Signaling in Muscle Contraction

Ivana Y. Kuo1 and Barbara E. Ehrlich1,2

1
Department of Pharmacology, School of Medicine, Yale University, New Haven, Connecticut 06520
2
Department of Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven,
Connecticut 06520
Correspondence: barbara.ehrlich@yale.edu

SUMMARY

Signaling pathways regulate contraction of striated (skeletal and cardiac) and smooth muscle.
Although these are similar, there are striking differences in the pathways that can be attributed
to the distinct functional roles of the different muscle types. Muscles contract in response to
depolarization, activation of G-protein-coupled receptors and other stimuli. The actomyosin
fibers responsible for contraction require an increase in the cytosolic levels of calcium, which
signaling pathways induce by promoting influx from extracellular sources or release from
intracellular stores. Rises in cytosolic calcium stimulate numerous downstream calcium-de-
pendent signaling pathways, which can also regulate contraction. Alterations to the signaling
pathways that initiate and sustain contraction and relaxation occur as a consequence of exer-
cise and pathophysiological conditions.

Outline

1 Introduction 8 Heart failure

2 Skeletal muscle contraction 9 Smooth muscle types
3 Skeletal muscle fiber types and exercise 10 The contractile process in smooth muscle
4 Malignant hyperthermia in skeletal muscle 11 Calcium sensitization
5 Cardiac muscle contraction 12 Vascular smooth muscle in disease
6 Exercise hypertrophy in cardiac muscle 13 Concluding remarks
7 Pathophysiological cardiac hypertrophy References

Editors: Lewis Cantley, Tony Hunter, Richard Sever, and Jeremy Thorner
Additional Perspectives on Signal Transduction available at www.cshperspectives.org

Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a006023
Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023 1
I.Y. Kuo and B.E. Ehrlich
1 INTRODUCTION
Muscle can be subdivided into two general categories: stri-
ated muscle, which includes skeletal and cardiac muscles;
and nonstriated muscle, which includes smooth muscle
such as vascular, respiratory, uterine, and gastrointestinal
muscles. In all muscle types, the contractile apparatus
consists of two main proteins: actin and myosin. Striated
muscle is so called because the regular arrangement of
alternating actomyosin fibers gives it a striped appearance.
This arrangement allows coordinated contraction of the
whole muscle in response to neuronal stimulation through
a voltage- and calcium-dependent process known as exci-
tation contraction coupling. The coupling enables the
rapid and coordinated contraction required of skeletal
muscles and the heart. Smooth muscle does not contain
regular striations or undergo the same type of excitation
contraction coupling. Instead, it typically uses second mes-
senger signaling to open intracellular channels that release
the calcium ions that control the contractile apparatus.
These processes, in contrast to excitation contraction cou-
pling, are slow and thus suitable for the slower and more
sustained contractions required of smooth muscle. The
actomyosin contractile apparatus is both calcium- and
A Striated muscle
Neuronal stimulus
(e.g., neurotransmitter release)
Activation of neurotransmitter receptors
Depolarization
Skeletal muscle Cardiac muscle
Activation of L-type Activation of L-type
Ca2+ channels Ca2+ channels
Tugging motion Opening of RyR
opens RyR via CICR
Increased intracellular Ca2+
Activation of contractile apparatus
Figure 1. Overview of muscle contraction signals in striated (A), and smooth (B) muscle.
2 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023
phosphorylation-dependent, and restoration of basal cal-
cium levels or its phosphorylation status returns an actively
contracting muscle to a noncontractile state. Muscle-spe-
cific signals modulate these processes, depending on the
type of muscle, its function, and the amount of force
required.
In all muscle cells, contraction thus depends on an in-
crease in cytosolic calcium concentration (Fig. 1). Calcium
has an extracellular concentration of 24 mM and a resting
cytosolic concentration of 100 nM. It is also stored inside
cells within the sarcoplasmic (SR, referring to skeletal and
cardiac muscle) and endoplasmic reticulum (ER, referring
to smooth muscle) at a concentration of 0.4 mM (Boot-
man 2012). In striated muscle, the increase in calcium levels
is due to its release from the SR stores via ryanodine re-
ceptor (RyRs). Neurotransmitters such as acetylcholine
bind to receptors on the muscle surface and elicit a de-
polarization by causing sodium/calcium ions to enter
through associated channels. This shifts the resting mem-
brane potential to a more positive value, which in turn
activates voltage-gated channels, resulting in an action
potential (the excitation part). The action potential stim-
ulates L-type calcium channels (also known as dihydropyr-
idine receptors). In skeletal muscle, these are mechanically
B Smooth muscle
Depolarization Hormonal/neuronal stimulus
(or window current) (e.g., vasopression, neurotransmitter)
Activation of Gq-coupled receptors
Activation of Gq
PLC
Activation of L-type
PIP2 IP3 and DAG
Ca2+ channels
IP3 binds IP3R and opens channel
Increased intracellular Ca2+
Activation of contractile apparatus

coupled to the SR RyRs and open them directly. In cardiac
muscle, calcium influx through the L-type channels opens
RyRs via calcium-induced calcium release (CICR) (Boot-
man 2012). The RyR is a large tetrameric six-transmem-
brane-span calcium-release channel. Of the three RyR
subtypes, RyR1 is predominantly found in skeletal muscle
(see review by Klein et al. 1996), and RyR2 is predominant-
ly found in cardiac muscle (Cheng et al. 1993).
Smooth muscle also contains voltage-gated calcium
channels and RyRs responsible for increases in intracel-
lular calcium concentration (see below). Depolarization
causes L-type calcium channels to open, enabling calcium
to enter down its concentration gradient into the cell (Fig.
1B). Opening of RyRs is usually associated with CICR.
As the intracellular calcium concentration rises, calcium
binds to RyRs, whose consequent opening further en-
hances the increase in cytoplasmic calcium concentration.
Another major mechanism controlling contraction in these
cells, however, involves a different tetrameric six-trans-
membrane-span calcium channel: the inositol 1,4,5-tri-
sphosphate (IP3) receptor (IP3R). Circulating hormones
(e.g., vasopressin and bradykinin) and neurotransmitters
released by sympathetic nerves (e.g., endothelin and nor-
epinephrine) act through G-protein-coupled receptors
(GPCRs) to generate the second messenger IP3 via acti-
vation of phospholipase C (PLC). IP3 binds to and opens
IP3Rs on the ER/SR, causing the calcium release that drives
contraction. IP3Rs are present in both skeletal and cardiac
muscle; however, they do not contribute significantly to the
excitation contraction coupling in striated muscle. Note
that both RyRs and IP3Rs are stimulated by low concentra-
tions of cytoplasmic calcium but close when the concen-
tration gets higher, showing bell-shaped response curves
(Bezprozvanny et al. 1991; Finch et al. 1991).
Once intracellular calcium levels are raised, calcium
binds to either troponin C on actin filaments (in striated
muscle) or calmodulin (CaM), which regulates myosin fil-
aments (in smooth muscle). In striated muscle, calcium
causes a shift in the position of the troponin complex on
actin filaments, which exposes myosin-binding sites (Fig.
2A). Myosin bound by ADP and inorganic phosphate (Pi)
can then form cross-bridges with actin, and the release of
ADP and Pi produces the power stroke that drives contrac-
tion. This force causes the thin actin filament to slide past
the thick myosin filament and shortens the muscle. Bind-
ing of ATP to myosin then releases myosin from actin, and
myosin hydrolyzes ATP to repeat the process (Fig. 2B).
In smooth muscle, by contrast, calcium binds to CaM,
which then interacts with myosin light-chain kinase
(MLCK), causing it to phosphorylate the myosin light-chain
(MLC) at S19 or Y18. The phosphorylated MLC then
forms cross-bridges with actin, producing phosphorylated
Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023
Signaling in Muscle Contraction
actomyosin, which leads to contraction (Fig. 2C). Note
that striated muscle contraction can also be regulated by
calcium-bound CaM and MLCK; however, this is not the
dominant mechanism. Finally, calcium and calcium-CaM
also bind to various other proteins in muscle cells, includ-
ing the phosphatase calcineurin and protein kinases such
as CaMKIV, respectively. These regulate other cellular tar-
gets, including transcription factors such as NFAT and
CREB, which control gene expression programs that can
have longer-term effects on muscle physiology.
These different calcium-release mechanisms all also
stimulate the pumping of calcium from the cytoplasm
back into intracellular stores via the SR/ER calcium ATPase
(SERCA) pump. The plasma membrane calcium ATPase
(PMCA) pump and the sodium/calcium exchanger
(NCX), both of which reside on the plasma membrane,
can also remove calcium from the cytosol. Calcium dis-
sociates from troponin C or calmodulin as the cytosolic
calcium concentration decreases as a consequence, which
terminates the contraction process.
The main pathways promoting muscle relaxation in-
volve the second messengers cAMP and cyclic guanosine
monophosphate (cGMP). cAMP is generated by adenylyl
cyclases, downstream from the b-adrenergic GS-coupled
receptor, which is activated by noradrenaline. Note that
the cAMP pathway generally promotes contraction in car-
diac muscle; however, in smooth muscle, activation of
cAMP causes relaxation. The cGMP pathway can be acti-
vated either by nitric oxide (NO) or natriuretic peptides
(NPs). In the case of blood vessels and other smooth mus-
cles, NO produced by endothelial NO synthase (eNOS)
diffuses across the muscle cell membrane to activate solu-
ble guanylyl cyclase (sGC), which in turn increases levels
of cGMP. NPs, such as atrial (ANP, released by the heart
atria under high blood pressure), brain (BNP, primarily
released by the heart ventricle), and c-type (CNP, mainly
involved in pathological conditions, and released by the
vascular and central nervous system), instead bind to trans-
membrane guanylyl cyclase, whose intracellular domain
possesses the enzymatic activity (Nishikimi et al. 2011).
The cAMP and cGMP generated act via the protein ki-
nase PKA and PKG on the contractile process in multiple
ways: (1) their phosphorylation of calcium pumps leads
to increased activity; (2) activation of MLC phosphatase
(MLCP) by PKG antagonizes MLCK; and (3) both PKA
and PKG cause a reduction in the sensitivity of the con-
tractile machinery by inhibiting the GTPase RhoA (this
increases MLCP activity and causes MLC dephosphoryla-
tion and muscle relaxation). The levels of cAMP and cGMP
are in turn regulated through their degradation by phos-
phodiesterases to yield the inactive metabolites 5 -AMP
and 5 -GMP.
3
I.Y. Kuo and B.E. Ehrlich

A B
1. Tropomyosin Actin 1. Ca2+ binds troponin C, exposing
myosin-binding sites on actin.
Myosin Actin

Troponin Actin Tropomyosin ADP

complex 6. ADP is released.
2. ATP binds to myosin.

Ca2+
Troponin T Troponin I ADP
Troponin C ATP 5. Pi is released; myosin
head changes confor-
3. ATP is hydrolyzed. Pi
2. Ca2+ Ca2+ Myosin is in the
mation to produce the
power stroke. Filaments
cocked state.
slide past each other.

Myosin
ATP ADP + Pi
ADP + Pi
Myosin-binding 4. Cross-bridge forms and myosin
sites on actin are exposed. binds to a new position on actin.

C
Ca2+ CaM

Ca2+ CaM

ATP
MLCK
MLC MLC -P

Actin
Actin

P-Actomyosin
MLC -P

Actomyosin
Pi
Contraction
MLC

Figure 2. Calcium triggers contraction in striated muscle. (A) Actomyosin in striated muscle. (1) Striated muscle in
the relaxed state has tropomyosin covering myosin-binding sites on actin. (2) Calcium binds to troponin C, which
induces a conformational change in the troponin complex. This causes tropomyosin to move deeper into the actin
groove, revealing the myosin-binding sites. (B) Cross-bridge cycle in striated muscle. (1) Calcium binds to troponin
C, causing the conformational shift in tropomyosin that reveals myosin-binding sites on actin. (2) ATP then binds to
myosin. (3) ATP is then hydrolyzed. (4) A cross-bridge forms and myosin binds to a new position on actin. (5) Pi is
released and myosin changes conformation, resulting in the power stroke that causes the filaments to slide past each
other. (6) ADP is then released. (C) Contraction in smooth muscle. In smooth muscle, calcium binds to calmodulin
and causes the activation of myosin light chain (MLC) kinase (MLCK). This phosphorylates MLC, which then binds
to actin to form phosphorylated actomyosin, enabling the cross-bridge cycle to start.

Below, we examine the key differences between the sig-
naling mechanisms controlling contraction of skeletal,
cardiac, and smooth muscle, and how these relate to their
differing functions. In addition, we discuss the changes to
the signaling pathways that occur as a consequence of ex-
ercise and pathological situations.
2 SKELETAL MUSCLE CONTRACTION
Skeletal muscles comprise multiple individual muscle fi-
bers that are stimulated by motor neurons stemming from
the spinal cord. They are grouped together to form mo-
tor units and more than one type of muscle fiber can be

4 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023

 Signaling in Muscle Contraction

present within each motor unit. Muscle fibers can be di-
vided into fast- and slow-twitch muscles. Fast-twitch mus-
cles use glycolytic metabolism and are recruited for phasic
activity (an active contraction). Slow-twitch muscles (also
known as red muscles) are rich in myoglobin, mitochon-
dria, and oxidative enzymes and specialized for sustained
or tonic activity. See Schiaffino and Reggiani (2011) for a
more complete discussion of skeletal muscle types and the
types of myosin isoforms that make up fast- and slow-
twitch muscles.
The neuromuscular junction (NMJ) that connects skel-
etal muscle with the nerves that innervate them consists
of three distinct parts: the distal motor nerve ending, the
synaptic cleft, and the postsynaptic region, located on the
muscle membrane. Motor neurons branch into multiple
termini, which are juxtaposed to motor endplates, special-
ized regions of muscle where neurotransmitter receptors
are concentrated (Fig. 3A). The transfer of information
between the nerve and muscle is mediated by the release
of acetylcholine from the motor neuron, which diffuses
across the synaptic cleft, and binds to and activates the
ligand-gated, nicotinic acetylcholine receptors (nAChRs)
on the endplate. Activation of the nAChR leads to an influx
of cations (sodium and calcium) that causes depolarization
of the muscle cell membrane. This depolarization in turn
activates a high density of voltage-gated sodium channels
on the muscle membrane, eliciting an action potential.
The action potential runs along the top of the mus-
cle and invades the T-tubules (specialized invaginations of
the membrane containing numerous ion channels). The
opening of voltage-gated sodium channels activates L-
type voltage-gated calcium channels lining the T-tubule.
A conformational change in these enables release of cal-
cium on the closely apposed SR via activation of RyR1.

A B

Nerve
ending
Action Ca2+
potential
propagates
Neuromuscular Na+ channels
junction K+ p38MAPK CaMKIV AMPK MLCK
ACh
T-tubule
Synaptic AChR Na+ CREB
cleft

Na+ SR
PGC1
RyR
L-type Ca2+
Ca2+
channel
Ca2+ FOXO MEF2 NRF PPAR

SERCA Mitochondrial biogenesis:

glucose and lipid homeostasis changes
Contraction Promotes
relaxation
Actomyosin
Cytoplasm

Figure 3. Skeletal muscle contraction and changes with exercise. (A) Neurotransmitter (acetylcholine, ACh) released
from nerve endings binds to receptors (AChRs) on the muscle surface. The ensuing depolarization causes sodium
channels to open, which elicits an action potential that propagates along the cell. The action potential invades T-
tubules and causes the L-type calcium channels to open, which in turn causes ryanodine receptors (RyRs) in the SR
to open and release calcium, which stimulates contraction. Calcium is pumped back into the SR by (SR/ER calcium
ATPase SERCA) pumps. The decreasing cytosolic calcium levels cause calcium to disassociate from troponin C and,
consequently, tropomyosin reverts to a conformation that covers the myosin-binding sites. (B) Signaling in exercised
skeletal muscle. Both calcium and calcium-independent signals stimulate the transcriptional coactivator PGC1a.
This activates a number of transcription factors that regulate genes associated with mitochondrial biogenesis,
glucose, and lipid homeostasis.

Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023 5

I.Y. Kuo and B.E. Ehrlich
Calcium then binds to troponin as described above, initi-
ating the contraction process. Calcium-bound CaM also
activates MLCK, whose phosphorylation of the MLC
changes cross-bridge properties. This modulates the tropo-
nin-dependent contraction, although there is no effect on
the ATPase activity of MLC. MLC phosphorylation instead
enhances force development at submaximal saturating cal-
cium concentrations (see below). The phosphate group is
subsequently removed by protein phosphatase 1 (PP1).
3 SKELETAL MUSCLE FIBER TYPES AND EXERCISE
Skeletal muscle is plastic. Exercise can lead to pronounced
changes in its metabolic properties and, sometimes, a
change in the fiber type. Physical differences between
fast- and slow-twitch muscles underlie the functional roles
of these fibers, including the type of myosin used and dif-
fering resting calcium levels. The free calcium level is two-
fold higher in slow-twitch muscle, even though the SR
volume is greater. The level of MLC phosphorylation is
higher in fast-twitch muscle, however, because of higher
levels of expression of MLCK (Bozzo et al. 2005). The force
enhancement produced by MLC phosphorylation, under
submaximal saturating calcium concentrations, counter-
acts the reduction in force caused by fatigue in fast-twitch
muscle fibers (Schiaffino and Reggiani 2011).
Fast- and slow-twitch fibers also have different calcium-
sequestering and -buffering systems. Different SERCA iso-
forms are present: SERCA2A is the main isoform in slow-
twitch muscle fibers, whereas SERCA1A is expressed in
fast-twitch muscle fibers. Similarly, different cytosolic cal-
cium buffers are expressed. Calsequestrin (CSQ) is the
main SR-luminal calcium-buffering protein. It is a high-
capacity, low-affinity calcium-binding protein that binds
calcium cooperatively (Campbell et al. 1983). When the
muscle is at rest, the SR is primed to release large amounts
of calcium, because CSQ is polymerized, which reduces its
ability to bind calcium. In cardiac muscle, only CSQ2 is
expressed. In skeletal muscle, CSQ1 and CSQ2 are found in
slow-twitch muscle fibers, but only CSQ1 is found in fast-
twitch muscle fibers. The two isoforms differ in their car-
boxy-terminal tail; functionally, CSQ1 reduces the activity
of RyR1, whereas CSQ2 increases the open probability of
RyR1 and RyR2 (Wei et al. 2009).
Other differences between the muscle fiber types in-
clude posttranslational modifications such as phosphory-
lation of RyR by PKA, and interactions between RyR and
other proteins, such as CaM and FK506-binding protein
(FKBP) 12 and FKBP12.6. Phosphorylation of RyR by
either PKA or CaMKII fully activates the channel. PKA
and CaMKII can also phosphorylate phospholamban, a
protein that inhibits SERCA; phosphorylation causes
6 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023
phospholamban to dissociate from SERCA. The FKBPs
are immunophilins that bind to immunosuppressants
such as rapamycin and FK506. FKBP12 and FKBP12.6
have differing expression levels in muscle tissue, but both
bind all three forms of the RyR and stabilize its closed state.
Collectively, these calcium-dependent differences between
fast- and slow-twitch muscle fibers, in addition to differ-
ences in the myosin isoform used and the number of mi-
tochondria, account for the different functional outputs of
the two muscle fiber types.
Long-term exercise causes a general shift in muscle fiber
type from slow twitch to fast twitch. It induces a number of
changes, including altered expression and activity of mem-
brane transporters and mitochondrial metabolic enzymes,
together with increased blood supply to skeletal muscle
(Hardie 2012). These, in turn, enhance the oxidative ca-
pacity and increase expression of enzymes preventing dam-
age by reactive oxygen species (ROS). One major signaling
pathway is through the peroxisome-proliferator-activated
receptor (PPAR) g coactivator (PGC) 1a (Fig. 3B). PGC1a
coactivates a number of transcription factors that regu-
late genes important for muscle function. These include
PPARs (which regulate glucose and lipid homeostasis, pro-
liferation, and differentiation), nuclear respiratory factors
(NRFs, which regulate metabolism and mitochondrial
biosynthesis), myocyte enhancer factor 2 (MEF2, which
is involved in development and hematopoesis), and Fork-
head box O (FoxO) family transcription factors (which
counter oxidative stress and promote cell-cycle arrest and
apoptosis) (Handschin and Spiegelman 2006; Ronnebaum
and Patterson 2010). In addition to PGC1a, calcium-de-
pendent processes are also involved. Rises in cytosolic
calcium result in the activation of calcineurin, which then
dephosphorylates NFAT. Translocation of NFAT to the nu-
cleus results in activation of slow-fiber gene expression.
Rises in nuclear calcium levels also cause calcium-depen-
dent signaling molecules to become active. These include
the phosphorylation of histone deacetylases (HDACs) by
nuclear calmodulin-dependent protein kinase. HDACs re-
press transcription by causing DNA to be tightly wrapped
around histones. Removal of HDACs enables transcrip-
tion factors such as MEF2 to bind and enable induction
of genes encoding proteins found in slow fibers (Liu et al.
2005).
As mentioned above, exercise induces an increase in
the levels of mitochondrial metabolic enzymes to com-
pensate for the increased metabolic demand on skeletal
muscle. Unsurprisingly, PGC1a is a potent stimulator of
mitochondrial biogenesis (see review by Olesen et al. 2010).
This was shown elegantly by experiments in which over-
expression of PGC1a in white, glycolytic skeletal muscle
could turn it into red, oxidative muscle by increasing the

levels and activity of a number of mitochondrial proteins
(Lin et al. 2002; Wenz et al. 2009). These proteins include
most components of the mitochondrial respiratory chain
and ATP synthase, as well as several enzymes in the Krebs
cycle and enzymes involved in fatty acid oxidation.
4 MALIGNANT HYPERTHERMIA
IN SKELETAL MUSCLE
Mutations in RyR and CSQ isoforms cause malignant
hyperthermia, demonstrating the importance of proteins
involved in calcium signaling in skeletal muscle. The mu-
tations in RyR1 appear to increase its open probability
when levels of luminal calcium are low and account for
the majority of malignant hyperthermia cases (80%); the
remainder are caused by mutations to CSQ1.
In the case of RyR1 mutations, volatile anesthetics (in-
haled anesthetics such as isoflurane or halothane) lead to a
rapid opening of RyR1 and an uncontrolled release of cal-
cium from the SR, which in turn leads to sustained skeletal
muscle contraction (Robinson et al. 2006). In response to
the elevated calcium levels, there is activation of SERCA to
pump calcium, using ATP, back into the SR. However, the
continual activation of SERCA consumes excessive ATP,
leading to hypermetabolism. This then leads to a drop in
ATP levels, acidosis, tachycardia, and an abnormal increase
in body temperature. These symptoms can be treated with
dantrolene, an inhibitor of the RyR signaling pathway. The
mutations in RyR1 associated with malignant hyperther-
mia are clustered in three hot spots on the 500 kDa protein
(Lanner et al. 2010). The first cluster is near the amino
terminus and the second cluster is in the middle of the
protein. The third cluster lies in the carboxy-terminal re-
gion surrounding the channel-forming domains. How mu-
tations in all three regions exert similar effects is yet to be
determined.
Mutations in CSQ can also result in malignant hyper-
thermia. A lack of buffering causes uncontrolled calcium
transients that lead to lethal malignant hyperthermia in
response to heat stress and volatile anesthetics (Dainese
et al. 2009).
5 CARDIAC MUSCLE CONTRACTION
In cardiac muscle, depolarization starts in the pacemaker
cells (modified cardiac myocytes that set the heart rate and
are rich in signaling molecules) in the sinoatrial node,
which is innervated by both parasympathetic and sympa-
thetic nerves. The external stimuli modulate the activity of
the pacemaker cellsthey undergo spontaneous self-de-
polarization to produce action potentials. This is achieved
by a slow leak of potassium ions and a concurrent influx
Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023
Signaling in Muscle Contraction
of sodium and calcium ions. The action potential then
traverses to the cardiac myocytes, where it invades the
T-tubule. However, unlike skeletal muscle, where L-type
calcium channels are directly coupled to RyRs, in cardio-
myocytes the influx of calcium across the plasma mem-
brane elicits calcium release from the SR via RyRs by
CICR (Fig. 4B). The predominant isoform in the heart is
RyR2. As in skeletal muscle, contraction is controlled by
phosphorylation of troponin but can also be modulated by
calcium-CaM and MLCK. Mice with a nonphosphory-
latable MLC in ventricular myocytes display depressed
contractile function and develop atrial hypertrophy and
dilatation (Sanbe et al. 1999).
Catecholamines, such as adrenaline and noradrenaline,
act on b-adrenergic receptors (metabotropic GPCRs) to
release cAMP that in turn activates PKA. PKA can be
viewed as a primary regulator of the contractile pathway, as
it phosphorylates a number of targets, including L-type
calcium channels and RyRs. In most cases, phosphory-
lation of these proteins increases calcium release (for
example, phosphorylation of RyR increases its open prob-
ability), and thus the outcome is to stimulate contraction
(Ibrahim et al. 2011). Another target of PKA is phospho-
lamban (an inhibitor of SERCA), which, when phosphor-
ylated, loses its inhibitory effect on SERCA.
6 EXERCISE HYPERTROPHY IN CARDIAC MUSCLE
Cardiac hypertrophy is an abnormal enlargement of the
heart that occurs because of increases in cell size and pro-
liferation of nonmuscle cells. These changes can either
be beneficial (e.g., exercised hearts), in which changes are
correlated with increased contractility, or detrimental, in
which changes lead to decreased contractility and subse-
quent heart failure.
Exercised hearts develop a form of mild cardiac hy-
pertrophy that does not lead to cardiac failure. The main
structural changes include a thickening of the ventricle wall,
which leads to increased contractility and thus a greater
ability to pump blood. Within myocytes, expression of the
a myosin heavy chain increases, which leads to high ATPase
activity and increased contractility (Fig. 4B). Various signals
are involved, including growth factors such as insulin-like
growth factor (IGF1), vascular endothelial growth factor
(VEGF), and hepatocyte growth factor (HGF) (Fig. 4B)
(Hemmings and Restuccia 2012). There is increased signal-
ing through the phosphoinositide 3 kinase (PI3K)/Akt
pathway, which leads to proliferation and growth of cardio-
myocytes (Matsui et al. 2003). Transcription factors up-
regulated in exercised hearts include GATA4, which regu-
lates genes involved in myocardial differentiation. Other
pathways, such as the calcineurin/NFAT pathway are
7
I.Y. Kuo and B.E. Ehrlich

A Adrenaline,
noradrenaline
-Adrenergic
receptor
Depolarization
G AC Contraction-
Gs ATP promoting
pathway

cAMP

SR L-type
PKA Ca2+ channel

RyR ClCR

SERCA
Relaxation-
promoting Ca2+
pathway

Ca2+
PKA Contraction

-P
Phospholamban

B External factors:
IGF1, VEGF, HGF

Signaling pathways:

PI3K Calcinuerin
Akt NFAT

Transcription factors
PGC1 GATA4

Proliferation
Growth
Myocardial differentiation

Figure 4. Cardiac muscle contraction and changes with exercise. (A) Cardiac muscle contraction can occur as a
consequence of calcium entry through L-type calcium channels, which activate ryanodine receptor (RyR) channels
in the SR. Alternatively, b-adrenergic receptors on the cell membrane lead to activation of adenylyl cyclase (AC),
which stimulates PKA. This can promote contraction by phosphorylating RyR and L-type calcium channels or
relaxation by phosphorylating the SERCA pump inhibitor phospholamban. (B) Changes with exercise lead to an
activation of the PI3K/Akt pathway, and a down-regulation of NFAT and calcinurin.

down-regulated (Oliveira et al. 2009). Cardiac muscle, like 7 PATHOPHYSIOLOGICAL CARDIAC

skeletal muscle, consumes tremendous amounts of ATP. HYPERTROPHY
Thus, PGC1a is also up-regulated in exercised hearts, facil-
itating transcription of metabolic and oxidative genes (Ven- The main pathways driving pathological cardiac hypertro-
tura-Clapier et al. 2007; Watson et al. 2007). phy are overstimulation of the sympathetic nervous system,

8 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023


increased oxidative stress, and inflammatory signaling (Ba-
lakumar and Jagadeesh 2010). These collectively lead to
induction of fetal isoforms of heart proteins and a corre-
sponding decrease in adult forms (Chien 1999), including
the myosin heavy chain (see below). The signals responsible
include the GPCR agonist endothelin 1, peptide growth
factors such as platelet-derived growth factor (PDGF), epi-
dermal growth factor (EGF), and cytokines such as cardio-
trophin and leukemia inhibitory factor (LIF). Mechanical
stress can also induce hypertrophy. In each case, activation
of the ERK mitogen-activated protein kinase (MAPK) path-
way (Morrison 2012) is often observed in hypertrophy and
leads to regulation of transcription factors that alter expres-
sion of the myosin heavy chain, IP3R2, and other proteins
(see below).
In hypertrophy, paracrine and autocrine neurohor-
monal factors that activate the heterotrimeric G protein
Gq, and consequently PLCb, are released. This results
in an increase in cytosolic calcium levels and activation
of PKC by diacylglycerol (DAG) as well as activation of
CaMKII (Mishra et al. 2010). The importance of the Gq
pathway in hypertrophy has been shown in studies of trans-
genic mice: mice overexpressing Gq have heart failure
(DAngelo et al. 1997), whereas mice with reduced Gq levels
are protected against hypertrophy (Wettschureck et al.
2001). There is also a switch from the a form of the myosin
heavy chain to the fetal b isoform (Miyata et al. 2000). This
has a lower ATPase activity and a lower rate of contraction.
Other changes include increased SERCA2A activity (Ha-
senfuss et al. 1994; Meyer et al. 1995), up-regulation of
IP3R2 (Harzheim et al. 2010), and changes to a neuronal
calcium sensor (NCS1). NCS1 is a calcium-binding protein
that also interacts with IP3R (Schlecker et al. 2006).
8 HEART FAILURE
The structural organization of the T-tubules breaks down
in heart failure. This breakdown, caused by myocardial
insults (such as myocardial infarction causing ischemia)
among other factors, leads to impaired contractility owing
to reduced, asynchronous, and chaotic calcium release.
Several signaling pathways are compromised in heart fail-
ure. Initially, there can be reorganization of the b-adrener-
gic system. Activation of the b2-adrenergic receptor is
normally limited to the T-tubule, whereas in heart failure,
there is a redistribution of the receptor across the entire
plasma membrane (Nikolaev et al. 2010). With chronic
adrenergic activation, the hyperphosphorylation of RyRs
results in leaky RyR channels, leading to a reduction of SR
calcium and, thus, weaker contractions.
Other alterations to RyR2 that are observed include
increased nitrosylation and loss of the regulatory protein
Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023
Signaling in Muscle Contraction
FKBP12.6 (Andersson and Marks 2010). Both of these
changes result in increased RyR activity. Moreover, muta-
tions in RyR2 that result in leaky channels have been linked
to catecholaminergic polymorphic ventricular tachycardia
(CPVT) and arrhythmogenic right ventricular dysplasia
type 2.
Mutations in CSQ2 cause CPVT (Postma et al. 2002)
by lowering buffering capacity within the SR, which results
in premature calcium release and thus arrhythmias. Anoth-
er important modulator of RyRs is junctin. Its levels are
reduced in heart failure, which may be a compensatory
mechanism to increase contractility (Pritchard and Kranias
2009).
Heart failure also leads to up-regulation of molecules
that may have a protective function. One pathway is through
cGMP, which promotes relaxation. The cGMP pathway is
regulated by cGMP-targeted phosphodiesterases, of which
one, PDE5A, looks to be a promising target for protective
therapy against hypertrophy.
9 SMOOTH MUSCLE TYPES
Smooth muscle is found lining the walls of various organs
and tubular structures in the body, including the intestine,
bladder, airway, uterus, blood vessels, and stomach. It re-
ceives neural innervation from the autonomic nervous
system, and its contractile state is also controlled by hor-
monal and autocrine/paracrine stimuli. Smooth muscle
can be divided into two types: unitary and multiunit
smooth muscles. In unitary smooth muscle, individual
smooth muscle cells are coupled to neighboring cells by
gap junctions. These gap junctions permit cell-to-cell pas-
sage of small molecules such as ATP and ions. These include
those mobilized in response to electrical signals causing
depolarization, which enable the whole area (known as a
syncytium) to coordinate activity. In contrast, in multiunit
smooth muscle, cells are not coupled to each other and are
intermingled with connective tissue. Smooth muscle can
undergo tonic (sustained) or phasic contractions. In the
case of vascular smooth muscle, a sustained contraction is
required to provide vessel tone. This enables the regulation
of blood flow. Blood vessels are divided into the larger di-
ameter conduit vessels (e.g., the thoracic aorta) and the
smaller diameter resistance vessels. The resistance blood
vessels display a myogenic response, in which increasing
pressures over the physiological range (70 100 mmHg)
result in a sustained contractile state. However, overcon-
striction of the vessels leads to hypertension (see below).
Other smooth muscle, such as that found in the gut, in-
cluding the stomach, small intestine, or gall bladder, shows
variable tone and rhythmic contractions known as slow
waves.
9
I.Y. Kuo and B.E. Ehrlich

10 THE CONTRACTILE PROCESS
IN SMOOTH MUSCLE
The important distinction between striated muscle and
smooth muscle is that calcium mediates contraction by
regulating the availability of actin filaments in striated mus-
cle, whereas in smooth muscle MLC is the target (Fig. 5).
The source of the increase in cytosolic calcium levels can be
extracellular or intracellular, or a combination of the two.
In the case of tonic constriction of blood vessels, a constant
supply of calcium comes from influx via the L-type calcium
channels. The resting membrane potential of smooth mus-
cle (between 250 and 240 mV) is such that it lies in an
overlap (the window current) between the activation and
inactivation curves of the L-type channel. Thus a small
population of the L-type channels is always open. An alter-
natively spliced high-voltage-gated form of T-type chan-
nels may also contribute to calcium influx (Kuo et al. 2011),
along with stretch-activated channels residing on the plas-
ma membrane, such as TRPC6.
In stomach muscle, the rhythmic contractions are due
to the activity of pacemaker cells, but activation of voltage-
gated calcium channels can trigger calcium entry and con-
traction. Sympathetic nerves run along the vascular smooth
muscle and can release stimuli such as acetylcholine, nor-
epinephrine, angiotensin, and endothelin. Moreover, circu-
lating blood factors such as cytokines and diffusible factors
such as nitric oxide can also act on receptors in the plasma
membrane or cross the plasma membrane, respectively, to
regulate pathways controlling intracellular calcium levels.

Agonist (e.g., ATP, vasopressin)

ANP GPCR
NO L-type Ca2+
channel

GC Rho PLC PIP2

Gq

GTP
cGMP IP3
PKC DAG Ca2+
GC ROCK
IP3R

PKG ER store

CPI-17 Ca2+

Relaxation CaM

MLC
MLCP MLCK

MLC -P

Contraction

Figure 5. Smooth muscle contraction. Calcium released by L-type calcium channels or IP3Rs downstream from Gq-
coupled cell-surface receptors causes smooth muscle contraction. It binds to calmodulin (CaM) and the resulting
complex stimulates myosin light-chain (MLC) kinase (MLCK). This phosphorylates MLC to promote contraction.
A RhoA/ROCK pathway and a diacylglycerol (DAG) pathway contribute to calcium sensitization by altering the
phosphorylation status of myosin light-chain phosphatase (MLCP). Relaxation is mediated through the cGMP/
PKG pathway downstream from nitric oxide (NO) and agonists such as atrial natriuretic peptide (ANP).

10 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023


The activation of receptor-operated channels (ROCs) also
causes calcium influx, which enables additional calcium
release from intracellular stores. GPCRs activate PLCb to
generate IP3, which releases calcium via IP3Rs. In vascular
smooth muscle and the circular smooth muscle of the gut,
the main isoform is IP3R1. Note, however, that there is some
heterogeneity. In longitudinal smooth muscle of the gut,
RyRs, rather than IP3Rs, are expressed. Agonists such as
cholecystokinin bind to the GPCR cholecystokinin A recep-
tor (CCK-AR), which activates phospholipase A2, which in
turn produces arachidonic acid. Arachidonic acid (AA) can
also be generated through the cleavage of DAG. AA activates
chloride channels, which depolarize the cell membrane,
enabling the opening of voltage-gated calcium channels
and an initial influx of calcium. This calcium can either
act directly on the RyR causing CICR or enable the release
of cyclic ADP ribose, which interacts with RyRs to enhance
CICR.
In all smooth muscle, calcium-bound CaM then binds
to MLCK, stimulating phosphorylation of MLC, which
leads to muscle contraction. The necessity for MLCK has
been shown in MLCK-knockout mice, in which smooth
muscle MLC cannot be phosphorylated by other kinases
(He et al. 2008; Zhang et al. 2010). The dephosphorylation
of MLC is catalyzed by MLCP and a complex of the myosin-
targeting protein MYPT1 and the phosphatase PP1 and
results in relaxation.
11 CALCIUM SENSITIZATION
Calcium sensitization is an essentially calcium-indepen-
dent process that enables the amount of constriction in
smooth muscle to be tuned by an alteration in the sensi-
tivity of MLC to calcium (Fig. 5). This process enables the
muscle to sustain a contraction once the initial calcium
transient has dissipated. There are two mechanisms for
calcium sensitization: a DAG-PLC-PKC pathway and a
RhoA pathway (Lincoln 2007).
Diacylglycerol (DAG) is produced by PLCb down-
stream from certain GPCRs and activates the conventional
and novel protein kinase C (cPKC and nPKC), but not
atypical PKC (aPKC) (Steinberg 2008). PKC has a variety
of downstream targets, such as MLCK and C-kinase poten-
tiated protein phosphatase 1 inhibitor, molecular mass
17 kDa (CPI-17), both of which enhance constriction.
CPI-17 is a smooth-muscle-specific inhibitor of MLCP
that binds to its catalytic subunit and inhibits phosphatase
activity, allowing contraction to persist.
Several agonists, including angiotensin II, norepine-
phrine, and endothelin, activate the small G protein
RhoA. RhoA in turn activates Rho kinase (ROCK), which
can mediate calcium sensitization through two main
Cite this article as Cold Spring Harb Perspect Biol 2015;7:a006023
Signaling in Muscle Contraction
pathways. First, ROCK stimulates phosphorylation of
MYPT1 (Feng et al. 1999). This can be direct, at T695 or
T853, with a preference for T853. Alternatively, it can phos-
phorylate another kinase, zipper-interacting protein kinase
(ZIPK, also known as DAPK3), which phosphorylates
MYPT1 primarily at T695 (Kiss et al. 2002). ZIPK also
phosphorylates MLC at T18/S19. Phosphorylation of
MYPT1 interferes with binding of MLCP to MLC, and
thus is believed to decrease phosphatase activity. ROCK
can also phosphorylate CPI-17 (MacDonald et al. 2001).
The preference for the MYPT1 or CPI-17 pathway de-
pends on the type of smooth muscle. Whereas MYPT1 is
ubiquitously expressed in smooth muscle, CPI-17 is differ-
entially expressed. Moreover, RhoA and associated proteins
are expressed at lower levels in phasic smooth muscle com-
pared with tonic smooth muscle (Patel and Rattan 2006).
Note that PKC can also phosphorylate CPI-17 to prevent
MLCP activity. Within resistance arteries, an increase in
vascular pressure also activates the RhoA pathway; how-
ever, the signaling intermediates linking the change in vas-
cular pressure and the activation of RhoA remain unknown
(Cole and Welsh 2011).
12 VASCULAR SMOOTH MUSCLE IN DISEASE
Smooth muscle cells are remarkably plastic, altering their
phenotype in response to conditions such as vascular in-
jury, altered blood flow conditions, or disease states. The
changes in phenotype that can occur include cell prolifer-
ation, apoptosis, and cell migration and are induced by
many factors, including cytokines and growth factors, me-
chanical forces, neuronal stimuli, and genetic factors. Here
we limit our discussion to hypertension.
In hypertension, there is often a change in the sympa-
thetic nervous system and the renin angiotensin system
that leads to increased blood pressure. Angiotensinogen
is converted to angiotensin I by renin, which in turn is
converted to angiotensin II by angiotensin-converting en-
zyme (ACE). Increased circulating angiotensin II acts on
the angiotensin receptors (AT1 and AT2), which, when
activated, cause increased peripheral resistance. The con-
sequence for smooth muscle cells is they become hy-
percontractile. Treatments include ACE inhibitors (which
inhibit the conversion of angiotensin I to angiotensin
II), a1-adrenergic antagonists (which block the AT1 and
AT2 GPCRs), and calcium channel blockers (such as dihy-
dropyridines, which inhibit the voltage-gated calcium
channels). All of these treatments aim to reduce the con-
tractility of smooth muscle. Interfering with downstream
targets such as RhoA signaling in hypertensive animals has
also been shown to be effective (Uehata et al. 1997; Seko
et al. 2003; Moriki et al. 2004).
11
I.Y. Kuo and B.E. Ehrlich
The sustained contractile state of vascular smooth mus-
cle is associated with the activation of calcium-dependent
transcription factors. These include SRF, FOS, NFAT,
and CREB. SRF, which is activated by the RhoA pathway,
promotes the expression of genes encoding components
of the contractile apparatus. Calcium-stimulated CaMKII
activates and causes the translocation of CaMKIV to the
nucleus, where it can activate CREB, which promotes tran-
scription of components of the contractile apparatus and
other targets. However, CaMKII can also activate a phos-
phatase that dephosphorylates and thus inactivates CREB
(Matchkov et al. 2012). NFAT is activated on dephosphor-
ylation by calcium-activated calcineurin, which induces
genes associated with proliferation and migration.
NO produced by eNOS in endothelial cells protects
against the changes observed in hypertension: the cGMP
pathway inhibits DNA synthesis, mitogenesis, and cell
proliferation (Forstermann and Sessa 2012). However, en-
dothelial dysfunction is a hallmark of vascular disease, in-
cluding hypertension. In many types of vascular diseases,
eNOS is up-regulated but owing to reduced oxygen avail-
ability it is converted to a dysfunctional enzyme that pro-
duces superoxides, which contribute to vascular oxidative
stress (Forstermann and Sessa 2012).
In some disease states, smooth muscle cells adopt a non-
contractile phenotype. Although these cells still have signal-
ing machinery that increases intracellular calcium levels,
they have significantly reduced calcium influx through
voltage-gated calcium channels. Thus, there is a shift to
intracellular-store-operated calcium release, similar to the
changes observed in cardiac hypertrophy. Concomitant
with decreases in the levels of SERCA, RyR2, PMCA1, and
the sodium/calcium exchanger, the levels of STIM, ORAI
( proteins associated with refilling of intracellular cal-
cium stores; see Bootman 2012), SERCA2B, and IP3R in-
crease and there is a change in RyR receptor subtypes
from RyR2 to RyR3 (Lipskaia and Lompre 2004; Berra-
Romani et al. 2008; Baryshnikov et al. 2009; Matchkov
et al. 2012). These changes collectively reflect a less contrac-
tile phenotype.
13 CONCLUDING REMARKS
Signal transduction is essential for the function of con-
tractile cells. The stimulatory signal results in an in-
crease in cytosolic calcium levels, which activates muscle
contraction. We now know the main contributors to the
various types of muscle contraction, and have a better ap-
preciation of the changes that occur to the contractile
apparatus under exercise and pathophysiological condi-
tions. For example, the identification of PGC1a as a master
regulator of transcription factors up-regulated in both
12
exercised and pathological striated muscle provides new
avenues to modulate muscles in a therapeutic setting. It is
also apparent that many signaling proteins in both smooth
and striated muscles are activated by changes in cytosol-
ic calcium levels, and these signaling pathways often lead
to alterations in gene expression. Because we now have a
better appreciation of the changes that occur to the con-
tractile apparatus under pathophysiological conditions,
this knowledge can be harnessed to allow us to treat disease
strategically.
ACKNOWLEDGMENTS
Research in the Ehrlich Laboratory is supported by Nation-
al Institutes of Health funds. I.Y.K. is an American Heart
Association postdoctoral fellow.
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