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

Cardiolipin (CL) is a four acyl chain, 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 chain monolysocardiolipin (MLCL) as well as CL species with saturated acyl chains (CLsat). While the presence of MLCL destabilizes electron transport chain (ETC) complexes and IMM-shaping proteins, the contributions of CLsat to mitochondrial dysfunction have not been elucidated. Here, we find that treatment of TAZ knockout cells with exogenous saturated fatty acids causes accumulation of CLsat and loss of IMM structure despite only minimal changes in MLCL composition. Cells with elevated CLsat show reduced fluidity of the inner membrane measured by a solvatochromic probe. 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 CLsat as a potential driver of mitochondrial dysfunction and an additional therapeutic target in mitigating BTHS pathology.