Saturated cardiolipins are potent disruptors of inner mitochondrial membrane structure and function

Cardiolipin (CL) is a four-acyl chained, mitochondrial-specific phospholipid crucial for maintenance of inner mitochondrial membrane (IMM) structure and function. In healthy tissues, CL acyl chains are highly unsaturated and maintained by a conserved remodeling pathway. However, dysregulation of CL acyl chain composition can arise from mutations in the CL transacylase, Tafazzin (TAZ), resulting in Barth syndrome (BTHS), where patients exhibit heightened mitochondrial dysfunction. Cells lacking TAZ accumulate three-acyl chained monolysocardiolipin (MLCL) as well as CL species with saturated acyl chains (CL_sat). While the presence of MLCL destabilizes electron transport chain (ETC) complexes and IMM-shaping proteins, the contributions of CL_sat to mitochondrial dysfunction have not been elucidated. Here, we find that treatment of TAZ knockout cells with exogenous saturated fatty acids causes accumulation of CL_sat and loss of IMM structure despite only minimal changes in MLCL composition. Imaging of cells with elevated CL_sat showed reduced fluidity of the inner membrane. Biophysical measurements and molecular dynamics analyses showed that di-saturated (C16:0 18:1)₂ CL species order and rigidify membranes, while also losing the intrinsic lipid curvature characteristic of tetra-unsaturated CL. These results implicate CL_sat as a potential driver of mitochondrial dysfunction and an additional therapeutic target in mitigating BTHS pathology.