Comparative lipidomic and metabolomic profiling of mdx and severe mdx-apolipoprotein e-null mice

Abstract

Despite its notoriously mild phenotype, the dystrophin-deficient mdx mouse is the most common model of Duchenne muscular dystrophy (DMD). By mimicking a human DMD-associated metabolic comorbidity, hyperlipidemia, in mdx mice by inactivating the apolipoprotein E gene (mdx-ApoE) we previously reported severe myofiber damage exacerbation via histology with large fibro-fatty infiltrates and phenotype humanization with ambulation dysfunction when fed a cholesterol- and triglyceride-rich Western diet (mdx-ApoE^W). Herein, we performed comparative lipidomic and metabolomic analyses of muscle, liver and serum samples from mdx and mdx-ApoE^W mice using solution and high-resolution-magic angle spinning (HR-MAS) ¹H-NMR spectroscopy. Compared to mdx and regular chow-fed mdx-ApoE mice, we observed an order of magnitude increase in lipid deposition in gastrocnemius muscle of mdx-ApoE^W mice including 11-fold elevations in -CH₃ and -CH₂ lipids, along with pronounced elevations in serum cholesterol, fatty acid, triglyceride and phospholipids. Hepatic lipids were also elevated but did not correlate with the extent of muscle lipid infiltration or differences in serum lipids. This study provides the first lipometabolomic signature of severe mdx lesions exacerbated by high circulating lipids and lends credence to claims that the liver, the main regulator of whole-body lipoprotein metabolism, may play only a minor role in this process.

Keywords: Duchenne; Fibrofatty infiltration; HR-MAS; Liver; Metabolism; Muscle; NMR.

Fig. 1

Representative Mason’s trichrome image of severely affected gastrocnemius muscle from Western diet (W)-fed mdx and mdx-ApoE KO mice, scale bar 1 mm. HR-MAS spectra for gastrocnemius, quadriceps and tibialis anterior muscle samples (aliphatic region) showing lipids and small metabolites resonance for four different groups: (1) mdx-ApoE^W (brown; A, E, & I), (2) mdx-ApoE^R (blue; B, F, & J), (3) mdx^R (black; C, G, & K), and (4) mdx^W (green; D, H, & L)

Fig. 2

Box and Whisker plots showing relative abundance of lipid CH₃ and lipid CH₂ in gastrocnemius (Gastroc), quadriceps(Quad), and tibialis anterior muscles (TA) via ¹H HR-MAS spectra. Significance was determined by two way ANOVA with “p” = 0.05 − 0.01, “p” ≤ 0.05 is denoted with “*”/”^#”/”⁺”, “p” between 0.01 − 0.001 is denoted with “**”/”^##”/”⁺⁺”, and “p” ≤ 0.001 is denoted with “***”/”^###”/”⁺⁺⁺”. “Asterisks” means significantly different within the same strain, “hash tag” means significantly different than the other strain within the regular (Reg) diet group, and “plus” means significantly different than the other high-fat Western diet strain. Numbers were mdx-ApoE^R (n = 5), mdx-ApoE^W (n = 7–9), mdx^R (n = 4), and mdx^W (n = 5–8)

Fig. 3

Heatmap representing the log₂-transformed fold changes with respect to mdx^R group for few lipid classes in six tissue samples obtained via ¹H HR-MAS spectra. A Gastroc, B Quad, C TA, D Soleus, E EDL, and F Liver. The number of samples per group were as follows: mdx-ApoE^R (n = 5), mdx-ApoE^W(n = 7–9), mdx^R(n = 4), and mdx^W(n = 5–8). Gastroc: gastrocnemius, quad: quadriceps, TA: tibialis anterior, EDL: extensor digitorum longus, and FC: fold-change

Fig. 4

Representative Box and Whisker plots (with error bars) for lipid components obtained from ¹H NMR spectra of organic phase extract of serum for four diet-dependent groups of mdx mice: A Cholesterol/cholesterol ester (CH₃, C18), B Phospholipid/Triglyceride (-CH₂-CH=CH-CH₂), C Fatty acids (terminal CH₃), D Fatty acids (-CH=CH-) E Phospholipid/Triglyceride (CH₂-CCO-), and F Phosphatidylcholine (N⁺(CH₃)₃). Significance was determined by two way ANOVA with “p” = 0.05-0.01, “p” ≤ 0.05 is denoted with “*”/”^#”/”⁺”, “p”≤ 0.001 is denoted with “***”/”^###”/”⁺⁺⁺”, and “p” ≤ 0.0001 is denoted with “****”/”^####”/”⁺⁺⁺⁺”. “Asterisk/s” means significantly different within the same strain, “hash tag” means significantly different than the other strain within the regular diet group, and “plus” means significantly different than the other high-fat diet strain. The number of samples per group were as follows: -ApoE^R (n=5), mdx-ApoE^W(n=9), mdx^R(n=4), and mdx^W(n=8)

Fig. 5

¹H NMR metabolomics profiles for four diet-dependent groups of mdx mice for organic phase sera samples obtained from: A principal component analysis B Partial least square discriminant analysis. mdx-ApoE^R (red), mdx-ApoE^W(green), mdx^R (blue), and mdx^W (cyan). Number of samples per group were as follows: mdx-ApoE^R (n = 5), mdx-ApoE^W(n = 9), mdx^R(n = 4), and mdx^W(n = 8). PC: principal component

Fig. 6

Heat maps representing the log₂-transformed fold changes compared with the mdx^R group for organic and aqueous phase serum samples obtained via 1D ¹H NOESY spectra. A organic phase, B aqueous phase. FC: fold-change