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Proteomic Profiling of the Dystrophin-Deficient MDX Heart Reveals Drastically Altered Levels of Key Metabolic and Contractile Proteins.

Lewis C, Jockusch H, Ohlendieck K - J. Biomed. Biotechnol. (2010)

Bottom Line: The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood.Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains.Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, National University of Ireland, Maynooth, Kildare, Ireland.

ABSTRACT
Although Duchenne muscular dystrophy is primarily classified as a neuromuscular disease, cardiac complications play an important role in the course of this X-linked inherited disorder. The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood. We therefore carried out a fluorescence difference in-gel electrophoretic analysis of 9-month-old dystrophin-deficient versus age-matched normal heart, using the established MDX mouse model of muscular dystrophy-related cardiomyopathy. Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains. Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins.

No MeSH data available.


Related in: MedlinePlus

2D gel electrophoretic analysis of the dystrophic heart using the pH 6–11 range. Shown are Cy3-labelled gels of the soluble fraction from normal (a) and dystrophic MDX (c) cardiac muscle, as well as Cy5-labelled gels containing pooled standards ((b), (d)). Representative fluorescent DIGE gels with electrophoretically separated proteins are shown for the pH 6–11 range. The pH-values of the first dimension gel system and molecular mass standards (in kDa) of the second dimension are indicated on the top and on the left of the panels, respectively.
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fig2: 2D gel electrophoretic analysis of the dystrophic heart using the pH 6–11 range. Shown are Cy3-labelled gels of the soluble fraction from normal (a) and dystrophic MDX (c) cardiac muscle, as well as Cy5-labelled gels containing pooled standards ((b), (d)). Representative fluorescent DIGE gels with electrophoretically separated proteins are shown for the pH 6–11 range. The pH-values of the first dimension gel system and molecular mass standards (in kDa) of the second dimension are indicated on the top and on the left of the panels, respectively.

Mentions: In contrast to non-fluorescent protein dye methodology, comparative studies using fluorescent CyDyes have an enhanced dynamic range of protein coverage [39]. DIGE analysis represents a highly accurate quantitative technique that enables multiple protein samples to be separated on the same two-dimensional gel. This reduces the introduction of potential artifacts due to gel-to-gel variations, making comparative DIGE approaches one of the most powerful analytical tools for conducting comparative biomedical investigations [40]. In order to determine global changes in the heart due to deficiency in the dystrophin isoform Dp427, we have carried out a DIGE analysis of crude tissue extracts from 9-month old normal versus age-matched dystrophic MDX mice. Due to the high cost of breeding and maintaining mice to old age, as well as the considerable cost of fluorescent tagging of large protein populations, the number of biological repeats and analytical DIGE gels was kept to a minimum. In order to satisfy the statistical requirements for the generation of proper proteomic data sets and perform an optimized DIGE analysis of normal versus affected proteomes, we have followed the recommendations by Urfer et al. [56] and Karp and Lilley [48], respectively. In the first dimensional separation step via isoelectric focusing, both a pH 4–7 and a pH 6–11 range were employed to cover as many cardiac proteins with differing charges as possible. Figure 1 illustrates the neutral and the more acidic range of protein species and Figure 2 shows the separation of neutral and more basic heart proteins. Shown are representative two-dimensional gels of Cy3-labelled normal muscle (Figure 1(a); Figure 2(a), Cy3-labelled dystrophic muscle (Figure 1(c); Figure 2(c) and corresponding Cy5-labelled pooled standards (Figures 1(b),1(d); Figures 2(b), 2(d), which were analysed with the help of a Typhoon Trio variable imager and Progenesis 2D analysis software. Overall, 2509 distinct protein species were recognized on the lower and higher pH-range gels. Recently Raddatz et al. [57] have catalogued the soluble murine heart protein complement. The protein spot pattern presented here agrees in large parts with their comprehensive 2D proteomic map of cardiac tissue. Out of 2048 detectable protein spots on the pH 4–7 gels and 487 detectable protein spots on pH 6–11 gels, 79 protein spots showed a drastic differential expression pattern, with 3 proteins being increased and 26 distinct protein species being decreased. Electrospray ionization MS/MS analysis was carried out to unequivocally identify the cardiac proteins with a changed abundance in dystrophic heart fibres.


Proteomic Profiling of the Dystrophin-Deficient MDX Heart Reveals Drastically Altered Levels of Key Metabolic and Contractile Proteins.

Lewis C, Jockusch H, Ohlendieck K - J. Biomed. Biotechnol. (2010)

2D gel electrophoretic analysis of the dystrophic heart using the pH 6–11 range. Shown are Cy3-labelled gels of the soluble fraction from normal (a) and dystrophic MDX (c) cardiac muscle, as well as Cy5-labelled gels containing pooled standards ((b), (d)). Representative fluorescent DIGE gels with electrophoretically separated proteins are shown for the pH 6–11 range. The pH-values of the first dimension gel system and molecular mass standards (in kDa) of the second dimension are indicated on the top and on the left of the panels, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2874991&req=5

fig2: 2D gel electrophoretic analysis of the dystrophic heart using the pH 6–11 range. Shown are Cy3-labelled gels of the soluble fraction from normal (a) and dystrophic MDX (c) cardiac muscle, as well as Cy5-labelled gels containing pooled standards ((b), (d)). Representative fluorescent DIGE gels with electrophoretically separated proteins are shown for the pH 6–11 range. The pH-values of the first dimension gel system and molecular mass standards (in kDa) of the second dimension are indicated on the top and on the left of the panels, respectively.
Mentions: In contrast to non-fluorescent protein dye methodology, comparative studies using fluorescent CyDyes have an enhanced dynamic range of protein coverage [39]. DIGE analysis represents a highly accurate quantitative technique that enables multiple protein samples to be separated on the same two-dimensional gel. This reduces the introduction of potential artifacts due to gel-to-gel variations, making comparative DIGE approaches one of the most powerful analytical tools for conducting comparative biomedical investigations [40]. In order to determine global changes in the heart due to deficiency in the dystrophin isoform Dp427, we have carried out a DIGE analysis of crude tissue extracts from 9-month old normal versus age-matched dystrophic MDX mice. Due to the high cost of breeding and maintaining mice to old age, as well as the considerable cost of fluorescent tagging of large protein populations, the number of biological repeats and analytical DIGE gels was kept to a minimum. In order to satisfy the statistical requirements for the generation of proper proteomic data sets and perform an optimized DIGE analysis of normal versus affected proteomes, we have followed the recommendations by Urfer et al. [56] and Karp and Lilley [48], respectively. In the first dimensional separation step via isoelectric focusing, both a pH 4–7 and a pH 6–11 range were employed to cover as many cardiac proteins with differing charges as possible. Figure 1 illustrates the neutral and the more acidic range of protein species and Figure 2 shows the separation of neutral and more basic heart proteins. Shown are representative two-dimensional gels of Cy3-labelled normal muscle (Figure 1(a); Figure 2(a), Cy3-labelled dystrophic muscle (Figure 1(c); Figure 2(c) and corresponding Cy5-labelled pooled standards (Figures 1(b),1(d); Figures 2(b), 2(d), which were analysed with the help of a Typhoon Trio variable imager and Progenesis 2D analysis software. Overall, 2509 distinct protein species were recognized on the lower and higher pH-range gels. Recently Raddatz et al. [57] have catalogued the soluble murine heart protein complement. The protein spot pattern presented here agrees in large parts with their comprehensive 2D proteomic map of cardiac tissue. Out of 2048 detectable protein spots on the pH 4–7 gels and 487 detectable protein spots on pH 6–11 gels, 79 protein spots showed a drastic differential expression pattern, with 3 proteins being increased and 26 distinct protein species being decreased. Electrospray ionization MS/MS analysis was carried out to unequivocally identify the cardiac proteins with a changed abundance in dystrophic heart fibres.

Bottom Line: The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood.Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains.Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, National University of Ireland, Maynooth, Kildare, Ireland.

ABSTRACT
Although Duchenne muscular dystrophy is primarily classified as a neuromuscular disease, cardiac complications play an important role in the course of this X-linked inherited disorder. The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood. We therefore carried out a fluorescence difference in-gel electrophoretic analysis of 9-month-old dystrophin-deficient versus age-matched normal heart, using the established MDX mouse model of muscular dystrophy-related cardiomyopathy. Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains. Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins.

No MeSH data available.


Related in: MedlinePlus