Background: Arrhythmogenic cardiomyopathy (ACM) is a myocardial disorder characterized by progressive fibro-fatty replacement of myocardium and life-threatening ventricular arrhythmias. A fully penetrant heterozygous missense mutation c.1073C>T (p.S358L) within the highly conserved gene of transmembrane protein 43 (TMEM43) has been genetically identified to cause a severe subtype of ACM. A second variant (c.332C>T; p.P111L) within TMEM43 is probably not 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 organization of the nuclear envelope.

Methods and Results: We generated 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. Cardiac phenotyping of the F3 incross generation at 72 hours post fertilization unveiled an increased proportion of cardiac developmental defects in TMEM43 mutant zebrafish. In addition, both TMEM43 mutant zebrafish lines displayed a decreased end-diastolic (TMEM43-WT: 13562 µm², TMEM43-p.P111L: 12875 µm², TMEM43-p.S358L-01: 10590 µm², p<0.001) and end-systolic ventricular area with overall enhanced contractile function, indicated by increased fractional shortening and fractional area change values. Interestingly, hypercontractility appeared to localize at certain areas of the ventricle. To determine the cause of the smaller ventricular size, whole-mount immunofluorescence (IF) staining with ALCAM revealed significantly smaller ventricular cardiomyocytes (TMEM43-WT: 58 µm², TMEM43-p.S358L-02: 44 µm², p<0.0001) and an unchanged number of overall nuclei in the p.S358L-mutant zebrafish suggesting that pathways affecting growth and proliferation are potentially affected. To investigate whether the mutation in TMEM43 affects its localization, we performed IF staining of fixed larvae and paraffin sections of adult hearts. In TMEM43-WT and -p.P111L, the TMEM43-eGFP fusion protein localized properly at the nuclear envelope and the endoplasmic reticulum. In contrast, TMEM43-p.S358L showed remarkably a delocalization in the cytoplasm and reduced overall expression. As ACM is a progressive disease, we evaluated heart morphology at 5 months of age. TMEM43-p.S358L ventricles of dissected hearts remain significantly smaller compared to controls as indicated by a reduced ventricular surface area/body length ratio (TMEM43-WT: 0.035, TMEM43-p.S358L-01: 0.029, TMEM43-p.S358L-02: 0.026, p<0.001). This implies that a compensation mechanism by expanding heart size is not required for maintaining cardiac function in adult zebrafish. To examine cardiac morphology at an ultrastructural level, we performed transmission electron microscopy on adult ventricular tissue. Nuclear morphology in TMEM43-p.S358L displayed an increased proportion of nuclei that exhibit enlarged perinuclear space and bulging of the outer nuclear membrane. 

Conclusion: Our data demonstrate that juvenile and adult transgenic zebrafish carrying the TMEM43-p.S358L mutation display an altered heart morphology and cardiac performance, indicating that abnormal nuclear integrity lead to early developmental cardiac defects. Further investigations will elucidate pathways involved in cardiomyocyte growth and proliferation.