It is essential to understand the role of cardiolipin (CL) in mitochondrial 
membrane organization given that changes in CL levels contribute to 
mitochondrial dysfunction in type II diabetes, ischemia-reperfusion injury, 
heart failure, breast cancer, and aging. Specifically, there are contradictory 
data on how CL influences the molecular packing of membrane phospholipids. 
Therefore, we determined how increasing levels of heart CL impacted molecular 
packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures 
found within the mitochondrial inner membrane, using merocyanine (MC540) 
fluorescence. We broadly categorized lipid vesicles of equal mass as loosely 
packed, intermediate, and highly packed based on peak MC540 fluorescence 
intensity. CL had opposite effects on loosely versus highly packed vesicles. 
Exposure of loosely packed vesicles to increasing levels of CL dose-dependently 
increased membrane packing. In contrast, increasing amounts of CL in highly 
packed vesicles decreased the packing in a dose-dependent manner. In vesicles 
that were categorized as intermediate packing, CL had either no effect or 
decreased packing at select doses in a dose-independent manner. Altogether, the 
results aid in resolving some of the discrepant data by demonstrating that CL 
displays differential effects on membrane packing depending on the composition 
of the lipid environment. This has implications for mitochondrial protein 
activity in response to changing CL levels in microdomains of varying 
composition.