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Cardiovascular Research Advance Access first published online on March 14, 2008
This version [Corrected Proof] published online on April 9, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn073
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms

Zhenyue Chen1,2, Wei Huang2, Tillman Dahme3, Wolfgang Rottbauer3, Michael J. Ackerman4,5,6 and Xiaolei Xu2,*

1 Department of Cardiology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
2 Department of Biochemistry and Molecular Biology, Division of Cardiovascular Diseases, Mayo Clinic College of Medicine, Stabile 4-10, 200 1st Street SW, Rochester, MN 55905, USA
3 Department of Medicine III, University of Heidelberg, Heidelberg, Germany
4 Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN, USA
5 Department of Pediatrics, Mayo Clinic College of Medicine, Rochester, MN, USA
6 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, MN, USA

* Corresponding author. Tel: +1 507 284 0685; fax: +1 507 538 6418. E-mail address: xu.xiaolei{at}mayo.edu

Received 29 August 2007; revised 14 February 2008; accepted 11 March 2008

Time for primary review: 42 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Mutations in the essential myosin light chain (ELC) and regulatory myosin light chain (RLC) genes have been linked to sarcomeric hypertrophic cardiomyopathies in humans; however, the specific functions of the different myosin light chains during cardiogenesis in a vertebrate animal are not well understood.

Methods and results: Using zebrafish (Danio rerio) as a model organism, we have identified cmlc1 and cmlc2 as the main ELC and RLC orthologues, respectively, and have furthermore characterized their functions during cardiogenesis by morpholino technology. Depletion of either cmlc1 or cmlc2 using morpholino-modified antisense oligonucleotides leads to a disruption in sarcomere structure and compromises cardiac function as well, although through seemingly distinct mechanisms. While myosin still assembles into a novel rod-like structure in both morphants, the sarcomere length is longer in cmlc1 morphants than that in wild-type embryos, whereas it is shorter in cmlc2 morphants. In addition, cardiomyocyte size and number are increased upon depletion of cmlc1, resulting in a larger ventricular chamber volume; in contrast, depletion of cmlc2 leads to a reduction in cardiomyocyte size and number.

Conclusion: Our data have elucidated distinct roles for cmlc1 and cmlc2 during zebrafish cardiogenesis, suggesting that cardiomyopathies resulting from human mutations in ELCs vs. RLCs may have distinct pathological characteristics during disease progression.

KEYWORDS Cardiomyopathy; Contractile apparatus; Contractile function; Hypertrophy; Ventricular function


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Muscle myosin is a hexamer that consists of two myosin heavy chains (MHCs), two regulatory light chains (RLCs) and two essential light chains (ELCs).1 There are three different domains in the MHC: (i) an N-terminal motor domain, which interacts with actin and hydrolyzes MgATP; (ii) a C-terminal tail domain consisting of {alpha}-helical coiled-coil motifs that aid in oligomerization and the formation of bipolar myosin filaments; and (iii) a middle ‘lever arm’ domain consisting of multiple IQ motifs (IQxxxRGxxxR) that differentially bind the ELCs and RLCs.2 Mutations in genes encoding for both ELCs and RLCs have been causally linked to ~1% of human cardiomyopathies.3,4 Thus, it is of great interest and importance to understand the functions of these different myosin light chains during normal cardiogenesis and cardiomyocyte remodelling as well as how mutation of the ELCs and RLCs might lead to cardiomyopathies.

Multiple genes encoding for different ELC and RLC family members (MYLs) have been identified in mammals, and in addition, these different isoforms exhibit distinct expression profiles.2,4 Within the ELC family, MYL3 (ELCv) is mainly detected in ventricles and slow skeletal muscle; MYL4 (ELCa) in the adult atrium and foetal atria, ventricle and skeletal muscle; MYL1 in fast skeletal muscle; and MYL6 in smooth muscle. Within the RLC family, MYL2 (MLC2v or RLCv) is expressed in the ventricle; MYL7 (MLC2a or RLCa) in the atrium; MYL5 in skeletal muscle; and MYL9 in smooth muscle. Recent studies using in vitro biochemical systems and in vivo transgenic models have suggested that MYLs are involved in force development during muscle contraction.2,5,6 As the ELCs and RLCs contain two Ca2+-binding EF-hand motifs, the different MYL isoforms may modulate the Ca2+ sensitivity of force generation and cross-bridge kinetics, thereby fine tuning cardiac contractility.7

A key distinguishing factor between ELCs and RLCs is the Ca2+-mediated regulation of RLCs through phosphorylation of a serine residue in an N-terminal peptide sequence present in RLCs but not ELCs. The Ca2+-binding protein calmodulin senses the Ca2+ concentration and, when appropriate, binds to and activates myosin light chain kinase (MLCK). Subsequent serine phosphorylation of myosin bound RLCs by MLCK then causes the myosin molecule to undergo a dramatic conformational change from a compact, folded form to an elongated form. Indeed, the contraction in smooth muscles is mainly modulated by the phosphorylation of RLCs.8,9 In contrast to the RLCs, most cardiac-specific ELCs contain a unique N-terminal domain that can bind actin, allowing ELCs to contribute to cross-bridge kinetics.10

Loss-of-function studies have been conducted for different RLCs in several model organisms. In agreement with the link between the mutations in RLCs and human cardiomyopathies, genetic analysis of RLC (MLC2a) in mice indicated that this RLC isoform is required for myofibril organization and atrial beating.11 Using another vertebrate model organism, electron microscopy analysis of a zebrafish RLC mutant (cmlc2) provided further support that RLCs are required for cardiac function and organized thick filament assembly.12 Although cardiomyopathies have also been linked to mutations in ELCs, loss-of-function studies analysing potential cardiac-specific roles of ELCs have not been reported.

Here, we have used zebrafish to investigate the specific functions of the two main ELC and RLC orthologues in heart development. By examining the expression pattern of the 12 zebrafish MYL orthologues, we identified cmlc1 and cmlc2 as the major ELC and RLC orthologues, respectively, that are expressed in the embryonic heart. Importantly, analysis of cmlc1 and cmlc2 morphants revealed that these two myosin light chains are both required for sarcomere assembly but serve distinct roles in modulating cardiac contractility, cardiomyocyte size and number during cardiogenesis. Thus, these data indicate a link between sarcomere structure, and the geometry and growth of cardiomyocytes, a potentially important aspect in the cardiac remodelling process during sarcomere-based cardiac diseases such as hypertrophic and dilated cardiomyopathy. In addition, our results suggest that mutations in ELCs and RLCs may result in cardiomyopathies with distinct pathological characteristics.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Bioinformatics
The human MYL4 peptide sequence was used to search the NCBI database, and the nucleotide and amino acid sequences for the zebrafish and human orthologues were aligned by ClustalW. The phylogeny tree was generated using DS Gene (Accelrys Inc.) based on nucleotide sequence identity.

2.2 Injection of morpholinos
Morpholino antisense oligonucleotides (morpholinos) were purchased from Gene Tools LLC. Morpholinos were prepared and injected as previously described.13 The sequences are as follows. The five nucleotides that are changed in MO-cmlc1Ct are underlined.

MO-cmlc1Ct: 5'-TGCGTTGCACTTTATTTGCTGCGAT-3'
MO-cmlc1 (targets translation start site): 5'-TGGGTTCCACTTTTTTTGGTGCCAT-3'
MO-cmlc2 (targets splice donor of exon 3): 5'-CTCAAGTGTACCTAGTTGTGCATAA-3'

2.3 Cardiac function analysis
Movies of beating hearts from embryos at 48 h post-fertilization (hpf) were recorded using a Zeiss microscope equipped with a Nikon camera. Images from movies were then used to measure the long axis length (a) and short axis length (b) between the myocardial borders of ventricles at diastole and systole, respectively. The percent shortening fraction (SF) was calculated using the formula: SF = (length at diastole – length at systole)/(length at diastole) x 100. End systolic or diastolic volumes were calculated using the formula: volume = 4/3Lcyab2. To measure the heart rate, the number of sequential heart contractions in a 15 s interval was counted.

2.4 Antibody staining
Whole-mount immunofluorescence staining was performed as previously described.14 The following antibodies were used at the indicated dilutions: anti-sarcomeric {alpha}-actinin (clone EA53; Sigma) at 1:1000, F59 at 1:10 and Zn5 at 1:500. Following staining, hearts were dissected using forceps and imaged using an AxioplanII Zeiss microscope equipped with an Apotome.

2.5 Quantification of cardiomyocyte cell size
The cell junctions of cardiomyocytes were labelled using the Zn5 antibody, which recognizes neurolin/DM-GRASP, a surface adhesin molecule,15 and 5–10 images were acquired as a Z-stack for each heart sample using an AxioplanII Zeiss microscope. The images were processed using ‘extended focus’ (Axiovision software), and the surface area of individual cardiomyocytes was measured. Only cells with clearly visible outlines after being rendered in the XY plane were chosen for measurement.

2.6 Quantification of cell number
Morpholinos were injected into the Tg(cmlc2:nuDsRed) transgenic line, where the nuclei of all cardiomyocytes are labelled with DsRed (kindly provided by Dr Geoff Burns, Massachusetts General Hospital). The hearts of the injected embryos were dissected at either 48 or 72 hpf, and 20–30 images were acquired as a Z-stack for each heart sample using an AxioplanII Zeiss microscope. After processing the images using ‘extended focus’, the cell number was counted.

2.7 Quantification of sarcomere length
Z-discs were labelled by immunofluorescence staining using the anti-sarcomeric {alpha}-actinin antibody. Embryos were incubated in relaxation buffer (20 mM imadazole, 5 mM EGTA, 7 mM MgCl2, 5 mM creatine phosphate, 10 mM ATP, 100 mM KCl)16,17 for 1.5 h before fixation. The hearts of injected embryos were dissected at either 48 or 72 hpf for imaging using an AxioplanII Zeiss microscope equipped with an Apotome. The distance between the midlines of {alpha}-actinin signals was measured using Axiovision software.

2.8 Statistics
Means and standard deviations of means (SD) were calculated from individual values, and a two-tailed t-test was used for comparison of two groups. Differences were considered significant for P < 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Identification of zebrafish myosin light chain genes with cardiac-specific expression
To identify zebrafish orthologues of human MYLs, we searched the zebrafish genomic and expression sequence tag databases. We identified 12 genes and, based on their sequence homology, categorized them into two groups, namely ELCs and RLCs (Figure 1A). The expression patterns of these genes were then characterized using whole-mount in situ hybridization (Figure 1B–D). Importantly, within the group of five zebrafish ELC orthologues and seven RLC orthologues, only cmlc1, an ELC, and cmlc2, an RLC, are expressed at high levels in a cardiac-specific manner, as shown by whole-mount in situ hybridization (Figure 1B). In addition, we identified two genes that exhibit weak cardiac expression, but strong somite expression (Figure 1C); four genes that exhibit strong somite expression only (Figure 1D); and four genes without detectable expression in striated muscle during embryogenesis. Here, we have focused on cmlc1 and cmlc2 to gain insights into the roles of myosin light chains during cardiogenesis.


Figure 1
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  		Figure 1 Identification of cardiac myosin light chain genes in zebrafish. Listed are the 12 MYL genes that have been examined in this paper using sequence analysis (A). The representative images for each expression group are shown in (B–D). These images represent lateral views of 48 hpf embryos after in situ hybridization. (B) Embryo after in situ staining using cmlc1 riboprobe. (C) Embryo after in situ staining using a riboprobe derived from the gene encoding AAO50214. The embryo was overdeveloped to reveal the weak cardiac expression. (D) Embryo after in situ staining using a riboprobe derived from the gene encoding XP_692285. Insets are ventral views of high magnification images in the heart region. Arrows indicate the location of hearts.

 
The cmlc1 gene is located on chromosome 5 and contains no introns, whereas cmlc2 is encoded by seven exons that are located on chromosome 8. Based on nucleotide and peptide sequence similarity between zebrafish and human myosin light chains (see Supplementary material online, Figure S1A–D), cmlc1 is most closely related to human MYL4, also named ELCa, while cmlc2 is probably the zebrafish orthologue of human MYL7, also named RLCa or MLC2a.12 During embryogenesis, the expression of both cmlc1 and cmlc2 in the cardiac progenitor regions of the anterior lateral plate mesoderm is initiated around the 16 somite (S) stage. This timing of expression is later than that of two other major components of heart muscle, titin (ttna) and ventricular myosin heavy chain (vmhc), with ttna expression being detected at the 5 S stage and vmhc expression at the 10 S stage (Figure 2). The onset of expression suggests that myosin light chains play a later role in sarcomere assembly than both titin and myosin.


Figure 2
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  		Figure 2 mRNA expression of cmlc1 and cmlc2. Images acquired following whole-mount in situ hybridization using the indicated riboprobes are shown. Dorsal views are shown of embryos at 5S, 10S and 16S stages, while ventral views (D, H, L and P) or a lateral view (T) are shown of embryos at 24 hpf. A riboprobe recognizing MyoD was always included to help determine the developmental stage. Expression of cmlc1 and cmlc2 is initiated at the 16S stage (C and G), whereas expression of ttna_N2B and vmhc begins around the 5S (M) and 10S (J) stages, respectively. The onset of cardiac expression for each gene is indicated by arrowheads.

 
3.2 cmlc1 and cmlc2 differentially affect sarcomere assembly
To investigate the functions of ELCs and RLCs, we used the technology of morpholino antisense oligonucleotide (morpholino)-mediated knockdown to reduce the expression of the respective genes in zebrafish. Since cmlc1 is an intronless gene, we designed a morpholino that targets the ATG translation start site, whereas a morpholino targeting a splice donor site of cmlc2 was used. Injection of 50 pg of MO-cmlc1 was observed to dramatically reduce the expression of cmlc1 with an estimated 80% efficacy as measured by quantification of the reduction of cmlc1-GFP fusion protein (see Supplemental material and methods and see Supplementary material online, Figure S2 for details on quantitation methods), and injection of 75 pg of MO-cmlc2 reduced the level of properly spliced cmlc2 mRNA to 3.08 ± 1.8% that of wild-type embryos, as quantified by real-time RT–PCR analysis (see Supplementary material online, Material and methods). In addition to the disrupted translation or splicing as expected from an ATG MO or a splice donor MO, whole-mount in situ hybridization staining of cmlc1 morphants (Figure 3E) and cmlc2 morphants (Figure 3J) indicated a reduction in the transcript levels of the respective genes. Thus, these two morpholinos, MO-cmlc1 and MO-cmlc2, effectively reduce the expression of their target genes, and will be useful in analysing the specific roles of these myosin light chains in cardiogenesis and cardiac function. As a control, we designed a scrambled morpholino, named MO-cmlc1Ct, which harbours five nucleotide changes in MO-cmlc1. In addition, we designed two more morpholinos against cmlc1, MO-cmlc1ATG3 and MO-cmlc1ATG4 (see Supplementary material online, Figure S3). Injection of these two morpholinos resulted in the same phenotypes as those from injection of MO-cmlc1 (for detailed phenotypic analysis of MO-cmlc1ATG3, see Supplementary material online, Figure S4), which confirmed the specificity of MO-cmlc1. If unspecified, 200 pg MO-cmlc1Ct, 50 pg of MO-cmlc1, or 75 pg of MO-cmlc2 were used throughout this paper.


Figure 3
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  		Figure 3 Transcriptional response of sarcomere genes upon reduction of a myosin light chain gene. Ventral views of 48 hpf wild-type (WT; A–D), cmlc1 morphant (MO-cmlc1; E–H) and cmlc2 morphant (MO-cmlc2; I–L) embryos after in situ hybridization staining with the indicated riboprobes are shown. Reduction of cmlc1 leads to a reduction of cmlc1 expression in the heart (E) and induction of cmlc2 in the atrium (I). Similarly, reduction of cmlc2 leads to a reduction of cmlc2 expression in the heart (J) and induction of cmlc1 in the atrium (F). Neither morphants affect the expression of vmhc or ttna in the heart. N, the number of embryos analysed.

 
We next examined whether reduction of cmlc1 or cmlc2 affects the expression of the untargeted myosin light chain. Interestingly, we noticed a mild elevation of cmlc1 expression in the atrium of cmlc2 morphants and a mild elevation of cmlc2 expression in the atrium of cmlc1 morphants (Figure 3F and I). However, the expression levels of other sarcomere genes such as vmhc and ttna in the heart did not appear to be altered upon reduction of either cmlc1 or cmlc2 (Figure 3C, D, G, H, K, and L). Thus, the expression levels of cmlc1 and cmlc2 may be co-regulated, such that down-regulation of one of these myosin light chains results in a slight increase in the expression of the other.

To examine the functions of cmlc1 and cmlc2 in sarcomere assembly, we first performed antibody staining to monitor the assembly of thick filaments and Z-discs in wild-type and morphant embryos. At 48 hpf, a network of assembled sarcomeres can be detected in the hearts of wild-type embryos (Figure 4A, B, and N). In addition, Z-bodies have assembled laterally to form Z-discs, while myosin has assembled into myosin filaments that align with each other to form A-bands, as revealed by the doublet band in each sarcomere following immunofluorescence staining with the F59 antibody. In both cmlc1 and cmlc2 morphants, dotted Z-bodies failed to further assemble laterally to form broader Z-bands (Figure 4E and G), but myosin still assembled into rod-like structures (Figure 4F and H). Furthermore, a similar phenotype was also observed in tel–/– homozygous embryos, a null mutant in cmlc2 gene (Figure 4C and D). The density of the thick filament network in cmlc1 morphant hearts (Figure 4F) was less than that observed in wild-type hearts (Figure 4B), whereas in cmlc2 morphants and cmlc2 null mutants (Figure 4D and H) the nascent thick filament network was comparable to that of age-matched wild-type embryos (Figure 4B). In support of these observations at the light microscopy level, ultrastructural analysis revealed that polymerized myosin filaments assemble in both morphants; however, these thick filaments fail to align to assemble into A-bands (Figure 4O and P).


Figure 4
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  		Figure 4 cmlc1 and cmlc2 are differentially required for sarcomere assembly in zebrafish heart. (A–L) Images of hearts from 48 hpf embryos of the indicated genotype following antibody staining. Z-discs were revealed by staining for {alpha}-actinin (A, C, E, G, I, K), whereas A-bands were revealed by staining for myosin using the F59 antibody (B, D, F, H, J, L). Insets are high magnification images. Scale bar = 20 µm. (M) Mean ± SD of sarcomere length as measured using images of samples stained for {alpha}-actinin. *P < 0.01, if compared with WT at 48 hpf; Figure 4P < 0.01, if compared with the corresponding morphants at 48 hpf; #P < 0.01, if compared with WT at 72 hpf. (N–P) Electron micrographs of ventricles at 48 hpf. Arrowheads indicate thick filament fibres. Sarcomere structures that contain Z-discs and A-bands can be detected in WT embryos (N), but not in MO-cmlc1 (O) or MO-cmlc2 (P) morphants. However, polymerized myosin filaments are still present in both morphants. Scale bar = 1 µm.

 
As part of the effort to analyse sarcomeric defects, we quantified the resting sarcomere length in wild-type and morphant embryos using images acquired following immunofluorescence staining with the anti-{alpha}-actinin antibody. The resting sarcomere length in wild-type embryos was 1.8 ± 0.1 and 1.8 ± 0.2 µm at 48 and 72 hpf, respectively (Figure 4M). Interestingly, sarcomere length was increased in cmlc1 morphants, but not in cmlc1Ct morphants, at both 48 and 72 hpf (2.1 ± 0.3 and 2.1 ± 0.2 µm, respectively), whereas depletion of cmlc2 resulted in a decrease in sarcomere length at similar developmental stages (1.6 ± 0.3 and 1.5 ± 0.3 µm, respectively; Figure 4M). Importantly, the changes in sarcomere length induced by depletion of cmlc1 or cmlc2 were reversed when the corresponding mRNA was co-injected with the morpholino (Figure 4M). Thus, the effects of depleting either cmlc1 or cmlc2 on sarcomere length are specific. In addition, the integrity of A-bands and Z-discs can be restored by cmlc2 mRNA in cmlc2 morphants (Figure 4K and L), while the restoration was not apparent in cmlc1 morphants co-injected with cmlc1 mRNA (Figure 4I and J). The latter observation suggested an incomplete rescue of sarcomere integrity, which might be due to limitations of the mRNA rescue technology. In summary, depletion of cmlc1 vs. cmlc2 results in distinct effects on sarcomere length, suggesting differential functions of these two genes.

3.3 Depletion of cmlc1 vs. cmlc2 results in similar effects on cardiac contractility but distinct effects on ventricular chamber volume
To gain further insight into the roles of ELCs and RLCs in cardiogenesis, we compared the cardiac functions of cmlc1 and cmlc2 morphants. Although depletion of cmlc1 or cmlc2 alone or in combination resulted in severe pericardiac oedema (Figure 5), we did not detect any significant change in heart rate for either morphant (Figure 6A). Thus, neither gene appears to be required for the establishment of cardiac rhythm. In contrast, cardiac contractility was reduced in more than 90% of injected embryos (524 out of 568 for cmlc1; 569 out of 612 for cmlc2), as quantified by measuring the SF of the ventricular chamber at 48 hpf (Figure 6A, see also Supplementary material online, Movies). Injection of 12 or 50 pg of MO-cmlc1, but not 200 pg of MO-cmlc1Ct, resulted in a reduction of the SF from 26 ± 5% in wild-type embryos to 12 ± 5% and 5 ± 4%, respectively (Figure 6A). Importantly, co-injection of 50 pg MO-cmlc1 with cmlc1 mRNA lead to a partial restoration of the SF (13 ± 8%), whereas co-injection with cmlc2 mRNA was not able to rescue the SF defect in cmlc1 morphants. Similarly, injection of 27 or 75 pg of MO-cmlc2 resulted in a reduction of the SF to 10 ± 4% and 4 ± 4%, respectively. In addition, co-injection of 75 pg MO-cmlc2 with cmlc2 mRNA restored the SF in cmlc2 morphants to 18 ± 8%. Co-injection of cmlc1 mRNA, however, could not compensate for the reduction in SF observed upon depletion of cmlc2. Thus, the roles of cmlc1 and cmlc2 in establishing cardiac contractility are not redundant. Indeed, depletion of cmlc1 and cmlc2 simultaneously further reduced the SF from 12 ± 5% (MO-cmlc1) and 10 ± 4% (MO-cmlc2) to 2.4 ± 1.7% (Figure 6A). Collectively, these results demonstrate that both cmlc1 and cmlc2 are required for the establishment of cardiac contractility, but not heart rhythm, during zebrafish embryogenesis.


Figure 5
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  		Figure 5 Depletion of cmlc1 and cmlc2 results in cardiac defects. Lateral views of live 48 hpf. embryos of the indicated genotype with anterior to the left are shown. High magnification images are shown in the right panels (B, D, F, H, J). Note the severe oedema in the heart region and chamber size difference between ventricles. Arrows indicate the position of the heart. A, atrium; V, ventricle.

 


Figure 6
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  		Figure 6 Depletion of either cmlc1 or cmlc2 reduces cardiac function, although with different effects on end systolic and diastolic ventricular volumes. (A) Mean ± SD of the shortening fraction (SF) of ventricular chambers and heart rate (HR) in wild-type (WT), and cmlc1 and cmlc2 morphants. *P < 0.01, if compared with WT; #P < 0.01, if compared with morphants injected with the same dose of morpholino. Ventricular contractility is severely reduced in cmlc1 and cmlc2 morphants, whereas HR is only slightly affected. SF can be recovered by co-injection of the corresponding mRNA. (B) Mean ± SD of end systolic and diastolic ventricular volume (mESV and mEDV, respectively) at 48 and 72 hpf. *P < 0.05, if compared with WT at 48 hpf; &P < 0.01, if compared with the corresponding morphants at 48 hpf; #P < 0.05, if compared with WT at 72 hpf. N, number of hearts that were quantified.

 
In addition to the severe pericardiac oedema visible in cmlc1 and cmlc2 morphants, observation of live fish suggested that the ventricular chamber volume was also affected in both of these morphants (Figure 5). Therefore, we quantified the mean end-diastolic volume (mEDV) and the mean end-systolic volume (mESV) of ventricles in 48 hpf wild-type and morphant embryos. As shown in Figure 6B, the ventricular mEDV of hearts in cmlc1 morphants, but not cmlc1Ct morphants, was slightly larger than that of age-matched wild-type embryos [(4.2 ± 2.5) x 10–4mm3 vs. (3.4 ± 1.1) x 10–4mm3], while the mESV in cmlc1 morphants, but not cmlc1Ct morphants, was approximately two-fold that of wild-type embryos [(3.2 ± 1.5) x 10–4mm3 vs. (1.3 ± 0.3) x 10–4 mm3]. Importantly, co-injection of cmlc1 mRNA with MO-cmlc1 was capable of fully or partially rescuing the mEDV and mESV cmlc1 morphant phenotypes [(3.3 ± 1.5) x 10–4 vs. (4.2 ± 2.5) x 10–4 mm3, and (2.4 ± 1.0) x 10–4 vs. (3.2 ± 1.5) x 10–4 mm3, respectively]. In contrast to cmlc1 morphants, the mEDV in cmlc2 morphants was less than one-third that of wild-type embryos [(0.9 ± 0.4) x 10–4 vs. (3.4 ± 1.1) x 10–4 mm3]. In addition, the mESV was also slightly decreased as compared to wild-type embryos [(0.8 ± 0.4) x 10–4 vs. (1.3 ± 0.3) x 10–4 mm3]. Similar to the cmlc1 morphant rescue experiments, co-injection of MO-cmlc2 with cmlc2 mRNA was able to increase both the mEDV and mESV [(1.5 ± 0.3) x 10–4 vs. (0.9 ± 0.4) x 10–4 mm3, and (1.0 ± 0.3) x 10–4 vs. (0.8 ± 0.4) x 10–4 mm3, respectively]. The changes in ventricular volume could simply be a result of a delay in development; however, we detected similar changes in the mEDV and mESV of 72 hpf cmlc1 and cmlc2 morphants, suggesting this is not the case (Figure 6B). Thus, both mEDV and mESV are affected in cmlc1 and cmlc2 morphants, but in an opposing manner, supporting that depletion of ELCs (cmlc1) vs. RLCs (cmlc2) results in distinct cardiac phenotypes.

3.4 Depletion of cmlc1 vs. cmlc2 results in distinct effects on ventricular cardiomyocyte size and number
We hypothesized that the effects of cmlc1 and cmlc2 depletion on chamber size might at least be partially attributable to a change in the size of individual cardiomyocytes. Therefore, we quantified the surface area of individual cardiomyocytes in cmlc1 and cmlc2 morphants (see Section 2) and compared this to the size of cardiomyocytes in wild-type embryos (Figure 7D–I and K). The individual cardiomyocytes in cmlc1 morphants, but not cmlc1Ct morphants, did indeed appear to be bigger than those in age-matched wild-type embryos (119 ± 34 vs. 111 ± 23 µm2 at 48 hpf, 134 ± 33 vs. 107 ± 29 µm2 at 72 hpf), while the individual cardiomyocytes in cmlc2 morphants were smaller (88 ± 22 vs. 111 ± 23 µm2 at 48 hpf, 77 ± 16 vs. 107 ± 29 µm2 at 72 hpf). Moreover, the change in cardiomyocyte size seemed to be a specific consequence of morpholino injection, as the cell size at 48 hpf was recovered by co-injection with the corresponding mRNA (106 ± 24 vs. 119 ± 34 µm2 and 102 ± 19 vs. 88 ± 22 µm2). Further analysis of cardiomyocytes from wild-type and morphant embryos using low magnification electron microscopy provided additional support to the concept that cardiomyocyte size is altered upon depletion of cmlc1 or cmlc2 (Figure 7G–I, see Supplementary material online, Material and methods). Interestingly, in addition to the effects on cardiomyocyte size, the shape of cardiomyocytes also appeared to be abnormal in both cmlc1 and cmlc2 morphants, as indicated by Zn5 staining (Figure 7D–F). Thus, cmlc1 and cmlc2 may mediate proper cardiac development at least in part through their effects on cardiomyocyte size, shape and organization.


Figure 7
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  		Figure 7 Depletion of cmlc1 and cmlc2 leads to differential changes in the size and number of ventricular cardiomyocytes. (A–C) Images of ventricular cardiomyocytes from wild-type (WT, A) and morphant embryos (MO-cmlc1, B; MO-cmlc2, C) where nuclei have been labelled using the Tg(cmlc2:nuDsRed) transgenic fish line. (D–F) Images of cell borders of ventricular cardiomyocytes from WT (D) and morphant (E, F) embryos as revealed by Zn5 antibody staining. Dashed lines provide an outline of the entire ventricle region. Scale bar = 20 µm. (G–I) Electron micrographs of single ventricular cardiomyocytes from WT (G) and morphant (H, I) embryos. Scale bar = 2 µm. Ventricular cardiomyocytes in cmlc1 morphants (E, H) appear larger than those in cmlc2 morphants (F, I). Quantification of cardiomyocyte number for the indicated conditions is summarized in (J), while quantification of the surface area of individual cardiomyocytes is summarized in (K). Mean ± SD is shown. *P < 0.01, if compared with WT at 48 hpf; Figure 7P < 0.01, if compared with the corresponding morphants; §P < 0.05, if compared with WT at 48 hpf; #P < 0.01, if compared with WT at 72 hpf.

 
The effects of depleting cmlc1 and cmlc2 on ventricular chamber size might not only be a result of alterations in cardiomyocyte morphology, but rather also cardiomyocyte number. To examine this possibility, transgenic embryos where the nuclei of all cardiomyocytes are labelled by DsRed [Tg(cmlc2:nuDsRed)] were injected with cmlc1 or cmlc2 morpholinos and analysed for cardiomyocyte number (see Section 2). Injection of MO-cmlc1, but not MO-cmlc1Ct, led to an increase in the number of ventricular cardiomyocytes as compared to wild-type embryos (233 ± 44 vs. 175 ± 41), whereas no significant change in cardiomyocyte number was observed for 48 hpf cmlc2 morphants (151 ± 35; Figure 7A–C and J). Interestingly, at 72 hpf when the cardiomyocyte number had almost doubled in wild-type embryos, the cardiomyocyte number in both cmlc1 and cmlc2 morphants was similar to that detected at 48 hpf (232 ± 38 for cmlc1, 155 ± 30 for cmlc2 vs. 308 ± 42 for wild-type; Figure 7J). The effects of MO-cmlc1 and MO-cmlc2 injection on cardiomyocyte number seem to be a result of cmlc1 and cmlc2 depletion, respectively, as the number can be rescued at both 48 and 72 hpf by co-injection of the corresponding mRNA (205 ± 42 and 281 ± 79 at 48 and 72 hpf in cmlc1 morphants, respectively; 162 ± 51 and 306 ± 39 at 48 and 72 hpf in cmlc2 morphants, respectively; Figure 7J). The reduced cell number in both morphants is not a consequence of cell death, since no apparent apoptosis can be detected by TUNEL assay (see Supplementary material online, Figure S5, Material and methods). In summary, these results indicate another aspect of cardiogenesis where cmlc1 and cmlc2 might play differing roles—cardiomyocyte proliferation. The effects of cmlc1 vs. cmlc2 depletion on cardiomyocyte number might contribute to the observed changes in ventricular chamber volume.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
Here, we have used zebrafish as a model organism to gain insights into the roles of ELCs and RLCs in vertebrate heart development and the effects of depleting an ELC family member vs. an RLC family member on cardiac function. Importantly, in characterizing the expression pattern of the 12 known MYL orthologues in zebrafish, we observed that only one ELC family member (cmlc1) and one RLC family member (cmlc2) exhibit strong cardiac expression, allowing us to focus on these two specific myosin light chains for our analyses. In contrast, there are two cardiac RLCs in mice, where one gene compensates the disruption of the other, which complicates interpretation of the results from loss-of-function studies.18 Interestingly, although depletion of either cmlc1 or cmlc2 compromises cardiac function, as indicated by a decrease in cardiac contractility, this appears to be through distinct mechanisms. Indeed, cmlc1 morphants exhibit an increase in cardiomyocyte size and number, sarcomere length, and ventricular chamber volume, whereas the exact opposite phenotypes, namely a decrease in these parameters, are observed in cmlc2 morphants (Figure 8). Thus, our results suggest that ELCs and RLCs serve distinct roles in mediating heart development and cardiac function.


Figure 8
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  		Figure 8 Summary of phenotypes observed upon depletion of an ELC (cmlc1) and a RLC (cmlc2) in zebrafish. Cartoons to summarize the phenotypes of cmlc1 and cmlc2 morphants at the cellular level are shown in (A–C). Depletion of cmlc1 leads to disrupted sarcomeres and longer sarcomere length, as well as an increase in cardiomyocyte size and number at 48 hpf. In contrast, depletion of cmlc2 results in non-organized sarcomeres, shorter sarcomere length and smaller as well as fewer cardiomyocytes.

 
4.1 Functions of essential myosin light chains and regulatory myosin light chains in sarcomere assembly
Our genetic analysis of cmlc1 in zebrafish provides the first loss-of-function evidence to reveal essential roles for an ELC family member in the assembly of thick filaments and thus proper sarcomere structure in the heart. The phenotypes observed in our cmlc1 and cmlc2 morphants suggest that both ELCs and RLCs are required for the assembly of A-bands, but not myosin polymerization, an earlier step in the thick filament assembly process. Myosin fibres were detected in cmlc1 and cmlc2 morphants at both the light and electron microscopy levels, similar to what has been reported for MLC2a homozygous mutant mice.11 In support of our observations, in vitro biochemical studies have indicated that a myosin fragment that contains only the C-terminal domain, but not MYL-binding domains, is capable of assembling into polymerized myosin filaments.19 Indeed, a 29 amino acid domain of the myosin rod is both necessary and sufficient for the assembly of myosin subfragments into higher order structures.20 In addition, in vivo genetics studies in Drosophila revealed that an N-terminally truncated myosin heavy chain mutant lacking the head region and the region that binds to ELCs is still able to assemble into thick filaments.21

4.2 Functions of essential myosin light chains and regulatory myosin light chains in cardiac contractility and ventricular chamber volume
A key discovery of this work is that ELC and RLC differentially affect cardiac contractility. The mEDV of cmcl1 morphants appeared normal, but the mESV was approximately two-fold of wild-type embryos, indicating the heart of cmlc1 morphants can dilate normally but cannot contract properly. In contrast, the mEDV of cmlc2 morphants was reduced to less than 30% of that in wild-type embryos, which indicates that the heart of cmlc2 morphants cannot dilate. These observations suggested distinct functions of ELC and RLC in fine tuning cardiac contractility, which could be explained by the different domain structures of ELC and RLC. ELC contains a unique N-terminal domain that interacts with actin to modulate cross-bridge kinetics, while RLC contains a unique phosphorylation site that modulates contraction by transmitting the MLCK pathway. In addition, the different cardiac contractility defects in ELC and RLC morphants may result from their distinct functions in sarcomere assembly. Sarcomeres without ELC tend to be longer than those in wild-type, which may preferentially affect contraction; while sarcomeres without RLC tend to be shorter, which may preferentially affect dilation. Since both ELC and RLC are molecular hinges that bind to the neck region of myosin, it will be interesting to examine whether defective ELC and RLC differentially affect the conformation of the myosin, which sequentially affects its function as a molecular motor.

At the cellular level, we observed that individual cardiomyocytes were larger in cmlc1 morphants and, additionally, that the proliferation of cardiomyocytes was altered. Thus, these two factors might account for or contribute to the increase in ventricular volume observed in embryos depleted of cmlc1. In contrast, depletion of cmlc2 resulted in smaller and fewer cardiomyocytes. Accordingly, cmlc2 morphants exhibited a reduction in ventricular chamber volume. It is likely that the change in chamber volume and the defective cellular responses are indirect consequences to the defective cardiac contractility. It has been previously demonstrated that proper contractility and fluid dynamics are important for normal cardiac development during zebrafish embryogenesis.22

4.3 Implication of essential myosin light chains and regulatory myosin light chains in pathogenesis of cardiomyopathy
Our results from the zebrafish model suggest that mutations in ELCs and RLCs may result in cardiomyopathy through distinct pathological processes. Although their sequences are highly conserved, the functions of ELCs and RLCs are different in sarcomere assembly and cardiac contractility. Sequentially, depletion of ELCs may activate a hypertrophic signalling pathway, while depletion of RLCs may impose a negative effect on the growth and size of cardiomyocytes. Indeed, a recent analysis of RLC (MYL1f) knockout mice indicated that new born homozygous mutant mice lack skeletal muscle, supporting a role for RLCs in muscle cell proliferation and/or survival.23 Zebrafish provides an ideal vertebrate model organism that is easily amenable to genetic manipulation to further examine the signalling pathways during the remodelling of individual cardiomyocytes. Further investigation is warranted to determine whether the cardiac remodelling events that we have discovered here in zebrafish are conserved in human cardiomyopathy patients and to help to identify potential therapies.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research Online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This work was supported by a grant from the Muscular Dystrophy Association, National Institute of Health grant HL81753, and a start up fund from Mayo Clinic Foundation to X. Xu.


   Acknowledgements
 
We are grateful to Jomok Beninio for maintaining our zebrafish facility and to Ruilin Zhang for making plasmid constructs. Thanks to Heather Thompson for helping to prepare the manuscript.

Conflicts of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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