High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis

Abstract

Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs) are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes.

Figure 1

Mesenchymal stem cells (MSCs) administration inhibits the decreased muscle strength induced by a high fat diet (HFD) in mice. C57BL/10J male mice were fed with a standard chow (control) or HFD for 38 weeks. At week 30, a subgroup of HFD mice received MSC injected through the tail-vein. At week 38, all mice were subjected to the following: (a) A weightlifting test to determine limb muscle strength: values represent the percentage of muscle strength reached by the mice with each weight and correspond to the mean ± SD (n = 8; ^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD, two-way ANOVA). (b) A weightlifting test: the values represent the scores normalized by body weight. The values correspond to the mean ± SD (n = 8; ^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD, two-way ANOVA). (c) Maximal isometric strengths (mN/mm²) against stimulation frequencies (Hz) in the tibialis anterior (TA) muscles: values represent the mean ± SD (n = 8; ^∗ P < 0.05 versus HFD, two-way ANOVA).

Figure 2

Systemic mesenchymal stem cells (MSCs) administration inhibits fiber type transitions in the tibialis anterior (TA) muscle of mice fed with a high fat diet (HFD). C57BL/10J male mice were fed with a standard chow (control) or HFD for 38 weeks. At week 30, a subgroup of HFD mice received MSC injected through the tail-vein. At week 38, all mice were sacrificed and the TA was analyzed to determine fiber type through the immunohistochemical detection of myosin heavy chain isoforms (IIa and IIb). Images obtained at 10x (a) and 40x (b) magnification show fiber types IIa (upper panel) and IIb (lower panel). Quantitative analysis of the fiber type is shown in (c). Graph representing the percentage of specific fiber types relative to the total fibers counted per field. Values represent the mean ± SD (n = 8; ^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD, two-way ANOVA).

Figure 3

Mesenchymal stem cells (MSCs) administration prevents the decreased fiber diameter of the tibialis anterior (TA) muscle in high fat diet- (HFD-) induced skeletal muscle atrophy. TA muscles were obtained from C57BL/10J male mice fed with standard chow (control), a HFD, or a HFD-MSC treated. Muscle cross sections were stained with wheat germ agglutinin to delimit muscle fiber sarcolemma. (a) Images were obtained at 10x and 40x magnification. Bars correspond to 150 μm. (b) Minimal Feret diameters were determined in TA cross sections from (b). Fiber diameters were grouped and ranged from 0 to 70 μm. Values are expressed as a percentage of the total quantified fibers. Values correspond to the mean ± SD (n = 8; ^∗ P < 0.05 versus control, two-way ANOVA).

Figure 4

Systemic mesenchymal stem cells (MSCs) administration prevents the decreased myosin heavy chain (MHC) levels in the tibialis anterior (TA) muscle of mice fed with a high fat diet (HFD). C57BL/10J male mice were fed with a standard chow (control) or HFD for 38 weeks. At week 30, a subgroup of HFD mice received MSC injected through the tail-vein. After eight weeks, all mice were sacrificed and the TA was excised and homogenized to evaluate the following: (a) MHC protein levels through Western blot analysis (GAPDH levels were used as the loading control; molecular weight markers are shown in kDa) and (b) quantitative analysis of the experiments from (a). The levels of MHC normalized to GAPDH are expressed relative to control mice (^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD).

Figure 5

Mesenchymal stem cells (MSCs) administration inhibits increases in ubiquitinated proteins and atrogin-1 and MuRF-1 gene expression levels induced by a high fat diet (HFD) in mice. C57BL/10J male mice were fed with a standard chow (control) or HFD for 38 weeks. At week 30, a subgroup of HFD mice received MSC injected through the tail-vein. At week 38, all mice were sacrificed and the TA was excised and homogenized to evaluate the following: (a) total ubiquitinated (Ub) protein levels through Western blot analysis (tubulin levels were used as the loading control; molecular weight markers are shown in kDa) and (b) quantitative analysis of the experiments from (a). The levels of Ub normalized to tubulin are expressed relative to control mice (^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD). Detection of atrogin-1 (c) and MuRF-1 (d) mRNA levels through RT-qPCR using 18S as the reference gene. Expressions are shown as the fold of induction relative to the TA from control mice, and the values correspond to the mean ± SD (^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD).

Figure 6

Systemic mesenchymal stem cells (MSCs) administration prevents increased reactive oxygen species (ROS) levels in the tibialis anterior (TA) of mice fed with a high fat diet (HFD). C57BL/10J male mice were fed with a standard chow (control) or HFD for 38 weeks. At week 30, a subgroup of HFD mice received MSC injected through the tail-vein. At week 38, all mice were sacrificed. (a) Cryosections obtained from the TA were incubated with a DCF probe for ROS detection through fluorescence microscopy. Nuclei were labelled via Hoechst staining. (b) Quantification of ROS levels from experiments showed in (a). The values are expressed as the fold of induction of the DCF probe intensity. Values correspond to the mean ± SD (n = 8; ^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD, two-way ANOVA).

Figure 7

Mesenchymal stem cells (MSCs) administration inhibits the increased myonuclear apoptosis induced by a high fat diet (HFD) in mice. C57BL/10J male mice were fed with a standard chow (control) or HFD for 38 weeks. At week 30, a subgroup of HFD mice received MSC injected through the tail-vein. At week 38, all mice were sacrificed and the tibialis anterior (TA) was excised and homogenized to evaluate (a) Bax and Bcl2 protein levels through Western blot analysis; (b) Bax/Bcl2 ratio analysis (the values are expressed as fold of induction relative to the control and correspond to the mean ± SD (n = 8; ^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD, two-way ANOVA)); (c) cleaved caspase-3 levels detected by Western blot analysis; (d) quantification of caspase-3 levels from experiments shown in (c). The values are expressed as fold of induction relative to the control and correspond to the mean ± SD (n = 8; ^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD, two-way ANOVA). For (a) and (c), the levels of tubulin are shown as the loading control. The molecular weights are shown in kDa. (e) Caspase-3 activity was measured and expressed as fold of induction relative to the control, with values corresponding to the mean ± SD (n = 8; ^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD; two-way ANOVA). (f) Cryosections of TA were used to perform the TUNEL assay. The bar corresponds to 50 μm. (g) Blinded quantification of TUNEL-positive nuclei per field in five randomly captured images. The values are expressed as the fold of induction relative to the control and correspond to the mean ± SD (n = 8; ^∗ P < 0.05 versus control; ^# P < 0.05 versus HFD, two-way ANOVA).