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INVITED REVIEW
Department of Molecular and Cellular Pharmacology, University of Miami, Leonard M. Miller School of Medicine, Miami Florida
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ABSTRACT |
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-helical lever arm domain of the myosin head, but its function in striated muscle contraction is poorly understood. Two ELC isoforms are expressed in fast skeletal muscle, a long isoform and its NH2-terminal 
40 amino acid shorter counterpart, whereas only the long ELC is observed in the heart. Biochemical and structural studies revealed that the NH2-terminus of the long ELC can make direct contacts with actin, but the effects of the ELC on the affinity of myosin for actin, ATPase, force, and the kinetics of force generating myosin cross-bridges are inconclusive. Myosin containing the long ELC has been shown to have slower cross-bridge kinetics than myosin with the short isoform. A difference was also reported among myosins with long isoforms. Increased shortening velocity was observed in atrial compared with ventricular muscle fibers. The common findings suggest that ELC provides the fine tuning of the myosin motor function, which is regulated in an isoform and tissue-dependent manner. The functional importance of the ELC is further implicated by the discovery of ELC mutations associated with Familial Hypertrophic Cardiomyopathy. The pathological phenotypes vary in severity, but more notably, almost all ELC mutations result in sudden cardiac death at a young age. This review summarizes the functional roles of striated muscle ELC in normal healthy muscle and in disease. Transgenic animal models and phenotypic characterization of ELC-mediated remodeling of the heart are also discussed.
cross-bridge kinetics; striated muscle contraction; familial hypertrophic cardiomyopathy; sarcomeric mutations; failing heart; sudden cardiac death
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STRIATED MUSCLE MYOSIN |
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-helical heavy chain, which contains two IQ motifs (IQxxxRGxxxR) that form the attachment sites for two myosin light chains, the essential light chain (ELC) and the regulatory light chain (RLC) (Fig. 1) (20, 76). The myosin tail region consists of -helical coiled-coil structures that form the thick filaments of muscle sarcomeres (14). This review focuses on the structure and function of the myosin ELC also called the alkali light chain. Consistent with the existing nomenclature, the ELC proteins of the fast skeletal muscle are abbreviated as MLC1 and MLC3, named after their gel mobility (7), and as ELCa and ELCv, depicting the atrial or ventricular isoform of the cardiac muscle, respectively. A summary of the biochemical, molecular biological, and physiological approaches that have to date been undertaken to elucidate the fundamental role of the ELC in striated muscle contraction in health and disease is presented.
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ELC ISOFORMS |
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150 amino acid long) are expressed in smooth muscle and non-muscle systems. They are similar in sequence to the short fast skeletal MLC3. This review focuses on the ELC isoforms expressed in skeletal and cardiac (striated) muscle.
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STRUCTURAL FEATURES OF ELC |
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-helical region of the MHC (Fig. 1). Whereas the functional significance of the myosin lever arm has been well documented and described, the specific roles of ELC and/or RLC in force development and the regulation of muscle contraction have yet to be elucidated (46, 93, 103). Both light chains belong to the EF-hand family of Ca2+ binding proteins, which contain homologous helix-loop-helix regions, each composed of a 12-residue Ca2+-binding loop flanked by two perpendicular -helices (EF-hand motifs) (Figs. 1, 3, and 4) (30, 40, 41, 90). The EF-hand Ca2+ binding proteins have a high sequence homology and structural similarity, yet their diverse response to Ca2+ binding results in different cellular functions, which they perform in various biological processes (12). Although striated and molluscan muscle myosin ELC isoforms share common structural similarities with other EF-hand Ca2+ binding proteins, the ability to bind Ca2+ has only been sustained in scallop myosin ELC (13, 43). The coordination of Ca2+ is facilitated by the complex designated the "myosin regulatory domain," consisting of ELC, RLC, and a portion of the MHC (accession no. 1WDC, Fig. 4) (33, 43, 112). The ELC-MHC protein-protein interactions schematically presented in Fig. 3, have been shown to be similar not only among muscle myosins [molluscan (33, 112), chicken skeletal (76)] but also in non-muscle systems (96).
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ROLE OF ELC IN STRIATED MUSCLE CONTRACTION |
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Direct Contacts Between ELC and Actin
There is a large body of evidence indicating that during striated muscle contraction, the positively charged NH2-terminus of the long ELC isoform makes direct contact with the negatively charged COOH-terminus of actin (3, 22, 32, 51, 52, 57, 61–63, 98, 99, 102). Some of the first evidence for this ELC-actin interaction came from comparisons of three-dimensional maps of vertebrate muscle thin filaments obtained by cryo-electron microscopy and image analysis (51). The authors determined the molecular structure of F-actin and predicted potential filament surface binding sites for MHC and the NH2-terminal portion of the myosin ELC (51). Utilizing 1H NMR techniques, Timson et al. (98) revealed that the major actin binding region of ELC is located in the first four NH2-terminal amino acid residues of the long ELC isoform (Fig. 2) (98). Numerous studies, comprehensively summarized in the recent review by Timson (97), demonstrated that the ELC-actin interaction may potentially affect the function of myosin as the molecular motor by changing binding of the myosin head (S1) to actin and thereby influencing the cross-bridge cycling kinetics. The importance of the ELC in the interaction of myosin with actin was demonstrated in the study of VanBuren et al. (104), where selective removal of ELC from chicken skeletal myosin resulted in a 50% reduction of force measured in an in vitro motility assay, whereas removal of RLC did not change the level of force. Removal of either or both ELC and RLC markedly reduced actin filament sliding velocity without a significant loss in actin-activated ATPase activity (46, 104).
Function of NH2-Terminus of ELC
The importance of ELC and its NH2-terminus in the regulation of striated muscle contraction was also shown in several experiments utilizing synthetic peptides of ELC (57, 75). An increase in isometric tension generation at both submaximal and maximal Ca2+ concentrations was observed in intact and skinned human cardiac muscle fibers reconstituted with NH2-terminal ELC peptides of different length, spanning amino acids: 5–14, 5–10, and 5–8 of the human ELCv sequence (Fig. 2B) (57). The strongest effect was observed for the longest 5–14 peptide, and much higher concentrations (micromolar) of shorter ELC peptides were required to achieve the same level of activation. Control peptides of random sequence had no effect on force in these reconstituted cardiac fibers (57). Similar effects were reported by Rarick et al. (75) with a peptide spanning the NH2-terminal 5–14 amino acids of the ELCv. Addition of the peptide to rat cardiac myofibrils resulted in a supermaximal increase in the MgATPase activity at submaximal Ca2+ levels with no effect at low and maximal Ca2+ levels. A control peptide of the same length but with a random sequence produced no effect at any Ca2+ concentration (75). The authors determined that the activation of MgATPase activity by this peptide required a full complement of thin filament regulatory proteins and that the effect was highly cooperative (75).
The functional significance of the NH2-terminal region of ELC was also shown with ELC deletion mutants. Using electron microscopy and synthetic myosin filaments, Podlubnaya et al. (69) showed that removal of 13 amino acids from the NH2-terminus of rabbit skeletal and/or cardiac muscle ELC is responsible for ablation of the Ca2+-induced mobility of the myosin subfragment-2 (S2) region. The movement of myosin S2 together with the myosin head (S1) on the surface of the synthetic myosin filaments was only observed for the native compared with truncated ELC mutant proteins (68–70). Using transgenesis, Miller et al. demonstrated that a ELCv missing the NH2-terminal residues 5–14 resulted in decreased maximum isometric tension without any change in Ca2+ sensitivity measured in skinned ventricular strips that were osmotically compressed to intact lattice spacing (50). However, no alterations in either unloaded shortening or maximum shortening velocities were observed when muscle strips were not compressed (80). A recent study by Haase et al. (31) showed that the expression of NH2-terminal human ELC peptides in transgenic rats correlated positively with improvements in the intrinsic contractile state of isolated perfused hearts.
ELC-Mediated Regulation of Actin-Myosin Interactions
An important question that arises while addressing the NH2-terminal ELC-actin interactions is whether these protein-protein contacts promote or inhibit binding of the myosin head to actin and whether they affect the rate of force generating myosin cross-bridges. Earlier studies showed that fast skeletal myosin S1 containing MLC1 (long ELC) had a lower apparent Km (higher affinity) for actin and a slower turnover rate of MgATP than myosin S1 containing MLC3 (short ELC) (45, 106, 108), suggesting that intermolecular contacts between the NH2-terminus of MLC1 and actin resulted in a stronger and slower interaction of myosin cross-bridges with actin. These results were supported by affinity chromatography studies (110) and by cosedimentation studies (101) showing that myosin S1 with MLC1 does indeed have a higher affinity for actin than myosin S1 with MLC3. Even though both cardiac ELC isoforms (ELCa and ELCv) are expressed with their long NH2-termini (Fig. 2B), ELC-actin interaction studies revealed isoform-specific differences. A weaker actin-binding affinity was observed in ELCa compared with the ELCv isoform (55). This difference in actin binding was thought to result from the faster cycling kinetics of cross-bridges containing ELCa (54, 55). Conflicting results were reported by Stepkowski et al. (89), where the NH2-terminal extension in the long ELC was shown to reduce rather than increase the affinity of myosin for actin. These experiments, however, were performed by utilizing HMM, a two-headed heavy meromyosin containing both myosin light chains, ELC and RLC, suggesting that a full understanding of the ELC-mediated actin-myosin interaction requires taking into account the cooperativity between the two myosin heads as well as potential interactions between ELC and RLC (83, 89). The complexity of these protein-protein interactions was further suggested by Pliszka et al. (67), who demonstrated that the NH2-terminal region of MLC1 not only interacts with actin but also with the MHC. Despite contradictory reports, the contribution of ELC to these intermolecular protein-protein interactions leading to force generation and muscle contraction becomes obvious (97).
Effect of ELC on Myosin Cross-Bridge Kinetics
In agreement with previous studies showing a slower turnover rate of MgATP by myosin S1 with MLC1 (106), Timson et al. (98, 99) demonstrated that skeletal S1 complexed with recombinant human atrial ELC (long ELCa, Fig. 2B) had actin-activated MgATPase kinetics similar to those observed for rabbit skeletal S1 with MLC1. Deletion of the first 45 amino acid residues from the ELCa resulted in the kinetics observed for the S1 with MLC3 (99). In addition, the kinetic properties of an ELCa truncation mutant, lacking the first NH2-terminal 11 residues, were somewhat intermediate with a phenotype that was slightly closer to that observed for myosin S1 with MLC3 (99). Sweeney et al. (92) reported that exchanging MLC1 for MLC3 in skinned rabbit psoas muscle fibers altered the maximal shortening velocity and showed that increasing ratios of MLC3 to MLC1 in fibers leads to an increase in maximal shortening velocity with approximately two-fold higher values observed for MLC3-exchanged fibers over that of MLC1 (92). These studies suggest that myosins containing long ELC isoforms demonstrate slower cross-bridge kinetics than those with short ELC isoforms.
A differential effect of ELC on myosin cross-bridge kinetics was also shown to be true among myosins with long ELC isoforms. The results from in vitro motility assays utilizing purified myosin from the atria and ventricles of young rats revealed that the velocity of actin filaments was greater in atrial compared with ventricular myosin (113). As shown in Fig. 2B, both myosin isoforms, in the atria and in the ventricles, contain ELC with long NH2-termini. Interestingly, faster kinetics of myosin cross-bridges with ELCa was shown to be associated with an ELCa-mediated improvement in overall cardiac function. Studies by Morano's group indicated that maximal shortening velocity, rate of tension development, and isometric force generation all increase upon partial replacement of ELCv by ELCa in human ventricles (56, 59). Experiments utilizing transgenic mice (25) and rats (1) (discussed in detail in ELC Transgenic Animal Models) supported these earlier findings obtained from human fibers and demonstrated an overall increase in the kinetic properties of the ventricular tissue expressing ELCa. Interestingly, increased levels of ELCa expression in rat ventricles due to chronic exercise were also shown to be associated with improvement of cardiac contractility (17–19). The ELCa-mediated enhancement in cardiac function was verified by Khalina et al. (38) at the myosin level, utilizing synthetic filaments of ventricular myosin reconstituted with recombinant human ELCa (38). In these studies, replacement of the endogenous ELCv with recombinant human ELCa at various levels (12%, 24%, and 42%) resulted in an increase of the actin-activated myosin ATPase activity in a dose-dependent manner, suggesting that ELCa can indeed enhance the function of ventricular myosin (38).
To assess the mechanism of the ELC-mediated changes in the kinetics of force generating myosin cross-bridges, Borejdo et al. (9, 21) compared the extent and kinetics of the angular motion of the proximal and distal ends of the myosin lever arm domain containing both myosin light chains ELC and RLC. Fluorescent ELC and RLC were exchanged with the native light chains in rabbit psoas muscle fibers and the transition, from rigor to relaxation was measured by polarized fluorescence after photogeneration of ATP. The results indicated that interactions of ELC with actin and/or myosin heavy chain do not inhibit rotation of the proximal end of the lever arm regulatory domain and that during contraction the whole domain rotates as a rigid body (9). However, the authors did not investigate whether the short ELC would produce different kinetics of the lever arm domain than those observed with the long ELC protein. Likewise, they did not address the differences observed for atrial versus ventricular ELC isoforms.
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POSTTRANSLATIONAL MODIFICATIONS AND PHOSPHORYLATION OF ELC |
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S led to an incorporation of thiophosphate into the ELC and tropomyosin, whereas the phosphate group was incorporated into the RLC when regular ATP was utilized. Recent proteomic analysis of preconditioned rabbit ventricular myocardium supported earlier findings and identified a phosphorylated form of the ELC (5). Two amino acids, Thr69 and Ser200 of rat ELCv corresponding to Thr64 and Ser 194/195 of human ELCv, were located as potential phosphorylation sites. The ELC phosphorylation was implicated as one of the stress-mediated posttranslational modifications in the contractile apparatus of the heart (5). Interestingly, a yeast two-hybrid screen for caspase-3 interacting proteins in failing cardiomyocytes by Moretti et al. (60) identified ELCv as a target for caspase-3. The authors suggested that a caspase-3-induced proteolytic cleavage of ELCv may be responsible for alterations in myosin-actin interactions, which could lead to cardiomyocyte apoptosis (60). Recently, ELCv was also identified as a new intracellular target for matrix metalloproteinase-2 (MMP-2) following ischemic reperfusion injury in perfused rat hearts (84). Future experiments will show whether ELC phosphorylation plays a role in the MMP-2-induced degradation of stressed myocardium. |
ROLE OF ELC IN HEART DISEASE |
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Numerous studies have been performed to unravel the functional significance of the differential expression of ELCa in cardiac disease (56, 59). The dominant role of the Ca2+-calmodulin-dependent signaling pathway in the regulation of human ELCa expression was shown by Woischwill et al. (111). The expression of ELCa in compartments other than the atrium has been associated with a diseased and/or hypertrophied heart (for review see Refs 54 and 65). Patients with hypertrophic and dilated cardiomyopathies, Tetralogy of Fallot, double outlet right ventricle, and congenital heart disease expressed ELCa in their ventricles, which partially replaced the endogenous ELCv (2, 59, 78). Interestingly, this ELCa for ELCv replacement was associated with an increase in the Ca2+ sensitivity of force and increased kinetics of force generating myosin cross-bridges, indicating an ELCa-mediated improvement in overall cardiac contractility (56, 59). An elevated accumulation of the higher performing ELCa in diseased hearts is considered a compensatory response in heart failure (2, 65).
Ventricular ELC
The human ELCv binds to the 
-MHC (gene: MYH7) IQ motif between amino acid residues 781–810 (LSRIITRIQAQSRGVLARMEYKKLLERRDS) (109). Clinical studies have revealed that ELCv is one of the contractile proteins of the heart associated with familial hyperthrophic cardiomyopathy (FHC). FHC is an autosomal dominant disease characterized by left ventricular and septal hypertrophy, myofibrillar disarray, and sudden cardiac death (SCD). The latter often occurs in young athletes with no previous symptoms or warning (48, 53, 88). The overall clinical phenotype of patients with FHC is broad, ranging from a complete lack of cardiovascular symptoms to exertional dyspnea, chest pain, and SCD. FHC is caused by missense or deletion mutations in the genes that encode for major sarcomeric proteins, such as -MHC; titin; actin; -tropomyosin; troponin T, I, and C; myosin binding protein C; ventricular myosin RLC; and ELC.
To date, five mutations in the human ELCv (encoded by the MYL3 gene) have been associated with FHC: E56G, A57G, E143K, M149V, and R154H (4, 8, 23, 44, 64, 71, 77). As shown schematically in Fig. 6, the MYL3 gene is composed of 7 exons, 6 of which encode the 195 amino acid long ELCv (8, 26). The FHC-linked mutations are located in exons 3 and 4 of the ELCv gene, which encode the EF-hand Ca2+-binding motifs of the ELCv protein (Fig. 6) (8). No FHC-linked mutations were found in other functional ELCv domains, such as the NH2-terminal actin-binding domain or the proline-rich region (Fig. 6). A three-dimensional representation of all identified FHC mutations in ELCv is demonstrated in Fig. 4. It was derived from the crystal structure of the scallop regulatory domain (accession no. 1WDC) (33). On the basis of sequence comparison of scallop and human ELCv, the E56G and A57G mutations are predicted to be in the -helix of the first EF-hand domain, whereas the E143K, M149V, and R154H mutations are located in the exposed loop (E143K), -sheet COOH-terminal region of the loop (M149V), and in the exiting helix (R154H) of the third EF-hand motif of ELCv (33) (Fig. 4). As shown in Fig. 5, all FHC ELCv mutations occur in highly conserved amino acid residues. The significance of each of these mutations is further discussed.
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No clinical information is available for the Glu56Gly (E56G) mutation of ELCv. It was identified together with other FHC-linked casual mutations in the genes encoding for sarcomeric proteins of the heart (77). The study, supported by the Eurogene Heart Failure Project, analyzed 197 unrelated cases of familial or sporadic hypertrophic cardiomyopathy and found 97 mutations in all examined genes. The majority of mutations occurred in the genes encoding myosin-binding protein C (accounted for 42% of all cases) and -MHC (40% of all cases) (77). The MYL3 gene accounted for <1% of all analyzed cases, and the E56G mutation in ELCv was found in one proband. No family history of the proband was reported nor the course of the disease for the patient (77). The E56G mutation is localized in exon 3 of ELCv (Fig. 6). A comparison of various ELC sequences demonstrates that the glutamic acid in position 56 is highly conserved across species and tissues (Fig. 5).
Ala57Gly
The Ala57Gly (A57G) mutation was found in two unrelated Korean families and in one Japanese patient diagnosed with hypertrophic cardiomyopathy (44). The phenotype associated with this mutation consists of a classic asymmetric septal hypertrophy and SCD, with pathology and disease progression varying among siblings and other family members. The study screened 38 probands of unrelated Korean families and eight genes associated with FHC encoding for different sarcomeric proteins were targeted. The A57G mutation was found in 14 of 29 genotyped members of two families and was not found in over 250 unrelated and unaffected control subjects (44). Among the affected subjects, seven individuals were below the age of eighteen. The younger group showed no phenotypic manifestation of the disease, whereas the older subjects displayed disease penetrance. A proband female (42-year-old) with few symptoms was diagnosed with asymmetric septal hypertrophy at age 28. Minimal disease progression was observed based on echocardiography examination at age 34 and 42. A male sibling diagnosed with FHC at age 29 died suddenly at the age of 34. Another 38-yr-old brother carrying the mutation was also diagnosed with asymmetric septal hypertrophy but was mostly asymptomatic. Two female siblings of 29 and 39 yr of age were also asymptomatic and had normal electrocardiograms and echocardiograms. In the second family, a 28-yr-old man died suddenly with no previous symptoms of the disease. His 60-yr-old father, diagnosed with a thickened interventricular septum and left ventricular posterior wall, had a previous history of syncope. The grandfather died at age 72 after a stroke, whereas his two brothers died suddenly at an early age. Little information was available on the Japanese patient or his family members. He was diagnosed with left ventricular hypertrophy and asymmetrical septal hypertrophy at the age of 54 yr and was not related to any of the Korean families. In summary, the A57G mutation of ELCv is associated with classic asymmetric septal hypertrophy, post-adolescent phenotypic manifestation, and a high incidence of SCD at a young age. It is located in exon 3 of the ELCv gene and, together with the adjacent E56G mutation, it is predicted to be situated in the -helix of the first EF-hand Ca2+ binding motif of ELCv (Figs. 4 and 6). It is highly conserved in other ELC isoforms across species (Fig. 5).
Glu143Lys
The Glu143Lys (E143K) mutation was identified in a young proband during a cardiac evaluation administered after the premature death of his two younger siblings (64). He was found to be homozygous for the E143K mutation and demonstrated a phenotype of left ventricular hypertrophy and ECG abnormalities. An unusual variant of hypertrophic cardiomyopathy was identified, characterized by midcavity left ventricular hypertrophy with mild dynamic obstruction in systole. The right ventricular apex was also hypertrophied (64). Echocardiograms for both deceased brothers were not available, but their medical records indicated they had cardiomyopathy with dilated atria, suggesting restrictive heart disease. The proband's sister, who was heterozygous for the E143K mutation, was negative for any symptoms of cardiomyopathy and her ECG findings were normal. The same was true for the proband's parents who were also heterozygous for the E143K mutation. No history of heart disease was found in the grandparents or other relatives. Some T-wave abnormalities were reported in the paternal grandmother, who was positive for the E143K mutation but showed no FHC phenotype. One incidence of an early death (7 yr old) from unknown causes of a distant relative was reported (64). Since no intermediate phenotype was observed with this E143K mutation, it was speculated that it could be associated with recessive hypertrophic cardiomyopathy (64). The E143K mutation results in a charge change from a negative glutamic acid to a positive lysine. As is true for other ELCv mutations, E143K occurs at an evolutionarily conserved residue among species and in various ELC isoforms (Fig. 5). It is found in the exposed loop region of the third EF-hand motif of ELCv (Figs. 4 and 6) (33, 76).
Met149Val
The Met149Val (M149V) mutation of ELCv is the most investigated since it was the first identified ELCv mutation shown to cause FHC (23, 71). DNA was screened from a large number (383) of unrelated families diagnosed with hypertrophic cardiomyopathy, and the M149V mutation was found in the members of one family. Six of the 13 family members positive for the mutation demonstrated a rare phenotype of mid-left ventricular chamber thickening due to the septal and papillary muscle hypertrophy (23, 71). Studies utilizing slow skeletal muscle fibers from the affected patients showed myopathy that was also cytochrome oxidase positive, consistent with mitochondrial disease. In vitro motility assays performed on ventricular myosin isolated from cardiac biopsies of three patients with this M149V mutation showed an 40% increase in the translocation of actin filaments on a M149V myosin-coated surface compared with control myosin from healthy cardiac tissue (71). The authors suggested that the higher filament velocity could be due to an increased myosin step size, duty cycle, and/or ATPase activity. The authors further hypothesized that the stretch-activation response of the papillary muscles in M149V patients might be impaired (24, 71, 105). A phenotype of nonobstructive hypertrophy localized to the cardiac apex associated with the M149V mutation was recently reported by Arad et al. (4) in the patients of European descent. Six of 12 family members had hypertrophy localized to the apex, and 6 had prototypic asymmetrical hypertrophy. Because the morphological classification of hypertrophy as midcavitary (71) or apical (4) may in part reflect the evolution of diagnostic imaging techniques from angiography, by which midcavitary hypertrophy was historically recognized, to echocardiography and magnetic resonance imaging, these may represent overlapping morphologies. Alternatively, midventricular hypertrophy may occur as a late manifestation of apical hypertrophy complicated by apical ischemia, infarction, and aneurysmal dilation (4). A family history of the proband positive for the M149V mutation revealed heart failure deaths in two individuals (at ages 35 and 54) and sudden cardiac death in three individuals (at ages 26, 33, and 35) (4, 49). The M149V mutation is located in exon 4 of the ELCv (Fig. 6). Similar to other ELCv-mutated residues, the M149 residue is shown to be highly conserved among species and in various tissues (Fig. 5). According to the crystal structure by Houdusse et al. (1WDC) (33), the mutated methionine is located to the -sheet COOH-terminal end of the exposed loop of the third ELCv EF-hand motif (Figs. 4 and 6).
Arg154His
The Arg154His (R154H) mutation of the ELCv was identified in parallel with the M149V mutation in the study by Poetter et al. (71). Similar to M149V, this mutation also caused a midcavitary hypertrophy (23). A young boy was diagnosed with a massive mid-left ventricular chamber thickening obstruction, a phenotype that occurs sporadically in children (16, 23, 71). No more information was available on this mutation, and the family history of this young proband was not available (71). As with the other ELCv mutations, the R154 residue is highly conserved in different ELC isoforms and in different species (Fig. 5). As shown in the crystal structure of scallop myosin (1WDC) (33), this mutation is predicted to be located in the exiting helix of the third EF-hand motif of ELCv (Figs. 4 and 6).
In summary, the FHC mutations in ELCv have been shown to cause various phenotypes in humans, from asymmetric septal hypertrophy (A57G), midcavitary, or apical hypertrophy (M149V, R154H) to no symptomatic course of the disease (E143K). Most of them have been associated with SCD at a young age (A57G, E143K, and M149V). The mechanisms by which these mutations in ELCv cause various phenotypes of FHC are not known. It has been hypothesized that the M149V and R154H mutations result in a midcavity obstruction phenotype due to the interference of the mutation with the stretch-activation response in the heart (23, 24). According to this hypothesis, the papillary muscles utilize the stretch-activation response to increase oscillatory power output, and ELCv mutations are speculated to impair this response by altering the properties of the myosin filaments of the papillary and adjacent ventricular muscles (16, 23, 105).
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TRANSGENIC ANIMAL MODELS FOR ELC |
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To address the functional differences between the atrial and ELCv isoforms, Fewell et al. (25) generated transgenic mice specifically replacing ELCv with ELCa in all compartments of the murine heart (25). Functional studies utilizing skinned cardiac muscle fibers and whole hearts from these transgenic animals demonstrated an overall increase in mouse cardiac function (25). Because of the ELCv to ELCa switch, the authors observed increased unloaded velocities of actin filaments translocating over transgenic myosins in an in vitro motility assay, as well as increased unloaded shortening velocities assessed in skinned muscle fibers. Cardiac function at the whole organ level, i.e., contractility and relaxation, was also significantly increased (25). Interestingly, no hypertrophic response due to the ELCa overexpression in these transgenic mouse hearts was observed. The latter was in contrast to earlier reports on humans where increased expression of the ELCa in the ventricles paralleled an increase in cardiac function (85).
To study the functional importance of the atrial isoform of ELC, Morano's group (1) generated a transgenic rat model overexpressing human ELCa in rat hearts. Twelve-week-old transgenic rat hearts were evaluated utilizing an intact perfused heart model. A statistically significant improvement was observed in the contractile parameters of the transgenic hearts compared with age-matched controls (1). Similar analyses were carried out using mice in which ELCa was replaced with ELCv (81). Mechanical analyses performed in skinned fiber strips from left atrial trabeculae fibers showed that atrial isometric force, shortening velocity, maximum relative power, and unloaded shortening velocity were reduced (81). These results with the use of transgenic animal models generally confirmed what was observed in humans, i.e., that the expression of the ELCa isoform in the ventricles positively correlates with improved cardiac function (2, 59).
Addressing the functional significance of the NH2-terminus of ELC in the direct interaction between ELC and actin during muscle contraction, Sanbe et al. (80) generated transgenic mice expressing truncated ELCa or ELCv (80). The mutated ELCa and ELCv proteins contained a 10-amino acid deletion at positions 5–14 at their NH2-termini. Surprisingly, when the kinetics of skinned fibers isolated from the ELCa
5–14 or ELCv5–14 mice were examined, no alterations in either the unloaded shortening velocity or the maximum shortening velocity were observed. Myofibrillar ATPase activity was also unchanged in these preparations (80). However, when skinned muscle strips from ELCv5–14 mice were osmotically compressed to intact lattice spacing, a decrease in maximum isometric tension without a change in calcium sensitivity was observed (50). These results suggested that the interaction between actin and the NH2-terminus of ELC and specifically with amino acid residues 5–14 plays an important role in cardiac muscle performance (50).
Similar aspects of the ELC-actin interaction in transgenic animal models were addressed in a recent study by Hasse et al. (31). The authors investigated whether the expression of NH2-terminal peptides of cardiac ELC could improve the intrinsic contractility at the whole heart level. Transgenic rats were generated overexpressing minigenes encoding the NH2-terminal 15 amino acids of the human ELCa or ELCv isoforms in cardiomyocytes. Synthetic NH2-terminal peptides revealed specific actin binding with a significantly lower dissociation constant for the ELCv compared with the ELCa. As was demonstrated, the expression of NH2-terminal human ELC peptides in transgenic rats correlated positively with improvements of the intrinsic contractile state (force generation and relaxation) in isolated perfused hearts (31).
To date, only the M149V mutation of ELCv shown to cause FHC (71) has been overexpressed in transgenic animal models (16, 82, 105). In one approach, a transgenic mouse model expressing the human cardiac ELCv gene with the M149V mutation was generated (105). Because of the promoter utilized for the ELCv gene expression, the M149V mutation was expressed in both the heart and in the slow skeletal muscle of transgenic mice. Interestingly, the disease phenotype observed in humans carrying this M149V mutation of ELCv was recapitulated in these transgenic animals whose hearts demonstrated a profound obliteration of the left ventricular cavity, especially visible in 1- to 1.5-yr-old mice (105). Surprisingly, this abnormal morphology was subsequently lost during backcross into pure C57BL/6J, suggesting that the observed phenotype depends on the mouse strain background (16). In contrast to using the human genomic sequence, when a mouse cDNA with the M149V mutation was used to generate transgenic mice, no phenotype of hypertrophic response, even in senescent animals, could be detected (82). The latter lack of hypertrophic response in transgenic mice prompted initiation of a study where the rabbit ELCv cDNA was placed under control of the -MHC promoter for specific expression in the rabbit ventricles (35). However, similar to the transgenic mice expressing the mouse M149V-ELCv, no visible pattern of disease was observed in young or adult transgenic rabbit hearts (35).
The lack of disease phenotype observed with the last two transgenic animal models for this M149V FHC mutation supports what is now well recognized, that a disease gene does not always cross species (10, 105). Moreover, beyond the interspecies effect, the phenotype may disappear/appear when a disease gene is bred into different mouse strains (16, 24, 105). Further testing and more animal models are necessary to fully understand the role of the ELC in the normal and a diseased heart.
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CLOSING REMARKS AND QUESTIONS TO PONDER |
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Numerous studies, including those reporting conflicting results, demonstrate the significance of ELC in the regulation of cross-bridge function and the effects of direct contacts between the NH2-terminus of ELC and actin on force generation in muscle. The common findings suggest that ELC provides the fine-tuning of the myosin motor function, which is regulated in an isoform and tissue-dependent manner. Contradictions may be due to the differential sensitivities of the specific methods utilized for measurements, such as steady-state versus kinetics measurements and different systems, e.g., fibers, solution studies, single molecule interactions, etc. In the end, much more testing, possibly in various animal models, will be needed to fully understand the modulatory effect of the ELC in striated muscle contraction. Finally, there are important questions to ponder regarding the role of ELC in the diseased state of the heart. Why have almost all of the relatively few ELCv FHC mutations compared with numerous FHC mutations in other sarcomeric proteins been associated with sudden cardiac death at a young age? What are the mechanisms underlying the ELC-mediated pathology of FHC? Hopefully, future experiments with new animal models will bring answers to these questions and to the inclusive role of the myosin ELC in healthy muscle and in disease.
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FOOTNOTES |
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ABSTRACT
STRIATED MUSCLE MYOSIN
