Molecular structural diversity of mitochondrial cardiolipins

Abstract

Current strategies used to quantitatively describe the biological diversity of lipids by mass spectrometry are often limited in assessing the exact structural variability of individual molecular species in detail. A major challenge is represented by the extensive isobaric overlap present among lipids, hampering their accurate identification. This is especially true for cardiolipins, a mitochondria-specific class of phospholipids, which are functionally involved in many cellular functions, including energy metabolism, cristae structure, and apoptosis. Substituted with four fatty acyl side chains, cardiolipins offer a particularly high potential to achieve complex mixtures of molecular species. Here, we demonstrate how systematically generated high-performance liquid chromatography-mass spectral data can be utilized in a mathematical structural modeling approach, to comprehensively analyze and characterize the molecular diversity of mitochondrial cardiolipin compositions in cell culture and disease models, cardiolipin modulation experiments, and a broad variety of frequently studied model organisms.

Keywords: cardiolipin; lipids; mass spectrometry; mathematical modeling; mitochondria.

Fig. 1.

Quantifying CLs by HPLC-MS/MS. (A) A typical mass-chromatogram obtained for a RAW 264.7 cell lipid extract is shown. (B) Integrated CL peak areas projected back onto their respective carbon chain length and double-bond components. Circle sizes indicate the CL mass-species peak areas, fill colors correspond to the respective total number of side-chain carbons. The ISTD CL(14:0)₄ is shown in gray. (C) Representation of the monoisotopic mass peak and the first three isotope peaks corresponding to CL70:4 (Upper) and as sum of the mass range 1,428.1 ± 0.3 (Lower Left), which elutes at least as double peak. (Lower Right) Hypothetical deconvolution of CL70:4 into contributions of individual molecular CL species.

Fig. 2.

Mathematical modeling of fragment spectra allows detailed structural characterization of individual CL species. (A) Initial fragmentation of CLs occurs primarily along the diphosphoglyceryl backbone, cleaving the CL into two PA fragments (PA1/2), which are substituted with two FAs, respectively (FA1–4). Three major PA fragment types are formed (SI Appendix, Fig. S5). (B) MS2 scans of CL70:4 (1,428.1 m/z, Left) and CL72:5 (1,454.2 m/z, Right) are shown, along with their manual PA fragment annotation. For example, the three major PA34:2 fragments correspond to 671.6, 727.6, and 807.6 m/z. (C) The PA fragment m/z distributions overlap between the three fragmentation paths and hamper an automated unique annotation. (D) CL characterization of one RAW 264.7 sample including an absolute quantitative CL profile (Top), and PA composition data projected onto the carbon chain length (Middle) and double-bond count (Bottom) components, respectively. (E) PA and (F) FA abundance profiles for the same sample as in D.

Fig. 3.

Impairment of CLs in BTHS patient fibroblasts. (A) Quantified CL composition observed in representative (i) control (n = 6) and (ii) BTHS patient fibroblasts (n = 3). Legend according to Fig. 1B. (B) BTHS cells exhibit (i) a reduced total CL content and increased (ii) absolute and (iii) relative MLCL levels. (C) The double-bond distribution in BTHS CLs is shifted toward a higher degree of saturation and (D) the same trend was observed at the FA level. Side-chain length distributions of (E) CLs and (F) FA in BTHS cells are enriched for lower carbon numbers. (G) Principal component analysis of FA profiles clearly separated BTHS cells from controls in dimension 1 (53.48% of variance, see Inset) and FA16:0, FA16:1, FA18:1, and FA18:2 were identified as major factors. (H) Two-way ANOVA was conducted on the influence of two variables (cell line, FA). The main effect for FA profiles resulted in an F-ratio of F(112, 1) = 167.6, P < 2e-16. Post hoc Bonferroni-corrected analysis revealed 2 of 112 possible fatty acids being significantly altered. We find a significant increase of FA16:0 (P = 2.11e-36) in BTHS cells while FA18:2 was strongly depleted (P = 3.24e-34). ***P < 0.001.

Fig. 4.

CL profile adaptation in response to lipid supplementation. (A) Quantified CL composition of HeLa cells grown in lipid and serum free (control, Left), standard (DMEM/FCS, Center), and pig heart lipids supplemented conditions (Heart, Right). One representative sample is shown (n = 4). Legend according to Fig. 1B. (B) FA composition in control (Top) and heart (Bottom) condition and their differential profile (Delta, Middle) (n = 4). (C) Oxygen flow in intact control and heart samples. (D) Doubling times of control and heart samples (n = 4). (E) Oxygen flow in permeabilized control and heart samples. NADH-pathway capacity (N-OXPHOS) was significantly increased upon heart lipid supplementation (n = 8, P = 0.01, Bonferroni-adjusted with m = 7). (F) Representative traces of oxygen flow for experiment shown in E used to calculate respiratory activities in different pathway-control states. Arrows indicate substrate-uncoupler-inhibitor titration steps: ADP, adenosine diphosphate; Ama, antimycin A; Ce, cells; Cyt, cytochrome c; Dig, digitonin; ET, maximal electron transfer capacity in presence of CCCP uncoupler (n = 4, Bonferroni-adjusted with m = 9); Glu, glutamate; LEAK, respiration after inhibition of ATP synthase; N, NADH-pathway; NS, convergent N- and succinate-pathway; PM, pyruvate and malate; Rot, rotenone; ROUTINE, cell respiration in presence of endogenous substrates; S, succinate; SP, succinate-pathway; U, uncoupler (CCCP). If not otherwise stated, data are shown as mean ± SD. *P < 0.05, **P < 0.01.

Fig. 5.

CL compositions across the domains of life. (A) PLS-DA visualizes the differences among CL profiles recorded for species and conditions. Components 1 and 2 captured 19% and 11% of variability, respectively. Clear species and condition-specific differences were observed and samples grouped according to their biological replicates. (B) FA profiles calculated for CL profiles shown in A. Bars represent the mean profile abundance of each FA in all biological replicates measured for the respective condition. Double-bond number per FA is depicted in color code. Complete data can be found in Dataset S1 and illustrated in SI Appendix, Fig. S18.