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

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.

Figure 1. Similar expression profiles in OPMD and elderly muscles

(A) Venn diagram shows the number of affected genes (p ≤0.01) in OPMD, elderly overlapping genes. The percentage of similar gene direction and the direction (up or down) are listed. For each group the mean age ± standard deviation are denoted. (B) Cumulative distribution function (CDF) plots show the distribution of normalized literature association-weights of commonly deregulated genes between OPMD and elderly with the concepts: Aging, Muscle contraction, Oxidative phospho-rylation, insulin signalling, TGFβsignalling, and the ubiquitin-proteasome system (UPS). Arrowheads indicate the maximum association weights. The further the curve shifts to the right, the higher the associations of the affected genes with the indicated concepts are. (C) Bar-chart shows fold-change of selected known age-regulated genes in elderly and in OPMD. P-values are indicated: * P≤0.01, ** P<0.005, *** P<0.0005.

Figure 2. The transcriptome of the OPMD mouse model is highly associated with aging

(A) Volcano plot shows the distribution of significantly deregulated genes (P = 0.05; indicated with a dashed line) in 6 week-old A17.1 mice against fold-change. Smaller P-value and higher fold-change suggests a higher impact in OPMD. For every gene a literature-associated weight with the ‘Aging' concept is assigned, and a normalized association-weight is presented with a circle on a linear scale between 0.05 and 1, where 1 equals the highest association. (B) Hierarchical clustering arrangements of 104 datasets in a literature-aided meta-analysis. Shades of blue indicate degree of similarities: from weak (white) to strong (dark blue). Three skeletal muscle aging-related datasets cluster together with the OPMD dataset of 6 week-old mice (highlighted in red). The clusters associated with muscular dystrophies and other myopathies are highlighted in green and blue, respectively.

Figure 3. Age regulated gene expression trends change faster in OPMD

(A) Expression trends in controls (grey) and expPABPN1 carriers (red). Similar expression trends were identified with k-means clustering using probe ID. (B) Examples of expression trends of 8 genes from clusters in A, in healthy controls (grey) or in exPABPN1 carriers at pre-symptomatic and symptomatic stages (red). Pre-symptomatic and symptomatic stages of OPMD are indicated.

Figure 4. Deregulation of the Spliceosome in both OPMD and elderly

(A) Bar-chart shows fold-change of spliceosome genes in OPMD and elderly from the transcriptome studies. P-values are indicated: * P<0.05, ** P<0.005, *** P<0.0005. In bold are gene-hubs in the OPMD-affected spliceosome network (B). A schematic gene network presentation of OPMD-regulated genes that are grouped in the spliceosome GO category. PABPN1 is highlighted

Figure 5. RT-qPCR analysis of PABPN1 expression trends in OPMD and during muscle aging

(A) Box plot shows PABPN1expression in Vastus lateralis from expPABPN1 carriers at a pre-symptomatic (pre-symp) or symptomatic (OPMD) stages, and age-matching control groups. (B) Box plot showsPABPN1expression in blood form OPMD patients and controls. (C) Scatter plot shows PABPN1expression in 78 healthy controls age 17-89 years. Male and female samples are indicated in black and grey, respectively. A quadratic fit is shown with a black line (a), and linear fits are for the age groups: 17-42 years (b) or 43-89 years (c) are denoted in grey. The table summarizes p-values and Beta ± standard errors, which were calculated after gender correction. Fold-changes were calculated after normalization of GUSB housekeeping gene and to control group age 17-22 years (A, C) or age matching controls (B). N denotes the number of samples in each group.

Figure 6. PABPN1-DR in human myotubes causes myogenic defects

Human 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.

Figure 7. PABPN1-DR induces cellular aging in muscle cells

(A) PABPN1-down regulation induces a decrease in mitochondrial metabolic rate. (i) Bar chart shows the ratio of J-aggregates to monomers in JC-1-labeled myoblasts or myotube cultures. Cell cultures are of control (7304) or PABPN1-DR (sh122 and sh121) cultures, before (mock) or after transducion with CFP-PABPN1 lentivirus particles. Averages and SD are from six replicates, and the number of cells per sample is indicated within each bar. Significant effect in PABPN1-DR from control cultures (P<0.05 or P<0.005) is indicated with one or two asterisks, respectively. (ii) Plot shows the ratio of J-aggregates to monomers in JC-1-labeled cultures after 1 hour H2O2, in concentrations as indicated in the chart. Averages and SD are from three replicates. (B) A decrease in cell growth is caused by PABPN1-down regulation. (i) Bar chart shows cell growth (24 hours) in control (7304) and PABPN1-DR (sh121) cultures, before (mock) or after transduction with CFP-PABPN1 lentivirus particles. Averages and SD are from three replicates. (ii) Heterochromatic foci are formed in PABPN1-DR cells. Heterochromatic foci are visualized with DAPI (indicated with arrowheads), cells are visualized with Desmin. Scale bar is 20 μm.