Magnetic resonance quantification of skeletal muscle lipid infiltration in a humanized mouse model of Duchenne muscular dystrophy

Abstract

Rodent models of Duchenne muscular dystrophy (DMD) often do not recapitulate the severity of muscle wasting and resultant fibro-fatty infiltration observed in DMD patients. Having recently documented severe muscle wasting and fatty deposition in two preclinical models of muscular dystrophy (Dysferlin-null and mdx mice) through apolipoprotein E (ApoE) gene deletion without and with cholesterol-, triglyceride-rich Western diet supplementation, we sought to determine whether magnetic resonance imaging and spectroscopy (MRI and MRS, respectively) could be used to detect, characterize, and compare lipid deposition in mdx-ApoE knockout with mdx mice in a diet-dependent manner. MRI revealed that both mdx and mdx-ApoE mice exhibited elevated proton relaxation time constants (T₂ ) in their lower hindlimbs irrespective of diet, indicating both chronic muscle damage and fatty tissue deposition. The mdx-ApoE mice on a Western diet (mdx-ApoE^W ) presented with greatest fatty tissue infiltration in the posterior compartment of the hindlimb compared with other groups, as detected by MRI/MRS. High-resolution magic angle spinning confirmed elevated lipid deposition in the posterior compartments of mdx-ApoE^W mice in vivo and ex vivo, respectively. In conclusion, the mdx-ApoE^W model recapitulates some of the extreme fatty tissue deposition observed clinically in DMD muscle but typically absent in mdx mice. This preclinical model will help facilitate the development of new imaging modalities directly relevant to the image contrast generated in DMD, and help to refine MR-based biomarkers and their relationship to tissue structure and disease progression.

Keywords: Duchenne muscular dystrophy; fibro-fatty infiltration; magnetic resonance imaging and spectroscopy; mouse; muscle tissues; time domain nuclear magnetic resonance.

Figure 1.

Box and Whisker plots showing the body weight, fat percentage and lean mass percentage of the mice among four different groups obtained from TD NMR. Significance was determined by Two way ANOVA with Tukey’s multiple comparisons, and considered significant if “p” ≤ 0.05. “p” ≤ 0.05 is denoted with “*”/“^#”/“⁺”, “p” between 0.01–0.001 is denoted with “**”/“^##”/“⁺⁺”, and “p” ≤ 0.001 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 high-fat diet of the other strain. The number of samples per group were as follows: mdx-ApoE^R (n=5), mdx-ApoE^W(n=7), mdx^R(n=4), and mdx^W(n=5).

Figure 2.

Representative T₂-weighted MRI images of mdx and mdx-ApoE mice on Western high-fat diet or normal chow diet. A) mdx-ApoE^W, B) mdx-ApoE^R, C) mdx^W, and D) mdx^R groups. The number of samples per group were as follows: mdx-ApoE^R (n=5), mdx-ApoE^W(n=7), mdx^R(n=3), and mdx^W(n=5). Arrows indicating area of T₂ hyperintensity.

Figure 3.

In vivo 1D ¹H spectra showing lipid CH_2, creatine and taurine peaks. The lipid CH₂ level is elevated in mdx-ApoE^W group. A) Green square in the insert showing the location of the voxel utilized. B) Box and Whisker plot for Lipid CH₂/Creatine. Significance was determined by Two way ANOVA with Tukey’s multiple comparisons, and considered significant if “p” ≤ 0.05. “p” ≤ 0.05 is denoted with “*”/“^#”/“⁺”, “p” between 0.01–0.001 is denoted with “**”/“^##”/“⁺⁺”, and “p” ≤ 0.001 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 high-fat diet of the other strain. The mdx-ApoE^W group was found statistically different than everybody else. The number of samples per group were as follows: mdx-ApoE^R (n=5), mdx-ApoE^W(n=7), mdx^R(n=4), and mdx^W(n=5). All the spectra were normalized to the creatine resonance at 3.02 ppm.

Figure 4.

Representative chemical shift encoded fat fraction and water maps (fat fraction – top row and water second row) from mdx^R, mdx^W, mdx-ApoE^R, and mdx-ApoE^W groups. White arrow in mdx_ApoE^Win dicate areas of fat deposits, which were visible on multiple images from each individual animal of that group. Third row shows the localized proton spectra, scaled to the total creatine resonance at 3.02 ppm, from the posterior compartment of each mouse.

Figure 5.

Box and Whisker plots showing muscle fat fraction (%) in the entire posterior, anterior, and middle compartment of the lower hind-limb. A) Fat fraction (%) in gastroc (posterior), B) in TA (anterior), and C) in MID (medial). Significance was determined by Two way ANOVA with Tukey’s multiple comparisons, and considered significant if “p” ≤ 0.05. “p” ≤ 0.05 is denoted with “*”/“^#”/“⁺”, “p” between 0.01–0.001 is denoted with “**”/“^##”/“⁺⁺”, and “p” ≤ 0.001 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 high-fat diet of the other strain. The number of samples per group were as follows: mdx-ApoE^R (n=4), mdx-ApoE^W(n=7), mdx^R(n=3), and mdx^W(n=3). RCD: regular chow diet, HFD: high-fat diet, Gastroc: gastrocnemius, TA: Tibialis anterior, MID: medial.

Figure 6.

A portion of 1D ¹H HR-MAS representative spectra for gastrocnemius muscles showing some lipids and metabolite resonances. The elevation in lipid level can be clearly seen on Western high-fat diet samples.

Figure 7.

Box and Whisker plots showing relative abundance of lipids for gastrocnemius samples via ¹H HRMAS spectra. Significance was determined by Two way ANOVA with Tukey’s multiple comparisons, and considered significant if “p” ≤ 0.05. “p” ≤ 0.05 is denoted with “*”/“^#”/“⁺”, “p” between 0.01–0.001 is denoted with “**”/“^##”/“⁺⁺”, and “p” ≤ 0.001 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 high-fat diet of the other strain. The mdx-ApoE^W group was found statistically different than everybody else. The number of samples per group were as follows: mdx-ApoE^R (n=5), mdx-ApoE^W(n=8), mdx^R(n=4), and mdx^W(n=8). RCD: regular chow diet, HFD: high-fat diet.