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A decline in PABPN1 induces progressive muscle weakness in oculopharyngeal muscle dystrophy and in muscle aging.

Anvar SY, Raz Y, Verway N, van der Sluijs B, Venema A, Goeman JJ, Vissing J, van der Maarel SM, 't Hoen PA, van Engelen BG, Raz V - Aging (Albany NY) (2013)

Bottom Line: Major expression changes were found to be associated with age rather than with expression of expanded-PABPN1, instead transcriptomes of OPMD and elderly muscles were significantly similar (P<0.05).Reduced PABPN1 levels (30% to 60%) in muscle cells induced myogenic defects and morphological signatures of cellular aging in proportion to PABPN1 expression levels.We suggest that PABPN1 levels regulate muscle cell aging and OPMD represents an accelerated muscle aging disorder.

View Article: PubMed Central - PubMed

Affiliation: Center for Human and Clinical Genetics, Leiden University Medical Center, the Netherlands.

ABSTRACT
Oculopharyngeal muscular dystrophy (OPMD) is caused by trinucleotide repeat expansion mutations in Poly(A) binding protein 1 (PABPN1). PABPN1 is a regulator of mRNA stability and is ubiquitously expressed. Here we investigated how symptoms in OPMD initiate only at midlife and why a subset of skeletal muscles is predominantly affected. Genome-wide RNA expression profiles from Vastus lateralis muscles human carriers of expanded-PABPN1 at pre-symptomatic and symptomatic stages were compared with healthy controls. Major expression changes were found to be associated with age rather than with expression of expanded-PABPN1, instead transcriptomes of OPMD and elderly muscles were significantly similar (P<0.05). Using k-means clustering we identified age-dependent trends in both OPMD and controls, but trends were often accelerated in OPMD. We report an age-regulated decline in PABPN1 levels in Vastus lateralis muscles from the fifth decade. In concurrence with severe muscle degeneration in OPMD, the decline in PABPN1 accelerated in OPMD and was specific to skeletal muscles. Reduced PABPN1 levels (30% to 60%) in muscle cells induced myogenic defects and morphological signatures of cellular aging in proportion to PABPN1 expression levels. We suggest that PABPN1 levels regulate muscle cell aging and OPMD represents an accelerated muscle aging disorder.

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PABPN1-DR in human myotubes causes myogenic defectsHuman myotubes were transduced with shRNA specific to PABPN1 (sh121, sh122, or sh123) or H1 empty vector. Non-transduced (NT) cells were used as controls. (A) i Bar-chart shows PABPN1 mRNA expression in stably-transduced myoblasts. Fold change was normalized to GapDH housekeeping gene and to a non-transduced culture. Averages are of 6 biological replicates. ii Western blot analysis of PABPN1, MHC1 and muscle actin (MSA), as a loading control in sh121, sh122 or H1 myotube cultures. iii Immunofluorescence of PABPN1 (labelled with Alexa-594) and MHC1 (labelled with Alexa-488) in sh121 or H1 myotube cultures. Scale bar 10 μm. (B) Bar chart shows fold change of MHC1, DMD, and CAV3 in 121-, 122-, 123-, and H1- myoblast cultures. Fold change was normalized to GapDH and to a non-transduced culture. Averages are of 3 biological replicates. Significant down-regulation (P<0.05) is indicated with asterisks. (C) i- images of H1 controls and the PABPN1 down regulation sh121 fused cultures. Scale bar is 50 μm. ii- Chart bar shows the percentage of nuclei in fused myotubes that express MHC1 (cell fusion) in H1 sh123 sh122 or sh121 myotube cultures. Averages and SD are from six replicates and the number of nuclei that were quantified per sample is indicated within each bar. Significant effect in PABPN1-DR cultures from control cultures (P<0.05 or P<0.005) is indicated with one or two asterisks, respectively.
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Figure 6: PABPN1-DR in human myotubes causes myogenic defectsHuman myotubes were transduced with shRNA specific to PABPN1 (sh121, sh122, or sh123) or H1 empty vector. Non-transduced (NT) cells were used as controls. (A) i Bar-chart shows PABPN1 mRNA expression in stably-transduced myoblasts. Fold change was normalized to GapDH housekeeping gene and to a non-transduced culture. Averages are of 6 biological replicates. ii Western blot analysis of PABPN1, MHC1 and muscle actin (MSA), as a loading control in sh121, sh122 or H1 myotube cultures. iii Immunofluorescence of PABPN1 (labelled with Alexa-594) and MHC1 (labelled with Alexa-488) in sh121 or H1 myotube cultures. Scale bar 10 μm. (B) Bar chart shows fold change of MHC1, DMD, and CAV3 in 121-, 122-, 123-, and H1- myoblast cultures. Fold change was normalized to GapDH and to a non-transduced culture. Averages are of 3 biological replicates. Significant down-regulation (P<0.05) is indicated with asterisks. (C) i- images of H1 controls and the PABPN1 down regulation sh121 fused cultures. Scale bar is 50 μm. ii- Chart bar shows the percentage of nuclei in fused myotubes that express MHC1 (cell fusion) in H1 sh123 sh122 or sh121 myotube cultures. Averages and SD are from six replicates and the number of nuclei that were quantified per sample is indicated within each bar. Significant effect in PABPN1-DR cultures from control cultures (P<0.05 or P<0.005) is indicated with one or two asterisks, respectively.

Mentions: Knockdown of PABPN1 in mouse muscle cells causes myogenic defects [18]. Since in vivo we found a decrease in PABPN1 expression with age in both normal aging and in OPMD, we studied whether at with a moderate down-regulation of PABPN1 myogenic defects can also be observed. Three PABPN1 shRNA clones were selected for functional studies in immortalized human myoblast cultures using the lentivirus expression system for efficient delivery. Stable cultures were generated and down-regulation was evaluated with RT-qPCR and western blot analyses. Compared with controls (H1 empty vector and non- transduced cells), the three PABPN1 shRNA clones, 121, 122 and 123, led to a 70%, 40% and 20% decrease in PABPN1 expression, respectively (Figure 6Ai). This decrease was also reflected in the residual protein levels (Figure 6Aii). Immunofluorescence microscopy with antibody to PABPN1 revealed a decrease in the accumulation of nuclear PABPN1 in the sh121-transduced cells (Figure 6Aiii).


A decline in PABPN1 induces progressive muscle weakness in oculopharyngeal muscle dystrophy and in muscle aging.

Anvar SY, Raz Y, Verway N, van der Sluijs B, Venema A, Goeman JJ, Vissing J, van der Maarel SM, 't Hoen PA, van Engelen BG, Raz V - Aging (Albany NY) (2013)

PABPN1-DR in human myotubes causes myogenic defectsHuman myotubes were transduced with shRNA specific to PABPN1 (sh121, sh122, or sh123) or H1 empty vector. Non-transduced (NT) cells were used as controls. (A) i Bar-chart shows PABPN1 mRNA expression in stably-transduced myoblasts. Fold change was normalized to GapDH housekeeping gene and to a non-transduced culture. Averages are of 6 biological replicates. ii Western blot analysis of PABPN1, MHC1 and muscle actin (MSA), as a loading control in sh121, sh122 or H1 myotube cultures. iii Immunofluorescence of PABPN1 (labelled with Alexa-594) and MHC1 (labelled with Alexa-488) in sh121 or H1 myotube cultures. Scale bar 10 μm. (B) Bar chart shows fold change of MHC1, DMD, and CAV3 in 121-, 122-, 123-, and H1- myoblast cultures. Fold change was normalized to GapDH and to a non-transduced culture. Averages are of 3 biological replicates. Significant down-regulation (P<0.05) is indicated with asterisks. (C) i- images of H1 controls and the PABPN1 down regulation sh121 fused cultures. Scale bar is 50 μm. ii- Chart bar shows the percentage of nuclei in fused myotubes that express MHC1 (cell fusion) in H1 sh123 sh122 or sh121 myotube cultures. Averages and SD are from six replicates and the number of nuclei that were quantified per sample is indicated within each bar. Significant effect in PABPN1-DR cultures from control cultures (P<0.05 or P<0.005) is indicated with one or two asterisks, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: PABPN1-DR in human myotubes causes myogenic defectsHuman myotubes were transduced with shRNA specific to PABPN1 (sh121, sh122, or sh123) or H1 empty vector. Non-transduced (NT) cells were used as controls. (A) i Bar-chart shows PABPN1 mRNA expression in stably-transduced myoblasts. Fold change was normalized to GapDH housekeeping gene and to a non-transduced culture. Averages are of 6 biological replicates. ii Western blot analysis of PABPN1, MHC1 and muscle actin (MSA), as a loading control in sh121, sh122 or H1 myotube cultures. iii Immunofluorescence of PABPN1 (labelled with Alexa-594) and MHC1 (labelled with Alexa-488) in sh121 or H1 myotube cultures. Scale bar 10 μm. (B) Bar chart shows fold change of MHC1, DMD, and CAV3 in 121-, 122-, 123-, and H1- myoblast cultures. Fold change was normalized to GapDH and to a non-transduced culture. Averages are of 3 biological replicates. Significant down-regulation (P<0.05) is indicated with asterisks. (C) i- images of H1 controls and the PABPN1 down regulation sh121 fused cultures. Scale bar is 50 μm. ii- Chart bar shows the percentage of nuclei in fused myotubes that express MHC1 (cell fusion) in H1 sh123 sh122 or sh121 myotube cultures. Averages and SD are from six replicates and the number of nuclei that were quantified per sample is indicated within each bar. Significant effect in PABPN1-DR cultures from control cultures (P<0.05 or P<0.005) is indicated with one or two asterisks, respectively.
Mentions: Knockdown of PABPN1 in mouse muscle cells causes myogenic defects [18]. Since in vivo we found a decrease in PABPN1 expression with age in both normal aging and in OPMD, we studied whether at with a moderate down-regulation of PABPN1 myogenic defects can also be observed. Three PABPN1 shRNA clones were selected for functional studies in immortalized human myoblast cultures using the lentivirus expression system for efficient delivery. Stable cultures were generated and down-regulation was evaluated with RT-qPCR and western blot analyses. Compared with controls (H1 empty vector and non- transduced cells), the three PABPN1 shRNA clones, 121, 122 and 123, led to a 70%, 40% and 20% decrease in PABPN1 expression, respectively (Figure 6Ai). This decrease was also reflected in the residual protein levels (Figure 6Aii). Immunofluorescence microscopy with antibody to PABPN1 revealed a decrease in the accumulation of nuclear PABPN1 in the sh121-transduced cells (Figure 6Aiii).

Bottom Line: Major expression changes were found to be associated with age rather than with expression of expanded-PABPN1, instead transcriptomes of OPMD and elderly muscles were significantly similar (P<0.05).Reduced PABPN1 levels (30% to 60%) in muscle cells induced myogenic defects and morphological signatures of cellular aging in proportion to PABPN1 expression levels.We suggest that PABPN1 levels regulate muscle cell aging and OPMD represents an accelerated muscle aging disorder.

View Article: PubMed Central - PubMed

Affiliation: Center for Human and Clinical Genetics, Leiden University Medical Center, the Netherlands.

ABSTRACT
Oculopharyngeal muscular dystrophy (OPMD) is caused by trinucleotide repeat expansion mutations in Poly(A) binding protein 1 (PABPN1). PABPN1 is a regulator of mRNA stability and is ubiquitously expressed. Here we investigated how symptoms in OPMD initiate only at midlife and why a subset of skeletal muscles is predominantly affected. Genome-wide RNA expression profiles from Vastus lateralis muscles human carriers of expanded-PABPN1 at pre-symptomatic and symptomatic stages were compared with healthy controls. Major expression changes were found to be associated with age rather than with expression of expanded-PABPN1, instead transcriptomes of OPMD and elderly muscles were significantly similar (P<0.05). Using k-means clustering we identified age-dependent trends in both OPMD and controls, but trends were often accelerated in OPMD. We report an age-regulated decline in PABPN1 levels in Vastus lateralis muscles from the fifth decade. In concurrence with severe muscle degeneration in OPMD, the decline in PABPN1 accelerated in OPMD and was specific to skeletal muscles. Reduced PABPN1 levels (30% to 60%) in muscle cells induced myogenic defects and morphological signatures of cellular aging in proportion to PABPN1 expression levels. We suggest that PABPN1 levels regulate muscle cell aging and OPMD represents an accelerated muscle aging disorder.

Show MeSH
Related in: MedlinePlus