Supplementation with a selective amino acid formula ameliorates muscular dystrophy in mdx mice

Duchenne muscular dystrophy (DMD) is one of the most common and severe forms of muscular dystrophy. Oxidative myofibre content, muscle vasculature architecture and exercise tolerance are impaired in DMD. Several studies have demonstrated that nutrient supplements ameliorate dystrophic features, thereby enhancing muscle performance. Here, we report that dietary supplementation with a specific branched-chain amino acid-enriched mixture (BCAAem) increased the abundance of oxidative muscle fibres associated with increased muscle endurance in dystrophic mdx mice. Amelioration of the fatigue index in BCAAem-treated mdx mice was caused by a cascade of events in the muscle tissue, which were promoted by endothelial nitric oxide synthase (eNOS) activation and vascular endothelial growth factor (VEGF) expression. VEGF induction led to recruitment of bone marrow (BM)-derived endothelial progenitors (EPs), which increased the capillary density of dystrophic skeletal muscle. Functionally, BCAAem mitigated the dystrophic phenotype of mdx mice without inducing dystrophin protein expression or replacing the dystrophin-associated glycoprotein (DAG) complex in the membrane, which is typically lost in DMD. BCAAem supplementation could be an effective adjuvant strategy in DMD treatment.


FACS analysis.
Peripheral blood (100 µl) from the retro orbital sinus was collected from each mouse, and red blood cells were lysed with ACK solution (150 mM NH4Cl, 10 mM KHCO3 and 0.1 mM Na2EDTA) to allow cytofluorimetric studies. Muscles were weighed and washed several times in PBS, finely minced with scissors, and incubated at 37°C for 45 min with 1 mg/mL collagenase type I (Sigma-Aldrich, USA), 80 g/mL DNase I (Roche), and 2.5% trypsin (1:3) (Gibco) in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen). Most of the skeletal muscle stem cells were released from the tissue after this step. The cell extract was filtered with a 70-µm nylon mesh (BD Biosciences, Immunocytometry Systems, Mountain View, CA) and labelled for FACS analysis as described above. After each incubation, performed at 4°C for 20 min, cells were washed in PBS containing 1% heat-inactivated foetal calf serum (FCS) and 0.1% sodium azide. Isotypematched immunoglobulins were added to each control sample. The cells were analysed using a Cytomics FC500 instrument and CXP 2.1 software (BC, Beckman-Coulter). Each analysis included at least 50000-200000 events for each gate. A light-scatter gate was set to eliminate cell debris from the analysis. The percentage of positive cells was assessed after correction for the percentage of cells reactive to an isotype control conjugated to relative fluorochromes.
Bone marrow was collected from treated and untreated mdx and C57BL6/J mice by flushing femurs and tibias with saline solution, and red blood cells were lysed with ACK solution.
Nucleated cells were labelled in PBS containing 2% FCS for 45 min at 4°C with the previously described antibodies.
Endothelial colony formation assay.
Blood-circulating EGFP+ and EGFP-cells were isolated by FACS for co-expression of Sca1 and CD34 and incubated in an endothelial growth medium containing medium 199 (Gibco-BRL, Life Technologies, Thermo Fisher Scientific) supplemented with 50 ng/ml VEGF. Cells were then seeded at a density of 30,000 cell/cm 2 on six-well plates coated with Matrigel (Corning, Corning, NY, https://www.corning.com/medium)/199 medium (1:1) and incubated in a humidified atmosphere with 5% CO2/95% air, at 37°C. For each condition, cells from at least three different mice were seeded in triplicate wells. After 24-48 hours, we observed the formation of colonies by using an inverted microscope (Leica DMIRE2). To evaluate endothelial colony formation potential, three different operators counted colonies with more than 20 cells (Mandò et al., 2016).

SUPPLEMENTARY FIGURES AND FIGURE LEGENDS
Supplementary Figure 1 Changes in plasma branched-chain amino acids after BCAAem bolus administration and evaluation of daily drinking water Plasma samples of untreated male and female C57BL6/J (n=4) and mdx (n=4) mice were obtained before (t0) and at different time intervals after (t1, 30 min; t2, 60 min; t3, 120 min) a single bolus of BCAAem, corresponding to the daily supplementation dose (1.5 g/kg body weight), was dissolved in tap water and administered by gavage (a). Water consumption was measured daily (b). Data are expressed as the mean ± s.e.m. Statistical error analysis was performed by two-way ANOVA with Bonferroni correction. BCAAem increases fatigue resistance in mdx-EGFP mice. Muscle function of BCAAemtreated and untreated C57BL6/J-EGFP and mdx-EGFP mice was determined by an incremental treadmill exhaustion test. Exhaustion time is expressed in min (n=5 for each group) (a). In vivo endurance time is expressed in min (b). Muscle function was evaluated in vitro, by measuring the tetanic force (g/µL) of single fibres isolated from TA strips (c). Fatigue index, defined as the decrease in tetanic force at different stimulation frequencies (from 0.03 to 0.3 Hz), is compared to the maximal tetanic force (Tf) and expressed in percentage (d). **** p<0.0001 at 0.09 and 0.3 Hz indicates the comparison between BCAAem-treated or untreated C57BL6/J-EGFP and mdx-EGFP mice, respectively; *p<0.05 at 0.3 Hz indicates the comparison between BCAAem-treated and untreated mdx-EGFP mice. All data are presented as the mean ± s.e.m. Statistical error analysis was performed by two-way ANOVA with Bonferroni correction; ****p<0.0001 indicate comparisons that reflect significant differences relative to the untreated group.