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Chronic hypoxia impairs muscle function in the Drosophila model of Duchenne's muscular dystrophy (DMD).

Mosqueira M, Willmann G, Ruohola-Baker H, Khurana TS - PLoS ONE (2010)

Bottom Line: To understand the effects of CH on dystrophin-deficient muscle in vivo, we exposed the Drosophila model for DMD (dmDys) to CH during a 16-day ascent to the summit of Mount Denali/McKinley (6194 meters above sea level).Interestingly, a number of genes (e.g. heat shock proteins) were discordantly regulated in response to CH between dmDys and WT.Impaired performance was noted for CH-dmDys compared to normoxic dmDys or WT flies (rank order: Normoxic-WT ≈ CH-WT> Normoxic-dmDys> CH-dmDys).

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Duchenne's muscular dystrophy (DMD) is a severe progressive myopathy caused by mutations in the DMD gene leading to a deficiency of the dystrophin protein. Due to ongoing muscle necrosis in respiratory muscles late-stage DMD is associated with respiratory insufficiency and chronic hypoxia (CH). To understand the effects of CH on dystrophin-deficient muscle in vivo, we exposed the Drosophila model for DMD (dmDys) to CH during a 16-day ascent to the summit of Mount Denali/McKinley (6194 meters above sea level). Additionally, dmDys and wild type (WT) flies were also exposed to CH in laboratory simulations of high altitude hypoxia. Expression profiling was performed using Affymetrix GeneChips® and validated using qPCR. Hypoxic dmDys differentially expressed 1281 genes, whereas the hypoxic WT flies differentially expressed 56 genes. Interestingly, a number of genes (e.g. heat shock proteins) were discordantly regulated in response to CH between dmDys and WT. We tested the possibility that the disparate molecular responses of dystrophin-deficient tissues to CH could adversely affect muscle by performing functional assays in vivo. Normoxic and CH WT and dmDys flies were challenged with acute hypoxia and time-to-recover determined as well as subjected to climbing tests. Impaired performance was noted for CH-dmDys compared to normoxic dmDys or WT flies (rank order: Normoxic-WT ≈ CH-WT> Normoxic-dmDys> CH-dmDys). These data suggest that dystrophin-deficiency is associated with a disparate, pathological hypoxic stress response(s) and is more sensitive to hypoxia induced muscle dysfunction in vivo. We hypothesize that targeting/correcting the disparate molecular response(s) to hypoxia may offer a novel therapeutic strategy in DMD.

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Validation of differential expression of five genes detected on WT profile by real time RT-PCR.Five out of top 10 differentially expressed genes were amplified using cDNA from four independent RNA preparations and analyzed by qPCR. Graph shows concordant changes of gene expression levels for various genes noted on microarrays (unfilled bars) and validated by qPCR (filled bars).
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pone-0013450-g004: Validation of differential expression of five genes detected on WT profile by real time RT-PCR.Five out of top 10 differentially expressed genes were amplified using cDNA from four independent RNA preparations and analyzed by qPCR. Graph shows concordant changes of gene expression levels for various genes noted on microarrays (unfilled bars) and validated by qPCR (filled bars).

Mentions: Having established the gene expression profile of dmDys subjected to CH we also determined the gene expression profile of CH wild type (WT) Drosophila in order to compare hypoxia-induced gene changes in dmDys to those in WT. We screened the Affymetrix Drosophila Genome 2.0 GeneChip® platform and identified gene expression profiles of four individual CH-WT (Hypoxia protocol provided in Table 1) flies with four individual normoxic WT flies. The expression levels of all probe sets represented on individual microarrays was plotted on a scatter graph (Figure 3A) and shows the overall pattern of differences in gene expression. The overall r2 for the normoxic sample data sets compared amongst themselves was 0.98, for the hypoxic sets 0.93 and the r2 between the normoxic and hypoxic sample data sets was 0.93, demonstrating the low intra-variability among the samples from normoxic and hypoxic transcriptomes in the WT flies. After imposing statistical and 2 fold expression levels cutoffs, 56 genes were found to be differentially expressed (Figure 3A; Table S6); 55 were up-regulated and only one gene was down-regulated. Figure 3B shows the heat map representation of hierarchical clustering of the entire profile. Branch-length analysis of data (Figure 3B) demonstrated that the four CH-WT samples were more similar to each other than to normoxic-WT samples, and vice versa demonstrating that the gene expression profiles between CH- and normoxic-WT were significantly different from each other. Similar results were obtained using principal component analysis (data not shown). To independently validate the microarray analysis randomly chose five (4 up-regulated and 1 down-regulated) out of the top 10 differentially expressed genes (Table S7) and analyzed them by qPCR from biologically independent samples using TaqMan® Gene Expression probe sets. All five genes showed significant fold-changes in a direction concordant to that as observed in the microarray, confirming the validity of the results (Figure 4). Functional clustering analysis using DAVID was performed only on the up-regulated gene list, and show an enrichment of the category related with response to stress (Table S8).


Chronic hypoxia impairs muscle function in the Drosophila model of Duchenne's muscular dystrophy (DMD).

Mosqueira M, Willmann G, Ruohola-Baker H, Khurana TS - PLoS ONE (2010)

Validation of differential expression of five genes detected on WT profile by real time RT-PCR.Five out of top 10 differentially expressed genes were amplified using cDNA from four independent RNA preparations and analyzed by qPCR. Graph shows concordant changes of gene expression levels for various genes noted on microarrays (unfilled bars) and validated by qPCR (filled bars).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958114&req=5

pone-0013450-g004: Validation of differential expression of five genes detected on WT profile by real time RT-PCR.Five out of top 10 differentially expressed genes were amplified using cDNA from four independent RNA preparations and analyzed by qPCR. Graph shows concordant changes of gene expression levels for various genes noted on microarrays (unfilled bars) and validated by qPCR (filled bars).
Mentions: Having established the gene expression profile of dmDys subjected to CH we also determined the gene expression profile of CH wild type (WT) Drosophila in order to compare hypoxia-induced gene changes in dmDys to those in WT. We screened the Affymetrix Drosophila Genome 2.0 GeneChip® platform and identified gene expression profiles of four individual CH-WT (Hypoxia protocol provided in Table 1) flies with four individual normoxic WT flies. The expression levels of all probe sets represented on individual microarrays was plotted on a scatter graph (Figure 3A) and shows the overall pattern of differences in gene expression. The overall r2 for the normoxic sample data sets compared amongst themselves was 0.98, for the hypoxic sets 0.93 and the r2 between the normoxic and hypoxic sample data sets was 0.93, demonstrating the low intra-variability among the samples from normoxic and hypoxic transcriptomes in the WT flies. After imposing statistical and 2 fold expression levels cutoffs, 56 genes were found to be differentially expressed (Figure 3A; Table S6); 55 were up-regulated and only one gene was down-regulated. Figure 3B shows the heat map representation of hierarchical clustering of the entire profile. Branch-length analysis of data (Figure 3B) demonstrated that the four CH-WT samples were more similar to each other than to normoxic-WT samples, and vice versa demonstrating that the gene expression profiles between CH- and normoxic-WT were significantly different from each other. Similar results were obtained using principal component analysis (data not shown). To independently validate the microarray analysis randomly chose five (4 up-regulated and 1 down-regulated) out of the top 10 differentially expressed genes (Table S7) and analyzed them by qPCR from biologically independent samples using TaqMan® Gene Expression probe sets. All five genes showed significant fold-changes in a direction concordant to that as observed in the microarray, confirming the validity of the results (Figure 4). Functional clustering analysis using DAVID was performed only on the up-regulated gene list, and show an enrichment of the category related with response to stress (Table S8).

Bottom Line: To understand the effects of CH on dystrophin-deficient muscle in vivo, we exposed the Drosophila model for DMD (dmDys) to CH during a 16-day ascent to the summit of Mount Denali/McKinley (6194 meters above sea level).Interestingly, a number of genes (e.g. heat shock proteins) were discordantly regulated in response to CH between dmDys and WT.Impaired performance was noted for CH-dmDys compared to normoxic dmDys or WT flies (rank order: Normoxic-WT ≈ CH-WT> Normoxic-dmDys> CH-dmDys).

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Duchenne's muscular dystrophy (DMD) is a severe progressive myopathy caused by mutations in the DMD gene leading to a deficiency of the dystrophin protein. Due to ongoing muscle necrosis in respiratory muscles late-stage DMD is associated with respiratory insufficiency and chronic hypoxia (CH). To understand the effects of CH on dystrophin-deficient muscle in vivo, we exposed the Drosophila model for DMD (dmDys) to CH during a 16-day ascent to the summit of Mount Denali/McKinley (6194 meters above sea level). Additionally, dmDys and wild type (WT) flies were also exposed to CH in laboratory simulations of high altitude hypoxia. Expression profiling was performed using Affymetrix GeneChips® and validated using qPCR. Hypoxic dmDys differentially expressed 1281 genes, whereas the hypoxic WT flies differentially expressed 56 genes. Interestingly, a number of genes (e.g. heat shock proteins) were discordantly regulated in response to CH between dmDys and WT. We tested the possibility that the disparate molecular responses of dystrophin-deficient tissues to CH could adversely affect muscle by performing functional assays in vivo. Normoxic and CH WT and dmDys flies were challenged with acute hypoxia and time-to-recover determined as well as subjected to climbing tests. Impaired performance was noted for CH-dmDys compared to normoxic dmDys or WT flies (rank order: Normoxic-WT ≈ CH-WT> Normoxic-dmDys> CH-dmDys). These data suggest that dystrophin-deficiency is associated with a disparate, pathological hypoxic stress response(s) and is more sensitive to hypoxia induced muscle dysfunction in vivo. We hypothesize that targeting/correcting the disparate molecular response(s) to hypoxia may offer a novel therapeutic strategy in DMD.

Show MeSH
Related in: MedlinePlus