Discovery of potential urine-accessible metabolite biomarkers associated with muscle disease and corticosteroid response in the mdx mouse model for Duchenne.

Urine is increasingly being considered as a source of biomarker development in Duchenne Muscular Dystrophy (DMD), a severe, life-limiting disorder that affects approximately 1 in 4500 boys. In this study, we considered the mdx mice-a murine model of DMD-to discover biomarkers of disease, as well as pharmacodynamic biomarkers responsive to prednisolone, a corticosteroid commonly used to treat DMD. Longitudinal urine samples were analyzed from male age-matched mdx and wild-type mice randomized to prednisolone or vehicle control via liquid chromatography tandem mass spectrometry. A large number of metabolites (869 out of 6,334) were found to be significantly different between mdx and wild-type mice at baseline (Bonferroni-adjusted p-value < 0.05), thus being associated with disease status. These included a metabolite with m/z = 357 and creatine, which were also reported in a previous human study looking at serum. Novel observations in this study included peaks identified as biliverdin and hypusine. These four metabolites were significantly higher at baseline in the urine of mdx mice compared to wild-type, and significantly changed their levels over time after baseline. Creatine and biliverdin levels were also different between treated and control groups, but for creatine this may have been driven by an imbalance at baseline. In conclusion, our study reports a number of biomarkers, both known and novel, which may be related to either the mechanisms of muscle injury in DMD or prednisolone treatment.

Dramatic elevation in urinary amino terminal titin fragment excretion quantified by immunoassay in Duchenne muscular dystrophy patients and in dystrophin deficient rodents.

Enzyme-linked and electrochemiluminescence immunoassays were developed for quantification of amino (N-) terminal fragments of the skeletal muscle protein titin (N-ter titin) and qualified for use in detection of urinary N-ter titin excretion. Urine from normal subjects contained a small but measurable level of N-ter titin (1.0 ± 0.4 ng/ml). A 365-fold increase (365.4 ± 65.0, P = 0.0001) in urinary N-ter titin excretion was seen in Duchene muscular dystrophy (DMD) patients. Urinary N-ter titin was also evaluated in dystrophin deficient rodent models. Mdx mice exhibited low urinary N-ter titin levels at 2 weeks of age followed by a robust and sustained elevation starting at 3 weeks of age, coincident with the development of systemic skeletal muscle damage in this model; fold elevation could not be determined because urinary N-ter titin was not detected in age-matched wild type mice. Levels of serum creatine kinase and serum skeletal muscle troponin I (TnI) were also low at 2 weeks, elevated at later time points and were significantly correlated with urinary N-ter titin excretion in mdx mice. Corticosteroid treatment of mdx mice resulted in improved exercise performance and lowering of both urinary N-ter titin and serum skeletal muscle TnI concentrations. Low urinary N-ter titin levels were detected in wild type rats (3.0 ± 0.6 ng/ml), while Dmdmdx rats exhibited a 556-fold increase (1652.5 ± 405.7 ng/ml, P = 0.002) (both at 5 months of age). These results suggest that urinary N-ter titin is present at low basal concentrations in normal urine and increases dramatically coincident with muscle damage produced by dystrophin deficiency. Urinary N-ter titin has potential as a facile, non-invasive and translational biomarker for DMD.

Decreased major urinary protein in male Bar Harbor 129 REJ dystrophic mice indicates a hormonal deficiency.

A significant decrease in major urinary protein (MUP) in adult male Bar Harbor 129REJ dystrophic mice correlated with a marked decrease in the amount of translatable MUPmRNA in the liver. Previous investigations have shown that MUP synthesis is under complex multihormonal regulation suggesting that the dystrophic mouse may have a hormonal deficiency.

Accelerated protein turnover in the skeletal muscle of dystrophic mice.

The excretion of 3-methylhistidine increased in the urine of dystrophic mice C57BL/6J. The content of 3-methylhistidine residue decreased in the muscle proteins of dystrophic mice, but not in other organs. Methylated proteins in the skeletal muscle, actin and myosin, were partially purified from the dystrophic and control muscles. The amount of 3-methylhistidine residue in unit weight of the actin and myosin preparations was normal in dystrophic muscle. These three facts indicate that the turnover rates of actin and myosin are increased in the muscle of the dystrophic mice.

Creatinine excretion as an index of myofibrillar protein mass in dystrophic mice.

1. Daily creatinine excretion in the urine of normal and dystrophic mice was determined and then the carcass proteins were quantitatively extracted into soluble, myofibrillar and collagenous fractions. 2. On a live body-weight basis, total carcass protein was 15% lower in dystrophic than in normal mice. Relative to carcass weight, however, the amount of protein was significantly lower only in male dystrophic mice. 3. The myofibrillar protein fraction comprised 36 . 3 and 32 . 5% of the total protein in male and female dystrophic mice and 48 . 8 and 45 . 0% respectively in normal mice. The decrease in myofibrillar protein in dystrophic mice was accompanied by an increase in the residual collagenous fraction of proteins. 4. The rate of excretion of creatinine was strongly correlated (r = +0 . 98) with the myofibrillar protein mass in each mouse. This relationship was the same for both normal and dystrophic mice, each gram of myofibrillar protein being associated with 3 . 6 mumol of creatinine excreted/day. 5. The creatinine excretion rate is a valid index of contractile muscle mass in murine dystrophy.

Myofibrillar protein degradation in the chicken. 3-Methylhistidine release in vivo and in vitro in normal and genetically muscular-dystrophic chickens.

Myofibrillar protein degradation was measured in 4-week-old normal (line 412) and genetically muscular-dystrophic (line 413) New Hampshire chickens by monitoring the rates of 3-methylhistidine excretion in vivo and in vitro. A method of perfusing breast and wing muscles was developed and the rate of 3-methylhistidine release in vitro was measured between 30 and 90min of perfusion. During this perfusion period, 3-methylhistidine release from the muscle preparation was linear, indicating that changes in 3-methylhistidine concentration of the perfusate were the result of myofibrillar protein degradation. Furthermore, the viability of the perfused muscle was maintained during this interval. After 60min of perfusion, ATP, ADP and creatine phosphate concentrations in pectoral muscle were similar to muscle freeze-clamped in vivo. Rates of glucose uptake and lactate production were constant during the perfusion. In dystrophic-muscle preparations, the rate of 3-methylhistidine release in vitro (nmol/h per g of dried muscle) was elevated 2-fold when compared with that in normal muscle. From these data the fractional degradation rates of myofibrillar protein in normal and dystrophic pectoral muscle were calculated to be 12 and 24% respectively. Daily 3-methylhistidine excretion (nmol/day per g body wt.) in vivo was elevated 1.35-fold in dystrophic chickens. Additional studies revealed that the anti-dystrophic drugs diphenylhydantoin and methylsergide, which improve righting ability of dystrophic chickens, did not alter 3-methylhistidine release in vitro. This result implies that changes in myofibrillar protein turnover are not the primary lesion in avian muscular dystrophy. From tissue amino acid analysis, the myofibrillar 3-methylhistidine content per g dry weight of muscle was similar in normal and dystrophic pectoral muscle. More than 96% of the 3-methylhistidine present in pectoral muscle was associated with the myofibrillar fraction. Dystrophic myofibrillar protein contained significantly less 3-methylhistidine (nmol/g of myofibrillar protein) than protein from normal muscle. This observation supports the hypothesis that there may be a block in the biochemical maturation and development of dystrophic muscle after hatching. Free 3-methylhistidine (nmol/g wet wt.) was elevated in dystrophic muscle, whereas blood 3-methylhistidine concentrations were similar in both lines. In summary, the increased myofibrillar protein catabolism demonstrated in dystrophic pectoral muscle correlates with the increased lysosomal cathepsin activity in this tissue as reported by others.

Abnormal levels of urinary catecholamines in dystrophic mice and hamsters.

Twenty-four-hour urine was collected from normal and dystrophic mice and hamsters for catecholamine determinations. A new method of analysis was used whereby 3,4-dihydroxyphenylalanine (DOPA), dopamine (DA), norepinephrine (NE), and epinephrine (E) were measured simultaneously. The procedure is based on a combination of liquid-solid extraction, cation exchange chromatography, and controlled potential electrochemistry. The results of these experiments indicated that while DA levels were similar in both normal and pathological animal urine, DOPA levels decreased slightly in the dystrophic mouse but not the hamster, and NE and E levels in dystrophic groups were two and four times greater than normal in both species. The data supports the concept of biochemical alterations in tissue other than muscle. While not necessarily supportive to catecholamine abnormality as the primary cause of muscular dystrophy, the present data cast doubt that this disease is a primary muscle disease.