Signaling to Myosin Regulatory Light Chain in Sarcomeres*

Myosin regulatory light chain (RLC) phosphorylation in skeletal and cardiac muscles modulates Ca2+-dependent troponin regulation of contraction. RLC is phosphorylated by a dedicated Ca2+-dependent myosin light chain kinase in fast skeletal muscle, where biochemical properties of RLC kinase and phosphatase converge to provide a biochemical memory for RLC phosphorylation and post-activation potentiation of force development. The recent identification of cardiac-specific myosin light chain kinase necessary for basal RLC phosphorylation and another potential RLC kinase (zipper-interacting protein kinase) provides opportunities for new approaches to study signaling pathways related to the physiological function of RLC phosphorylation and its importance in cardiac muscle disease.

In myocytes from striated cardiac and skeletal muscles, sarcomeric contraction results from the Ca 2ϩ -regulated binding of the myosin motor to actin (Fig. 1). Ca 2ϩ binds to troponin in actin thin filaments, thereby allowing myosin cross-bridges in thick filaments to bind actin for the development of force and cell shortening with ATP hydrolysis (1). Myosin cross-bridges, containing the actin-binding surface and ATP pocket in the motor domain, taper to an ␣-helical neck that connects to the myosin rod region responsible for the self-assembly into thick filaments. Two small protein subunits, the essential light chain and regulatory light chain (RLC), 2 wrap around each ␣-helical neck region, providing mechanical stability.
Ca 2ϩ released to the sarcomeres during contraction may also activate Ca 2ϩ /calmodulin-dependent protein kinases that phosphorylate RLC ( Fig. 1) (2). RLC phosphorylation has no significant effect on actin-activated myosin ATPase activity but changes myosin cross-bridge properties, resulting in modulation of Ca 2ϩ /troponin-dependent contractions. This minireview describes RLC phosphorylation in heart and skeletal muscles, emphasizing integration of molecular and biochemical properties with physiological performance and pathophysiological adaptations.

Distinct Myosin Light Chain Kinases Phosphorylate Sarcomeric RLCs
A family of myosin light chain kinases (MLCKs) catalyzes the Ca 2ϩ /calmodulin-dependent phosphorylation of different RLCs in the myosin II family (Fig. 2). The MYLK gene expresses short MLCK in smooth muscles, where it is sufficient and necessary for initiating contraction (2)(3)(4)(5)(6). The kinase has a catalytic core, a regulatory segment containing the autoinhibitory and calmodulin-binding sequences, structural Ig and fibronectin modules, and three repeating DFRXXL actin-binding motifs in the N terminus. A long smooth muscle MLCK (smMLCK) expressed from the same gene via an alternative promoter is identical to the short smMLCK except for the additional structural modules at the N terminus (Fig. 2). It is found in epithelial, endothelial, and other kinds of nonmuscle cells, where it plays functional roles in cell adhesion and other cellular processes (7,8). Thus, the kinases expressed from MYLK are ubiquitous.
The MYLK2 gene expresses a distinct skeletal muscle MLCK (skMLCK) selectively in skeletal muscle and most abundantly in fast-twitch fibers (2,9). skMLCK was reported to be present in the heart (10), but the amount is too low to physiologically maintain RLC phosphorylation (9). Ablation of the MYLK2 gene had no effect on RLC phosphorylation in the heart (9). skMLCK has a catalytic core and regulatory segment similar to those of smMLCK. Although both are dedicated protein kinases with RLCs recognized as the only physiological substrate, there are differences in the catalytic properties in terms of substrate recognition determinants (2,11), including respective signature consensus phosphorylation sequences around the phosphorylatable Ser in addition to RLC subdomain interactions with the catalytic core (12).
Recently, a novel cardiac MLCK (cMLCK) was identified in human heart failure and found to be regulated by the cardiac homeobox protein Nkx2.5 during development (13,14). The kinase is expressed by the MYLK3 gene only in cardiac ventricular and atrial myocytes. Both overexpression and knockdown of cMLCK in cultured neonatal cardiomyocytes suggested that the kinase may regulate sarcomere organization and cardiomyocyte contraction (14). In cultured neonatal cardiomyocytes, knockdown of cMLCK by siRNA also impaired epinephrine-induced activation of sarcomere reassembly (13). Knockdown of cMLCK expression using morpholino antisense oligonucleotides in zebrafish resulted in dilated cardiac ventricles and immature sarcomere structures, suggesting a significant role for cMLCK and RLC phosphorylation in cardiogenesis. However, ablation of cMLCK expression in mice did not impair cardiac development or sarcomere formation (15). Nevertheless, the extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles of adult mice. Importantly, adult mice lacking cMLCK and RLC phosphorylation had diminished cardiac performance culminating in heart failure (15). Collectively, these studies show that cMLCK is the primary kinase that maintains basal RLC phosphorylation required for normal physiological cardiac performance. cMLCK has a catalytic core and regulatory segment that are conserved in smMLCK and skMLCK (Fig. 2). Like skMLCK, it does not have the structural modules or motifs found in smMLCK.
ZIPK expressed by the DAPK3 gene is a highly conserved Ca 2ϩ -independent protein kinase that belongs to the deathassociated protein kinase family (16,17). ZIPK has its catalytic core in the N terminus of the protein with a C-terminal putative leucine zipper domain (Fig. 2). ZIPK is ubiquitously expressed in different tissues, including the heart (18,19). It plays a role in apoptosis and autophagy in nonmuscle cells and modulates contraction in smooth muscle. Biochemical studies have identified smooth muscle myosin RLC and the regulatory subunit of smooth muscle myosin light chain phosphatase (MYPT1 (myosin protein-targeting subunit 1)) as substrates for ZIPK (18,20,21). An unbiased substrate search on heart homogenates with ZIPK identified RLC of ventricular myosin as a prominent substrate (22). ZIPK phosphorylated cardiac RLC at Ser-15 with a V max value 2-fold greater than the value for smooth/nonmuscle RLC, indicating that cardiac RLC was a favorable biochemical substrate. Knockdown of ZIPK in neonatal cardiac myocytes by siRNA significantly decreased by 35% the extent of RLC Ser-15 phosphorylation. Thus, ZIPK may act as a cardiac RLC kinase and thereby affect contractility. ZIPK does not appear to be involved in basal RLC phosphorylation because ablation of cMLCK results in loss of RLC phosphorylation. However, ZIPK may be activated to phosphorylate RLC by upstream kinases responsive to different signaling pathways (17,20,21,23,24). Biochemically, the activity of ZIPK is regulated by several phosphorylation sites and an autoinhibitory segment (18,23,24). Thr-225 and Thr-265 are potential autophosphorylation sites, and Thr-265 may also be phosphorylated by ROCK1. The kinase that phosphorylates the Thr-180 main activation site is not known, although it appears to be agonist-dependent in smooth muscle. No information on ZIPK function or regulation in adult cardiac muscle is available at this time.

Sarcomeric Myosin RLCs Are Phosphorylated by Different Physiological Mechanisms
skMLCK is dependent on both Ca 2ϩ and calmodulin for activity (25). Upon complex formation with a peptide of the calmodulin-binding sequence of skMLCK, Ca 2ϩ /calmodulin undergoes a conformational collapse, with its two domains wrapping around the peptide through the bending of a flexible central helix (26). The calmodulin-binding sequence of skMLCK forms an amphiphilic ␣-helix.
Detailed enzymatic and structural studies of the binding interactions among Ca 2ϩ , calmodulin, and skMLCK show an ordered sequence of events that culminate in skMLCK activation (25,(27)(28)(29)(30)(31)(32). The active site of the kinase is in a catalytic cleft between the N-terminal domain that binds ATP and the larger C-terminal domain that opens and closes about a glycine hinge connecting the two domains. The autoinhibitory sequence extends from the C terminus of the catalytic core and folds back on its surface to prevent RLC but not ATP binding in the catalytic site. Results from protein fragmentation comple-FIGURE 1. Ca 2؉ -dependent phosphorylation of myosin RLC in striated muscles. A, skMLCK is inactive due to the regulatory segment containing autoinhibitory (yellow) and calmodulin-binding (red) sequences binding in the catalytic cleft between the N-and C-terminal domains of the catalytic core (green). Ca 2ϩ (F) binds to four Ca 2ϩ -binding sites in calmodulin (CaM), and the complex binds to MLCK to displace the regulatory segment from the catalytic cleft for phosphorylation of RLC (2). This simplified scheme may be more complex for cMLCK. Protein structures are discussed with references in the text. MLCP, myosin light chain phosphatase. B, striated muscle myosin heads (green) contain essential light chains (blue) and RLCs (red) with weak head-head interactions in the resting position or disordered and extended from the thick filament (49,50). Phosphorylation of striated RLC increases the mobility of myosin heads such that they move away from the thick filament surface toward actin thin filaments in skeletal and cardiac fibers. FIGURE 2. Structural elements in different protein kinases that phosphorylate RLCs. Short smMLCK contains a catalytic core (green) structurally similar to protein kinases with a contiguous regulatory segment composed of autoinhibitory (yellow) and calmodulin (CaM)-binding sequences (red). It also has three repeating actin-binding motifs (gray) as well as Ig (burnt orange) and fibronectin (blue) structural modules. Long smMLCK is identical to smMLCK with an additional N-terminal extension. skMLCK and cMLCK contain catalytic cores and regulatory segments similar but not identical to those of smMLCK or to each other. ZIPK is regulated by autophosphorylation (P) as well as phosphorylation by upstream signaling protein kinases at multiple sites in the catalytic core. ZIPK also contains a leucine zipper domain (light blue).
mentation analyses indicate that the principal autoinhibitory motif is contained within the sequence between the catalytic core and the calmodulin-binding sequence, consistent with previous results obtained with truncation mutants (33,34). In the absence of Ca 2ϩ , the kinase is autoinhibited, and calmodulin is not bound.
When Ca 2ϩ binds to calmodulin, its C-terminal domain binds to the N terminus of the calmodulin-binding sequence in skMLCK, with the subsequent binding of the N-terminal domain to the C terminus of the calmodulin-binding sequence (35,36). Ensuing calmodulin interactions with the catalytic core per se appear to be necessary for activation. The regulatory segment is subsequently displaced from the catalytic site, with calmodulin collapsed at a position near the base but adjacent to the catalytic core. The exposed catalytic site of the kinase allows the N terminus of RLC to bind, with closure of the cleft and transfer of phosphate from ATP to RLC.
Repetitive increases in cytosolic Ca 2ϩ necessary for contractile responses would also result in Ca 2ϩ binding to calmodulin and then Ca 2ϩ /calmodulin binding and activating skMLCK (25,37). Based on the known biochemical properties of skM-LCK, a quantitative model predicted 1) the importance of the interpulse interval for fractional kinase activation; 2) limiting kinase activity so that RLC was phosphorylated in seconds, not milliseconds; and 3) the much greater kinase activity relative to the phosphatase activity so that the activation of a small fraction of kinase would result in RLC phosphorylation that was sustained (38,39). Ca 2ϩ binds to troponin and calmodulin at a similar rate, but skeletal muscle force development increases at a faster rate than calmodulin binding to skMLCK (40). Thus, excitation-contraction coupling events distal to Ca 2ϩ binding to troponin occur more rapidly than the formation of the Ca 2ϩ / calmodulin-skMLCK complex. Rapid kinetic measurements indicate a diffusion-controlled bimolecular association of Ca 2ϩ /calmodulin with skMLCK (41). Furthermore, the association of Ca 2ϩ /calmodulin with skMLCK results in rapid activation without an apparent conformational latency, similar to the rapid activation of smMLCK (6). Importantly, the rate of dissociation of Ca 2ϩ from calmodulin bound to skMLCK is slow, resulting in dissociation of calmodulin from the kinase at 3 s Ϫ1 in vitro (11) and in vivo (40). This slow rate of calmodulin dissociation allows for a longer period of skMLCK activity after brief high frequency stimulation as well as between the Ca 2ϩ transients at low frequencies of contraction, and thus provides a biochemical memory by which RLC phosphorylation is sustained while muscle fibers are not contracting (Fig. 3).
Information on Ca 2ϩ /calmodulin regulation of cMLCK is limited (13,14). The kinase has a predicted high affinity calmodulin-binding sequence similar to smMLCK and skMLCK, and RLC was phosphorylated by recombinant cMLCK in a Ca 2ϩ /calmodulin-dependent manner (13). We found that cMLCK bound to calmodulin with high affinity in a Ca 2ϩ -dependent manner and that its activity was Ca 2ϩ /calmodulindependent, similar to skMLCK. 3 However, another study reported that cMLCK phosphorylated RLC similarly in the presence of EGTA or Ca 2ϩ /calmodulin (14). The reasons for the disparate results are not clear but could be related to differences in phosphorylation that affect Ca 2ϩ /calmodulin activation, similar to results obtained with a related protein kinase, titin kinase (42). Physiological studies have provided indirect evidence that basal cardiac RLC phosphorylation may be Ca 2ϩdependent. Inhibition of contraction, including removal of Ca 2ϩ from the buffer in perfused hearts, results in dephosphorylation, and restoration of contractions results in rephosphorylation of RLC (43,44). Additional biochemical investigations on the regulation of cMLCK activity are needed.
The maximal activity of cMLCK is much lower than that of skMLCK or smMLCK (12)(13)(14). If cMLCK is regulated by Ca 2ϩ / calmodulin, cMLCK would be saturated with bound Ca 2ϩ /calmodulin, with continuous contractions at high frequencies such as those found in rodent hearts (6,39,40). However, the low specific activity results in a slow turnover of phosphate in RLC (t1 ⁄ 2 ϭ 250 min), with 0.4 mol of phosphate/mol of RLC basal phosphorylation (Fig. 3) (44, 45). Thus, kinase activity may be a primary limiting factor for RLC phosphorylation (15).

RLC Phosphorylation Is Reversed by Myosin Light Chain Phosphatase
RLCs are dephosphorylated by a myosin light chain phosphatase composed of distinct subunits: a catalytic subunit of the type 1 protein phosphatase ␦ isoform (PP1c␦), one of two isoforms of myosin-binding regulatory subunits (MYPT1 or MYPT2), and a small subunit of undefined function (46).  Physiologically, RLC phosphorylation is transient in response to brief neurostimulation of smooth (red) and fast skeletal (green) muscles, whereas it is stable in response to continuous beating of the heart (blue). Neurostimulation for 1 s leads to the rapid binding of Ca 2ϩ /calmodulin to both smMLCK and skMLCK. The smMLCK activity results in rapid phosphorylation of RLC (red), which is rapidly dephosphorylated by robust myosin light chain phosphatase activity in smooth muscle. skMLCK activation also results in rapid RLC phosphorylation (green), but the low myosin light chain phosphatase activity prolongs RLC phosphorylation after skMLCK is inactivated by dissociation of Ca 2ϩ /calmodulin in skeletal muscle. Constant contraction of the heart results in a steady-state basal RLC phosphorylation (blue) due to equilibrium between cMLCK and myosin light chain phosphatase activities. If the heart does not contract for 30 min or longer, RLC is slowly dephosphorylated. Restoration of normal contractions results in a slow rephosphorylation of RLC (inset). MYPT1 and MYPT2 have several important functions, including localization, activation, and regulation of phosphatase activity. MYPT2 is expressed predominantly in striated muscles. Adenovirus-mediated gene transfer of MYPT2 and PP1c␦ into neonatal cardiac myocytes reduced RLC phosphorylation following stimulation with A23187 and blocked the angiotensin II-induced sarcomere organization in cultured cardiomyocytes (47). Transgenic mice overexpressing MYPT2 showed an increase in PP1c␦ forming the holoenzyme and decreased basal RLC phosphorylation and cardiac contractility (48).

RLC Phosphorylation Modulates Force Development in Skinned Fibers
Ca 2ϩ -dependent phosphorylation of smooth and nonmuscle myosin RLCs increases actin-activated myosin ATPase activity that supports a variety of functions in many different cells, including smooth muscle contraction and aspects of cell motility and adhesion (2,3). During relaxation of smooth muscle, dephosphorylated cross-bridges are in an "off" state due to head-head interactions (49). RLC phosphorylation disrupts the multiple weak, predominantly ionic interactions to release the cross-bridges to bind to actin. The heads of striated muscle myosins can undergo similar but much weaker interactions to produce an ordered array in relaxed muscle with unphosphorylated RLC. The head-head interaction does not switch activity off but may represent a resting position, where the weak headhead interactions do not inhibit myosin function (50). Phosphorylation of striated RLC increases the mobility of myosin cross-bridges such that they move away from the thick filament surface toward actin thin filaments in skeletal and cardiac fibers ( Fig. 1) (51-57). RLC phosphorylation increases the number of cross-bridges entering the contractile cycle by up-regulation of actin-induced phosphate release from the weakly bound actinmyosin-ADP-P i state (58). This displacement leads to a leftward shift in the force-pCa relationship, thus increasing Ca 2ϩ sensitivity of the myofilaments (Ca 2ϩ sensitization) and increasing the rate of force development by increasing cross-bridge transition to the strongly bound, force-generating state while slowing the rate of decay of the force-generating state. Interestingly, cardiac myosin-binding protein C phosphorylation by cAMP-dependent protein kinase also increases the proximity of cross-bridges to actin and thus modulates the kinetics of force development by structural mechanisms similar to RLC phosphorylation (54).

RLC Phosphorylation Modulates Contraction of Fast Skeletal Muscle
Repetitive stimulation of mammalian fast skeletal muscle causes a transitory increase in the peak force of the isometric twitch response (59). This twitch force potentiation follows a brief (1-2 s) high frequency (150 Hz) stimulation sufficient to induce a tetanus (post-tetanic potentiation), or it follows continuous low frequency (5-10 Hz) stimulation (staircase effect). Manning and Stull (60,61) noted a temporal correlation between the extent of RLC phosphorylation and potentiation of isometric twitch force. This correlation was prominent in fast, but not slow, skeletal muscles (38,40). Reduced RLC phosphorylation in slow-twitch muscles may be due to lower skMLCK and greater myosin phosphatase compared with fast skeletal muscle. In skMLCK knock-out mice, RLC is not phosphorylated in response to electrical stimulation, and the knockout is accompanied by an ablation of post-tetanic twitch force potentiation and a markedly reduced staircase effect (9). Additionally, overexpression of skMLCK in fast skeletal muscle in transgenic mice increased the rate of RLC phosphorylation and force potentiation (40). Thus, RLC phosphorylation by skM-LCK is a primary biochemical mechanism for physiological force potentiation intrinsic to fast skeletal muscle fibers.
Physiological studies in animals show that phosphorylation of RLC increases the extent of twitch force development in fast skeletal muscles fatigued by prolonged conditioning stimuli (62). RLC phosphorylation may also potentiate concentric work when isolated extensor digitorum longus muscle is rhythmically shortened and lengthened to simulate contractions in vivo (63). Furthermore, RLC phosphorylation-induced force potentiation is increased by alterations in excitation-contraction coupling noted for muscular dystrophy and aging (64).
Our understanding of the role of skMLCK and RLC phosphorylation in human skeletal muscle is limited. Post-activation potentiation in human skeletal muscle has recently received attention for its potential to affect human performance in strength and endurance exercise (65,66). Post-activation potentiation is induced by an intense voluntary contraction that increases both peak force and rate of force development during subsequent twitch contractions. The proposed mechanisms underlying post-activation potentiation are phosphorylation of myosin RLCs and increased recruitment of motor units. Post-activation potentiation may thus be induced by conditioning contractions to enhance power and performance during subsequent explosive muscle activities involving jumping, sprinting, etc.

RLC Phosphorylation Affects Force Development of Cardiac Contraction
In a beating heart under basal physiological conditions, RLC in ventricular myocytes is phosphorylated at ϳ0.4 mol of phosphate/mol of RLC in a variety of animal species, including humans (43,44,(67)(68)(69). This basal RLC phosphorylation plays a role in setting the kinetics of force development as well as the Ca 2ϩ sensitivity of force in skinned cardiac fibers, and therefore, its physiological importance is predicted (70).
Initial studies on RLC phosphorylation in ventricular myocytes examined its potential role in mediating positive inotropic effects of ␤-adrenergic agents (71)(72)(73)(74). Collectively, these studies found no significant changes in RLC phosphorylation after ␤-adrenergic stimulation for 20 -40 s in isolated perfused heart preparations. Thus, RLC phosphorylation (and its modulatory contribution to myofibrillar contraction) was not a predominant contributor to the positive inotropic response. The extent of RLC phosphorylation is determined by the opposing activities of kinases that phosphorylate RLC relative to dephosphorylation by myosin light chain phosphatase. Studies show that these reactions are much slower than observed in skeletal and smooth muscles, and thus, it has been difficult to study the physiological contributions of RLC phosphorylation to contraction in intact ventricular myocytes (43)(44)(45). Measurements of RLC phosphorylation 5 min after initiating perfusion with epinephrine resulted in a small increase similar to that observed with ischemia; however, cardiac performance was increased with epinephrine and decreased with ischemia (74). Recently, RLC phosphorylation increased in mouse hearts following dobutamine infusion for 5 min, thus suggesting a role for RLC phosphorylation in prolonged ␤-adrenergic stimulation (75). In general, these experimental approaches have not revealed specifically a function for RLC phosphorylation in physiological contractions of ventricular muscle primarily because changes in RLC phosphorylation do not occur or are modest relative to phosphorylation of other myofibrillar proteins, including troponin I and myosin-binding protein C (1, 76 -78).
Genetic approaches have provided insights into the function of RLC phosphorylation in cardiac performance. Transgenic mice overexpressing skMLCK specifically in cardiomyocytes demonstrated markedly increased phosphorylation of sarcomeric cardiac RLC and cytoplasmic nonmuscle RLC in their hearts (79). Quantitative measures of RLC phosphorylation revealed no spatial gradients, in contrast to immunostaining results from a previous study (10). No significant cardiac hypertrophy or structural abnormalities were observed up to 6 months of age. Hearts and cardiomyocytes from wild-type animals subjected to voluntary running exercise and isoproterenol treatment showed hypertrophic cardiac responses, but the responses for transgenic mice were attenuated. Thus, increased RLC phosphorylation may inhibit physiological and pathophysiological hypertrophy responses by contributing to enhanced contractile performance and efficiency.
In another model, transgenic mice overexpressed ventricular RLC that was mutated to prevent phosphorylation and that replaced endogenous phosphorylatable RLC (80). In cardiac skinned fibers from these transgenic animals, Ca 2ϩ sensitivity of force development was attenuated. Adult mice showed both atrial hypertrophy and dilatation with severe tricuspid valve insufficiency. Isolated hearts from these transgenic mice consistently developed a reduced systolic pressure and decreased contractility with an additional decrease in troponin I phosphorylation (81). Functional studies performed in vivo on the hearts of these transgenic mice showed decreases in base-line loadindependent measures of contractility and power and an increase in ejection duration (75). There was also a decrease in myosin-binding protein C and troponin I phosphorylation. Thus, RLC phosphorylation appears to be critical for normal left ventricular ejection.
Other genetic approaches in mice have focused on cMLCK and the MYPT2 regulatory subunit of myosin light chain phosphatase. Ablation of cMLCK expression did not result in any obvious developmental problems for the heart (15). The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles, with attenuation of RLC phosphorylation associated with ventricular myocyte hypertrophy, necrosis, and fibrosis. Echocardiography showed increases in left ventricular mass as well as enddiastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased RLC phosphorylation, culminating in failure in hearts lacking significant RLC phosphorylation. Ablation of cMLCK did not affect troponin I phosphorylation. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation, which is required for normal physiological cardiac performance in vivo.
Transgenic mice overexpressing MYPT2 showed a concomitant increase in endogenous PP1c␦, resulting in an increase in the myosin phosphatase holoenzyme (48). Ventricular RLC phosphorylation was reduced, resulting in left ventricular enlargement with associated impairment of contractile performance. Permeable fibers from the transgenic hearts showed decreased Ca 2ϩ sensitization of force development, and ultrastructural examination showed cardiomyocyte degeneration. Ablation of MYPT2 did not affect troponin I or phospholamban phosphorylation. Thus, MYPT2 is the primary regulatory subunit of cardiac myosin light chain phosphatase in vivo. This alternative genetic approach showed that basal RLC phosphorylation plays a significant role in modulating cardiac function.
Atrial RLC phosphorylation is also attenuated with ablation of cMLCK expression, but no apparent pathology develops as it does in ventricular muscle (15). Phosphorylation of RLC in permeable atrial fibers increases Ca 2ϩ sensitivity of force development, similar to that found in ventricular and skeletal muscle fibers (82). In electrically paced human atria, ␣-adrenergic receptor stimulation results in a prominent increase in contractile force that depends on a MLCK activity and is accompanied by an increase in RLC phosphorylation (83). Phenylephrine does not increase RLC phosphorylation in human ventricular muscle strips. Stretch also increases RLC phosphorylation in human atria by the release and actions of angiotensin II (84). Atrial RLC phosphorylation appears to be more dynamic than ventricular RLC phosphorylation.

RLC Phosphorylation May Be Involved in Heart Diseases
Familial hypertrophic cardiomyopathy is characterized by genetic changes in cardiac proteins of the sarcomere, leading to myofibrillar disarray and thickening of ventricular muscle with impairment of contractile performance (85)(86)(87). There is a correlation between decreased ventricular RLC phosphorylation and expression in mice of some RLC mutations linked to familial hypertrophic cardiomyopathy (88,89). Mutation of Glu-22 to Lys in human RLC is also associated with hypertrophic cardiomyopathy. This mutation inhibits phosphorylation and Ca 2ϩ binding to the light chain (90). Additionally, it increases Ca 2ϩ sensitivity of myofibrillar ATPase activity and force development, probably related to effects on Ca 2ϩ binding (91).
Heart failure is a complex disease resulting from many cellular changes, including changes in phosphorylation of multiple myofibrillar proteins that affect contractile responses (92). In heart failure, myofilament Ca 2ϩ desensitization, through increased troponin I phosphorylation, contributes to the negative force-frequency relation but is counteracted by frequencydependent RLC phosphorylation. Increased Ca 2ϩ responsiveness of myofibrillar contraction in end-stage failing human hearts results from a complex interplay between changes in the phosphorylation of RLC and troponin I (93). Considering that cMLCK was identified in human heart failure (14), it may play a role not only in the physiological performance of the heart but also in adaptive responses to pathophysiological stresses. Infu-sion of neuregulin, a peptide that activates ErbB receptor tyrosine kinases in cardiac myocytes, increases expression of cMLCK, with increased RLC phosphorylation associated with improved cardiac performance after myocardial infarction in rats (94). Administration of neuregulin to patients with stable chronic heart failure improves hemodynamic responses acutely and chronically (95). It is predicted that cMLCK mutations that cause a loss of kinase activity and reduced RLC phosphorylation will lead to sarcomeric dysfunction and cardiac failure. The identification of the central importance of cMLCK provides a new clinical target for discovery of its role in human cardiac pathophysiology.