Dynamin-related protein 1 regulates substrate oxidation in skeletal muscle by stabilizing cellular and mitochondrial calcium dynamics

Mitochondria undergo continuous cycles of fission and fusion to promote inheritance, regulate quality control, and mitigate organelle stress. More recently, this process of mitochondrial dynamics has been demonstrated to be highly sensitive to nutrient supply, ultimately conferring bioenergetic plasticity to the organelle. However, whether regulators of mitochondrial dynamics play a causative role in nutrient regulation remains unclear. In this study, we generated a cellular loss-of-function model for dynamin-related protein 1 (DRP1), the primary regulator of outer membrane mitochondrial fission. Loss of DRP1 (shDRP1) resulted in extensive ultrastructural and functional remodeling of mitochondria, characterized by pleomorphic enlargement, increased electron density of the matrix, and defective NADH and succinate oxidation. Despite increased mitochondrial size and volume, shDRP1 cells exhibited reduced cellular glucose uptake and mitochondrial fatty acid oxidation. Untargeted transcriptomic profiling revealed severe downregulation of genes required for cellular and mitochondrial calcium homeostasis, inhibition of ATP-stimulated calcium flux, and impaired substrate oxidation stimulated by calcium levels. The insights obtained herein suggest that DRP1 regulates fatty acid oxidation by altering whole-cell and mitochondrial calcium dynamics. These findings are relevant to the targetability of mitochondrial fission and have clinical relevance in the identification of treatments for fission-related pathologies such as hereditary neuropathies, inborn errors in metabolism, cancer, and chronic diseases. Overall design: Differentiated cells were lysed and total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was quantified using NanoDrop and normalized to 200 ng/µL in nuclease-free water. RNA integrity was assessed with Agilent Bioanalyzer 2100. Libraries were constructed and sequenced using Lexogen QuantSeq. Briefly, library generation was performed using an oligodT primer, and double-stranded cDNA was purified with magnetic beads. Libraries were amplified using PCR, and transcripts were forward sequenced at 75 bp using NextSeq 500 (Illumina). BlueBee software was used to analyze alignment and DESeq2 was used for differential expression analysis. n=5-6 per group