The Role of MYH6 Variants in atrial cardiomyopathy: insights from clinical observations and zebrafish models

Figures

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  Fig. 1. Investigation of three families with MYH6 variants. A Pedigrees of three families. Arrows indicate the probands. ‘+’ indicates individuals carrying the MYH6 variant and ‘-’ indicates gene-negative individuals. Individuals without annotation were not genetically tested. Dark-filled symbols indicate individuals with documented atrial fibrillation. Grey-filled symbols indicate family members who exhibited cardiac-related symptoms or abnormal findings (palpitation or premature atrial contractions). Non-filled symbols represent unaffected family members. Squares and circles denote males and females, respectively. B Representative ECG and (C) echocardiography of the proband in family 3 carrying the MYH6 p.A1327T variant. Abbreviations: AF, atrial fibrillation; PAC, premature atrial contraction; HCM, hypertrophic cardiomyopathy; DCM, dilated cardiomyopathy.

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  Fig. 2. Establishment and validation of myh6 knockout zebrafish. A Establishment of myh6+/− and myh6−/− zebrafish lines labelled by eGFP under myl7 promoter. Representative images illustrating the cardiac chambers of 72-hour postfertilization (hpf) larvae. B CRISPR/Cas9 technique was used to knockout exon1 in the myh6 gene. C DNA sequence alignment of WT and mutant myh6 alleles showing 8 bp loss. D Cross-sectional genotype distribution of surviving zebrafish at different developmental stages. At each indicated time point, a subset of surviving fish was randomly sampled and genotyped. Total numbers of fish analyzed were n = 60 at 72 hpf, n = 52 at 14 days, and n = 58 at 6 months. E, F Western Blot and quantification of myh6 and myh7 protein expression levels in whole-heart samples from 1-month zebrafish of three genotypes (n = 3 vs. 3). G Video-based cardiac function analysis in WT (n = 10), myh6+/− (n = 20), and myh6−/− (n = 10) zebrafish; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 estimated using one-way ANOVA. Abbreviations: FS, fractional shortening; FAC, fractional area change.

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  Fig. 3. Electrical and structural abnormalities in adult myh6+/− zebrafish. A Representative ECG in 6-month zebrafish showed lower heart rate, prolonged PR interval, increased P wave and QRS complex duration in myh6+/− mutants compared to WT controls. B Heart rate, PR interval, P wave duration, QRS complex duration, P wave amplitude, and R wave amplitude of WT (n = 10) vs. myh6+/− mutants (n = 10). C Doppler flow imaging in zebrafish hearts demonstrates the blood flow in the chambers of adult zebrafish. D Representative M-mode frame of WT and myh6+/− zebrafish. (E) Heart rate, atrial FAC, ventricular FAC, E peak velocity, A peak velocity, and velocity time integral of WT (n = 5) vs. myh6+/− mutants (n = 10). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 estimated using the Student t test. F Histological abnormalities of myh6+/− mutants in atria and ventricle. TEM of the zebrafish atria or ventricle obtained from WT and myh6+/− mutant. The mutants showed disrupted Z-discs (yellow), unclear I-bands (red), missing M-lines (blue), and blurred mitochondria (green) (scale bar = 1 μm). G Quantification of sarcomere length in the atrium and ventricle samples from WT (n = 20) and…

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  Fig. 4. Gene expression profiles in 6-month-old zebrafish atrium samples from WT and myh6+/− zebrafish (n = 3 vs. 3). A Volcano map of the expression profile of 1318 differentially expressed genes (DEGs) (|Log2(FoldChange)| ≥ 0.5 and P value ≤ 0.05). DEGs in the GO term of ‘muscle tissue development’ were annotated. B, C Heat maps of DEGs related with the GO function of ‘sarcomere’ and ‘muscle tissue development’. D String analysis of top 10 genes functionally associated with myh6. GO enrichment analysis of downregulated genes (n = 594) in myh6+/− mutant atrium samples illustrated by dot plot with cardiac-related terms highlighted (E) and gene-concept network plot (F). G GSEA analysis of ‘structural molecule activity’.