Sex-Specific Patterns of Diaphragm Phospholipid Content and Remodeling during Aging and in a Model of SELENON-Related Myopathy

Abstract

Growing evidence shows that the lipid bilayer is a key site for membrane interactions and signal transduction. Surprisingly, phospholipids have not been widely studied in skeletal muscles, although mutations in genes involved in their biosynthesis have been associated with muscular diseases. Using mass spectrometry, we performed a phospholipidomic profiling in the diaphragm of male and female, young and aged, wild type and SelenoN knock-out mice, the murine model of an early-onset inherited myopathy with severe diaphragmatic dysfunction. We identified 191 phospholipid (PL) species and revealed an important sexual dimorphism in PLs in the diaphragm, with almost 60% of them being significantly different between male and female animals. In addition, 40% of phospholipids presented significant age-related differences. Interestingly, SELENON protein absence was responsible for remodeling of 10% PL content, completely different in males and in females. Expression of genes encoding enzymes involved in PL remodeling was higher in males compared to females. These results establish the diaphragm PL map and highlight an important PL remodeling pattern depending on sex, aging and partly on genotype. These differences in PL profile may contribute to the identification of biomarkers associated with muscular diseases and muscle aging.

Keywords: aging; cardiolipin; mass spectrometry; myopathy; phospholipid; selenoprotein N; sexual dimorphism; sphingomyelin.

Figure 1

Anatomical parameters of WT and SelenoN KO 1-month (young) and 20-month-old (aged) male and female mice (TA for tibialis anterior). In the graph, p-value are from Tukey’s post hoc tests between pairwise groups. Next to the graph, p-values for sex, age and SelenoN effect are defined in Table 2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns not significant.

Figure 2

Diaphragm phospholipidomic investigation. (a) F-ratio analysis. (b) ANOVA analysis. (c) Number of significant PLs between male and female in young WT, young KO, aged WT and aged KO groups. (d) Number of significantly different PLs between young and aged in female WT, female KO, male WT and male KO groups. (e) Number of significantly different PLs between WT and KO in female young, female aged, male young and male aged groups.

Figure 3

Ethanolamine-containing PLs in diaphragm. (a) Analysis of m/z intensity of PE species (mean ± SEM; n = 4 for each group, except young male WT and KO with n = 5 and aged male KO with n = 3). (b) Analysis of m/z intensity of LPE species. In the graph, p-value are from Tukey’s post hoc tests between pairwise groups. Under the graph, p-values for sex, age and SelenoN effect are defined in Table 2. * p < 0.05 and ** p < 0.01.

Figure 4

PL containing long fatty acyl chains in diaphragm present sex dimorphism. (a) Analysis of m/z intensity of PL (40:6) species (mean ± SEM; n = 4 for each group, except young male WT and KO with n = 5 and aged male KO with n = 3). (b) Analysis of m/z intensity of PL (38:4) species. In the graph, p-values are from Tukey’s post hoc tests between pairwise groups. Under the graph, p-values for sex, age and SelenoN effect are defined in Table 2. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 5

Sphingomyelin and PL (36:1) remodeling in a sex, age and/or SelenoN KO context. (a) Analysis of m/z intensity of SM species (mean ± SEM; n = 4 for each group, except young male WT and KO with n = 5 and aged male KO with n = 3). (b) Analysis of m/z intensity of PL (36:1) species. In the graph, p-value are from Tukey’s post hoc tests between pairwise groups. Under the graph, p-values for sex, age and SelenoN effect are defined in Table 2. * p < 0.05 and ** p < 0.01.

Figure 6

Cardiolipin remodeling in a sex, age and/or SelenoN KO context. (a) Analysis of m/z intensity of CL species with combined sex, age and SelenoN effects (mean ± SEM; n = 4 for each group, except young male WT and KO with n = 5 and aged male KO with n = 3). (b) Analysis of m/z intensity of CL species with sex, age or SelenoN significant differences. (c) Analysis of m/z intensity of CL containing 70 or 72 carbons. (d) Analysis of m/z intensity of CL containing 74 carbons. In the graph, p-value are from Tukey’s post hoc tests between pairwise groups. Under the graph, p-values for sex, age and SelenoN effect are defined in Table 2. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 6

Cardiolipin remodeling in a sex, age and/or SelenoN KO context. (a) Analysis of m/z intensity of CL species with combined sex, age and SelenoN effects (mean ± SEM; n = 4 for each group, except young male WT and KO with n = 5 and aged male KO with n = 3). (b) Analysis of m/z intensity of CL species with sex, age or SelenoN significant differences. (c) Analysis of m/z intensity of CL containing 70 or 72 carbons. (d) Analysis of m/z intensity of CL containing 74 carbons. In the graph, p-value are from Tukey’s post hoc tests between pairwise groups. Under the graph, p-values for sex, age and SelenoN effect are defined in Table 2. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 7

Lipid biosynthesis, remodeling and transport pathways. mRNA expression analysis in male and female, young and aged and WT and SelenoN KO diaphragm normalized to RPL19 and HPRT mean. (a) Fabp3 (fatty acid binding protein3) involved in FA transport. (b) Sms1 (sphingomyelin synthase 1) involved in SM biosynthesis. (c) Crls1 (cardiolipin synthase1) involved in CL biosynthesis. (d) AcadM (acyl-coA dehydrogenase medium chain), AcadVL (acyl-coA dehydrogenase very long chain) and Hadha (hydroxyacyl-coA dehydrogenase alpha) involved in FA beta oxidation. (e) Hacd1 (3-hydroxyacyl-coA dehydratase 1), Elov1 (fatty acid elongase1) and Elov3 (fatty acid elongase3) involved in FA elongation. (f) Fas (fatty acid synthase), Scd1 (stearoyl-coA desaturase 1) and Scd2 (stearoyl-coA desaturase 2) involved in lipogenesis. Mean ± SEM; n = 3 for KO aged male and female, n = 4 for WT young and aged female and WT aged male, and n = 6 for young WT male and female and young KO male). In the graph, p-values are from Tukey’s post hoc tests between pairwise groups. Next to the graph, p-values for sex, age and SelenoN effect are defined in Table 2.