small metabolic substrate, and signaling molecules. So far little is known about the role of Panx1 channels in muscles but skeletal muscles of Panx1(-/-) mice do not show an evident phenotype. Innervated adult fast and slow skeletal myofibers show Panx1 reactivity in close proximity to dihydropyridine receptors in the sarcolemma of T-tubules. These Panx1 channels are activated by electrical stimulation and extracellular ATP. Panx1 channels play a relevant role in potentiation of muscle contraction because they allow release of ATP and uptake of glucose, two molecules required for this response. In support of this notion, the absence of Panx1 abrogates the potentiation of muscle contraction elicited by repetitive electrical stimulation, which is reversed by exogenously applied ATP. Phosphorylation of Panx1 Thr and Ser residues might be involved in Panx1 channel activation since it is enhanced during potentiation of muscle contraction. Under denervation, Panx1 levels are upregulated and this partially explains the reduction in electrochemical gradient, however its absence does not prevent denervation-induced atrophy but prevents the higher oxidative state. Panx1 also forms functional channels at the cell surface of myotubes and their functional state has been associated with intracellular Ca(2+) signals and regulation of myotube plasticity evoked by electrical stimulation. We proposed that Panx1 channels participate as ATP channels and help to keep a normal oxidative state in skeletal muscles.Frontiers in Physiology 01/2014; 5:139.
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ABSTRACT: Abstract Context: Ginsenosides are primary active ingredients of ginseng, which are believed to have various health benefits. It is found that the biotransformation of ginsenosides mainly takes place in the gastrointestinal tract and the information about ginsenosides-exerted effects on intestinal contractility is not sufficient. Aims: The present study proposed that ginsenosides could exert stimulatory or inhibitory effects on intestinal motility depending on the assay condition-related intestinal contractile states and was to characterize the effects of ginsenosides on intestinal motility. Methods: Jejunal contractility determination, Western blotting analysis, and real-time polymerase chain reaction were performed to test the effects of total ginsenosides isolated from Panax ginseng C. A. Mey (Araliaceae) root. Results: The results showed that ginsenosides at the fixed concentration of 20 mg/L exerted bidirectional regulation (BR) on the contractility of isolated jejunal segment (IJS), depending on the contractile states. The contractility of IJS was increased by ginsenosides in low contractile states, which were correlated to the cholinergic activation, and the contractility of IJS was decreased by ginsenosides in high contractile states, which were correlated to the adrenergic activation and nitric oxide related mechanisms. Ginsenosides-induced BR was abolished in the absence of Ca(2+) or by using tetrodotoxin, implicating the requirement of Ca(2+) and the enteric nervous system. Effects of ginsenosides on myosin light chain phosphorylation and the mRNA expression of myosin light chain kinase were also bidirectional. Discussion and conclusion: Results suggest that ginsenosides may have the potential clinical implication for reliving the symptoms of alternative hypo- and hyper-intestinal motility.Pharmaceutical Biology 09/2013; · 1.21 Impact Factor
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ABSTRACT: The contractile performance of mammalian fast twitch skeletal muscle is history dependent. The effect of previous or ongoing contractile activity to potentiate force, i.e. increase isometric twitch force, is a fundamental property of fast skeletal muscle. The precise manifestation of force potentiation is dependent upon a variety of factors with two general types being identified; staircase potentiation referring to the progressive increase in isometric twitch force observed during low frequency stimulation while posttetanic potentiation refers to the step-like increase in isometric twitch force observed following a brief higher frequency (i.e. tetanic) stimulation. Classic studies established that the magnitude and duration of potentiation depends on a number of factors including muscle fiber type, species, temperature, sarcomere length and stimulation paradigm. In addition to isometric twitch force, more recent work has shown that potentiation also influences dynamic (i.e. concentric and/or isotonic) force, work and power at a range of stimulus frequencies in situ or in vitro, an effect that may translate to enhanced physiological function in vivo. Early studies performed on both intact and permeabilized models established that the primary mechanism for this modulation of performance was phosphorylation of myosin, a modification that increased the Ca(2+) sensitivity of contraction. More recent work from a variety of muscle models indicates, however, the presence of a secondary mechanism for potentiation that may involve altered Ca(2+) handling. The primary purpose of this review is to highlight these recent findings relative to the physiological utility of force potentiation in vivo.Journal of Muscle Research and Cell Motility 10/2013; · 1.36 Impact Factor
Myosin light chain kinase and myosin
phosphorylation effect frequency-dependent
potentiation of skeletal muscle contraction
Gang Zhi*, Jeffrey W. Ryder*, Jian Huang*, Peiguo Ding*, Yue Chen†, Yingming Zhao†, Kristine E. Kamm*,
and James T. Stull*‡
Departments of *Physiology and†Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390
Edited by Edward D. Korn, National Institutes of Health, Bethesda, MD, and approved October 10, 2005 (received for review August 8, 2005)
Repetitive stimulation potentiates contractile tension of fast-
twitch skeletal muscle. We examined the role of myosin regulatory
light chain (RLC) phosphorylation in this physiological response by
ablating Ca2??calmodulin-dependent skeletal muscle myosin light
chain kinase (MLCK) gene expression. Western blot and quantita-
tive-PCR showed that MLCK is expressed predominantly in fast-
twitch skeletal muscle fibers with insignificant amounts in heart
and smooth muscle. In contrast, smooth muscle MLCK had a more
ubiquitous tissue distribution, with the greatest expression ob-
served in smooth muscle tissue. Ablation of the MYLK2 gene in
mice resulted in loss of skeletal muscle MLCK expression, with no
change in smooth muscle MLCK expression. In isolated fast-twitch
skeletal muscles from these knockout mice, there was no signifi-
cant increase in RLC phosphorylation in response to repetitive
electrical stimulation. Furthermore, isometric twitch-tension po-
tentiation after a brief tetanus (posttetanic twitch potentiation) or
low-frequency twitch potentiation (staircase) was attenuated rel-
ative to responses in muscles from wild-type mice. Interestingly,
the site of phosphorylation of the small amount of monophospho-
by MLCK, indicating a potential alternative signaling pathway
affecting contractile potentiation. Loss of skeletal muscle MLCK
expression had no effect on cardiac RLC phosphorylation. These
results identify myosin light chain phosphorylation by the dedi-
cated skeletal muscle Ca2??calmodulin-dependent MLCK as a pri-
mary biochemical mechanism for tension potentiation due to
repetitive stimulation in fast-twitch skeletal muscle.
calcium ? calmodulin ? twitch
transverse tubule that triggers Ca2?release from the sarcoplas-
mic reticulum through the intracellular ryanodine receptor (1,
2). The Ca2?binds to troponin in thin filaments, thereby
allowing myosin cross bridges to bind actin and generate muscle
tension (3). However, muscle contractions involve more complex
mechanisms that affect performance. Since Ranke noted in 1865
(4) that, with stimuli uniform in strength the later twitch
contractions were stronger than the first, there has been con-
siderable interest in identifying the mechanisms involved in
isometric twitch potentiation during trains of stimuli at low
frequency (staircase) or after a tetanus (posttetanic potentia-
tion). Considerations have been given to changes in compliance
of the series elastic elements, to activation of more fibers within
a muscle, to increased Ca2?release within a single fiber to
activate fully the contractile proteins, and to changes in excita-
tion–contraction coupling processes (5–8).
Ca2?released during muscle contraction can also activate the
dedicated protein kinase Ca2??calmodulin-dependent skeletal
muscle myosin light chain kinase (skMLCK) to initiate myosin
regulatory light chain (RLC) phosphorylation (9, 10). RLC
phosphorylation has no significant effect on skeletal muscle
keletal muscle contraction depends on a voltage-driven
conformational change in the L-type Ca2?channel in the
actin-activated myosin ATPase activity (9, 11). However, Man-
ning and Stull (12) noted a temporal correlation between the
extent of RLC phosphorylation and potentiation of peak iso-
metric twitch tension. This correlation was prominent in fast-
twitch, but not slow-twitch, muscles (13). Subsequent studies
myosin thick filaments, with movement of phosphorylated my-
osin cross bridges away from the thick filament backbone,
resulting in an increase in the rate at which cross bridges enter
force-producing states (9, 11, 14). Nevertheless, it is still not
known how much RLC phosphorylation quantitatively contrib-
utes to potentiation of twitch tension in intact skeletal muscle
fibers. Additional biochemical investigations reveal calmodulin
modulation of the L-type Ca2?channel and ryanodine receptor
(1, 2, 15), raising the possibility that stimuli with repetitive Ca2?
release could affect the functions of these two proteins and
thereby alter excitation–contraction coupling properties. Thus,
during repetitive motor unit firing at physiological frequencies
that initiate unfused tetanus, muscle force may be enhanced by
multiple mechanisms involving Ca2?. In terms of RLC phos-
phorylation per se, there are additional complications, including
the presence of smooth muscle MLCK (smMLCK) in skeletal
muscle fibers (16) and the potential for RLC phosphorylation by
other kinases, similar to the case found in smooth muscle and
non-muscle cells (17).
To resolve these issues regarding contributions of different
biochemical mechanisms involved in contraction potentiation, as
well as RLC phosphorylation, we disrupted the MYLK2 gene to
eliminate expression of skMLCK.
Generation of skMLCK-Deficient Mice. The homologous short arm
of the skMLCK gene (MYLK2) used in the targeting vector was
a 2.5-kb NotI and BamHI fragment amplified from genomic
DNA from mouse embryonic stem cells (Strain 129?SvJ) by
PCR. The length of 5? terminal arm in the construct was ?2.6
1). The length of the 3? terminal arm was ?6.8 kb and included
introns 7–13 and exons 8–14, followed by a herpes virus thymi-
dine kinase cassette to permit negative selection with gancyclo-
vir. The 1.7-kb Neo coding region replaced the targeted locus
region, which included part of exon 6, intron 6, and exon 7. The
targeted deletion included 660 bp that encode the ATP-binding
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: MLCK, myosin light chain kinase; RLC, regulatory light chain of myosin;
Q-PCR, quantitative real-time PCR; skMLCK, skeletal muscle MLCK; smMLCK, smooth
‡To whom correspondence should be addressed at: Department of Physiology, University
of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-
9040. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
November 29, 2005 ?
vol. 102 ?
no. 48 ?
sequence of the catalytic core. The targeting vector was subse-
quently linearized with NotI and transfected into 129?SvEvTac
embryonic stem cells by electroporation. Clones surviving G418
by Southern blotting. Selected clones were then injected into
C57BL?B6 blastocysts and implanted into pseudopregnant C57?
ICR females by the Transgenic Core Facility at the University of
Texas Southwestern Medical Center at Dallas. The chimeric
founder mice were bred to C57BL?6 for transmission of the
mutant skMLCK allele. Animals were housed on a 12-h light and
12-h dark cycle with free access to water and standard mouse
diet. Animal experiments were conducted following protocols
approved by the Institutional Animal Care and Use Committee.
Genotyping and Southern Analysis of Mice. DNA was extracted
from mouse tail samples by proteinase K digestion, phenol
extraction, and isopropanol precipitation. DNA was digested
overnight with HincII restriction endonuclease and separated on
an agarose gel. DNA was immobilized on a Nylon membrane by
Rapid Downward Transfer Systems (Schleicher & Schuell) and
probed with an ?-32P-labeled 503-bp DNA probe located outside
of the targeting vector (Fig. 1). The probe primers used for PCR
Western Blots. For analysis of skeletal and smooth muscle
MLCK expression, tissues were collected and homogenized in
SDS sample buffer (18). Lysate protein concentration was
measured by bicinchoninic acid (BCA) protein assay reagent,
and 40 ?g of protein was loaded per well for 7.5% SDS?PAGE,
followed by transfer to nitrocellulose membranes. Smooth and
skeletal muscle MLCK were probed with K36, a monoclonal
antibody against smMLCK (Sigma) or polyclonal goat anti-
body against skMLCK (31).
RLC phosphorylation was measured in isolated mouse exten-
sor digitorum longus muscles quick-frozen in clamps prechilled
in liquid nitrogen by urea?glycerol-PAGE and immunoblotting
where the nonphosphorylated RLC is separated from mono-
phosphorylated RLC (19, 20). RLC phosphorylation was mea-
sured similarly in ventricular heart samples quickly dissected and
frozen in liquid nitrogen from anesthetized mice. Changes in
RLC phosphorylation occur on the order of 20–30 min in heart
so immediate in situ fixation is not essential to measure the
extents of phosphorylation that reflect in vivo values (21).
Antibodies raised in rabbits to purified skeletal or cardiac RLCs
were used for the appropriate analyses. Horseradish peroxidase
the Amersham Pharmacia ECL system, and quantitative mea-
surements were processed on a Storm PhosphorImager and
analyzed by IMAGEQUANT software.
Quantitative Real-Time PCR (Q-PCR). Total RNA was extracted from
mouse tissues by using RNAStat 60 (Tel-Test, Friendswood, TX).
Two micrograms of total RNA was DNase I (Roche) treated and
reverse transcribed by using random hexamers (Roche Applied
Science) and SuperScript II Reverse Transcriptase (Invitrogen).
The following primers used for real-time PCR analysis were de-
signedbyusing PRIMER EXPRESS 2.0software(AppliedBiosystems):
mouse smMLCK, forward 5?-AGAAGTCAAGGAGGTAAA-
GAATGATGT-3?, reverse 5?-CGGGTCGCTTTTCATTGC-3?;
mouse skMLCK, forward 5?-ACATGCTACTGAGTGGCCT-
CTCT-3?, reverse 5?-GGCAGACAGGACATTGTTTAAGG-3?;
mouse 36B4, forward 5?-CACTGGTCTAGGACCCGAGAAG-
3?, reverse 5?-GGTGCCTCTGGGAGATTTTCG-3?. Real-time
PCRs were performed on an Applied Biosystems Prism 7000.
SYBR green Master Mix (Applied Biosystems), and 150 nM
forward and reverse primer sets in a total volume of 20 ?l. Smooth
and skeletal MLCK RNA expression was calculated by using the
comparative CTmethod with 36B4 used as the normalizer. Final
results are expressed as the percentage of the mean for the highest
expressing tissue from wild-type mice.
Isolated Extensor Digitorum Longus Muscle Preparation. The effects
of skMLCK gene ablation on the contractile and RLC phos-
phorylation properties were studied in isolated extensor digito-
rum longus muscles as described (19, 22). Briefly, the muscles
were surgically isolated from 8- to 12-week-old mice and
mounted on Grass FT03.C force transducers, bathed in physio-
logical salt solution at 30°C, and gassed continuously with 98%
O2-5% CO2. Muscles were stimulated with two platinum wire
electrodes to establish optimal stimulus intensity and muscle
muscle length to Lo or 0.7 Lo (length equal to 0.7 of the maximal
force development), they remained quiescent for at least 30 min
before isometric twitches were elicited three times at one twitch
per min to establish control tension values. Muscles were then
stimulated at 150 Hz for 2 s, and the posttetanic twitch response
was measured at 10 s. A separate set of muscles were quick-
frozen at 12 s after tetanus for RLC phosphorylation measure-
ments. In a different experiment, muscles were stimulated at 10
Hz for 15 s to elicit a progressive increase in isometric twitch
tension (staircase), followed by muscle freezing for RLC phos-
Phosphorylation Site Mapping for Skeletal Muscle RLC. Extensor
digitorum longus muscles from wild-type and skMLCK knock-
out mice were excised and subjected to mechanical?hypoxic
stress by manually rolling the muscle between the thumb and
of the targeting vector and proposed homologous recombination in the
Southern blot analysis of germ-line mice. HincII-digested genomic DNA was
prepared from mouse tail and hybridized with the probe as described under
and skMLCK?/?mice whereas a 10.1-kb fragment representing the wild-type
of skMLCK expression in extensor digitorum longus muscles from wild-type
(???), heterozygous (???), and knockout (???) mice.
www.pnas.org?cgi?doi?10.1073?pnas.0506846102 Zhi et al.
index finger for 5 s, a handling procedure that routinely increases
RLC phosphorylation. Muscles were then frozen with aluminum
tongs cooled in liquid nitrogen and stored at ?80°C. Muscles
were homogenized on ice in 400 ?l of 50 mM Tris, 1 mM EDTA,
1 mM DTT, 1% Nonidet P-40, 0.25 mM trans-epoxysuccinyl-L-
orophosphate. One milliliter of 10% trichloroacetic acid was
added to 150-?l aliquots of homogenate immediately upon
completion of homogenization or after overnight incubation at
room temperature to allow for protein dephosphorylation. The
precipitated proteins were collected by centrifugation and sol-
ubilized in urea sample buffer for RLC phosphorylation mea-
surements for the Western blot analysis as described above. For
mass spectrometry analysis, 30–90 ?g of protein was subjected
to electrophoresis on the urea?glycerol gels, followed by staining
with Colloidal Blue (Invitrogen) for the visualization of the
monophosphorylated RLC band.
The protein band was cut into small pieces and destained with
25 mM ammonia bicarbonate solution [pH 8.0, methanol?water,
50:50 (vol?vol)] three times for 10 min each. The destained gels
were cleaned in an acidic buffer (acetic acid?methanol?water,
10:50:40, vol?vol?vol) three times for 1 h each, and swollen in
water two times for 20 min each. The gel pieces were dehydrated
in acetonitrile before drying in a SpeedVac (Thermo Savant,
at the concentration of 10 ng??l in 50 mM ammonia bicarbonate
(pH 8.0) were added to the gel pieces for overnight digestion.
The resulting tryptic peptides were extracted sequentially with
buffer (acetonitrile?water?TFA, 75:24:1, vol?vol?vol). The pep-
tide extracts were combined and dried in a SpeedVac.
Tryptic digests of the protein were analyzed by MALDI-TOF
mass spectrometry (Voyager DE Pro mass spectrometer, Ap-
plied Biosystems), nanospray precursor ion scan in QStar XL
mass spectrometer (Applied Biosystems), and HPLC?LTQ 2-D
ion trap mass spectrometer (ThermoFinnigan, San Jose, CA),
coupled with Agilent 1100 nano-flow HPLC system (Agilent,
Palo Alto, CA) for identification of phosphopeptide and precise
localization of phosphorylation sites.
Disruption of the skMLCK Gene. We ablated the functional expres-
sion of skMLCK in fast-twitch skeletal muscle of mice. Southern
screening showed only a 12.1-kb DNA fragment obtained after
HincII digestion in homozygous knockout mice with no expres-
sion of skMLCK protein (Fig. 1). In wild-type mice, only a
showed both DNA fragments, with half as much MLCK protein
expressed relative to wild-type mice. No obvious phenotypes
were noted for the knockout mice, including litter size, fertility,
or viability relative to wild-type mice as old as 6 months.
Amounts of mRNA Expression for Skeletal and Smooth Muscle MLCKs
in Different Tissues. Expression levels of skeletal and smMLCK
mRNAs were determined in fast-twitch extensor digitorum longus
muscle, slow-twitch soleus muscle, heart (mid-left ventricle), and
urinary bladder from wild-type and skMLCK?/?mice by using
Q-PCR. In wild-type mice, expression of skMLCK mRNA was
greatest in extensor digitorum longus muscle, where amounts were
5-fold greater than in soleus muscle (Fig. 2). Minimal amounts of
skMLCK mRNA (?0.1% of extensor digitorum longus muscle)
were detected in heart and bladder from wild-type mice, consistent
expression in these tissues (see below). To determine whether
ablation of skMLCK could lead to a compensatory increased
expression of smMLCK, the mRNA abundance of the latter was
determined in tissues from wild-type and the skMLCK?/?mice
(Fig. 2). In wild-type mice, expression of smMLCK mRNA was
greatest in bladder, and amounts were not significantly altered in
bladders from knockout mice. Expression of smMLCK mRNA in
extensor digitorum longus, soleus, and heart tissues of wild-type
significantly altered in skMLCK?/?mice.
Expression of MLCKs in Tissues from Wild-Type and skMLCK Knockout
Mice. Quantitative Western blotting of different tissues from
wild-type mice showed abundant expression of the skMLCK in
fast-twitch extensor digitorum longus muscles, with substantially
less kinase expressed in slow-twitch soleus muscle (Fig. 3). The
kinase was not detected in cardiac or bladder smooth muscle
tissues or in nonmuscle tissues such as liver, lung, or kidney (data
not shown). Considering the previous report that skMLCK was
expressed in cardiac myocytes (23), we did additional probing
with gels overloaded with 200 ?g of protein, but still no kinase
was detected. These results on protein expression of skMLCK in
different tissues are consistent with results obtained for the
We also measured smMLCK expression in different tissues
from wild-type mice (Fig. 3). This MLCK was abundantly
expressed in the urinary bladder smooth muscle with lower but
significant amounts in the other muscle tissues. The extent of
smMLCK expression in these different tissues was not signifi-
cantly changed in the skeletal muscle knockout mouse, thus
showing that there was not a compensatory increased expression
of the smooth muscle kinase with disrupted expression of the
skMLCK (Fig. 3). There were similar amounts of smMLCK
expressed in soleus and cardiac muscle tissues.
RLC Phosphorylation and Contractile Tension Potentiation in Fast-
Twitch Skeletal Muscle from skMLCK Knockout Mice. The effect of
repetitive stimulation on RLC phosphorylation in isolated
types of muscles. Relative amounts of skMLCK and smMLCK mRNA were
determined by Q-PCR in tissues from wild-type and skMLCK?/?mice as de-
scribed under Methods. Values are reported as the means ? SEM for three
mice of each genotype.
Expression of skeletal and smooth muscle MLCK mRNA in different
Zhi et al.PNAS ?
November 29, 2005 ?
vol. 102 ?
no. 48 ?
extensor digitorum longus muscle of mice was measured with
two modes of stimulation. The isometric twitch tension in-
creased 75% at 10 s after a tetanic stimulation at 150 Hz for
2 s in muscles from wild-type mice (posttetanic potentiation)
but not from skMLCK?/?mice (Figs. 4 and 5). Similarly, RLC
phosphorylation increased in muscles from wild-type mice
after the tetanus but not in muscles from the skMLCK?/?mice
(Figs. 4 and 5). These results show that the posttetanic
potentiation of the isometric twitch tension and RLC phos-
phorylation responses are significantly attenuated with abla-
tion of skMLCK expression.
The effect of repetitive stimulation at a constant low fre-
quency on isometric twitch properties and RLC phosphory-
lation was also examined. Stimulation of isolated muscles from
wild-type animals at 10 Hz for 15 s produced the characteristic
staircase increase in isometric twitch potentiation (Figs. 4 and
5). After a transient small decline, isometric twitch tension
progressively increased, reaching a maximum 55% increase by
15 s. RLC phosphorylation also increased by 15 s in response
to the low frequency stimulation. In the muscles from the
skMLCK?/?mice, the transient decrease in isometric tension
was still present followed by a recovery phase with a 30%
increase in tension. The decrease in isometric twitch potenti-
ation response in skMLCK?/?mice was associated with at-
tenuation of RLC phosphorylation in response to low-
frequency repetitive stimulation.
Interestingly, the extent of RLC phosphorylation in resting,
isolated extensor digitorum longus muscles from the skMLCK?/?
mice varied from 0.05 to 0.09 mol of phosphate per mol of RLC,
as a monophosphorylated RLC because of the specific increase in
protein migration by urea?glycerol-PAGE that is associated with
monophosphorylation (Fig. 6; refs. 12 and 13). Furthermore, the
monophosphorylated RLC band disappeared in a muscle homog-
enate prepared with a nondenaturing buffer without protein phos-
phatase inhibitors. The gel shown in Fig. 6 is overexposed to show
clearly the monophosphorylated RLC in skMLCK?/?muscle sam-
ples. The identity of the band intermediate between the nonphos-
phorylated and monophosphorylated RLCs is not clear, but, in
quantitative blots, it represents ?5% or less of the total immuno-
reactivity and does not change with electrical stimulation. The
apparent increase in density of the intermediate band with phos-
phatase treatment may be due to limited proteolysis of the non-
types of muscles. Relative expression amounts of skMLCK and smMLCK pro-
tein were determined by Western blotting in tissues from wild-type and
skMLCK?/?mice as described under Methods. Values are reported as the
means ? SEM for at least four mice of each genotype.
Expression of skeletal and smooth muscle MLCK protein in different
ric twitch potentiation in isolated mouse extensor digitorum longus muscles.
Shown are representative force tracings of isometric twitches showing single
twitches before and after a tetanus (150 Hz, 2 s) (Left) and with continuous
electrical stimulation at 10 Hz for 15 s (Right) in muscles from wild-type
(Upper) and skMLCK?/?mice (Lower). The measured responses are relative to
single isometric twitches before repetitive stimulation.
Effect of repetitive contractions on RLC phosphorylation and isomet-
ric twitch potentiation in isolated mouse extensor digitorum longus muscles.
Average isometric twitch tension (Lower) and RLC phosphorylation (Upper)
responses after a tetanus (Left) and with continuous electrical stimulation at
10 Hz for 15 s (Right) for wild-type (WT) and skMLCK?/?(KO) mice. The values
represent the means ? SEM for at least six muscle responses.
Effect of repetitive contractions on RLC phosphorylation and isomet-
www.pnas.org?cgi?doi?10.1073?pnas.0506846102Zhi et al.
phosphorylated RLC. Analysis of the purified monophosphory-
lated RLC from MLCK?/?mouse muscle led to detection of
peptides that cover ?95% sequence of the protein (Fig. 7). Colli-
sion-induced fragmentation spectrum (MS?MS) of the phos-
phopeptide (residues 10–31) conclusively determined the phos-
phorylation site at residue serine 15, adjacent to the site
phosphorylated by MLCK (24). These observations suggest that
another kinase that is not activated by repetitive contractions may
phosphorylate skeletal muscle RLC.
Effect of skMLCK Ablation on Cardiac RLC Phosphorylation and
Growth. Because skMLCK was reported to be significantly
cardiac RLC phosphorylation (23), we examined the potential
effects of skMLCK gene ablation on heart properties (Table 1).
There were no significant differences in the extent of cardiac
RLC phosphorylation in knockout animals compared with
wild-type animals. Furthermore, the extents of phosphorylation
were similar to previously reported values (21, 25, 26). Addi-
tionally, there were no significant differences in heart weight,
body weight, and, thus, heart weight?body weight ratio in
wild-type and skMLCK?/?mice. These results are consistent
with the lack of detection of any significant amounts of skMLCK
mRNA or protein in cardiac muscle.
The extents of expression of skeletal and smooth muscle MLCKs
are tissue-dependent, with skMLCK showing a more restricted,
tissue-specific expression. During differentiation of skeletal
muscle myoblasts to myotubes, skMLCK is expressed to amounts
found in adult skeletal muscles (16). Because the kinase was
found only in adult skeletal muscle but not heart, smooth, or
nonmuscle cells by Western blotting, it was proposed that it was
the only tissue-specific MLCK. In contrast, smMLCK was found
in all adult tissues, including cardiac and skeletal muscle myo-
cytes, and thus should not be considered as a smooth muscle-
heart. With antibodies raised to the expressed kinase, they
showed a substantially lesser amount of an immunoreactive band
in mouse heart than in skeletal muscle by Western blotting.
Because this band did not have the same migration as MLCK
from mouse skeletal muscle, it may not have been skMLCK
protein. Although we and others (16) were not able to detect
skMLCK protein in cardiac or smooth muscle by Western
blotting, Q-PCR showed skMLCK mRNA present in heart tissue
in an amount substantially lower (?1,000 fold) than that ob-
served in skeletal muscle but similar to that observed in smooth
muscle tissue. Thus, skMLCK may have been cloned from a rare
amount of mRNA in cardiac muscle (23). Our results from the
skMLCK knockout mice also show that gene ablation had no
effect on the extent of cardiac RLC phosphorylation, indicating
that it is not a functional kinase in cardiac myocytes.
In agreement with the results of Herring et al. (16), we found
smMLCK to be expressed in all three kinds of muscles, although
the amounts measured by Western blotting and Q-PCR were
much greater in smooth muscle than skeletal or cardiac muscle.
Although we cannot exclude contributions from smooth muscle
in blood vessels in these measurements, Herring et al. (16)
reported smMLCK expression in differentiated skeletal and
cardiac myocytes in culture. Perhaps smMLCK is responsible for
cardiac RLC phosphorylation. This kinase phosphorylates car-
diac RLC, but the catalytic efficiency is substantially less than
smooth muscle RLC (27). However, mouse hearts beat at a high
rate (?600 beats per minute). Because MLCK is inactivated
slowly (1 s?1; refs. 9 and 13), it is predicted that the kinase is not
inactivated during the short relaxation time before the heart
contracts again with increased cytoplasmic Ca2?concentrations.
Thus, in a beating heart, the kinase would be activated contin-
uously. Additional experiments are needed to identify clearly
whether smMLCK or another kinase phosphorylates cardiac
Biochemically, the skeletal muscle RLC is also not a good
substrate for the smMLCK because of a Glu at the p-3 position
relative to the phosphorylatable serine, which is an Arg in
smooth and nonmuscle RLCs (24). Even though skeletal muscle
fibers contain smMLCK in the skMLCK?/?mice, this kinase
apparently does not phosphorylate the skeletal muscle RLC in
response to repetitive stimulation probably because of poor
substrate properties (10, 24). However, it is interesting to note
that there is a small amount of monophosphorylated muscle
RLC in muscles from knockout mice. Phosphorylation of the
serine near the serine phosphorylated by skMLCK indicates
another kinase capable of the phosphorylation. Although it will
be important to identify this kinase and the signaling pathways
that lead to its physiological activation, the effect on cross-bridge
movement and enhanced contractile tension will be the same as
phosphorylation by skMLCK.
Our results show a biochemical mechanism for the physiolog-
ical potentiation of twitch contractions by repetitive stimuli in
fast-twitch skeletal muscles. Ablation of the skMLCK expression
results in inhibition of RLC phosphorylation in response to
electrical stimulation in skeletal muscle. These results are con-
glycerol-PAGE. Muscle samples were prepared as described in Methods and
subjected to urea?glycerol-PAGE followed by Western blotting for RLC.
Shown are results without (?) or with (?) dephosphorylation treatment.
Separation of nonphosphorylated and phosphorylated RLC by urea?
y designate the N- and C-terminal fragments of the peptide produced by
residue number from either the N or C terminus.*, Those daughter ions with
loss of 98-Da peaks.
Collision-induced fragmentation spectrum of the phosphorylated
Zhi et al.PNAS ?
November 29, 2005 ?
vol. 102 ?
no. 48 ?
sistent with the hypothesis that Ca2?released during membrane
excitation stimulates contraction by binding to troponin on thin
filaments but also activates skMLCK by means of Ca2?binding
to calmodulin to effect skeletal muscle RLC phosphorylation. A
small portion of MLCK is activated by the intracellular Ca2?
transient associated with a single twitch. However, with repet-
itive contractions, more activated kinase accumulates due to the
slow rate (1 s?1) of inactivation induced by Ca2??calmodulin
dissociation (9). The phosphorylation of myosin RLC by the
Ca2??calmodulin-dependent MLCK increases the number of
cross bridges in the strong-binding state by their displacement
away from the myosin thick filament toward the actin thin
a potential mechanism for potentiation of contractions with
repetitive stimuli, it did not rule out effects of Ca2??calmodulin
on the L-type Ca2?channel and ryanodine receptor that are
manifested physiologically in other ways (1, 2). The contribution
of RLC phosphorylation to tension potentiation seems to be
greater in posttetanic twitch potentiation than the staircase, and
previous investigations suggested a linear relationship between
the extent of RLC phosphorylation and isometric twitch poten-
tiation (9, 13). Thus, effects on Ca2?handling may be greater
with low frequency stimulation for longer durations as observed
in the staircase. Prolonged repetitive Ca2?release may affect
excitation processes through calmodulin effects on the dihydro-
pyridine and ryanodine receptors with a greater amount of Ca2?
released for contractile protein activation (1, 2). On the other
hand, the twitch potentiation could involve Ca2?independent
mechanisms such as compression of skeletal muscle fibers with
a decrease in the distance between thin and thick filaments. This
effect would mimic the effect of RLC phosphorylation (3, 9, 11).
The kinetic properties of myosin RLC phosphorylation in
skeletal muscle are suited for modulation of contraction prop-
erties because the rate of phosphorylation is reasonably fast
(requiring a few seconds) whereas the dephosphorylation is slow
(minutes) (12, 13, 28). Thus, a short period of contractile activity
during a warm-up period results in RLC phosphorylation that
will be maintained during intermittent periods of relaxation but
poised for potentiation of subsequent contractions. This history-
dependent phosphorylation will initially increase the rate and
extent of tension development at physiological frequencies of
motor nerve firing that result in unfused tetanus, thus increasing
the integral of the tension–time relationship. Force enhance-
ment for a submaximal voluntary contraction in humans may be
However, the motor nerve frequency required for force main-
tenance will decrease due to force potentiation by RLC phos-
phorylation. Thus, RLC phosphorylation will also contribute
secondarily to the maintenance of force at a lower dynamic level
of Ca2?, with continuous nerve firing thereby increasing muscle
efficiency due to lower energy consumption for removal of
cytosolic Ca2?. This general pattern of motor unit activity is
commonly observed in working muscles during exercise (30) and
provides a model for investigating the role of RLC phosphory-
lation in exercise responses (29).
We are grateful for the expert advice and assistance from Joyce Repa for
Q-PCR and the expert technical assistance of Tara Billman with the
mice. This work was supported by National Institutes of Health (NIH)
Grants HL29043 and HL26043 and by the Moss Heart Fund. J.T.S. is the
Fouad A. and Val Imm Bashour Distinguished Chair in Physiology.
J.W.R. was supported as a postdoctoral fellow by NIH Training Grant
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Table 1. Cardiac properties of mice with ablation of skMLCK
MouseBody weight, gHeart weight, mg
mol P?mol RLC
22.13 ? 1.36
24.21 ? 1.07
111 ? 14.7
132 ? 9.1
5.07 ? 0.12
5.45 ? 0.11
0.44 ? 0.02
0.40 ? 0.01
Heart weight, body weight, and cardiac RLC phosphorylation were measured as described in Methods. Values
are means ? SEM for at least five values. P, phosphate.
www.pnas.org?cgi?doi?10.1073?pnas.0506846102Zhi et al.