Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Aberrant localization of p.S358L-mutant TMEM43 affects cardiac development and function in transgenic zebrafish

M. Zink1, A. Seewald1, M. C. Nguyen1, T. Williams1, D. Liedtke2, C. Stigloher3, S. Childs4, B. Gerull1

1Deutsches Zentrum für Herzinsuffizienz, Universitätsklinikum Würzburg, Würzburg; 2Institut für Humangenetik, Universität Würzburg, Würzburg; 3Zentrale Abteilung für Mikroskopie, Universität Würzburg, Würzburg; 4Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, CA;

Background: Arrhythmogenic cardiomyopathy (ACM) is caused by the fully penetrant heterozygous missense mutation c.1073C>T (p.S358L) within the highly conserved gene of transmembrane protein 43 (TMEM43) which leads to arrhythmias, heart failure and sudden cardiac death. A second variant (c.332C>T; p.P111L) within TMEM43 is possibly pathogenic, but has not been functionally validated yet. TMEM43 is an integral protein of the inner nuclear membrane and is involved in maintaining the structural integrity of the nuclear envelope. Methods and Results: We established cardiomyocyte-restricted transgenic zebrafish lines that overexpress eGFP-linked full-length human wildtype TMEM43 and the respective mutations using the Tol2-system. Mutant transgenic zebrafish are viable and survive until adulthood. At 72 hours post fertilization cardiac phenotyping of the F3 incross generation revealed an increased number of TMEM43 mutant zebrafish larvae with at least one or more cardiac developmental defects. Furthermore, both mutant zebrafish lines showed a decreased end-diastolic ventricular area. In addition, the p.S358L-mutant displayed overall enhanced systolic function, indicated by increased fractional shortening and fractional area change values. Whole-mount immunofluorescence (IF) staining unveiled a significantly reduced area of the ventricular cardiomyocytes in the p.S358L-mutant as the underlying cause for the smaller ventricular size, suggesting pathways involved in cell growth and differentiation are potentially affected. To analyse whether the mutation in TMEM43 has an impact on its localization, we performed IF staining on fixed larvae and paraffin sections of adult hearts. Unlike, TMEM43-WT and TMEM43-p.P111L, which is mainly localized at the nuclear envelope and the endoplasmic reticulum, TMEM43-p.S358L displayed strikingly a delocalization in the cytoplasm and a reduced overall protein level. Since ACM develops with increasing age, heart morphology at 5 months of age was examined. TMEM43-p.S358L ventricles of dissected hearts remain significantly smaller compared to controls as indicated by a reduced ventricular surface area/body length ratio, but no apparent myocardial degeneration or fibrotic replacement could be observed. On ultrastructural level ventricular tissue of TMEM43-p.S358L displayed mitochondria with altered shape as well as an elevated proportion of nuclei that exhibit expanded perinuclear space and bulging of the outer nuclear membrane. In contrast, ventricular tissue of TMEM43-p.P111L showed an increased distance of the cell membranes at the desmosomal junctions. Finally, transcriptomic analysis of adult ventricular tissue demonstrated differences in gene expression patterns between TMEM43-WT and the respective mutant, with differentially expressed genes involved in adrenergic signaling in cardiomyocytes, cardiac muscle contraction and calcium signaling. Conclusion: Altogether, these findings suggest that the p.S358L mutation provokes aberrant localization of TMEM43, thereby disrupting the integrity of the nuclear membrane and affecting gene expression within the myocardium of transgenic zebrafish.

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