Heiko Meyer

The directionality of blood flow is regulated by heart valves, among other things. While the heart valves in vertebrates are multicellular and complex, the valve in the Drosophila heart consists of exactly two highly specialized cells. They arise during early larval development from two cardiomyocytes, those that form the boundary between the aorta and the posterior ventricle. Here, we show that the conserved PDGF signaling pathway is involved in the determination of heart valve cells. RNAi‐mediated knockdown of the Pvr receptor and one of its ligands, Pvf2, leads to an inhibition of valve cell differentiation. In contrast, the simultaneous expression of the Pvr and Pvf2 in the entire heart tube leads to the formation of additional heart valve cells in ectopic positions. A single expression of the receptor or the ligand does not lead to heart valve formation.

The endolysosomal system is crucial for the degradation of cellular waste in the lysosomal lumen. Within this pathway, endosomes mature prior to their fusion with lysosomes. This process relies on the sequential action of the CORVET and HOPS tethering complexes, guided by Rab5 and Rab7 GTPases, respectively. CORVET acts on early endosomes (EEs), whereas HOPS acts on maturing late endosomes and/or multivesicular bodies (LEs/MVBs) for lysosomal fusion. This process is finely tuned by the Rab-activating guanine nucleotide exchange factor (GEF) and the inactivating GTPase-activating protein (GAP). The BuMC1–GEF complex (Bulli–Mon1–Ccz1) uniquely activates Rab7 in metazoans and interacts with Rab5, which stimulates its activity. Here, we have identified GAPsec as a previously unreported GAP that activates Rab5 and is required for endosomal maturation in fruit fly nephrocytes. Inactivation of GAPsec results in enlarged, dysfunctional endosomes that are unable to reach lysosomes for degradation. Our study highlights the importance of coordinated Rab regulation for efficient endosomal trafficking.

Arrhythmogenic right ventricular cardiomyopathy type 5 is caused by the missense mutation S358L in the gene TMEM43 in humans. To date, the molecular mechanisms underlying the disease remain poorly understood. We established a CRISPR/Cas9 knock-in Drosophila model carrying the orthologous Tmem43p.S333L mutation to investigate these mechanisms in vivo. The resulting flies were viable but displayed reduced lifespan, smaller body size, lipid droplet accumulation, and mitochondrial defects. Proteomic and lipidomic profiling revealed a dosage-dependent misregulation of the energy metabolism, concomitant with reduced fatty acid synthesis and ß-oxidation rates, altered peroxisomal pathways, and changes in membrane phospholipid composition. Notably, phosphatidylethanolamine (PE) and phosphatidylinositol (PI) levels were elevated, while triacylglycerols were reduced. Ultrastructural analyses confirmed mitochondrial degradation in the muscle tissue of corresponding mutants. These findings establish Tmem43p.S333L knock-in flies as a robust in vivo model of ARVC-5 and support a role for TMEM43 in linking lipid homeostasis to mitochondrial energy metabolism and integrity. Mutation-derived impairments in these processes result in cardiomyopathy.

BACKGROUND: TMEM43 (transmembrane protein 43) is a ubiquitously expressed 4-transmembrane-protein localized in the endoplasmic reticulum and nuclear lamina. The missense mutation TMEM43 -p.S358L causes fully penetrant ARVC5 (arrhythmogenic right ventricular cardiomyopathy type 5) especially in males. The TMEM43 function of the protein and the pathomechanisms of TMEM43-p.S358L remain poorly understood. We analyzed carrier-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), human myocardial tissue from TMEM43-wild-type, and TMEM43-p.S358L and identified differentially interacting proteins. Here we provide evidence for a novel pathomechanism contributing to the onset of ARVC5. METHODS: Microsomes of human wild-type myocardium were separated by sucrose-gradient ultracentrifugation and characterized by mass-spectrometry to identify potential interacting proteins. Proteome and metabolome analyses of a TMEM43 -p.S358L explanted human myocardium were performed. hiPSC-derived cardiomyocytes of TMEM43 -p.S358L carrier and a corresponding isogenic control were generated. A 3’-end HA-Tag was introduced in TMEM43 for pull-down experiments under optimized conditions. Lipidomics, proteomics, contractility, and ATP-content were measured in hiPSC-CMs. RESULTS: Pull-down analyses of TMEM43-WT and mutant showed altered interacting proteins involved in metabolic pathways. Lipidomics revealed the accumulation of lipids and decreased lipid metabolism capacity in mutant hiPSC-CMs. The ATP to ADP ratio was lower in mutant hiPSC-CMs and could be associated with diminished contraction frequency. The human TMEM43 -p.S358L myocardial proteome revealed altered protein-expression of metabolic pathways comparable to mutant hiPSC-CMs. Metabolic remodeling was also found in the mutant human myocardium. Ultracentrifugation fraction with the highest protein amount of TMEM43 and pull-down experiments of hiPSC-CMs revealed differentially interacting proteins of TMEM43-p.S358L from endoplasmic reticulum and mitochondrial membranes. CONCLUSIONS: We suggest differential interaction of mutant TMEM43 with proteins of mitochondria and endoplasmic reticulum influences endoplasmic reticulum-mitochondrial contact sites. TMEM43 -p.S358L primarily contributes to changes in mitochondrial function affecting lipid homeostasis and energy supply.

The endolysosomal system is crucial for the degradation of cellular waste in the lysosomal lumen. Within this pathway, endosomes mature prior to their fusion with lysosomes. This process relies on the sequential action of the CORVET and HOPS tethering complexes, guided by Rab5 and Rab7 GTPases, respectively. CORVET acts on early endosomes (EEs), transitioning to HOPS on maturing late endosomes/multivesicular bodies (LEs/MVBs) for lysosomal fusion. This process is finely tuned by the Rab activating guanine nucleotide exchange factor (GEF) and the inactivating GTPase activating protein (GAP). The BuMC1 GEF complex (Bulli-Mon1-Ccz1) uniquely activates Rab7 in metazoans and interacts with Rab5, which stimulates its activity. Here, we identified GAPsec as a novel GAP with activity for Rab5 required for endosomal maturation in fruit fly nephrocytes. Inactivation of GAPsec results in enlarged, dysfunctional endosomes that are unable to reach lysosomes for degradation. Our study highlights the importance of coordinated Rab regulation for efficient endosomal trafficking.

The acrosome is a highly conserved organelle located at the anterior tip of the mature sperm. It is essential to successful fertilization across species. Herein, we provide evidence that the peptidase Neprilysin 4 localizes to the acrosome during Drosophila sperm maturation and that activity of this enzyme is essential to proper acrosome structure, sperm tip partitioning and male fertility. In contrast to controls, sperm from animals with impaired Nep4 function exhibited a severely disturbed tip structure that affected intracellular and sperm surface components. These effects were secondary to structural defects in the acrosome morphology. Corresponding sperm were quickly discarded by the females, exhibited a reduced ability to fertilize eggs, and did not initiate embryonic development, even if sperm entry had occurred. This study demonstrates an essential role for a neprilysin in the structural integrity of the acrosome and the sperm tip, and thus introduces a new and crucial function of neprilysins in ensuring male fertility.

Arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC-5) is a fully penetrant form of ARVC caused by the missense mutation in the gene TMEM43 p.S358L. Despite extensive research, the molecular basis underlying the detrimental effects of TMEM43 p.S358L still needs to be discovered. TMEM43 is a phylogenetically conserved protein. We previously analysed the Drosophila homologue (CG8111 or Dmel\Tmem43) to understand the protein's physiological relevance and the mutation p.S358L. Drosophila Tmem43 is localised at the ER/SR membrane and interacts with the outer mitochondrial membrane protein Porin/VDAC. This interaction is lost in a Tmem43p.S333L mutant that resembles the human p.S358L mutation. In addition, Tmem43p.S333L caused a breakdown in mitochondrial membrane potential and increased cellular reactive oxygen species, suggesting impaired mitochondrial function as a major pathomechanism. Complementary ultrastructural analyses revealed severe structural defects in the affected mitochondria, including degeneration of the organelles. Highly similar ultrastructural defects were observed in the human right ventricular myocardium of a TMEM43p.S358L trait carrier, suggesting a common molecular basis for the detrimental effects of the mutation in flies and humans. We propose that both the p.S358L mutation in humans and the p.S333L mutation in Drosophila impair TMEM43/VDAC interaction, which affects the stability of ER/SR-mitochondrial contact sites and, thus, proper mitochondrial function and oxidative phosphorylation rates. The consequential undersupply of ATP likely results in cardiac cell death and, ultimately, heart failure.

Drosophila nephrocytes are specialised cells that share critical functional, morphological, and molecular features with mammalian podocytes. Accordingly, nephrocytes represent a preferred invertebrate model for human glomerular disease. Here, we established a method for cell-specific isolation of the two types of Drosophila nephrocytes, garland and pericardial cells, from animals of different developmental stages and ages. Mass spectrometry-based proteomics and RNA-Seq-based transcriptomics were applied to characterise the proteome and transcriptome of the respective cells in an integrated and complementary manner. We observed characteristic changes in the proteome and transcriptome due to cellular ageing. Furthermore, functional enrichment analyses suggested that larval and adult nephrocytes, as well as garland and pericardial nephrocytes, fulfil distinct physiological functions. In addition, the pericardial nephrocytes were characterised by transcriptomic and proteomic profiles suggesting an atypical energy metabolism with very low oxidative phosphorylation rates. Moreover, the nephrocytes displayed typical signatures of extensive immune signalling and showed an active antimicrobial response to an infection. Factor-specific comparisons identified novel candidate proteins either expressed and secreted by the nephrocytes or sequestered by them. The data generated in this study represent a valuable basis for a more specific application of the Drosophila model in analysing renal cell function in health and disease.

Drosophila, like all insects, has an open circulatory system for the distribution of haemolymph and its components. The circulation of the haemolymph is essentially driven by the pumping activity of the linear heart. The heart is constructed as a tube into which the haemolymph is sucked and pumped forward by rhythmic contractions running from the posterior to the anterior, where it leaves the heart tube. The heart harbours cardiac valves to regulate flow directionality, with a single heart valve differentiating during larval development to separate the heart tube into two chambers. During metamorphosis, the heart is partially restructured, with the linear heart tube with one terminal wide-lumen heart chamber being converted into a linear four-chambered heart tube with three valves. As in all metazoan circulatory systems, the cardiac valves play an essential role in regulating the direction of blood flow. We provide evidence that the valves in adult flies arise via transdifferentiation, converting lumen-forming contractile cardiomyocytes into differently structured valve cells. Interestingly, adult cardiac valves exhibit a similar morphology to their larval counterparts, but act differently upon heart beating. Applying calcium imaging in living specimens to analyse activity in valve cells, we show that adult cardiac valves operate owing to muscle contraction. However, valve cell shape dynamics are altered compared with larval valves, which led us to propose our current model of the opening and closing mechanisms in the fly heart.

Neprilysins are highly conserved ectoenzymes that hydrolyze and thus inactivate signaling peptides in the extracellular space. Herein, we focus on Neprilysin 4 from Drosophila melanogaster and evaluate the existing knowledge on the physiological relevance of the peptidase. Particular attention is paid to the role of the neprilysin in regulating feeding behavior and the expression of insulin-like peptides in the central nervous system. In addition, we assess the function of the peptidase in controlling the activity of the sarcoplasmic and endoplasmic reticulum Ca²⁺ ATPase in myocytes, as well as the underlying molecular mechanism in detail.