Cardiolipin (CL), the signature phospholipid of the inner mitochondrial membrane, accounts for approximately 12–15% of cardiac phospholipids and is dominated by tetralinoleoyl species ((18:2)₄CL; approximately 80–90% linoleate) with highly conserved acyl symmetry in healthy mammalian hearts. This linoleate-rich CL supports electron transport chain organisation (complexes I/III/IV and their supercomplexes), proton handling, substrate transport, ATP synthase activity, cytochrome-c anchoring, protein import, mitochondrial dynamics, and lipid signalling. Mass spectrometry consistently shows progressive remodelling of CL and the broader myocardial phospholipid pool in rodent models of heart failure and aging, as well as in human failing hearts. Linoleic acid (18:2n-6) declines, while highly unsaturated fatty acids (HUFAs), such as arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3), accumulate, particularly within cardiolipins. Importantly, this membrane polyunsaturated fatty acid (PUFA) shift is at least partially reversible with mechanical unloading (in patients supported with a left ventricular assist device), suggesting plasticity in the remodelling process.