Regulatory light chains of striated muscle myosin. Structure, function and malfunction.
ABSTRACT Striated (skeletal and cardiac) muscle is activated by the binding of Ca(2+) to troponin C and is regulated by the thin filament proteins, tropomyosin and troponin. Unlike in molluscan or smooth muscles, the myosin regulatory light chains (RLC) of striated muscles do not play a major regulatory role and their function is still not well understood. The N-terminal domain of RLC contains a 'Ca(2+)-Mg(2+)'-binding site and, analogous to that of smooth muscle myosin, also contains a phosphorylation site. During muscle contraction, the increase in Ca(2+) concentration activates the Ca(2+)/calmodulin-dependent myosin light chain kinase and leads to phosphorylation of the RLC. In agreement with other laboratories we have demonstrated that phosphorylation and Ca(2+) binding to the RLC play an important modulatory role in striated muscle contraction. Furthermore, the ventricular isoform of human cardiac RLC has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. Our recent studies have demonstrated that phosphorylation and Ca(2+) binding to human ventricular RLC are significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation, whether located in the proximity of the RLC 'Ca(2+)-Mg(2+)'-binding site or the phosphorylation site (Serine 15). We have also shown that there is a functional coupling between Ca(2+) and/or Mg(2+) binding to the RLC and phosphorylation and that the FHC mutations can affect this relationship. Further in vivo studies are necessary to investigate the mechanisms involved in the pathogenesis of RLC-linked FHC.
- SourceAvailable from: Wenrui Huang[Show abstract] [Hide abstract]
ABSTRACT: We have examined, for the first time, the effects of the familial hypertrophic cardiomyopathy (HCM)- associated Lys104Glu mutation in the myosin regulatory light chain (RLC). Transgenic mice expressing the Lys104Glu substitution (Tg-MUT) were generated and the results compared to Tg-WT (wild-type human ventricular RLC) mice. Echocardiography with pulse wave Doppler in 6month-old Tg-MUT showed early signs of diastolic disturbance with significantly reduced E/A transmitral velocities ratio. Invasive hemodynamics in 6month-old Tg-MUT mice also demonstrated a borderline significant prolonged isovolumic relaxation time (Tau) and a tendency for slower rate of pressure decline, suggesting alterations in diastolic function in Tg-MUT. Six month-old mutant animals had no LV hypertrophy; however, at >13months they displayed significant hypertrophy and fibrosis. In skinned papillary muscles from 5-6 month-old mice a mutation induced reduction in maximal tension and slower muscle relaxation rates were observed. Mutated cross-bridges showed increased rates of binding to the thin filaments and a faster rate of the power stroke. In addition, ~2-fold lower level of RLC phosphorylation was observed in the mutant compared to Tg-WT. In line with the higher mitochondrial content seen in Tg-MUT hearts, the MUT-myosin ATPase activity was significantly higher than WT-myosin, indicating increased energy consumption. In the in vitro motility assay, MUT-myosin produced higher actin sliding velocity under zero load, but the velocity drastically decreased with applied load in the MUT vs. WT myosin. Our results suggest that diastolic disturbance (impaired muscle relaxation, lower E/A) and inefficiency of energy use (reduced contractile force and faster ATP consumption) may underlie the Lys104Glu-mediated HCM phenotype.Journal of Molecular and Cellular Cardiology 06/2014; · 5.22 Impact Factor
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ABSTRACT: Postactivation potentiation (PAP) is known to enhance force production. Maximal isometric strength assessment protocols usually consist of two or more maximal voluntary isometric contractions (MVCs). The objective of this study was to determine if PAP would influence isometric strength assessment. Healthy male volunteers (n = 23) performed two five-second MVCs separated by a 180-seconds interval. Changes in isometric peak torque (IPT), time to achieve it (tPTI), contractile impulse (CI), root mean square of the electromyographic signal during PTI (RMS), and rate of torque development (RTD), in different intervals, were measured. Significant increases in IPT (240.6 ± 55.7 N·m versus 248.9 ± 55.1 N·m), RTD (746 ± 152 N·m·s(-1) versus 727 ± 158 N·m·s(-1)), and RMS (59.1 ± 12.2% RMSMAX versus 54.8 ± 9.4% RMSMAX) were found on the second MVC. tPTI decreased significantly on the second MVC (2373 ± 1200 ms versus 2784 ± 1226 ms). We conclude that a first MVC leads to PAP that elicits significant enhancements in strength-related variables of a second MVC performed 180 seconds later. If disconsidered, this phenomenon might bias maximal isometric strength assessment, overestimating some of these variables.BioMed Research International 01/2014; 2014:126961. · 2.71 Impact Factor
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ABSTRACT: The responsible mechanisms for post-activation potentiation have been a popular research issue during the past years. One commonly mentioned mechanism is the force enhancement via phosphorylation of myosin regulatory light chains, which affects the sensitivity of the actin – myosin to Ca 2+Journal of Physical Education and Sport. 06/2014; 14(21):134-137.
Current Drug Targets - Cardiovascular & Haematological Disorders, 2003, 3, 187-197187
1568-0061/03 $41.00+.00© 2003 Bentham Science Publishers Ltd.
Regulatory Light Chains of Striated Muscle Myosin. Structure, Function and
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, FL 33136,
Abstract: Striated (skeletal and cardiac) muscle is activated by the binding of Ca2+ to troponin C and is
regulated by the thin filament proteins, tropomyosin and troponin. Unlike in molluscan or smooth muscles, the
myosin regulatory light chains (RLC) of striated muscles do not play a major regulatory role and their function
is still not well understood. The N-terminal domain of RLC contains a 'Ca2+-Mg2+'-binding site and, analogous
to that of smooth muscle myosin, also contains a phosphorylation site. During muscle contraction, the increase
in Ca2+ concentration activates the Ca2+/calmodulin-dependent myosin light chain kinase and leads to
phosphorylation of the RLC. In agreement with other laboratories we have demonstrated that phosphorylation
and Ca2+ binding to the RLC play an important modulatory role in striated muscle contraction. Furthermore,
the ventricular isoform of human cardiac RLC has been shown to be one of the sarcomeric proteins associated
with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left
ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. Our recent studies have demonstrated
that phosphorylation and Ca2+ binding to human ventricular RLC are significantly altered by the FHC
mutations and that their detrimental effects depend upon the specific position of the missense mutation,
whether located in the proximity of the RLC 'Ca2+-Mg2+'-binding site or the phosphorylation site (Serine 15).
We have also shown that there is a functional coupling between Ca2+ and/or Mg2+ binding to the RLC and
phosphorylation and that the FHC mutations can affect this relationship. Further in vivo studies are necessary
to investigate the mechanisms involved in the pathogenesis of RLC-linked FHC.
Key Words: Regulatory Light Chains of Myosin (RLC), Phosphorylation, Ca2+-binding, Skinned Fibers, Familial
Hypertrophic Cardiomyopathy (FHC) Mutations.
It is well established that the myosin regulatory light
chains (RLC) play a major regulatory role in scallop and
smooth muscle contraction, but their functional role in
mammalian striated (skeletal and cardiac) muscle is still not
entirely understood. The regulation of contraction in
molluscan muscle occurs via myosin, which binds Ca2+
directly [1,2], while in smooth muscle contraction is
initiated by phosphorylation of the RLC with
Ca2+/calmodulin (CaM) activated myosin light chain kinase
(MLCK) [3-5]. The activation and regulation of striated
muscle contraction occurs via thin filament proteins,
tropomyosin (Tm) and troponin (Tn) (see reviews [6-8]),
Fig. (1). Binding of Ca2+ to the Ca2+-specific, low affinity
sites of troponin C (TnC), activates contraction and leads to
its interaction with the inhibitory subunit of Tn, troponin I
(TnI) and the Tm-binding subunit, troponin T (TnT), Fig.
(1). These intermolecular Tn interactions promote
translocation of the Tm-Tn complex away from the outer
domain of the actin filaments enabling the binding of the
myosin heads (S1) to actin [9,10], Fig. (1). At the
sarcomeric level, these cyclic cross-bridge (actin-S1)
interactions, coupled with the hydrolysis of MgATP, result
in sliding of the thin and thick filaments against each other
*Address correspondence to this author at the Department of Molecular
& Cellular Pharmacology, University of Miami School of Medicine
Rosenstiel Medical Sciences Building R-189, Room 6113, USA;
Telephone: (305) 243-2908; FAX: (305) 243-4555; E-mail:
and lead to force generation and muscle contraction . The
RLC, a major regulatory subunit of molluscan and smooth
muscle myosins, does not play a key role in striated muscle
contraction; however, it has been thought to modulate the
Tn-controlled mechanism of force generation.
The physiological role of the RLC in skeletal and cardiac
muscle contraction as well as the functional coupling
between Ca2+ binding to the RLC and its phosphorylation
are the primary topics of this review. Moreover, the effect of
familial hypertrophic cardiomyopathy (FHC) mutations in
human ventricular RLC on cardiac contractility along with
the mechanisms involved in the pathogenesis of RLC-linked
FHC are being addressed.
STRUCTURE OF RLC
Myosin is a major protein of the thick filaments of
smooth, skeletal and cardiac muscles and together with actin
plays an essential role in force generation. The myosin
molecule is a hexamer composed of two heavy chains and
four light chains [12,13]. Each of the amino-termini of the
myosin heavy chains form a globular domain, called the
myosin head or subfragment 1 (S1), and contains a pair of
the regulatory and essential (ELC) light chains (In Fig. (2)
the RLC is colored magenta and the ELC yellow) . The
crystal structures of chicken skeletal S1, Fig. (2),  and
the regulatory domain of scallop myosin consisting of one
RLC, one ELC and a part of the myosin heavy chain 
have been solved to 2.8 Å resolution. These studies have
188 Current Drug Targets - Cardiovas. & Haemat. Dis., 2003, Vol. 3, No. 2Danuta Szczesna
Fig. (1). Graphic illustration of Ca2+-Tn·Tm regulated striated muscle contraction and the effect of RLC phosphorylation and Ca2+
binding. Repetitive stimulation of the striated muscle elevates the intracellular Ca2+ level that in turn activates the Ca2+/CaM
-dependent MLCK complex and leads to RLC phosphorylation. It is proposed that RLC phosphorylation may increase the maximal
level of force generation by regulating the transition from weakly to strongly bound cross-bridges. According to Davis et al., RLC
phosphorylation may increase the number of cross-bridges entering the contractile cycle by up-regulation of actin-induced
phosphate release state . Therefore, RLC phosphorylation could be controlling the transition from 'actin-S1·ADP·Pi' to 'actin-
S1·ADP' state. As was also hypothesized by Sweeney et al. , phosphorylation of RLC moves the average position of cross-bridges
away from the thick filament, increasing its interaction with actin. Abbreviations: S1, the myosin head domain; RLC, regulatory light
chain of myosin; CaM, calmodulin, MLCK, myosin light chain kinase; Tm, tropomyosin; Tn, troponin (C, I, T); ATP, adenosine
triphosphate; ADP, adenosine diphosphate; Pi, inorganic phosphate.
Fig. (2). The structure of the myosin head (S1). Stereo diagram of the myosin head domain (S1) containing the actin-binding site, ATP
hydrolysis site (active site), ELC (essential light chain) and RLC (regulatory light chain). From I. Rayment et al. .
Regulatory Light Chains of Striated Muscle Myosin Current Drug Targets - Cardiovas. & Haemat. Dis., 2003, Vol. 3, No. 2 189
Fig. (3). Conformational switch in the EF hands of the N lobe of TnC. Stereo diagram of the EF-hand domains I (helices A and B) and II
(helices C and D) in the Ca2+-free (gray) and Ca2+-bound (black) structures of the N lobe of TnC. The helices of a domain are
reoriented during the transition by conformational changes occurring in two hinge regions. From Houdusse et al. .
revealed that the RLC is localized at the head-rod junction of
the myosin heavy chain and, together with the ELC,
stabilizes the 8.5 nm long α-helical neck of the myosin
head, Fig. (2). These calmodulin (CaM) -like light chains
are non-covalently bound to the heavy chain of myosin, with
the RLC arranged such that its N-terminal domain wraps
around the C-terminus of the heavy chain between amino
acid residues Asn 825 and Leu 842 while its C-terminus
interacts with the heavy chain in the region between Glu 808
and Val 826 . The structure of the N-terminus of RLC is
similar to other EF-hand Ca2+ binding proteins such as
CaM, TnC, or ELC while the C-terminus is considerably
less similar . The N-terminal domain of the RLC
contains a divalent cation-binding site, located in the first
helix-loop-helix motif, which binds both Ca2+ and Mg2+,
Figs (1, 2). This site has a conformation similar to that of
TnC depicted in Fig. (3) . Shown are the Ca2+-free
(gray) and Ca2+-bound (black) conformations of the N-
terminal two regulatory Ca2+ specific sites of TnC. In the
Ca2+-free form, the two helices flanking the Ca2+ binding
site are arranged close together in a so-called 'closed
conformation'. Upon binding of Ca2+ a conformational
change occurs that leads to the 'open conformation' of the
Ca2+ binding site, in which the helices are nearly
perpendicular to each other, Fig. (3).
Analogous to smooth muscle myosin, the N-terminal
domain of RLC of skeletal and cardiac myosins also
contains a phosphorylation site (Serine) that is located in the
proximity of the cation-binding site, Figs (4, 5, 6). The
functional significance of the Ca2+ binding-site and the
phosphorylation-site in the RLC is discussed below.
Skeletal muscles are composed of a large variety of
functionally diverse fiber types . These different fiber
types correlate well with the myosin heavy chain isoforms.
During the first few weeks of life, fetal myosin is replaced
by slow myosin in slow muscle and by fast myosin in fast
muscle. Likewise, the RLC of skeletal muscle myosin
originating from different fibers could have various amino
acid sequences. There are three major adult forms of skeletal
RLC that are encoded by different genes: two fast isoforms:
type 1 (NCBI Accession No. P24732) and type 2 (NCBI
Accession No. P02608) and the slow isoform (NCBI
Accession No. A61567). Fig. (4) demonstrates the amino
acid sequences of five different skeletal and cardiac RLC
isoforms. The amino acid sequence analysis indicates that
the skeletal fast isoforms type 1 and type 2 are 98.8%
The ventricular RLC gene is expressed in both the
ventricular myocardium and slow-twitch muscle [18,19].
The gene expressing the human ventricular isoform of the
RLC as well as slow skeletal RLC has been mapped to
chromosome 12q23-q24 [20,21]. Three different isoforms of
the RLC have been identified in the human heart: atrial,
ventricular-a and ventricular-b. Atrial and ventricular RLC
differ electrophoretically with the atrial isoform being more
190 Current Drug Targets - Cardiovas. & Haemat. Dis., 2003, Vol. 3, No. 2Danuta Szczesna
Fig. (4). Amino acid sequences of the myosin regulatory light chains (RLC) of skeletal and cardiac muscles. 1) Fast skeletal isoform,
type 1 (rabbit), NCBI accession number: P24732. Theoretical pI/MW: 4.82 / 19028.55; 2) Fast skeletal isoform (rabbit), type 2, NCBI
Accession No. P02608. Theoretical pI/MW: 4.82 / 19026.54; 3) Slow skeletal isoform, fragment (rabbit), NCBI Accession No.
A61567. Theoretical pI/MW: 4.57 / 17378.59; 4) Human ventricular isoform, NCBI Accession No. P10916. Theoretical pI/MW: 4.92 /
18789.29; 5) Human atrial isoform, NCBI Accession No. M94547. Theoretical pI/MW: 4.83 / 19448.03.
acidic . Fig. (4) demonstrates the amino acid
composition of the human atrial (NCBI Accession No.
M94547) and ventricular (NCBI Accession No. P10916)
RLC isoforms. The two ventricular RLC isoforms have the
same molecular weight but different isoelectric points. The
more basic isoform-b is predominantly expressed in the
normal human heart with an a/b ratio of 2.3 [23,24]. No
differences are shown between the RLC from the right and
left ventricle, while there is a difference between the RLC
from right and left atria.
FUNCTION OF RLC
The regulatory light chains of myosin have been shown
to be important for both the proper structure of myosin and
the thick filaments in striated muscles and for regulation of
smooth and striated muscle contraction. The localization of
the RLC at the head-rod junction of the myosin molecule
implies their possible importance in cross-bridge cycling in
contracting muscle, Fig. (1). This region of the myosin
heavy chain, which contains the RLC, has been postulated
to undergo conformational changes that are important for
working muscle [25,26]. The motions of this myosin head
region were predicted by crystallographic models [25,27] and
studied further by fluorescence polarization spectroscopy
. It has been demonstrated that during active contraction,
the RLC binding domain of the myosin head undergoes
repetitive conformational changes (tilt and twist) and
therefore may play an active role during force generation in
The structural significance of the RLC in skeletal
muscles has been studied
extraction/reconstitution methods applied to skinned muscle
fibers. Studies of Moss et al. , Hofmann et al. ,
Metzger and Moss  and Patel et al.  have shown that
partial extraction of RLC from skeletal muscle fibers
increases the rate of tension re-development at submaximal
[Ca2+]. Our studies , however, have revealed that the
removal of the RLC decreases the rate of force development
by a factor of two and that this decrease could be reversed by
re-incorporation of the RLC. In another approach, utilizing
in vitro motility assays, it has been shown that removal of
the RLC from skeletal myosin also reduces the velocity of
single actin filaments migrating on a myosin-coated surface
. The velocity of actin movement could be restored upon
reconstitution of myosin with the RLC. Thus, there are
numerous observations that strongly suggest a role for the
RLC in the regulation of contraction although the exact
mechanism of these processes is not known.
During muscle contraction, the increase in Ca2 +
concentration activates the Ca2+/CaM-dependent myosin
light chain kinase (MLCK) and leads to phosphorylation of
the RLC, Fig. (1). There are contradictory reports with
regard to the effects of phosphorylation of the RLC on
skeletal muscle contraction. In vivo studies of intact skeletal
muscles have demonstrated that the level of myosin
phosphorylation significantly increases after tetanic
stimulation . RLC phosphorylation in skinned skeletal
muscle fibers has been shown to have only a small effect, if
any, on the Ca2+ sensitivity of isometric tension whereas it
has a significant effect on the rate of force re-development
[35-37]. On the other hand, several studies suggest that
phosphorylation of the RLC reduces cross-bridge cycling
rates [38,39]. In contrast to the above, no evidence
correlating phosphorylation of the RLC with skeletal muscle
regulation was found by Butler et al. . There are also
contradictory reports as to whether the phosphorylation
influences actin-activated myosin ATPase activity [41-43].
Our recent studies  demonstrate that phosphorylation of
the RLC by Ca2+/CaM-activated MLCK has a much bigger
Regulatory Light Chains of Striated Muscle MyosinCurrent Drug Targets - Cardiovas. & Haemat. Dis., 2003, Vol. 3, No. 2 191
effect on the maximal force development, the Ca2 +
sensitivity of force in skinned skeletal muscle fibers and on
the ATPase activity in reconstituted thin filaments, than
previously observed or appreciated (see below).
The N-terminal divalent cation-binding site of the RLC
is thought to be occupied by Mg2+  when muscles are in
the relaxed state and may become partially saturated with
Ca2+, depending on the length of the [Ca2+] transient ,
Fig. (1). Since the process of cation exchange may occur at a
much slower rate than muscle activation, the functional
importance of this site is unknown. Based on solution
binding studies, this site has been classified as a 'Ca2+-
Mg2+'-type site that binds either Ca2+ or Mg2+ [45,47,48].
Because of this, it has been suggested that this site does not
play a primary role in the Ca2+ activation of skeletal muscle
contraction, since, as a 'Ca2+-Mg2+'-site, its rate of Ca2+
binding would be slow, due to the slow dissociation of the
Mg2+ bound to the site during muscle activation. Although
this may be true for myosin in solution, it may not reflect
the true binding properties of myosin in muscle. One
possibility is that metal binding to this site is only
important for increasing the affinity of the RLC for the
myosin heavy chain and that this association may be
important for its proper function, e.g., influencing cross-
bridge cycling rate, etc.
Ca2+ Binding to RLC
The binding of Ca2+ to isolated phosphorylated or non-
phosphorylated RLC and the effect of Mg2+ on this binding
were studied by Szczesna et al.  using the flow dialysis
method. The KCa for non-phosphorylated rabbit skeletal
RLC was ≈ 1.5 x 105 M-1 and was slightly decreased upon
phosphorylation of the RLC, KCa ≈ 1.04 x 105 M-1. In the
presence of 2 mM Mg2+, the K’Ca was ≈ 6.02 x 104 M-1 for
non-phosphorylated RLC and ≈ 5.19 x 104 M-1 for the
phosphorylated protein. Similar KCa and K’Ca values were
obtained utilizing the tryptophan fluorescence of the single
Trp residue in the rabbit skeletal RLC . Low KCa values
monitored either by the flow dialysis or fluorescence method
and the low sensitivity to Mg2+ suggest that the binding of
Ca2+ to the isolated RLC may not reflect the physiological
situation observed in muscle. The Ca2+ affinity to the RLC
when bound to myosin has been reported to be a 100-fold
higher than to isolated RLC . It is possible that the
specificity for Ca2+ differs depending upon the complexity
of the system (isolated state, bound to myosin, bound to
myosin in muscle). Likewise, the effect of phosphorylation
of the RLC could be different in the isolated state than when
bound to myosin in the muscle cell.
Physiological Consequences of RLC Phosphorylation in
We have recently demonstrated a large effect of RLC
phosphorylation on the Ca2+ sensitivity of ATPase activity
measured in the reconstituted thin filaments . A shift
towards lower Ca2+ concentrations (∆pCa50 ≈ 0.25) in the
actin-Tm-Tn - activated myosin ATPase activity was
observed for the P-RLC reconstituted myosin compared with
non-phosphorylated RLC. Utilizing skinned skeletal muscle
fibers we have also demonstrated that the Ca2+ sensitivity of
force development dramatically increases as a result of RLC
phosphorylation . These results are in accord with the
work of Sweeney et al.  and Metzger et al.  although
the effect seen by these authors was much smaller than that
observed in our study. As we have shown, a wide range of
phosphorylation dependent changes in the Ca2+ sensitivity
of force development observed by various laboratories could
result from different levels of initial RLC phosphorylation
in skinned fiber preparations . In addition, consistent
with the reports of Metzger et al. and Sweeney et al.
[35,49], we have shown that kinetics of force activation
increased only slightly with RLC phosphorylation .
Beyond changes in the Ca2+ sensitivity of force, we have
also shown that phosphorylation of the RLC raises the
maximal steady state force . A phosphorylation-
dependent increase in maximal force in frog muscle fibers
under fatigue conditions was also reported by Godt and
Nosek . Recently, Davis et al. , demonstrated that
force of fully Ca2+-activated, but BDM (2,3-butanedione
monoxime)-inhibited, fast and slow skeletal muscle fibers
can increase further by about 40% upon R L C
phosphorylation. The RLC phosphorylation partially
reversed the inhibitory effect of BDM on fiber tension
possibly due to increase of inorganic phosphate release from
the actin-attached S1, which could facilitate strong cross-
bridge formation , Fig. (1).
Effects of RLC Phosphorylation in Cardiac Muscle
In the human heart the phosphorylation of the RLC
isoforms (atrial and ventricular) is not uniform. The two
ventricular isoforms in quickly frozen biopsy specimens
showed varied levels of RLC phosphorylation ranging from
0.26 to 0.39 mol Pi/mol of RLC . In patients with
severe cardiac failure the two ventricular RLC isoforms were
completely dephosphorylated . It has been postulated
that increasing or decreasing the levels of RLC
phosphorylation is associated with the positive or negative
inotropic state of the heart, respectively . Studies
utilizing various animal models also showed a correlation
between the level of RLC phosphorylation and cardiac
performance. Long-term treadmill exercise involving adult
rats resulted in an increase in their ventricular RLC
phosphorylation level compared to the control sedentary
group . An increased level of RLC phosphorylation was
observed in the European hamster during summer activity
that declined 2.5 times during hibernation . The level of
RLC phosphorylation was found to be uncoupled from the
systolic and diastolic cycles possibly because of a very low
turnover rate of phosphate exchange . The steady level of
RLC phosphorylation, monitored during both systole and
diastole, also did not significantly change upon
pharmacological intervention [56,57]. In another study, the
effect of RLC phosphorylation on cardiac mechanics was
studied with the use of slow fibers from rabbit soleus
muscle . These fibers could be utilized in such studies
because human and rabbit slow skeletal muscle fibers have
been shown to express cardiac myosin . A dramatic 2.5-
fold increase in isometric tension from 22% to 56% of the
maximal Ca2 + activated tension due to RLC
192 Current Drug Targets - Cardiovas. & Haemat. Dis., 2003, Vol. 3, No. 2Danuta Szczesna
phosphorylation was monitored in these slow muscle fibers
that were subjected to large stretches of 0.4-0.8% of muscle
length . Since the contracting heart works at half-
maximum Ca2+ activation, RLC phosphorylation could be
important in modulating cardiac contraction from low
tensions to full power at a fixed concentration of Ca2+ .
Modulation of Striated Muscle Contraction by RLC
Phosphorylation and Ca2+ Binding
The question remaining is how does phosphorylation of
RLC modulate skeletal muscle contraction? Also, the
relationship between phosphorylation of RLC and its Ca2+
-binding site is still under investigation. Our results suggest
that these two important RLC regions, the Ca2+-binding site
and the phosphorylation site are communicating with each
other and that this intramolecular communication is
important for the phosphorylation dependent effects on force
generation (alteration in Ca2+ sensitivity, maximal force,
etc.), Fig. (1). While the RLC phosphorylation-
dephosphorylation process is too slow to trigger striated
muscle contraction, it plays a role in maintaining a specific
level of force at a lower Ca2+ concentration and could be
important for working muscle, e.g. improving its
performance. At the molecular level, the phosphorylation-
dependent force potentiation may simply result from the
recruitment of more strongly bound cross-bridges as the
phosphorylation of the RLC causes cross-bridges to move
away from the thick filament backbone and become more
accessible to actin , Fig. (1). The latter was illustrated
by electron micrographs of permeabilized rabbit psoas fibers
containing phosphorylated thick filaments whose near-
helical, periodic arrangement characteristic of the relaxed
state was lost due to RLC phosphorylation . One could
speculate that a phosphorylation related charge change in the
N-terminus of RLC increases myosin head mobility and its
accessibility to actin. These changes may affect the overall
ordered array of myosin heads on actin filaments that is
observed under relaxing conditions . According to Davis
et al., RLC phosphorylation may increase the number of
cross-bridges entering the contractile cycle by up-regulation
of actin-induced phosphate release state . RLC
phosphorylation could regulate the transition from 'actin-
S1·ADP·Pi' to 'actin-S1·ADP' state, Fig. (1). Observed
increases in tension mediated by RLC phosphorylation are
suggested to function independently of the state of the Ca2+-
Tn·Tm regulatory switch .
RLC MUTATIONS ASSOCIATED WITH FAMILIAL
Recent studies have revealed that the ventricular RLC is
one of the sarcomeric proteins associated with Familial
Hypertrophic Cardiomyopathy (FHC) [62-64]. FHC is an
autosomal dominant disease, characterized by left ventricular
hypertrophy, myofibrillar disarray and sudden death. It is
caused by missense mutations in various genes that encode
for β-myosin heavy chain , myosin-binding protein C
, the ventricular RLC and ELC [62-64,67], troponin T
, troponin I , troponin C , α-tropomyosin ,
actin  and titin . Depending on the affected gene, and
the site of the mutation, FHC has variable presentation with
regard to its degree, severity and to the extent of myocardial
disarray. The clinical manifestations of FHC range from
benign to severe heart failure and to sudden cardiac death
(SCD). The best characterized clinical cases include patients
with β-myosin heavy chain mutations, who show a high
level of cardiac hypertrophy and those with TnT mutations
who have less hypertrophy, but a higher incidence of SCD
in young adults.
The first three mutations identified in RLC, A13T,
E22K and P95A, Figs. (5, 6), were shown to be associated
with a particular subtype of cardiac hypertrophy defined by
mid left ventricular obstruction . Two other RLC
mutations, F18L and R58Q, Figs. (5, 6), identified by
Flavigny et al. , were associated with a typical form of
hypertrophic cardiomyopathy, which causes increased left
ventricular wall thickness and abnormal ECG findings with
no mid-cavity obliteration. Very recently, three novel RLC
FHC mutations were found in the Danish cohort, N47K,
K104E and a IVS6-1 splice site mutation, with the latter
being the first of this type described in RLC , Figs. (5,
6). The amino acid sequence analysis of cardiac RLC from
different organisms, Fig. (6), reveals a high sequence
homology among species and also demonstrates that FHC
RLC mutations occur at very conserved residues among all
presented species, Fig. (6).
Effect of FHC Mutations on the Properties of Human
The Ca2+ binding properties of human cardiac RLC
(HCRLC) and five recombinant RLC FHC mutants were
tested with the flow dialysis method . Similar to what
was found for rabbit skeletal RLC, the HCRLC wild type
(WT) bound Ca2+ with relatively low affinity, KCa = 6.67±
0.21 x 105 M-1. Three of the FHC mutations, A13T, F18L
and P95A, decreased the KCa approximately 3-fold, while
two other FHC RLC mutations, E22K and R58Q, changed
the Ca2+ binding properties in a more drastic way.
Compared to HCRLC-WT, the E22K mutation decreased the
KCa value by ≈17-fold whereas the R58Q mutation totally
eliminated Ca2+ binding to HCRLC .
Phosphorylation of WT with Ca2+-CaM activated
MLCK decreased its Ca2+ binding affinity by ≈7.4-fold.
However, the effects of FHC mutations on the properties of
HCRLC varied depending upon the location of the
mutation. The most dramatic effect was observed for the
E22K mutant. The E to K substitution in the proximity of
the HCRLC phosphorylation site (S15), Fig. (5), prevented
phosphorylation of the protein. Even a 20-fold increase of
the MLCK/E22K ratio and longer incubation time did not
result in phosphorylated E22K . Moreover,
phosphorylation of another FHC mutant, A13T, with the
mutation located even closer to the phosphorylation site
(S15) of HCRLC, increased the Ca2+ affinity from 2.06 ±
0.23 to 13.3 ± 0.2 x 105 M-1 . Interestingly,
phosphorylated A13T demonstrated a 15-fold greater affinity
for Ca2+ than phosphorylated WT, while non-
phosphorylated A13T bound Ca2+ with a 3-fold lower
affinity than non-phosphorylated-WT. Unpredictably,
substitution of the A to T residue also resulted in a large
increase in the α-helical content as monitored by far UV CD
Regulatory Light Chains of Striated Muscle MyosinCurrent Drug Targets - Cardiovas. & Haemat. Dis., 2003, Vol. 3, No. 2 193
Fig. (5). Amino acid sequence of human ventricular RLC (NCBI Accession No. P10916). Labeled: Familial Hypertrophic
Cardiomyopathy (FHC) mutations, the phosphorylation site, and the Ca2+-binding site. The first FHC RLC mutations identified in an
American population, A13T, E22K and P95A, were shown to be associated with a particular subtype of cardiac hypertrophy defined by
mid left ventricular obstruction . Two other RLC mutations, F18L and R58Q, identified in a French population, were associated
with a typical form of hypertrophic cardiomyopathy, which caused increased left ventricular wall thickness and abnormal ECG
findings with no mid-cavity obliteration . Three new RLC FHC mutations were recently found in the Danish cohort, N47K, K104E,
and an IVS6-1 splice site mutation that is the first of this type identified in the RLC .
Fig. (6). FHC RLC mutations in various cardiac isoforms of RLC. The sequence of rabbit skeletal RLC (as in Fig. 4) is also shown.
Labeled: FHC mutations, the phosphorylation site, and the Ca2+-binding site.
spectroscopy . This was quite surprising considering
alanine’s predisposition for stabilizing α-helical structures.
The α-helical content of the A13T mutant returned to a
normal level (that of WT) upon phosphorylation. Therefore,
phosphorylation of the A13T mutant attenuated whatever
sterical constraints were introduced by this FHC mutation.
An interesting effect of phosphorylation on Ca2+ binding
was observed for the R58Q mutant. This mutant could not
bind Ca2+ in the non-phosphorylated state but its ability to
bind Ca2+ was restored after phosphorylation (KCa =
3.04±1.02 x 105 M-1).
In summary, the Ca2+ binding studies as well as
investigations into the effects of FHC mutations on the
secondary structure of HCRLC revealed that the detrimental
effects of the FHC mutations in human cardiac RLC could
be compensated for by phosphorylation, Ca2+ binding or
both. Ca2+ binding was restored to R58Q upon
phosphorylation, E22K had decreased Ca2+ affinity and
could not be phosphorylated, Ca2+ binding to E22K or to
A13T decreased their α-helical content that was initially
increased by the FHC mutation, and phosphorylation of
A13T restored the amount of the α-helical content to the
194 Current Drug Targets - Cardiovas. & Haemat. Dis., 2003, Vol. 3, No. 2Danuta Szczesna
level of WT. Another study also demonstrated a correlation
between the location of the FHC mutation and the ATPase
activity measured in skinned cardiac muscle myofibrils
reconstituted with different FHC mutants . The E22K
and R58Q mutations, located in the helices flanking the
Ca2+ binding site of HCRLC altered the Ca2+ sensitivity of
myofibrillar ATPase activity, while A13T, F18L and P95A
mutations that are located in close proximity to the
phosphorylation site (S15), decreased the maximal level of
the ATPase activity .
Identification of Novel FHC RLC Mutations
Studies of Andersen et al.  analyzed consecutively
collected data on 68 FHC families from Denmark and 130
probands from South Africa. The subjects with an MVH
(mid-ventricular hypertrophy) phenotype in the Danish
families were identified and the segregation of this
phenotype among their relatives was investigated. The
probands of these families, as well as those with the
mutations, irrespective of their clinical phenotype, were
screened for the presence of mutations in the coding regions
of β-myosin heavy chain, myosin binding protein C, TnT,
TnI, α-Tm, α-actin and myosin ELC to assess the
correlation between the MVH phenotype and a causal
mutation. Mutations in the RLC gene in both exon 5 and
exon 7 were found, which generated a glutamic acid
substitution at the evolutionarily highly-conserved lysine
104 (K104E), and a G>C transversion in the acceptor splice
site of intron 6, IVS6-1, respectively, Fig. (5). No
mutations were detected when screening the other seven
FHC genes in this family.
A C>A transversion in exon 3 of the RLC gene,
resulting in an N47K substitution, Fig. (5), was identified
in the proband of another Danish family . The mutation
was not identified in the 150 healthy controls or in the other
197 probands. No other mutations were identified in this
patient for the additional seven FHC genes screened. The
affected proband was diagnosed with HCM at the age of 60
years, at which time, as well as during follow up, he had a
high, relatively fixed mid-ventricular flow gradient, as well
as diastolic filling abnormalities. The mid-ventricular flow
gradient was caused not only by the pronounced septal
hypertrophy, but also by a significant increase in the size of
the papillary muscle apparatus. Interestingly, a marked
progression in the septal hypertrophy, from 31 to 45 mm,
was seen over two years from age 60 to 62 years. There was
no family history of sudden death .
It will be very interesting to examine these new FHC
RLC mutations for their effects on the binding of Ca2+ to
the single 'Ca2+-Mg2+'-binding site and on the
phosphorylation-regulated function of the RLC.
MOUSE MODELS FOR CARDIAC RLC
Transgenic Mouse Models
Studies by Buck et al.  showed that total
replacement of the atrial RLC isoform with the ventricular
RLC isoform in atria resulted in an increase in unloaded
shortening velocity in atrial cardiomyocytes. This suggested
that the ventricular isoform of RLC might contribute to the
greater power-generating capabilities of the ventricles
compared with that of the atria. Another study from the
Robbins lab analyzed the effect of complete or partial
replacement of the cardiac RLC with the isoform that is
normally expressed in fast skeletal muscle fibers in both the
atria and ventricles . In atria, isoform replacement with
the skeletal protein was quite efficient, while the ventricle
was much more resistant to replacement. Despite very high
levels of RNA transcription in these transgenic mice, the
overall level of the RLC in both compartments remained
constant. The partial replacement of the ventricular with the
skeletal isoform reduced both left ventricular contractility
and relaxation, although the unloaded shortening velocity of
isolated ventricular cardiomyocytes was not significantly
different. Furthermore, Sanbe et al.  have shown that
RLC could control aspects of cross-bridge cycling and alter
force development. The authors demonstrated that fiber
kinetics were not affected when transgenically encoded
protein was expressed in its endogenous compartment, but
that ectopic replacement invariably led to changes in the
fiber's cross-bridge kinetics.
A role of RLC phosphorylation in modulating cardiac
function was also assessed in transgenic mice in which three
potentially phosphorylatable serines in the ventricular
isoform of RLC were mutated to alanines . The mice
showed a wide spectrum of cardiovascular changes. The
Ca2+ sensitivity of force development was dependent upon
the phosphorylation status of the RLC. Structural
abnormalities were detected by both gross histology and
transmission electron microscopic analyses. Mature animals
showed both atrial hypertrophy and dilatation. The authors
concluded that phosphorylation of the RLC plays an
important role in maintaining normal cardiac function .
The only animal model for FHC RLC mutations
generated to date was that of RLC-E22K . In humans,
this mutation was shown to be associated with a particular
subtype of cardiac hypertrophy defined by mid left
ventricular obstruction . Transgenic mice expressing
E22K showed no detectable phenotype and no hypertrophy
could be detected in mature adult animals either when
chamber weights were determined or at the cellular level
. It seems likely that mice do not always recapitulate
important aspects of human hypertrophy.
Knock-Out RLC Mouse Models
As shown by Chen et al., the total disruption of the
murine cardiac ventricular RLC gene resulted in sarcomeric
disassembly, an embryonic form of dilated cardiomyopathy
and the early death of homozygous embryos . Drastic
changes in ventricular structure and function were observed
in ventricle muscles expressing the atrial isoform of RLC.
This indicated that there was a selective requirement for the
ventricular RLC isoform in the normal development of
murine ventricular cardiac myocyte structure and function
. The authors concluded that the ventricular isoform of
RLC could have a unique function in the maintenance of
cardiac contractility and ventricular chamber morphogenesis
during mammalian cardiogenesis . Another study of
Regulatory Light Chains of Striated Muscle MyosinCurrent Drug Targets - Cardiovas. & Haemat. Dis., 2003, Vol. 3, No. 2 195
these RLC knock-out mice showed that the ventricles of
heterozygotes displayed a 50% reduction in RLC ventricular
mRNA, yet expressed normal levels of protein .
Heterozygotes exhibited cardiac function comparable to that
of wild-type controls and demonstrated no significant
differences in contractility and response to Ca2+ in unloaded
cardiomyocytes. Thus, heterozygous mice showed neither a
molecular nor a physiological cardiac phenotype suggesting
that post-transcriptional compensatory mechanisms may play
a major role in maintaining the level of the ventricular RLC
in murine hearts .
SUMMARY AND CONCLUSIONS
Our results as well as the results of others suggest that
phosphorylation of the regulatory light chains of myosin has
an important physiological role in the regulation of striated
muscle contraction. Moreover, a functional coupling between
phosphorylation and Ca2+ binding to RLC is implicated to
play a key role in the modulation of skeletal and cardiac
muscle contraction, Fig. (1). The latter correlation between
the effects brought about by the Ca2+ binding to and
phosphorylation of RLC could be especially important in
the working heart. Both of these processes may operate as
adaptive and/or protective mechanisms to either improve
performance of the contracting cardiac muscle or play a role
in attenuating the detrimental effects of FHC mutations.
Any alterations introduced by the FHC RLC mutations may
contribute to cardiac hypertrophy and malfunctioning of the
human heart. At the molecular level, cardiac hypertrophy
may develop as a compensatory mechanism to altered
interaction of the mutated RLC with the heavy chain of
myosin that in turn may affect the function of cycling cross-
bridges and force generation. As observed, the patients with
the FHC RLC mutations have developed a phenotype of
hypertrophic cardiomyopathy, although no sudden deaths
associated with these mutations were reported. Further in
vivo work is needed to assess the physiological consequences
of the RLC FHC mutations and to identify the mechanisms
involved in the pathogenesis of RLC-linked FHC.
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