The Newfoundland mutation TMEM43 p.S358L causes an impaired cardiac energy metabolism and mitochondrial function through altered protein-interaction

S. Ratnavadivel (Bad Oeynhausen)¹, A. Gärtner (Bad Oeynhausen)¹, N. Klinke (Osnabrück)², K. Jürgens (Osnabrück)², A. Malmendal (Roskilde)³, J. Groß (Bad Oeynhausen)¹, K. Klingel (Tübingen)⁴, S. Walter (Osnabrück)², H. Boen (Edegem)⁵, E. Van Craenenbroeck (Edegem)⁵, R. Schramm (Bad Oeynhausen)⁶, J. Gummert (Bad Oeynhausen)⁶, A. Kostareva (St. Petersburg)⁷, A. Kassner (Bad Oeynhausen)¹, H. Harten (Osnabrück)², A. Paululat (Osnabrück)², H. Milting (Bad Oeynhausen)^1
1Herz- und Diabeteszentrum NRW E.& H. Klessmann-Institut f. kardiovask. Forschung Bad Oeynhausen, Deutschland; ²Universität Osnabrück, Fachbereich Biologie/Chemie Zoologie und Entwicklungsbiologie Osnabrück, Deutschland; ³University Roskilde Department of Science and Environment Roskilde, Dänemark; ⁴Universitätsklinikum Tübingen Kardiopathologie Tübingen, Deutschland; ⁵Antwerp University Hospital Department of Cardiology Edegem, Belgien; ⁶Herz- und Diabeteszentrum NRW Klinik für Thorax- und Kardiovaskularchirurgie Bad Oeynhausen, Deutschland; ⁷Almazov National Medical Research Centre Institute of Molecular Biology and Genetics St. Petersburg, Russland

Introduction TMEM43 also known as LUMA is a ubiquitously expressed 4-transmembrane protein localized in the ER and the nuclear lamina. The variant TMEM43 p.S358L causes fully penetrant ARVC5 in male carriers. The function of the protein and the pathomechanisms of the ARVC5-associated mutation remain poorly understood. We analysed carrier derived hiPSC-cardiomyocytes (hiPSC-CMs), enriched TMEM43 in membrane preparations of human myocardial microsomes and identified differentially interacting proteins. Here we provide evidence for a novel pathomechanism of TMEM43 p.S358L that may contribute to the onset of ARVC5.

Methods hiPSC derived cardiomyocytes of TMEM43 p.S358L variant carriers were generated and the mutation was corrected using CRISPR-Cas9 to generate isogenic TMEM43-WT controls. For the analysis of the interacting proteins a HA-Tag was introduced at the 3’ end of TMEM43. Pull down analyses were performed under optimized conditions to extract TMEM43 and interacting proteins from the membrane. Explanted TMEM43-WT human myocardium was used for the generation of microsomes (MS) and separated by sucrose gradient ultracentrifugation (UC). All fractions were characterized for their protein composition using mass spectrometry. hiPSC-CMs were analysed by lipidomics, on contractility and ATP-content. TMEM43 p.S358L explanted human myocardium was characterized by proteome and metabolome analysis.

Results Pull down analyses of TMEM43 WT and mutant showed altered interaction of proteins involved in metabolic pathways. Lipidomics of mutant cells revealed accumulation of lipids, fatty acids, cholesterol esters and decreased lipid metabolism capacity upon supplementing linoleic and oleic acid as a substrate. ATP/ADP-ratios were significantly lower in the mutant hiPSC-CMs. Reduced ATP was associated with diminished contraction frequency of mutant hiPSC-CMs. These results imply that cells carrying TMEM43 p.S358L have an impaired fatty acid metabolism for energy production. Proteome analyses from left ventricular myocardium of a transplanted TMEM43 p.S358L carrier revealed altered expression of proteins involved in metabolic pathways as a major effect, which was also found in TMEM43 p.S358L hiPSC-CMs. Metabolic remodeling is also supported by the presence of metabolites that were measured in the myocardial samples from a TMEM43 p.S358L carrier when compared to non-failing control myocardium. Mass spectrometry data from UC-fractions containing the highest amount of TMEM43 and pull-down experiments of hiPSC-CMs revealed differentially interacting proteins of TMEM43 p.S358L from ER and mitochondrial membranes.

Conclusions We suggest that the interaction of mutant TMEM43 p.S358L with proteins of the mitochondria and the ER is disturbed, leading to functional impairment of ER-mitochondrial contact sites. In contrast to most ARVC-related variants affecting desmosomal proteins TMEM43 p.S358L primarily contributes to changes in mitochondrial function affecting lipid homeostasis and energy metabolism.