Discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to Duchenne muscular dystrophy patients

Abstract

It is expected that serum protein biomarkers in Duchenne muscular dystrophy (DMD) will reflect disease pathogenesis, progression and aid future therapy developments. Here, we describe use of quantitative in vivo stable isotope labeling in mammals to accurately compare serum proteomes of wild-type and dystrophin-deficient mdx mice. Biomarkers identified in serum from two independent dystrophin-deficient mouse models (mdx-Δ52 and mdx-23) were concordant with those identified in sera samples of DMD patients. Of the 355 mouse sera proteins, 23 were significantly elevated and 4 significantly lower in mdx relative to wild-type mice (P-value < 0.001). Elevated proteins were mostly of muscle origin: including myofibrillar proteins (titin, myosin light chain 1/3, myomesin 3 and filamin-C), glycolytic enzymes (aldolase, phosphoglycerate mutase 2, beta enolase and glycogen phosphorylase), transport proteins (fatty acid-binding protein, myoglobin and somatic cytochrome-C) and others (creatine kinase M, malate dehydrogenase cytosolic, fibrinogen and parvalbumin). Decreased proteins, mostly of extracellular origin, included adiponectin, lumican, plasminogen and leukemia inhibitory factor receptor. Analysis of sera from 1 week to 7 months old mdx mice revealed age-dependent changes in the level of these biomarkers with most biomarkers acutely elevated at 3 weeks of age. Serum analysis of DMD patients, with ages ranging from 4 to 15 years old, confirmed elevation of 20 of the murine biomarkers in DMD, with similar age-related changes. This study provides a panel of biomarkers that reflect muscle activity and pathogenesis and should prove valuable tool to complement natural history studies and to monitor treatment efficacy in future clinical trials.

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Figures

Figure 1.

Distribution of protein ratios identified in proteome profiling of sera samples of mdx-52 mice and wild-type BL6 mice spiked at 1:1 ratio with serum aliquots from SILAC-labeled BL6 mouse. Each serum aliquot containing 50 µg of total proteins from 3 weeks old mdx-52 mice (n = 3) or 3 weeks old BL6 mice (n = 3) was spiked with 50 µg serum aliquots from 13C6-Lys labeled BL10 serum and processed for proteome profiling and quantification as described in Materials and Methods. Top panel shows the overall log ratio distribution of all identified unlabeled and labeled peptide pairs in 1:1 serum mixtures of unlabeled BL10 mice relative to 13C6-Lys labeled BL6 internal standard and the bottom panel show similar ratios distribution in 1:1 serum mixture of unlabeled mdx-52 mice relative to 13C6-Lys labeled BL6 internal standard. All distributions showed a normal Gaussian shape with values centered around 1 in BL6 versus SILAC BL6 pairs and wilder distribution in mdx-52 versus SILAC BL6 pairs analyzed. An average of 180–200 proteins were identified and quantified in each analysis.

Figure 2.

Box plots showing levels of six representative proteins in sera samples of mdx-23 mice compared with its healthy counterpart wild-type BL10 mice. Serum samples from 3 weeks old mdx-23 mice (n = 3) and age-matched wild-type BL10 mice (n = 3) were each spiked with fixed amount of 15N-labeled serum samples from wild-type mice as internal standard. Sample mixtures were processed for protein identification and quantification as described in Materials and Methods. Tope panel shows an example of three proteins, aldoalse, cystochrom-c somatic and myoglobin whose level was significantly (P-value < 0.001) higher in serum of mdx-23 mice group compared with the wild-type BL10 mice group. Bottom panels show an example of two proteins, adiponectin and lumican whose level was significantly decreased in serum of the mdx-23 mice group compared with wild-type Bl10 mice (P-valued < 0.001) and transtherytin protein that remained unchanged between the two groups.

Figure 3.

Hierarchical clustering of selected candidate protein biomarkers in serum samples of mdx-23 mice across different age group. Serum samples from mdx-23 mice at 7 days, 3 weeks, 2 months, 4 months and 7 months of age (n = 3 per group) were spiked with fixed amount of 15N-labeled serum from the wild-type mouse as internal standard. Similarly serum samples from wild-type Bl10 mice at 7 days, 5 weeks and 2 months of age were spiked with 15N- labeled serum and used as controls. Sample mixtures were processed for proteomes profiling as described in Materials and Methods. Ratios of candidate biomarkers were measured in serum samples across different age groups and then uploaded into Partek software for clustering analysis. SILAC ratio values were natural log transformed, and the color scale is based on how many standard deviations each value deviates from the mean (white for values below the mean, and black for values above the mean). Clustering by columns shows that a select group of biomarkers is able to discriminate wild-type BL10 from mdx-23 mice, and age within each group. Clustering by rows shows consolidation of biomarker ‘classes’ described in the text.

Figure 4.

Western blot analysis showing levels of PGAM2 in sera samples of DMD patients and healthy controls. Serum aliquots containing 30 µg total proteins from DMD patients with different age groups (4 to 5, 6–9 and 11–15 years of age, n = 5 per group) and from healthy controls (n = 6) with age ranging from 6 to 15 years old were processed for western blot analysis against PGAM2 as described in Materials and Methods. Top panel shows the actual western blot results of PGAM2 which was detected around its expected molecular mass of 30 kDa and it was present only in serum samples of DMD patients (D1 to D15) and undetectable in serum of healthy controls (CT1 to CT6) . The bottom panel shows the density histogram plot of the PGAM2 band across different age groups. The data clearly show the rapid age-dependent decrease in the serum levels of PGAM2 in DMD patients.

Figure 5.

Serum levels of candidate biomarkers discovered by label-free proteome profiling in serum samples of DMD patients and healthy control across different age groups. Serum aliquots containing 100 µg total proteins from 12 DMD patients at 4, 8, 12 and 15 years of age (n = 3 per age group) and from 3 healthy controls at 6, 8 and 12 years of age were processed for label-free proteome profiling as described in Materials and Methods. (A) Levels of five glycolytic enzymes in DMD groups and control group. PYGM, glycogen phosphorylase; ALDOA, fructose-bisphosphate aldolase A; LDHB, lactate dehydrogenase B; LDHA, lactate dehydrogenase A; ENOB, beta enolase. (B) Levels of five myofibrillar proteins, in DMD groups and control group. MYOM3, myomesin-3; TITIN, titin; ACTS, alpha skeletal muscle actin; FLNC, filamin-C; MYL1, myosin light chain 1/3 in DMD groups and control group. (C) Levels of nine other muscle-specific proteins in DMD groups and control group. MYG, myoglonin; CAH3, carbonic anhydrase III; ADIPO, adiponectin; TSP4, thrombospondin-4; MDHC, cytosolic malate dehydrogenase; CYC, somatic cytochrome C; MMP9, matrix metalloproteinase-9; FIBG, fibrinogen gamma chain; FABPH, fatty acid-binding protein heart type. A CK level was added along with glycolytic enzyme in (A) and myofibrillar proteins in (B) for trends comparison. It was omitted in the other muscle-specific protein group in (C) due to the large dynamic range between the levels of CK and this class of proteins. Error bars represent standard error obtained from triplicate biological sample.