Analysis and Quantification of the Mitochondrial-ER Lipidome.

Mitochondria are vital organelles essential for cellular functions, but their lipid composition and response to stressors are not fully understood. Recent advancements in lipidomics reveal insights into lipid functions, especially their roles in metabolic perturbations and diseases. Previous methods have focused on the protein composition of mitochondria and mitochondrial-associated membranes. The advantage of our technique is that it combines organelle isolation with targeted lipidomics, offering new insights into the composition and dynamics of these organelles in pathological conditions. We developed a mitochondria isolation protocol for L6 myotubes, enabling lipidomics analysis of specific organelles without interference from other cellular compartments. This approach offers a unique opportunity to dissect lipid dynamics within mitochondria and their associated ER compartments under cellular stress. Key features • Analysis and quantification of lipids in mitochondria-ER fraction through liquid chromatography-tandem mass spectrometry-based lipidomics (LC-MS/MS lipidomics). • LC-MS/MS lipidomics provide precise and unbiased information on the lipid composition in in vitro systems. • LC-MS/MS lipidomics facilitates the identification of lipid signatures in mammalian cells.

Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism.

Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP3-receptor (IP3R) calcium channels are necessary for the mitochondrial Ca2+ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling). Methods: Mitochondria matrix and cytoplasmic Ca2+ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca2+ sensor (CEPIA3mt) or a cytoplasmic Ca2+ sensor (RCaMP). The role of intracellular Ca2+ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP3R and Dantrolene for RyR1) and a genetic approach (shIP3R1-RFP). O2 consumption was detected using Seahorse Extracellular Flux Analyzer. Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca2+ levels. Mitochondrial Ca2+ uptake required functional inositol IP3R and RyR1 channels. Inhibition of either channel decreased basal O2 consumption rate but only RyR1 inhibition decreased ATP-linked O2 consumption. Cell membrane depolarization-induced Ca2+ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca2+ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca2+-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber. Conclusion: Ca2+-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an "excitation-metabolism" coupling process in skeletal muscle fibers.