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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

Immunofluorescence survey of mitochondrial content, nuclei and cardiac marker proteins in dystrophic tissue. Confocal microscopy was used for the localization of nuclei, mitochondria and cellular markers in normal ((a), (c), (e), (g), (i), (k), (m), (o), (q), (s)) versus dystrophic MDX ((b), (d), (f), (h), (j), (l), (n), (p), (r), (t)) heart cryosections. Shown is labeling of nuclei with the DNA binding dye DAPI ((a), (b)), visualization of mitochondria with the red-fluorescent MitoTracker dye CMXRos ((e)-(h)), and antibody labeling of full-length dystrophin isoform Dp427 ((c), (d)), desmin DES ((i)-(l)), prohibitin PRO ((m)-(p)) and succinate dehydrogenase SDH ((q)-(t)). The number of nuclei and the labeling of mitochondria with the MitoTracker dye CMXRos were found not to be significantly different between normal and dystrophic preparations. The bars in panels (a) to (t) equal 30 μm.
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fig7: Immunofluorescence survey of mitochondrial content, nuclei and cardiac marker proteins in dystrophic tissue. Confocal microscopy was used for the localization of nuclei, mitochondria and cellular markers in normal ((a), (c), (e), (g), (i), (k), (m), (o), (q), (s)) versus dystrophic MDX ((b), (d), (f), (h), (j), (l), (n), (p), (r), (t)) heart cryosections. Shown is labeling of nuclei with the DNA binding dye DAPI ((a), (b)), visualization of mitochondria with the red-fluorescent MitoTracker dye CMXRos ((e)-(h)), and antibody labeling of full-length dystrophin isoform Dp427 ((c), (d)), desmin DES ((i)-(l)), prohibitin PRO ((m)-(p)) and succinate dehydrogenase SDH ((q)-(t)). The number of nuclei and the labeling of mitochondria with the MitoTracker dye CMXRos were found not to be significantly different between normal and dystrophic preparations. The bars in panels (a) to (t) equal 30 μm.

Mentions: Since the DIGE analysis of the normal versus the dystrophic MDX heart has revealed changes in a variety of mitochondrial components, it was important to determine whether this alteration was due to a reduced number of mitochondria in the dystrophic heart or internal abundance changes in the mitochondrial protein complement. Figure 7 shows representative results from an immunofluorescence survey of mitochondrial content, nuclei and cardiac marker proteins in dystrophic tissue. Cryosections of MDX tissue showed a complete absence of the dystrophin isoform Dp427 (Figures 7(c), 7(d)), confirming the mutant status of the dystrophic hearts used in this study. DAPI staining revealed 229 ± 6 and 270 ±18 nuclei per examined tissue section in normal versus dystrophic tissue sections, respectively (Figures 7(a), 7(b)). The slight increase of nuclei in MDX preparations was not statistically significant (n = 4). Thus the proteomic finding of a dystrophy-dependent increase in lamin-A/C is therefore not directly related to a drastic change in the number of nuclei per cardiac tissue unit. Fluorescent labeling of mitochondria with the MitoTracker dye CMXRos showed a characteristic internal staining pattern in cardiac fibres (Figures 7(e)–7(h)). Fluorescent intensity values of 1470 ± 36 and 1384 ± 40 for normal versus MDX preparations were found not to be significantly different (n = 4). Hence, lower levels of mitochondrial enzymes do not seem to be a consequence of a drastic decrease in mitochondrial density in the dystrophic MDX heart. In addition, confocal microscopy was employed for the localization of cardiac marker proteins. The immunofluorescence labeling of desmin (Figures 7(i)–7(l)), prohibitin (Figures 7(m)–7(p)) and succinate dehydrogenase (Figures 7(q)–7(t)) was shown to be comparable between control and pathological samples. The microscopical localization of desmin and succinate dehydrogenase revealed unexpectedly not a decrease of these proteins in dystrophic tissue sections, as found by DIGE analysis. Hence, although distinct subspecies of certain cardiac proteins are affected in X-linked muscular dystrophy as determined by mass spectrometry-based proteomics, the overall isoform complement of these elements is not drastically altered. This indicates that no major differences exist in the expression levels of desmin-containing intermediate filament structures and the population of cardiac mitochondria in normal versus dystrophin-deficient cells.


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)

Immunofluorescence survey of mitochondrial content, nuclei and cardiac marker proteins in dystrophic tissue. Confocal microscopy was used for the localization of nuclei, mitochondria and cellular markers in normal ((a), (c), (e), (g), (i), (k), (m), (o), (q), (s)) versus dystrophic MDX ((b), (d), (f), (h), (j), (l), (n), (p), (r), (t)) heart cryosections. Shown is labeling of nuclei with the DNA binding dye DAPI ((a), (b)), visualization of mitochondria with the red-fluorescent MitoTracker dye CMXRos ((e)-(h)), and antibody labeling of full-length dystrophin isoform Dp427 ((c), (d)), desmin DES ((i)-(l)), prohibitin PRO ((m)-(p)) and succinate dehydrogenase SDH ((q)-(t)). The number of nuclei and the labeling of mitochondria with the MitoTracker dye CMXRos were found not to be significantly different between normal and dystrophic preparations. The bars in panels (a) to (t) equal 30 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig7: Immunofluorescence survey of mitochondrial content, nuclei and cardiac marker proteins in dystrophic tissue. Confocal microscopy was used for the localization of nuclei, mitochondria and cellular markers in normal ((a), (c), (e), (g), (i), (k), (m), (o), (q), (s)) versus dystrophic MDX ((b), (d), (f), (h), (j), (l), (n), (p), (r), (t)) heart cryosections. Shown is labeling of nuclei with the DNA binding dye DAPI ((a), (b)), visualization of mitochondria with the red-fluorescent MitoTracker dye CMXRos ((e)-(h)), and antibody labeling of full-length dystrophin isoform Dp427 ((c), (d)), desmin DES ((i)-(l)), prohibitin PRO ((m)-(p)) and succinate dehydrogenase SDH ((q)-(t)). The number of nuclei and the labeling of mitochondria with the MitoTracker dye CMXRos were found not to be significantly different between normal and dystrophic preparations. The bars in panels (a) to (t) equal 30 μm.
Mentions: Since the DIGE analysis of the normal versus the dystrophic MDX heart has revealed changes in a variety of mitochondrial components, it was important to determine whether this alteration was due to a reduced number of mitochondria in the dystrophic heart or internal abundance changes in the mitochondrial protein complement. Figure 7 shows representative results from an immunofluorescence survey of mitochondrial content, nuclei and cardiac marker proteins in dystrophic tissue. Cryosections of MDX tissue showed a complete absence of the dystrophin isoform Dp427 (Figures 7(c), 7(d)), confirming the mutant status of the dystrophic hearts used in this study. DAPI staining revealed 229 ± 6 and 270 ±18 nuclei per examined tissue section in normal versus dystrophic tissue sections, respectively (Figures 7(a), 7(b)). The slight increase of nuclei in MDX preparations was not statistically significant (n = 4). Thus the proteomic finding of a dystrophy-dependent increase in lamin-A/C is therefore not directly related to a drastic change in the number of nuclei per cardiac tissue unit. Fluorescent labeling of mitochondria with the MitoTracker dye CMXRos showed a characteristic internal staining pattern in cardiac fibres (Figures 7(e)–7(h)). Fluorescent intensity values of 1470 ± 36 and 1384 ± 40 for normal versus MDX preparations were found not to be significantly different (n = 4). Hence, lower levels of mitochondrial enzymes do not seem to be a consequence of a drastic decrease in mitochondrial density in the dystrophic MDX heart. In addition, confocal microscopy was employed for the localization of cardiac marker proteins. The immunofluorescence labeling of desmin (Figures 7(i)–7(l)), prohibitin (Figures 7(m)–7(p)) and succinate dehydrogenase (Figures 7(q)–7(t)) was shown to be comparable between control and pathological samples. The microscopical localization of desmin and succinate dehydrogenase revealed unexpectedly not a decrease of these proteins in dystrophic tissue sections, as found by DIGE analysis. Hence, although distinct subspecies of certain cardiac proteins are affected in X-linked muscular dystrophy as determined by mass spectrometry-based proteomics, the overall isoform complement of these elements is not drastically altered. This indicates that no major differences exist in the expression levels of desmin-containing intermediate filament structures and the population of cardiac mitochondria in normal versus dystrophin-deficient cells.

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