RRAGD p.(Ser76Leu) Variant Causes Dysregulated Expression of Muscle Development and Cytoskeleton Genes in Cardiomyocytes.

Autosomal dominant kidney hypomagnesemia with RRAGD variants (ADKH-RRAGD) is a hereditary disorder characterized by kidney tubulopathy and dilated cardiomyopathy (DCM). RagD, encoded by the RRAGD gene, is a small GTPase involved in activating the mechanistic target of rapamycin complex 1 (mTORC1) by amino acids. Although several gain-of-function variants in the RRAGD gene have been identified, their contributions to DCM remain unclear. Here, we hypothesize that these RRAGD variants induce mTORC1 overactivation, thereby contributing to the manifestation of DCM. To investigate this, we established T-REx HeLa cell lines that overexpress the RRAGD p.(Ser76Leu) or the wild-type (WT) variant to assess the effects on mTORC1 signaling. Additionally, we developed the first cellular model of ADKH-RRAGD utilizing genetically edited human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that express the mutated variant. Our data indicate that the RRAGD p.(Ser76Leu) variant maintains the phosphorylation of mTORC1 targets (i.e., S6K, 4E-BP1, and TFEB) during amino acid starvation, in contrast to RRAGD WT in T-REx HeLa cells. The pharmacological inhibition of mTOR with Torin1 reversed these changes. In 2D-cultured RRAGD