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. 2008 Jul 1;79(1):97-108.
doi: 10.1093/cvr/cvn073. Epub 2008 Mar 14.

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

Zhenyue Chen  1 , Wei Huang, Tillman Dahme, Wolfgang Rottbauer, Michael J Ackerman, Xiaolei Xu
Affiliations

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

Zhenyue Chen et al. Cardiovasc Res. .

Abstract

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.

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Conflict of interest statement

Conflicts of interest: none declared.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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 α-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 α-actinin. *P < 0.01, if compared with WT at 48 hpf; ╬P < 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.
Figure 5
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
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.
Figure 7
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; ╬P < 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.
Figure 8
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.
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