In Drosophila, formation of the cardiac extracellular matrix (ECM) starts during embryogenesis. Assembly and incorporation of structural proteins such as Collagen IV, Pericardin, and Laminin A, B1, and B2 into the cardiac ECM is critical to the maintenance of heart integrity and functionality and, therefore, to longevity of the animal. The cardiac ECM connects the heart tube with the alary muscles; thus, the ECM contributes to a flexible positioning of the heart within the animal's body. Moreover, the cardiac ECM holds the larval pericardial nephrocytes in close proximity to the heart tube and the inflow tract, which is assumed to be critical to efficient haemolymph clearance. Mutations in either structural ECM constituents or ECM receptors cause breakdown of the ECM network upon ageing, with disconnection of the heart tube from alary muscles becoming apparent at larval stages. Finally, the heart becomes non-functional. Here, we characterised existing and new pericardin mutants and investigated biosynthesis, secretion, and assembly of Pericardin in matrices. We identified two new pericardin alleles, which turned out to be a null (pericardin3-548) and a hypomorphic allele (pericardin3-21). Both mutants could be rescued with a genomic duplication of a fosmid coding for the pericardin locus. Biochemical analysis revealed that Pericardin is highly glycosylated and forms redox-dependent multimers. Multimer formation is remarkably reduced in animals deficient for the prolyl-4 hydroxylase cluster at 75D3-4.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a severe cardiac disease that leads to heart failure or sudden cardiac death (SCD). For the pathogenesis of ARVC, various mutations in at least eight different genes have been identified. A rare form of ARVC is associated with the mutation TMEM43 p.S358L, which is a fully penetrant variant in male carriers. TMEM43 p.S358 is homologous to CG8111 p.S333 in Drosophila melanogaster. We established CRISPR/Cas9-mediated CG8111 knock-out mutants in Drosophila, as well as transgenic fly lines carrying an overexpression construct of the CG8111 p.S333L substitution. Knock-out flies developed normally, whereas the overexpression of CG8111 p.S333L caused growth defects, loss of body weight, cardiac arrhythmias, and premature death. An evaluation of a series of model mutants that replaced S333 by selected amino acids proved that the conserved serine is critical for the physiological function of CG8111. Metabolomic and proteomic analyses revealed that the S333 in CG8111 is essential to proper energy homeostasis and lipid metabolism in the fly. Of note, metabolic impairments were also found in the murine Tmem43 disease model, and fibrofatty replacement is a hallmark of human ARVC5. These findings contribute to a more comprehensive understanding of the molecular functions of CG8111 in Drosophila, and can represent a valuable basis to assess the aetiology of the human TMEM43 p.S358L variant in more detail.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG Objectives TMEM43 encodes for a transmembrane protein located in the ER membrane and the nuclear rim. TMEM43 has predicted four transmembrane domains. The missense variant TMEM43 p.S358L is fully penetrant in males and associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) type 5. Even though, TMEM43 is a phyllogenetically conserved protein throughout different species, little is known on the molecular mechanism of the mutation. TMEM43 is highly homologous to the single CG8111 gene in Drosophila melanogaster. The clincal relevant amino acid serine at position p358 is situated in the third transmembrane domain, the same applies to the corresponding amino acid p.S333L in the Drosophila homologue. Here, we established the first invertebrate model to study the pathophysiological effects of the human variant in the Drosophila homologue CG8111. Methods To investigate the effect of this missense mutation in flies, we introduced the variant CG8111 p.S333L in Drosophila and overexpressed the mutant variant CG8111p.S333L in transgenic flies, either ubiquitously or in a tissue specific manner. Cardiac specific physiological effects of the mutant variant were examined by Semi-automatic Optical Heartbeat Analysis (SOHA) for heart rate, systolic and diastolic interval, fractional shortening and arrhythmicity index. Mutant and wildtype flies were further compared by mass-spectrometry for differentially regulated proteins. Metabolites were analysed by NMR accordingly. Results Our studies showed that in comparison to the wild type the ubiquitously overexpressed variant leads to lethal effects at pupal stages. Individuals died from reduced food uptake and excessive lipid droplet accumulation in adipocytes. CG8111p.S333L animals up-regulate proteins, involved in fatty acid metabolism pathways. Metabolomic data revealed increased amounts of fatty acids accumulation in mutant versus wild-type animals. Furthermore, heart specific overexpression of CG8111p.S333L was associated with arrhythmias in five week old male flies. Conclusions CG8111p.S333L overexpression is a lethal genotype in Drosophila. Our data suggest a metabolic dysfunction associated with the mutant form of CG8111p.S333L, which shares pathophysiological characteristics of the human condition in TMEM43 p.S358L carriers.

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 450999875 Background Transmembrane protein 43 (TMEM43) is a phylogenetically highly conserved and ubiquitously expressed protein with unknown function. The protein contains four transmembrane domains located in the inner nuclear envelope and the endoplasmic reticulum (ER). A large acidic domain located between the first and second transmembrane domains, with unknown function, is exposed to the ER lumen. TMEM43-c.1073C>T is a rare fully penetrant mutation that leads to arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). ARVC5 is associated with myocardial fibrosis, fibrofatty replacement and progressive loss of right ventricular myocardial tissue and severe arrhythmias causing sudden cardiac death (SCD) especially in males. The pathomechanism of ARVC5 is not well understood. Methods We isolated and cultivated primary dermal fibroblasts of three different individuals carrying TMEM43-c.1073C>T (p.S358L). Proteins were isolated from cell cultures and analysed by Orbitrap mass spectrometry. Proteome data of the mutation carriers were compared to TMEM43 wildtype control fibroblasts. Additionally, TMEM43-p.S358L and control fibroblasts were investigated by transmission electron microscopy (TEM). Results A total of 9 proteins were upregulated in fibroblasts expressing TMEM43-p.S358L (p<0.003). Of note, proteins involved in fatty acid metabolism, such as acyl-coenzym A (CoA) thioesterase 1 encoded by the gene ACOT1, and Malonate-CoA ligase encoded by ACSF3 were strongly upregulated (FC>3 or >2, respectively). ACOT1 catalyses hydrolysis acyl-CoAs to long chain fatty acids and CoA. ACSF3 catalyses the formation of thioesters between fatty acids and CoA. ACOT1 and ACSF3 regulate the intracellular levels of free fatty acids and are expressed in the cardiomyocytes of the human myocardium. TEM images of TMEM43-p.S358L carriers show an accumulation of multilamellar vesicles, which may support the dysregulation of the lipid metabolism. Conclusion The differentially regulated proteins in dermal fibroblasts and the accumulation of multilamellar vesicles suggest a dysregulation of the lipid metabolism by TMEM43-p.S358L, which may also have relevance for the development of myocardial fibrofatty replacement contributing to ARVC5.