Daily Supplementation of D-ribose Shows No Therapeutic Benefits in the MHC-I Transgenic Mouse Model of Inflammatory Myositis

<div><p>Background</p><p>Current treatments for idiopathic inflammatory myopathies (collectively called myositis) focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1), leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg) over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures.</p><p>Results</p><p>Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose.</p><p>Conclusions</p><p>Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis.</p></div

Quantitative RT-PCR analysis suggests that a lack of RBKS prevents the utilization of D-ribose.

<p>After mice were sacrificed, RNA was isolated from the quadriceps muscle tissue and used to determine the expression of several genes. The expression of each gene was normalized to the level of GAPDH mRNA. QRT-PCR was performed to measure the expression of AMPD1 <i>(A)</i> and muscle creatine kinase (CKM) in the quadriceps muscle of mice (<i>B</i>). Results for the expression of ribokinase (RBKS) in the quadriceps muscle in mice are shown (<i>C</i>), as are results for hexokinase (HK2) in mice (<i>D</i>). HT mice showed an acquired deficiency of AMPD1 expression that was not significantly affected by daily oral D-ribose treatments. QRT-PCR analysis also revealed that RBKS was 5.53-fold less abundant than hexokinase in both healthy and diseased mice.</p

Mice with myositis are deficient for the breakdown metabolites of AMP.

<p>Hypoxanthine levels in mice with myositis (HT mice, n = 10) were significantly lower than in healthy (H mice, n = 10) littermates (<i>A</i>). IMP levels in HT mice were also lower in HT mice than in healthy controls (<i>B</i>). All mice involved in metabolite assays were 16-week-old females. Metabolite levels were measured in lysates from quadriceps muscle tissue.</p

IL-1Ra treatment decreases activity and increases rest time in <i>mdx</i> mice.

<p>Horizontal activity (A) was measured using an open-field Digiscan apparatus and did not differ significantly between IL-1Ra- and saline-treated mice. During the Digiscan measurements, the (B) movement time (26% decrease in the IL-1Ra-treated mice vs. control, p = 0.0075) and (C) total distance traveled (27% decrease in the IL-1Ra-treated mice vs. control, p = 0.008) were recorded and were significantly lower in the IL-1Ra-treated mice than in the controls. Graphs represent mean ± SEM and statistically significant differences were determined by parametric, unpaired, two- tailed, t-tests with a p≤0.05 being significant. (n = 6 saline, n = 10 IL-1Ra).</p

Treatment with oral doses of D-ribose does not improve body mass over time.

<p>Body weights for mice were recorded starting at the time of D-ribose administration. Mice were sacrificed at 16 weeks of age. The gap in bodyweight between healthy controls and untreated diseased mice widened over time (<i>A</i>). Treatment with D-ribose had no apparent failed to prevent weight loss in HT animals (<i>B</i>).</p

Histological analysis of mouse skeletal muscle showed no difference between untreated and treated animals.

<p>After mice were sacrificed at 16 weeks of age, quadriceps tissue was sectioned and stained with H&E. Untreated healthy mice (H) showed normal muscle histology (<i>A</i>). Healthy mice treated with daily oral D-ribose (H+Rib) also showed normal muscle histology (<i>B</i>). Untreated myositis mice (HT) showed variable muscle fiber diameter consistent with myositis (<i>C</i>). Myositis mice treated with daily oral D-ribose (HT+Rib) showed no apparent improvement at the histological level (<i>D</i>).</p

AMP catabolism can generate free D-ribose.

<p>The catabolism of AMP into hypoxanthine (Hpx) and ribose is illustrated, along with the utilization of ingested D-ribose (<i>A</i>). The treatment schedule of mice is depicted (<i>B</i>). Mice received a total of 6 weeks of daily oral D-ribose supplements. Behavioral assays require 5 days to conduct, and were therefore carried out one week prior to sacrificing the animals and performing electrophysiology. Abbreviations: IMP: inosine monophosphate, Hpx: hypoxanthine, D-ribose-1P: D-ribose-1-phosphate, D-ribose-5P: D-ribose-5-phosphate.</p

Treatment with daily oral D-ribose did not improve mouse body weight, grip strength and open field behavioral activity.

<p>Data from four treatment groups are shown: Healthy untreated mice (H, n = 8), healthy mice given daily oral D-ribose (H+Rib, n = 7), untreated myositis mice (HT, n = 6), and myositis mice given daily oral D-ribose (HT +Rib, n = 8). The body weight for all animals was measured at 16 weeks of age (<i>A</i>). Thoracic limb grip strength for all animals was measured over 5 days and normalized to body weight (<i>B</i>). Mouse voluntary movement in an open field was measured as movement in an hour averaged over 5 days. Horizontal motion measured how many times an infrared beam was broken in the open field (<i>C</i>). Distance traveled was measured by how many centimeters each mouse moved (<i>D</i>). Vertical movements indicate the number of times each mouse stood upright (<i>E</i>). Overall, the onset of myositis resulted in a significant loss of body weight, while grip strength measurement did not discriminate well between groups. Treatment with ribose showed no beneficial effect on any of the measured parameters.</p

IL-1Ra treatment does not alter EDL muscle force.

<p>Muscle force in the extensor digitorum longus (EDL) muscle did not differ in saline-treated and IL-1Ra-treated <i>mdx</i> mice. There was a decrease in the strength capacity of the mice treated with IL-1Ra when compared to the controls for all measurements recorded: (A) twitch force, mN (5% decrease), (B) muscle force, mN (4% decrease) and (C) specific force, kN/m² (12% decrease); however, none of these differences was statistically significant. Values in the graphs represent mean ± SEM. Statistical significance was determined by parametric, unpaired, two-tailed, t-tests (n = 4 saline and n = 6 IL-1Ra).</p

IL-1Ra blunted the effect of LPS on IL-1 secretion in splenocytes from <i>mdx</i> mice.

<p>Enzyme-linked immunosorbent assay (ELISA) was performed on medium from primary splenocytes isolated from <i>mdx</i> mice that had been treated with IL-1Ra or saline. Splenocytes from IL-1Ra- and saline-treated mice were isolated and stimulated with lipopolysaccharide (LPS). Medium was collected after 24 h to quantify the levels of IL-1 secreted into the medium by the splenocytes. LPS treatment significantly increased the IL-1 production in the splenocytes from control mice (groups 1–2) and from the IL-1Ra-treated mice (groups 3–4). Although not significant, this increase was blunted in the IL-1Ra and was 36% lower than the amount of secreted IL-1 in group 2. Values in the graphs represent mean ± SEM. Statistically significant differences were determined by using parametric, unpaired, two-tailed, t-tests with a p≤0.05 being significant (n = 4 for each group tested).</p