Proteolipid domains form in biomimetic and cardiac mitochondrial vesicles and are regulated by cardiolipin concentration but not monolyso-cardiolipin

Abstract

Cardiolipin (CL) is an anionic phospholipid mainly located in the inner mitochondrial membrane, where it helps regulate bioenergetics, membrane structure, and apoptosis. Localized, phase-segregated domains of CL are hypothesized to control mitochondrial inner membrane organization. However, the existence and underlying mechanisms regulating these mitochondrial domains are unclear. Here, we first isolated detergent-resistant cardiac mitochondrial membranes that have been reported to be CL-enriched domains. Experiments with different detergents yielded only nonspecific solubilization of mitochondrial phospholipids, suggesting that CL domains are not recoverable with detergents. Next, domain formation was investigated in biomimetic giant unilamellar vesicles (GUVs) and newly synthesized giant mitochondrial vesicles (GMVs) from mouse hearts. Confocal fluorescent imaging revealed that introduction of cytochrome into membranes promotes macroscopic proteolipid domain formation associated with membrane morphological changes in both GUVs and GMVs. Domain organization was also investigated after lowering tetralinoleoyl-CL concentration and substitution with monolyso-CL, two common modifications observed in cardiac pathologies. Loss of tetralinoleoyl-CL decreased proteolipid domain formation in GUVs, because of a favorable Gibbs-free energy of lipid mixing, whereas addition of monolyso-CL had no effect on lipid mixing. Moreover, murine GMVs generated from cardiac acyl-CoA synthetase-1 knockouts, which have remodeled CL acyl chains, did not perturb proteolipid domains. Finally, lowering the tetralinoleoyl-CL content had a stronger influence on the oxidation status of cytochrome than did incorporation of monolyso-CL. These results indicate that proteolipid domain formation in the cardiac mitochondrial inner membrane depends on tetralinoleoyl-CL concentration, driven by underlying lipid-mixing properties, but not the presence of monolyso-CL.

The cardiolipin-binding peptide elamipretide mitigates fragmentation of cristae networks following cardiac ischemia reperfusion in rats

“…5f). This aggregation effect was not seen in vesicles devoid of CL, and our previous work has shown that proteins that do not associate with CL do not have this effect on CL aggregation 30 . Additionally, we serendipitously discovered that CL-containing vesicles and elamipretide eventually precipitated when in solution together (Supplemental Fig.…”

Monolysocardiolipin (MLCL) interactions with mitochondrial membrane proteins

“…Using electron spray mass spectroscopy, MLCL was shown to enhance Bid and tBid mitochondrial membrane association, relative to CL [78]. MLCL also appeared to enhance the release of cytochrome c [78] and affect the oxidation state of cytochrome c [79]. Furthermore, MLCL was shown to be capable of inducing tBid dissociation from nBid (the other Bid-cleavage product) [77].…”

“…In cultured lymphoblast cells from people with BTHS, apoptosis was found not to be increased [4]. However, MLCL interactions with many proteins involved in apoptosis have been investigated demonstrating that MLCL interacts with tBid more tightly than CL [77,78], caspase-8 [29] less tightly than CL, and also modifies the release of cytochrome c [78,79].…”

The role of cardiolipin concentration and acyl chain composition on mitochondrial inner membrane molecular organization and function

“…Using a complex mitochondrial inner membrane mimicking model, our lab has also shown that cyt c and mtCK promote proteolipid microdomain formation. These domains are highly sensitive to a decreased CL levels but not substitution of MLCL or (18:1) 4 CL, suggesting that CL content is the more important factor in regulating mitochondrial proteolipid microdomains (161). However, extreme remodeling by the complete incorporation of DHA into CL's acyl chains [(22:6) 4 CL] abolishes proteolipid microdomain organization, which is driven by changes in the Gibbs free energy of lipid mixing (87).…”