Low Intensity, High Frequency Vibration Training to Improve Musculoskeletal Function in a Mouse Model of Duchenne Muscular Dystrophy

The objective of the study was to determine if low intensity, high frequency vibration training impacted the musculoskeletal system in a mouse model of Duchenne muscular dystrophy, relative to healthy mice. Three-week old wildtype (n = 26) and mdx mice (n = 22) were randomized to non-vibrated or vibrated (45 Hz and 0.6 g, 15 min/d, 5 d/wk) groups. In vivo and ex vivo contractile function of the anterior crural and extensor digitorum longus muscles, respectively, were assessed following 8 wks of vibration. Mdx mice were injected 5 and 1 days prior to sacrifice with Calcein and Xylenol, respectively. Muscles were prepared for histological and triglyceride analyses and subcutaneous and visceral fat pads were excised and weighed. Tibial bones were dissected and analyzed by micro-computed tomography for trabecular morphometry at the metaphysis, and cortical geometry and density at the mid-diaphysis. Three-point bending tests were used to assess cortical bone mechanical properties and a subset of tibiae was processed for dynamic histomorphometry. Vibration training for 8 wks did not alter trabecular morphometry, dynamic histomorphometry, cortical geometry, or mechanical properties (P≥0.34). Vibration did not alter any measure of muscle contractile function (P≥0.12); however the preservation of muscle function and morphology in mdx mice indicates vibration is not deleterious to muscle lacking dystrophin. Vibrated mice had smaller subcutaneous fat pads (P = 0.03) and higher intramuscular triglyceride concentrations (P = 0.03). These data suggest that vibration training at 45 Hz and 0.6 g did not significantly impact the tibial bone and the surrounding musculature, but may influence fat distribution in mice

Estradiol deficiency and skeletal muscle apoptosis : Possible contribution of microRNAs

Background Menopause leads to estradiol (E2) deficiency that is associated with decreases in muscle mass and strength. Here we studied the effect of E2 deficiency on miR-signaling that targets apoptotic pathways. Methods C57BL6 mice were divided into control (normal estrous cycle, n = 8), OVX (E2 deficiency, n = 7) and OVX + E2 groups (E2-pellet, n = 4). Six weeks following the OVX surgery, mice were sacrificed and RNA isolated from gastrocnemius muscles. miR-profiles were studied with Next-generation sequencing (NGS) and candidate miRs verified using qPCR. The target proteins of the miRs were found using in silico analysis and measured at mRNA (qPCR) and protein levels (Western blot). Results Of the apoptosis-linked miRs present, eleven (miRs-92a-3p, 122-5p, 133a-3p, 214-3p, 337-3p, 381-3p, 483-3p, 483-5p, 491-5p, 501-5p and 652-3p) indicated differential expression between OVX and OVX + E2 mice in NGS analysis. In qPCR verification, muscle from OVX mice had lower expression of all eleven miRs compared with OVX + E2 (p < 0.050). Accordingly, OVX had higher expression of cytochrome C and caspases 6 and 9 compared with OVX + E2 at the mRNA level (p < 0.050). At the protein level, OVX also had lower anti-apoptotic BCL-W and greater pro-apoptotic cytochrome C and active caspase 9 compared with OVX + E2 (p < 0.050). Conclusion E2 deficiency down regulated several miRs related to apoptotic pathways thus releasing their targets from miR-mediated suppression, which may lead to increased apoptosis and contribute to reduced skeletal muscle mass. Abbreviations AIFApoptosis inducing factorBCL2B-cell lymphoma-2 regulator proteinBCL-XLB-cell lymphoma-extra-large regulator proteinBCL-WB-cell lymphoma-like protein 2CASPCaspasecytCCytochrome CE2EstradiolFasLFas ligandGAPDHGlyceraldehyde 3-phosphate dehydrogenaseHSPHeat shock proteinmiRmicroRNANGSNext-generation sequencingOVXOvariectomypeerReviewe

Muscle contractile measures following 8 weeks of low intensity, high frequency vibration training in wildtype and <i>mdx</i> mice.

<p>Muscle contractile measures following 8 weeks of low intensity, high frequency vibration training in wildtype and <i>mdx</i> mice.</p

Eight weeks of vibration training affected fat pad masses but not body masses.

<p>Vibrated mice had smaller sized subcutaneous fat pads following 8-weeks of training. <i>Mdx</i> mice had a larger body mass but smaller fat pad masses compared to wildtype mice following 8-weeks of training. Body masses were not different in mice subject to vibration compared to non-vibrated control mice. Data are means ± SE. P-values associated with the main effects of two-way ANOVAs are indicated above each set of bars. Interactions between vibration and genotype P≥0.056.</p

Eight weeks of vibration training did not impact tibial cortical bone.

<p>Vibration training for 8 weeks did not influence the following tibial cortical bone properties: <b>A)</b> cross-sectional moment of inertia, <b>B)</b> ultimate load, or <b>C)</b> stiffness. <i>Mdx</i> mice had lower values for ultimate load and trends for lower stiffness compared to wildtype mice. Data are means ± SE. P-values associated with the main effects of two-way ANOVAs are indicated above each set of bars. Interactions between vibration and genotype P≥0.287.</p

Effects of low intensity, high frequency vibration training and genotype on muscle and muscle fiber characteristics.

<p>Effects of low intensity, high frequency vibration training and genotype on muscle and muscle fiber characteristics.</p

Eight weeks of vibration did not impact <i>in</i> <i>vivo</i> muscle strength or susceptibility to injury.

<p><b>A)</b> Maximal isometric torque was not different between vibrated and non-vibrated mice following 8-weeks of training; isometric torque was less in <i>mdx</i> than wildtype mice. Interaction between vibration and genotype P≥0.357. <b>B)</b> Vibration training for 8 weeks did not alter susceptibility to eccentric contraction-induced injury. As expected, <i>mdx</i> mice were more susceptible to eccentric injury relative to wildtype mice. Data are means ± SE. In Panel A, P-values associated with the main effect of two-way ANOVAs are indicated above the bars. In panel B, only a main effect of genotype was present, where * signifies a significant (P<0.05) difference between <i>mdx</i> and wildtype mice at that contraction number.</p

Eight weeks of vibration training did not impact <i>ex</i> <i>vivo</i> EDL muscle contractile function.

<p>Vibration training for 8 weeks did not influence the following EDL muscle contractile measures: <b>A)</b> maximal isometric tetanic force production, <b>B)</b> specific force, or <b>C)</b> susceptibility to eccentric contraction-induced injury compared to non-vibrated mice. As expected, <i>mdx</i> mice had lower values for each of the three measurements of EDL muscle function compared to wildtype mice. Data are means ± SE. P-values associated with the main effects of two-way ANOVAs are indicated above each set of bars in Panel A and B. In panel C, an interaction between genotype and eccentric contraction number was present, where the * signifies a significant (P<0.05) difference between <i>mdx</i> and wildtype mice from post-hoc testing. Interactions between vibration and genotype for panels A and B P≥0.329.</p