Structure of Lung-Mimetic Multilamellar Bodies with Lipid Compositions Relevant in Pneumonia

Abstract

The hierarchical assembly of lipids, as modulated by composition and environment, plays a significant role in the function of biological membranes and a myriad of diseases. Elevated concentrations of calcium ions and cardiolipin (CL), an anionic tetra-alkyl lipid found in mitochondria and some bacterial cell membranes, have been implicated in pneumonia recently. However, their impact on the physicochemical properties of lipid assemblies in lungs and how it impairs alveoli function is still unknown. We use small- and wide-angle X-ray scattering (S/WAXS) and solid-state nuclear magnetic resonance (ssNMR) to probe the structure and dynamics of lung-mimetic multilamellar bodies (MLBs) in the presence of Ca and CL. We conjecture that CL overexpressed in the hypophase of alveoli strongly affects the structure of lung-lipid bilayers and their stacking in the MLBs. Specifically, S/WAXS data revealed that CL induces significant shrinkage of the water-layer separating the concentric bilayers in multilamellar aggregates. ssNMR measurements indicate that this interbilayer tightening is due to undulation repulsion damping as CL renders the glycerol backbone of the membranes significantly more static. In addition to MLB dehydration, CL promotes intrabilayer phase separation into saturated-rich and unsaturated-rich lipid domains that couple across multiple layers. Expectedly, addition of Ca screens the electrostatic repulsion between negatively charged lung membranes. However, when CL is present, addition of Ca results in an apparent interbilayer expansion likely due to local structural defects. Combining S/WAXS and ssNMR on systems with compositions pertinent to healthy and unhealthy lung membranes, we propose how alteration of the physiochemical properties of MLBs can critically impact the breathing cycle.

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“…In contrast, the diseased lung extract films display considerably weaker lamellar peaks. This differs to the results obtained for bulk BLES systems in diseased states 17 where clear lamellar phases could be detected. This indicates that indeed BLES samples are not as well oriented with respect to the device substrate when compared to the model system.…”

“…In the healthy state, both the model membrane and BLES display clear lamellar GISAXS peaks (indicated as 001, and 002) with a repeat distance d ¼ 2π=q 001 ¼ 98 Å. Consistent with our previous findings in the same systems in bulk, a second set of lamellar peaks (marked by 001′, d 001 0 ¼ 80 Å) emerges due to domain formation within lung membranes and alignment of domains in registry, which is expected for this lipid mixture of saturated and unsaturated phospholipids 17,57 . In the diseased state of the model system, clear lamellar peaks are still present in the integrated profiles.…”

“…The thickness of these two membranes was comparable ($100 nm) as shown in the cross-section topography profiles. Compared to healthy mimetic membranes, diseased-mimetic membranes exhibit higher surface roughness, consistent with cardiolipin and Ca 2þ -induced morphological changes 17 .…”

“…We have previously demonstrated 17 that the combination of cardiolipin and calcium ions promotes in-plane domain formation in model lung membrane systems (here represented schematically by the green and light blue areas in Fig. 4b).…”

“…In addition, lung fluid from humans and mice infected with bacterial pneumonia displays significantly elevated contents of cardiolipin, a mitochondrial-specific phospholipid 16 . We have recently demonstrated that cardiolipin induces significant water loss in lung membrane extracts and model multilamellar bodies 17 .…”