Altered Expression of TMEM43 Causes Abnormal Cardiac Structure and Function in Zebrafish

Figures

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  Figure 1. The p.S358L variant changes protein conformation of TMEM43. In silico modeling of TMEM43 tertiary structure of wild-type (A,C) and proteins with the corresponding missense mutations (B) p.S358L, (D) p.P111L, (E) p.P111A, and (F) p.P111Q. Yellow indicates transmembrane domain (TMD) 1, blue indicates TMD2, rose indicates TMD3, and turquoise indicates TMD4. The residue of interest is indicated in red (p.S358 in A and B; p.P111 in C–F).

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  Figure 2. Reduced expression and altered localization of TMEM43-p.S358L. (A) Quantitative reverse transcription PCR demonstrates transcript reduction in human TMEM43 in adult ventricles of both TMEM43 variant transgenic lines compared to wild-type (WT). Each replicate consists of a pool of 2–7 ventricles and relative expression is calculated to TMEM43-WT expression levels. (B) Representative Western blot showing reduced occurrence of the human TMEM43-eGFP fusion-protein in adult ventricles of TMEM43-S358L compared to the TMEM43-WT transgenic line. For detection, both a TMEM43- and a GFP-antibody were used. Each biological replicate consists of a pool of 1–4 ventricles. (C) Western blot analysis using the TMEM43-antibody showed a reduced protein level of the human TMEM43-eGFP fusion-protein in adult ventricles of TMEM43-S358L compared to the TMEM43-WT transgenic line. Each data point represents relative Western blot band intensity of a technical replicate. (A,C) One-way ANOVA with Bonferroni’s multiple comparison test, * p ≤ 0.05, *** p ≤ 0.001. Error bars correspond to SEM. (D) Confocal images of whole-mount immunofluorescence staining of hearts expressing the transgene TMEM43-eGFP (green)…

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  Figure 3. Overexpression of TMEM43 variants induce altered cardiac morphology and function at 3 days post-fertilization (dpf). (A) Visual phenotyping demonstrates the percentage of embryos with zero to five different cardiac-specific defects. The defects were classified into looping defects, pericardial edemas, arrhythmias, altered ventricular shape, strong ventricular contractions, heterogenous ventricular contractions, and altered atrial shape. Both TMEM43 variant lines show an increased percentage of embryos with at least one cardiac defect. The number of phenotyped embryos is indicated at the bottom of the columns. (B) Confocal maximum intensity projections of immunofluorescence-stained hearts. Atria are labeled for S46 localization (red), ventricles for GFP localization (green), and nuclei with DAPI (blue). In contrast to TMEM43-WT hearts, both TMEM43 variant lines display developmental defects (looping defects and reduced ventricular chamber size in TMEM43-S358L, as well as altered atrial chamber shape in TMEM43-P111L). Ventral views, arterial pole to the top. Scale bar = 100 µm. (C) Representative histological images of hematoxylin and eosin-stained sagittal plastic sections showing…

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  Figure 4. Cardiac overexpression of stably folded TMEM43 leads to hypertrophic ventricular growth, but unchanged proliferation capacity in embryos. (A) Confocal maximum intensity projections of 3 days post fertilization (dpf) hearts immunostained with DAPI (blue) and Alcam (red) to highlight plasma membranes. Scale bar = 30 µm. (B) Quantification of cardiomyocyte (CM) area represented in (A) reveals larger CMs in TMEM43-WT and TMEM43-P111L compared to non-transgenic control (NTG) and TMEM43-S358L individuals (n ≥ 10 hearts per genotype). (C) Quantification of ventricular end-diastolic area shows the mean area of three individual sections of each fish from a time series of the embryonic cardiac cycle. Significantly increased end-diastolic ventricular areas were measured in TMEM43-WT and TMEM43-P111L compared to NTG and TMEM43-S358L individuals. (D) Representative confocal maximum intensity projection of a heart immunostained with PCNA (red), GFP (green), and DAPI (blue) at 3 dpf. Scale bar = 50 µm. In the graph, individual data points represent the percentage of PCNA+ nuclei in the ventricle of each fish (n ≥ 18 individuals per genotype). A slightly increased proliferation rate was observed…

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  Figure 5. TMEM43 wild-type (WT) and TMEM43-P111L transgenic lines display enlarged hearts in adult zebrafish. (A) Preserved myocardial structure in adult zebrafish at 5 months post-fertilization (mpf) (n ≥ 5 hearts per genotype). Scale bars = 500 µm. (a–d) Representative brightfield images of dissected fish hearts. (e–h) Histology was analyzed by hematoxylin and eosin (H&E) staining. (i–l) Fibrosis was analyzed by Masson’s Trichrome (MTC) staining. (B) TMEM43-WT and TMEM43-P111L individuals display larger ventricles as indicated by an increased ventricular area to body length ratio (VA/BL) compared to the non-transgenic control (NTG) and TMEM43-S358L. Brightfield images represented in (a–d) were used for assessment of ventricular surface area. (C) Quantification of body length reveals normal growth and development of adult transgenic zebrafish. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for shared differently expressed genes (DEGs) between “NTG vs. TMEM43-WT” and “NTG vs. TMEM43-P111L” (padj ≤ 0.05) using ShinyGO v0.76. The x-axis represents fold enrichment and y-axis represents enriched KEGG terms. The size of the dots represents the number of involved…

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  Figure 6. Transmission electron microscopy (TEM) reveals altered ultrastructure in ventricular heart tissue from TMEM43 transgenic lines at 5 months post-fertilization. (A) TEM images showing nuclei and mitochondria of the ventricular myocardium. Scale bars = 1 µm. (B) Analysis of the nuclear area and (C) circularity demonstrate no alterations between the genotypes. (D) Assessment of mitochondrial area uncovers significantly reduced mitochondrial size in both TMEM43 variant lines compared to TMEM43-WT. (E) Evaluation of mitochondrial circularity revealed altered mitochondrial shape in TMEM43-S358L and TMEM43-WT ventricles compared to the non-transgenic control (NTG). (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for shared differently expressed genes (DEGs) between “NTG vs. TMEM43-S358L” and “NTG vs. TMEM43-P111L” (padj. ≤ 0.05) using ShinyGO v0.76. The x-axis represents fold enrichment and y-axis represents enriched KEGG terms. The size of the dots represents the number of involved genes. The false discovery rate (FDR) value is indicated by colors. (G) Heatmap showing DEGs belonging to the KEGG pathway “Metabolic pathways”. (H) Representative TEM images of…

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  Figure 7. Enlarged adult hearts of homozygous tmem43wue4/wue4 mutant fish. (A) Phenotyping showing the percentage of embryos with zero to five different cardiac-specific defects. The defects were classified in looping defects, pericardial edemas, arrhythmias, altered ventricular shape, strong ventricular contractions, heterogenous ventricular contractions, and altered atrial shape. The number of analyzed embryos is indicated at the bottom of the columns. (B) Quantification of ventricular end-diastolic area showing the mean area of three individual sections of each fish from a time series of the embryonic cardiac cycle. (C) Analysis of cardiac function by quantification of ventricular fractional shortening (FS) from a time series of the embryonic cardiac cycle. Data points represent the average measurement obtained from three diastolic/systolic phases of each fish. The short axis of the ventricle was used for quantification. (A–C) Analyses were performed on embryos from three independent clutches per genotype. (B,C) Significance was determined by an unpaired t-test. Error bars correspond to SEM. (D) Adult cardiac morphology at 24 months post fertilization (mpf). Scale bars = 500 µm. (a–c)…