A Single Serine in the Carboxyl Terminus of Cardiac Essential Myosin Light Chain-1 Controls Cardiomyocyte Contractility In Vivo
Although it is well known that mutations in the cardiac essential myosin light chain-1 (cmlc-1) gene can cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of cMLC-1 in the heart are only poorly understood. We have isolated the zebrafish mutant lazy susan (laz), which displays severely reduced contractility of both heart chambers. By positional cloning, we identified a nonsense mutation within the zebrafish cmlc-1 gene to be responsible for the laz phenotype, leading to expression of a carboxyl-terminally truncated cMLC-1. Whereas complete loss of cMLC-1 leads to cardiac acontractility attributable to impaired cardiac sarcomerogenesis, expression of a carboxyl-terminally truncated cMLC-1 in laz mutant hearts is sufficient for normal cardiac sarcomerogenesis but severely impairs cardiac contractility in a cell-autonomous fashion. Whereas overexpression of wild-type cMLC-1 restores contractility of laz mutant cardiomyocytes, overexpression of phosphorylation site serine 195–deficient cMLC-1 (cMLC-1S195A) does not reconstitute cardiac contractility in laz mutant cardiomyocytes. By contrast, introduction of a phosphomimetic amino acid on position 195 (cMLC-1S195D) rescues cardiomyocyte contractility, demonstrating for the first time an essential role of the carboxyl terminus and especially of serine 195 of cMLC-1 in the regulation of cardiac contractility.
Mutations in the cardiac essential myosin light chain (cMLC) have been linked for some time to human hypertrophic cardiomyopathy, the leading cause of premature death in the young.1 However, the precise in vivo structural and functional roles of essential MLCs in the heart are still not well understood. Dissecting their biological role in mammals is especially complicated by the fact that in the mammalian heart at least 2 different essential MLC isoforms (ELCv, which is expressed mainly in ventricles and slow skeletal muscle; and ELCa, which is expressed in atria and ventricles during development and restrictedly expressed in atria in the adult) are coexpressed and can at least partially compensate for each others function.2 In contrast to mice and humans, in zebrafish only 1 isoform of essential MLC is expressed in the heart. Loss of cardiac essential MLC function in mice leads to early embryonic lethality attributable to impaired mesoderm development, whereas morpholino (MO) knockdown studies in zebrafish recently revealed an essential function of cMLC-1 in cardiomyocyte sarcomerogenesis.3,4
Both essential and regulatory MLCs bind to the neck region of myosin heavy chains (MHCs) and contain calcium-binding EF-hand motifs and hence might modulate the calcium sensitivity of cross-bridge cycling, thereby fine tuning cardiac contractility. Phosphorylation of cardiac regulatory MLCs at a unique serine on position 19 by MLC kinase (MLCK) can induce conformational changes and endorse myosin–actin interaction and thus enhance cardiomyocyte contraction.5,6 This crucial serine residue is not present in essential MLCs and accordingly essential MLCs are commonly thought to play a rather limited role in modulating cardiomyocyte contraction. However, in recent studies phosphorylation of essential MLCs at threonine 64 and serine 195 could be detected in rat and rabbit cardiomyocytes.7,8 Furthermore, essential MLCs contain a unique NH2-terminal domain that can directly bind actin, potentially enabling essential MLCs to modulate actin–myosin cross-bridge cycling via a mechanism distinct from regulatory MLCs.9–11 Interestingly, cardiomyocyte-specific overexpression of the NH2 terminus of human ELCa in rats leads to enhanced cardiac contractility.12,13 Similarly, in several in vitro studies both, synthetic peptides or DNA constructs of the NH2 terminus of ELCa are able to significantly enhance myofilament force generation.14
We sought here the molecular cause of the ethylnitrosourea (ENU)-induced recessive embryonic-lethal zebrafish mutant lazy susan (lazm647), which displays severely reduced contractility of both heart chambers. We identified a nonsense mutation within the zebrafish cmlc-1 gene to be responsible for the laz phenotype. The carboxyl-terminally truncated cMLC-1 in laz mutant hearts is still sufficient for normal cardiac sarcomerogenesis but leads to severe impairment of cardiac contractility in a cell-autonomous fashion. Overexpression of phosphorylation site–deficient cMLC-1S195A in laz mutant cardiomyocytes does not reconstitute cardiomyocyte contractility, whereas overexpression of wild-type cMLC-1 or phosphomimetic cMLC-1S195D rescues cardiomyocyte contractility of laz mutant embryos. Accordingly, dissection of the molecular cause of laz reveals an important role of the carboxyl terminus and especially of the phosphorylation site S195 of cMLC-1 in the regulation of cardiac contractility in vivo.
Materials and Methods
Genetic mapping; positional cloning; RNA antisense in situ hybridization; immunostaining; MO, mRNA, and plasmid injection procedures15; functional assessment; protein structure prediction; and statistical analysis are described in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
Laz Is Required for Embryonic Heart Contractility
Lazm647 is an ENU-induced, fully penetrant embryonic lethal recessive mutation in zebrafish that selectively perturbs cardiac contractility during early embryonic development. Similar to other zebrafish mutants with impaired cardiac contractility, laz mutant embryos develop progressive pericardial edema. Blood flow is never established in homozygous laz mutant embryos. Beside the cardiac-specific phenotype, development of other organ systems appears to be unaffected by the laz mutation and the absence of blood circulation (Figure 1A and 1B).
Laz mutants form an apparently morphologically normal heart, in anatomic correct position with defined endocardial and myocardial layers. Early steps of cardiac development proceed normally with the heart tube moved to the left and subsequent looping of the ventricle. By 36 hours postfertilization (hpf), the 2 heart chambers (atrium and ventricle) of laz mutants are distinct and separated by the atrioventricular ring. At 72 hpf, similar to the wild-type situation, in laz mutants, cardiac MHCs are found to be expressed in a chamber-specific pattern (Figure 1I and 1J). By 72 hpf, appropriate growth of the ventricular myocardium can be observed in laz mutant hearts (Figure 1G and 1H). However, in contrast to wild-type embryos, in which by 24 hpf vigorous peristaltic contraction waves traverse the heart tube, in laz mutants these contraction waves are very weak. By 48 hpf, although sequential and rhythmic contractions of first the atrium and then the ventricle can be observed in the properly developed laz hearts, cardiac force remains severely reduced (Figure 1C and 1D; supplemental movie I). By 48 hpf, fractional shortening (FS) of laz atria (laz, 19.8±6.6%; versus wild type, 45.4±9.9%; P<0.005) and ventricles (laz, 5.0±5.7%; versus wild type, 38.6±6.8%; P<0.00005) is strongly reduced compared to wild types. By 72 hpf, a further decline in systolic function of laz mutant hearts can be observed (Figure 1E and 1F). Heart rates are not significantly different from wild-type embryos at various developmental stages examined (data not shown).
Heterozygous laz mutant embryos show no obvious phenotype, and have a normal lifespan and reproductive cycle. At 72 hpf, FS is not altered in heterozygous laz mutant embryos in comparison to wild-type littermates excluding a dominant-negative effect of the laz mutation in heterozygous zebrafish embryos (FS: laz+/+, 36.1±3.7%; versus laz+/−, 36.3±3.5%; n=7; P=0.94; Figure I in the online data supplement).
The Laz Locus Encodes Zebrafish cMLC-1
We identified the ENU-induced mutation causing the recessive laz mutant phenotype by a positional walk (Figure 2A). The laz mutation maps to zebrafish chromosome 5 between microsatellite markers z3804 and z51132. By analyzing 796 laz mutant meiotic events and using custom-designed genetic markers, we identified on BAC DKEY-43F11 the polymorphic markers CL171 and CL173, which display no recombination events. The genomic interval between markers CL178 and CL124 contains 5 open reading frames: the amino terminus of a gene homologous to human PAPPA (pregnancy-associated plasma protein-A) (XM_001919080), cmlc-1 (NM_131692), a gene homologous to human AK5 (adenylate kinase-5) (NM_001017540), a gene predicted as pol polyprotein (XM_001343578), and edf1 (endothelin-related factor-1) (NM_200745). By subsequent sequence analysis of these open reading frames, we identified within laz mutant cDNA a cytosine-to-adenine substitution at nucleotide position 558 of zcmlc-1 predicted to lead to premature termination of translation at amino acid position 186 and truncation of zebrafish cMLC-1 by 11 aa (Figure 2B). No mutations were identified within the remaining open reading frames.
Zebrafish cmlc-1 is encoded by 1 exon. Conceptual translation of cDNA sequences reveals high homology (≈77%) to the human and mouse atrial essential MLC-1 protein (Figure 2C), which is found to be expressed throughout the embryonic heart but becomes restricted to the atrial chamber during adulthood of mice and humans.16 To define the spatial and temporal expression pattern of laz, we generated a RNA antisense probe against cmlc-1 and analyzed different zebrafish embryonic and larvae stages for laz transcript occurrence and distribution (Figure 3A through 3D). cmlc-1 expression is first detectable at approximately 16 somites, where bilaterally located cardiac progenitors of the anterior lateral plate mesoderm start to fuse at the embryonic midline.16 At 24 hpf, when cardiac activity in the heart tube is initiated, cmlc-1 expression is specifically detectable in the heart tube. cmlc-1 remains highly expressed in both heart chambers from 48 to 96 hpf and the adult zebrafish heart. Hence, similar to the zebrafish cardiac regulatory MLC-2 and in contrast to the mammalian situation, cMLC-1 in zebrafish remains expressed in both heart chambers throughout embryonic development and in adulthood.
The Laz Mutation Does Not Interfere With cMLC-1 Protein Stability
As shown above, laz mutants carry a nonsense mutation in cmlc-1 predicted to lead to carboxyl-terminal truncation of cMLC-1. Hence, to evaluate whether the laz mutation leads to nonsense-mediated cmlc-1 mRNA decay, similar to the situation observed for cmlc-2 in zebrafish tell tale heart mutants, we assayed cmlc-1 transcript levels in laz mutant hearts by whole-mount in situ hybridization.17 As shown in Figure 3D and 3E, by 72 hpf, equal amounts of cmlc-1 mRNA are present in both wild-type and homozygous laz mutant hearts, indicating that the laz nonsense mutation does not induce decay of the mutated RNA. To further test whether the predicted truncated protein in laz mutants is translated and stable and thereby might still fulfill some of cMLC-1 essential functions, we generated NH2-V5-tagged wild-type and laz mutant cMLC-1 constructs. After in vitro translation and immunoblotting, both cMLC-1wt and cMLC-1186Stop protein variants were detectable at comparable levels demonstrating that despite truncation by 11 aa, cMLC-1186Stop mutant protein remains stable (Figure 3F).
cmlc-1 mRNA Partially Restores Cardiac Function in Laz Mutants
To evaluate whether the laz mutant phenotype is indeed induced by carboxyl-terminal truncation of cMLC-1, we first tested whether capped mRNA encoding full-length cMLC-1 can suppress the laz phenotype. To do so, homozygous laz mutant embryos (laz−/−) derived from intercrossing heterozygous zebrafish were injected at the 1-cell stage with mRNA encoding wild-type cmlc-1. After injection of 1.2 ng of wild-type cmlc-1 mRNA 38.7±0.8% of genotypically homozygous laz mutant embryos (n=48) showed partially rescued heart function with improved atrial and ventricular chamber contractility and blood circulation through the vascular bed, whereas injection of 1.2 ng of laz mutant cmlc-1 mRNA neither rescued cardiac contractility nor blood circulation in laz homozygous mutant embryos (Figure 4A). These findings clearly indicate that the carboxyl-terminal truncation of cMLC-1 accounts for cardiac hypocontractility in laz mutants.
Overexpression of Laz Mutant cMLC-1 Impairs Cardiomyocyte Contractility
To further evaluate whether laz mutant cMLC-1 can compete with wild-type cMLC-1 in cardiomyocytes and thereby impair sarcomere function, we injected either wild-type (cMLC-1wt) or laz mutant (cMLC-1laz) cmlc-1 under control of the zebrafish cardiac cmlc-2 promoter and simultaneously expressing an independent cmlc-2–driven enhanced green fluorescent protein (GFP) reporter (cmlc-2-GFP) into wild-type zebrafish embryos and assayed the effect of these constructs on cardiomyocyte contractility by measuring FS of GFP-positive ventricular cardiomyocytes. cMLC-1laz overexpression significantly impairs contractility of wild-type cardiomyocytes (FS, 17.75±7.65%; n=12; P<0.05) resembling a situation comparable to homozygous laz mutants, whereas overexpression of cMLC-1wt does not change cardiomyocyte contractility (FS, 34.13± 18.56%; n=8; Figure 4B). Hence, exaggerated amounts of laz mutant cMLC-1 replace wild-type cMLC-1 in sarcomeres, thereby impairing cardiomyocyte function similar to the situation in homozygous laz mutant embryos.
The Laz Mutation Does Not Interfere With Cardiomyocyte Thick Filament Assembly
Knockdown of zebrafish cMLC-1 was recently shown to induce cardiac acontractility in zebrafish embryos as a result of disturbed sarcomerogenesis.4 As shown, the laz mutation leads to a rather discrete carboxyl-terminal truncation of 11 aa of cMLC-1 but also severely impairs cardiac contractility. Hence, to evaluate whether the essential function of cMLC-1 in thick filament assembly is primarily mediated by its carboxyl terminus and reduced cardiac contractility in laz mutants is attributable to disturbed cardiomyocyte sarcomerogenesis, we next analyzed ultrastructure in laz homozygous mutant hearts in comparison to wild-type littermates and cMLC-1 morphants, as well as MO control–injected zebrafish embryos (Figure 4D through 4G).
By 72 hpf, atrial and ventricular cardiomyocytes of wild-type littermates and control-injected zebrafish embryos contain highly organized sarcomeres with thin and thick myofilaments in well-aligned bundles and discernible A-, I-, and Z-bands (Figures 4F and 5⇓A). By contrast, in cardiomyocytes of MO-cmlc-1–injected embryos, organized sarcomeric units are completely absent, both in the ventricle and the atrium, and only an immature assembly of thin filament actin (z-bodies) can be observed (Figure 4G).17
Interestingly, in contrast to cMLC-1 morphants, in laz−/− hearts, mature and well-defined sarcomeric units are present in appropriate numbers in both the ventricle and the atrium. Similar to the situation in wild types, by 72 hpf, laz mutant cardiac sarcomeric units are appropriately elongated and laterally maturated, displaying well-organized bundles of overlapping thick and thin filaments, which are interconnected at the sarcomeric Z-disc (Figure 5A and 5B). Accordingly, the cardiac thin filament proteins α-tropomyosin (Figure 5C and 5D) and troponin T (data not shown) and the thick filament protein myosin are expressed at normal levels and localization in laz mutant hearts. In summary, the laz mutation does not abolish all cMLC-1 biological functions, such as coordination of thick filament assembly, but rather seems to interfere with a novel role of cMLC-1, namely the modulation of cardiomyocyte contractility by its carboxyl terminus via an as yet undefined mechanism.
The Carboxyl Terminus of cMLC-1 Is Crucial for Modulating Cardiomyocyte Force Generation
Recently, it was postulated that both the amino terminus, as well as a potential threonine and serine phosphorylation site, of cMLC-1 might modulate myosin–actin cross-bridge cycling.8,14 Interestingly, the carboxyl-terminal serine of cMLC-1 at amino acid position 195 is highly conserved between human, mouse, zebrafish, frog, and chick and abolished by the laz nonsense mutation (Figures 6A and 7⇓A). Because laz mutant cardiomyocytes did not show any sarcomeric abnormalities but severely impaired contractile force, we wondered whether only the loss of this carboxyl-terminal phosphorylation site might account for the laz phenotype.
Hence, we first generated a phosphorylation site–deficient cMLC-1 by substituting the evolutionary highly conserved amino acid serine 195 by alanine. Furthermore, we made constructs in which we exchanged serine 195 by aspartic acid, mimicking serine in its phosphorylated form. By computational analysis based on x-ray crystal structures of the chicken MLC-1, we obtained 3D models of wild-type and laz mutant cMLC-1 protein structures. As shown, neither the carboxyl-terminal truncation of cMLC-1 nor the replacement of S195 with either alanine or aspartic acid affect the main domain structure of zebrafish cMLC-1, such as the NH2-terminal globular domain, the central linker domain, and the COOH-terminal globular domain (Figure 6).
We then injected either wild-type (cMLC-1wt), laz mutant (cMLC-1laz), phosphorylation site–deficient (cMLC-1S195A), or phosphomimetic (cMLC-1S195D) cmlc-1 under control of the zebrafish cardiac cmlc-2 promoter, simultaneously expressing an independent cmlc-2-driven enhanced GFP reporter (Figure 7A) into homozygous laz mutants and assayed the effect of the various constructs on laz cardiomyocyte contractility by measuring FS of GFP-positive ventricular cardiomyocytes (Figure 7E).18 Overexpression of cMLC-1wt restores contractility of homozygous laz mutant cardiomyocytes to almost normal values in a cell-autonomous fashion (FS of GFP-positive cells, 29±15.14%; n=6). By contrast, overexpression of laz mutant cMLC-1laz does not restore cardiomyocyte contractility in laz mutant hearts by 60 hpf (FS, 11.2±7.79%; n=5; P<0.05). Similarly, overexpression of the phosphorylation site–deficient cMLC-1S195A does not improve contractility of laz−/− ventricular cardiomyocytes (FS, 9±7.48%; n=5; P<0.05). To further underline the importance of the phosphorylation site S195 in cMLC-1, we next analyzed cardiomyocyte contractility after introducing a phosphomimetic mutation into cMLC-1 by substituting serine 195 with aspartic acid (S195D). In contrast to laz mutant or phosphorylation site–deficient cMLC-1, overexpression of cMLC-1S195D significantly enhances contractility (FS, 28.67±4.04%; n=3; P<0.05) of laz−/− cardiomyocytes (Figure 7F through 7I).
These findings demonstrate that S195 of zebrafish cMLC-1 by itself is essential to restore laz mutant cardiomyocyte contractility and solely the ablation of this critical serine phosphorylation site by the laz mutation accounts for severe impairment of cardiac contractility in laz mutants. In summary, thorough characterization of laz mutant zebrafish reveals an essential role of serine 195 of cMLC-1 in maintaining vertebrate cardiomyocyte contractility in vivo.
Dissection of molecular mechanisms that allow the heart to adapt its force of contraction to various hemodynamic needs poses many challenges for in vivo studies, because mammalian development largely depends on intact heart function early on.19 Assessing embryonic heart function is facilitated in zebrafish embryos, because they are transparent and not dependent on intact cardiovascular function during the first 7 days of development.
Here we show, for the first time, that cardiac essential MLC-1 is indispensable to maintain cardiac contractility in the zebrafish embryonic heart independent of its role in cardiomyocyte sarcomerogenesis. In a forward genetic screen, we isolated the recessive lethal mutation in the zebrafish cmlc-1 gene lazy susan (laz).20 The laz mutation leads to expression of a carboxyl-terminally truncated cMLC-1 but does not affect cardiac myofibrillogenesis and sarcomere formation. However, heart contractility in laz mutant embryos is severely reduced. Overexpression of phosphorylation site–deficient cMLC-1 does not reconstitute cardiac contractility in laz mutants, whereas introduction of a charge mutation is able to preserve contractile force, demonstrating an essential role of the carboxyl terminus and especially of serine 195 of cMLC-1 in the regulation of vertebrate heart contractility.
Crystal structure analyses of cardiac myofilaments map the position of MLCs to the neck region of MHCs, implicating an imperative role of MLCs in modulating myosin function and actin–myosin cross-bridge cycling. However, in contrast to smooth muscle cells, where contraction is initiated by phosphorylation of regulatory MLCs by the MLCK, in cardiomyocytes, contraction is mainly dependent on Ca2+-troponin/α-tropomyosin interaction, and MLCs are thought to play a rather ancillary role. Nevertheless, it was found that phosphorylation of regulatory MLCs by a cardiac-specific MLCK can at least fine tune sarcomeric force generation and contractility of cardiomyocytes.6 Until now, the exact role of essential MLCs in cardiomyocyte force generation has not been well elucidated. Deletion of the mlc1/3 gene enhancer in mice results in early embryonic lethality caused by impaired mesoderm development, constraining the dissection of a potential regulatory role of cMLC-1 on cardiac contractility in the vertebrate heart. By contrast, MO knockdown of cMLC-1 in zebrafish does not impair early mesoderm development but selectively restrains sarcomerogenesis in cardiomyocytes.4 The laz mutation described here does not abolish all of cMLC-1 functions but rather leads to the expression of a carboxyl-terminally truncated cMLC-1, which only lacks 11 aa. Laz mutant zebrafish show neither abnormal mesoderm development nor impaired cardiac sarcomerogenesis; however, cardiac contractility is severely reduced. Hence, laz reveals a modulatory role of cMLC-1 on cardiomyocyte contractility in vivo which appears to be largely dependent on the carboxyl terminus and especially serine 195 of cMLC-1.
Besides our findings, a regulatory effect of cMLC-1 on cardiac contractility has been postulated previously, especially for its NH2 terminus. In the failing human heart atrial essential MLC, which expression is usually restricted to the atrium, becomes expressed in the ventricle.21,22 Accordingly, it was shown that 72% of patients with end-stage ischemic or dilative cardiomyopathy express significant amounts of atrial MLC-1 in the ventricular myocardium and that the amount of the atrial isoform directly correlates with Ca2+ responsiveness and force development of ventricular cardiomyocytes.22 The resulting changes in myosin–actin cross-bridge kinetics and positive inotropic effect is thought to be mainly mediated by the NH2 terminus of atrial MLC-1, which differs significantly from the ventricular isoform of cMLC-1.9,23,11 These findings are further supported by studies of transgenic mouse models and in vitro experiments, where the introduction of short NH2-terminal peptides of cMLC-1 led to improvement in cardiomyocyte contractility.13,14 The mutation in laz leads to a carboxyl-terminal truncation and has no direct effect on the NH2 terminus. Although the mutated protein is stably expressed and sufficient for normal sarcomerogenesis, cardiac contractility is severely impaired, pointing to an essential function of the carboxyl terminus of cMLC-1 in modifying contractility of the vertebrate heart. In this context, it is interesting to note that cMLC-1 was recently found to carry a noncanonical cleavage site for caspase-3, an enzyme for which expression is known to be upregulated in failing myocardium. Caspase-3 cleavage of MLC-1 at the carboxyl-terminal glutaminic acid 135 in vivo is associated with significant reduction in cardiomyocyte contractility and hence may contribute to depression of cardiomyocyte function by altering sarcomeric structure and myosin–actin cross-bridge kinetics.24
Besides the importance of the NH2- and carboxyl-terminal regions of cMLC-1, it was recently speculated that similar to regulatory MLCs, phosphorylation of essential cMLC-1 at residues threonine 64 and serine 195 could equally modify actin–myosin cross-bridge cycling and hence regulate cardiac contractility. In an in vitro study, incorporation of radiolabeled thiophosphate in cMLC-1 could be shown in porcine cardiac muscle.25 In 2 more recent publications, single and double phosphorylated forms of cMLC-1 were isolated from rat and rabbit cardiomyocytes.7,8 The corresponding phosphorylation sites were mapped to threonine 64 and serine 200 of rat cMLC-1, which corresponds to threonine 64 and serine 194 or serine 195 in human cMLC-1.7 Both residues are located in exposed regions of cMLC-1 near the lever arm of MHC and therefore might directly influence MHC function. Nevertheless, the functional consequences of cMLC-1 phosphorylation remained unknown, and although MLCK was shown to be capable to thiophosphorylate cMLC-1 in vitro, the kinase that mediates MLC-1 phosphorylation in vivo has not been identified to date. The here presented zebrafish mutant laz reveals a fundamental functional role of the essential cMLC-1 in the regulation of cardiac contractility. As shown here, solely the ablation of the putative phosphorylation site S195 of cMLC-1 accounts for severely diminished cardiac contractility in laz mutants, pointing toward an essential role of cMLC-1 phosphorylation in modulating actin–myosin cross-bridge cycling in the vertebrate heart. The molecular mechanism by which carboxyl-terminal phosphorylation of cMLC-1 modifies myofilament cross-bridge cycling remains subject to speculation. Based on structural analysis and antibody competition experiments against different domains of MLC-1, a recently published study suggests that the carboxyl terminus of MLC-1 indeed can modulate myosin–ATPase activity.26 Hence, phosphorylation of cMLC-1 might either induce minute conformational changes in cMLC-1 or affect protein–protein interactions, thereby influencing myosin–ATPase activity or actin–myosin interplay.
We recently demonstrated that the zebrafish mutant tel carries a mutation within regulatory cMLC-2.24 Ultrastructural analysis revealed complete absence of organized sarcomeres in mutant cardiomyocytes. Similar to tel mutants, complete loss of cMLC-1 function in zebrafish also leads to severe heart failure through disturbance of sarcomere assembly.5 By contrast, laz mutants are devoid of sarcomeric defects, and reduction of cardiac contractility rather is attributable to loss of carboxyl-terminal amino acid residues of cMLC-1, especially phosphorylation site S195.
Mutations in cardiac essential and regulatory MLCs have been identified as genetic causes of human hypertrophic cardiomyopathy.1 Besides myofibrillar disarray, cardiomyocyte hypertrophy is among the hallmarks of hypertrophic cardiomyopathy. The exact pathophysiology of how mutations in sarcomeric protein lead to cardiac hypertrophy is not well understood. It is speculated that disturbed calcium affinity, altered phosphorylation, or insufficient force generation leads to activation of the hypertrophic gene programs. As shown here, in zebrafish, complete loss of cMLC-1 function leads to disturbance of myofibrillar assembly and reduction of cardiac contractility, 2 mechanisms thought to play a key role in human cardiomyopathies.4 By contrast, laz mutant hearts show regular sarcomeric structures, but cardiac contractility is severely reduced because of carboxyl-terminal truncation of cMLC-1 and the loss of the phosphorylation site S195. Interestingly, none of the mutations in cMLC-1 identified to date that were found to be associated with human hypertrophic cardiomyopathy resides within the carboxyl terminus or affect cMLC-1 phosphorylation. Hence, in future studies, it will be interesting to dissect the impact of carboxyl-terminal cMLC-1 mutations in human heart diseases and to evaluate whether targeted modification of cMLC-1 phosphorylation at serine 195 might be a new therapeutic approach to human cardiomyopathies and heart failure.
We thank J. Rudloff, S. Kolb, S. Weber, and H. Hosser for excellent technical assistance.
Sources of Funding
This work was supported by Deutsche Forschungsgemeinschaft grants Ro2173/1-1, Ro2173/2-1, Ro2173/2-2, and Ro2173/3-1; Bundesministerium für Bildung und Forschung grants 01GS0108, 01GS0420, and 01GS0836 (NGFNplus); and Klaus-Georg and Sigrid Hengstberger Stipendium.
↵*These authors contributed equally to this work.
Original received September 8, 2008; revision received December 30, 2008; accepted January 8, 2009.
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