Deacetylation Inhibition Reverses PABPN1-Dependent Muscle Wasting

Abstract

Reduced poly(A)-binding protein nuclear 1 (PABPN1) levels cause aging-associated muscle wasting. PABPN1 is a multifunctional regulator of mRNA processing. To elucidate the molecular mechanisms causing PABPN1-mediated muscle wasting, we compared the transcriptome with the proteome in mouse muscles expressing short hairpin RNA to PABPN1 (shPab). We found greater variations in the proteome than in mRNA expression profiles. Protein accumulation in the shPab proteome was concomitant with reduced proteasomal activity. Notably, protein acetylation appeared to be decreased in shPab versus control proteomes (63%). Acetylome profiling in shPab muscles revealed prominent peptide deacetylation associated with elevated sirtuin-1 (SIRT1) deacetylase. We show that SIRT1 mRNA levels are controlled by PABPN1 via alternative polyadenylation site utilization. Most importantly, SIRT1 deacetylase inhibition by sirtinol increased PABPN1 levels and reversed muscle wasting. We suggest that perturbation of a multifactorial regulatory loop involving PABPN1 and SIRT1 plays an imperative role in aging-associated muscle wasting. VIDEO ABSTRACT.

Keywords: Biological Sciences; Molecular Biology; Physiology.

Graphical abstract

Figure 1

Reduced PABPN1 Levels Induce Muscle Regeneration (A) Schematic workflow of the ex vivo analyses in scram and shPab muscles. RNA expression profiles (RNA-seq) are compared with the shPab proteome of the same muscles. The shPab acetylome was then analyzed. Procedures were validated using qRT-PCR, western blot (WB), or muscle histology. Ex vivo experiments were performed on paired muscles (N = 4 mice). (B) qRT-PCR of Pabpn1 mRNA levels after normalization to Hprt housekeeping control. Paired dot plot is from N = 4 mice. (C) PABPN1 protein and levels in paired muscles. Representative western blot of PABPN1 and GAPDH loading control are shown. Paired dot plot shows PABPN1 levels after normalization to loading control, N = 4 mice. (D) Gomori trichrome tissue histology in cross sections. Images are of the mouse with highest PABPN1 fold change. White arrowheads point to ECM thickening, central myonuclei are depicted with red arrowheads, and split myofibers with black arrowheads. Scale bar, 50 μm. (E) Paired dot plot shows the mean number of myofibers per image frame, calculated from 5 frames per muscle (N = 8 muscles). (F) Paired dot plot shows the mean fraction of central nuclei in myofibers, calculated from 5 frames per muscle (N = 8 muscles). (G) Paired dot plot shows eMyh3 mRNA levels in scram and shPab muscles (N = 4 mice). Expression values were calculated after normalization to Hprt and to the average eMyh3 expression of all scram muscles. (H and I) (H) Representative fluorescent images for scram and shPab muscles stained with PAX7 antibody (green). Nuclei are counterstained with DAPI (blue). White arrowheads indicate nuclear PAX7. Scale bar, 7.5μm. (I) Paired dot plot shows the fraction of PAX7 positive nuclei in paired muscles. The percentage was calculated from over 1,000 nuclei per muscle (N = 6 muscles). In all dot plots paired muscles are connected with lines; blue or green lines mark the mouse with the highest or lowest PABPN1 fold change, respectively. Quantification of the muscle histology was performed over the entire muscle cross section. Means and standard deviations are depicted. Statistical difference was assessed with a paired test.

Figure 2

Reduced PABPN1 Levels Result in a Greater Effect on Proteome Compared with Transcriptome (A) A volcano plot of the average mRNA fold change (FC) against p values in the shPab transcriptome. mRNAs that pass the cutoff p value < 0.05 or p values corrected for false discovery rate are depicted in blue or red, respectively. Examples of validated genes are highlighted. (B) Volcano plot of the average protein fold change against p values in the shPab proteome. Proteins that pass the cutoff (p < 0.05) are depicted in blue. (C) Scatterplot shows the fold changes between shPab proteome and transcriptome. Correlation (indicated as gray dashed line) was assessed with a Spearman rank test and compared with the diagonal (solid gray line). Average protein fold changes of 1.5 and −1.5 are indicated with red lines. Proteins with an average fold changes higher than 1.5 or lower than −1.5 are depicted in pink or black, respectively. (D) Bar chart shows fold change direction of shPab-affected genes (average fold change cutoff > |1.5|) sorted for RNA or proteins. (E) Bar chart shows the percentage of proteins (average fold change cutoff > |1.5|) with similar fold change direction or opposite fold change direction compared with RNA direction. Fold change higher than 1.5 is denoted in black and fold change lower than −1.5 in pink. Both transcriptome and proteome were determined from the same muscles (N = 4 mice).

Figure 3

PABPN1 Levels Affect Muscle Proteome of Which a Large Part Is Linked to Acetylation (A) Heatmap of protein abundance for the differentially expressed proteins (p < 0.05). Muscles are paired; the mice with the highest and lowest PABPN1 fold change are depicted in blue and green, respectively. Western blot shows PABPN1 expression for each mouse. GAPDH was used as loading control. Mitochondrial proteins are highlighted with green lines, and ribosomal proteins are highlighted with red lines. Color key indicates the Z scores (based on rows). (B) Pie chart shows the significantly enriched protein groups (UP-keyword, David) of the differentially expressed proteins (p < 0.05, n = 248). The transparent pies (outer circle) indicate the number of proteins within the protein groups that are linked to protein acetylation group. Numbers indicate the percentage per group. (C) The protein-protein interaction map shows protein networks for 248 proteins that pass the cutoff (p < 0.05). Mitochondrial (green), translation (red), cytoskeletal (purple), and glycolysis (pink) clusters are highlighted. Within the mitochondrial cluster, the oxidative phosphorylation group is highlighted in dark green. Unconnected proteins are not shown, and networks with n < 4 are removed. Proteins that are found in the acetylation group are highlighted in yellow, and those that were not found in the acetylation group are gray.

Figure 4

The shPab-Dependent Acetylome (A) A workflow of the shPab acetylome procedure. Tibialis anterior muscles (N = 7 mice, contralateral injection of scram or shPab AAVs) were used. Protein extracts were pooled per genotype. Tryptic digestion, followed by immunoprecipitation with an anti-acetylated lysine (AcK) antibody, and mass spectrometry (MS) were carried out separately for the scram and shPab. Peptide analysis pipeline included an MS quality control (1% FDR) and coefficient of variation (CV) < 0.3. The differentially acetylated peptides were considered with a 1.5-fold change (fold change) cutoff. (B) Scatterplot shows average peptide fold change (log2) versus average CV (-log2). Peptides with CV < 0.3 and a fold change of >1.5 and < -1.5 are depicted with blue and red, respectively. Peptides that are only found in either shPab or scram muscle are lined as INF (infinite). Unchanged peptides are depicted in gray. The dashed line marks CV < 0.3. (C and D) (C) Western blot of acetylated proteins in scram and shPab muscle protein extracts. GAPDH is loading control. (D) Immunofluorescence with AcK antibody in scram and shPab muscle cryosections. Left panels show an overlay between bright-field and DAPI staining of the nuclei (blue). The middle panels show AcK signal (yellow). Right panels show an enlarged AcK and DAPI overlay around the nuclei that are marked with an arrow. Intensity distribution plots for DAPI and AcK signal are made from the nucleus, which is marked with a line. Examples of AcK-positive and AcK-negative nuclei are in the upper and lower plots, respectively. Scale bar, 5 μm. (E) Bar chart shows the percentage of AcK-positive nuclei in scram and shPab muscles from four mice (unpaired). The number of measured nuclei is depicted within each bar. Trend for statistical difference was assessed with the Mann-Whitney test. (F) Fold change direction of nuclear proteins in the shPab acetylome: positive and negative fold changes are depicted in blue and red, respectively. (G) Pie chart shows the significantly affected gene ontology terms in the shPab acetylome. (H) Schematic representation of the acetylated and the shPab differentially acetylated oxidative phosphorylation subunits. The acetylated subunits in tibialis anterior muscle are highlighted in light green, and the shPab differentially acetylated are in dark green. The number of affected proteins in each complex is depicted.

Figure 5

Sirt1 mRNA Levels Are PABPN1-Regulated via Alternative PAS in the 3′ UTR (A and B) SIRT1 levels in muscles of four mice. (A) A representative western blot in scram and shPab muscles. GAPDH is used as a loading control and for normalization. (B) Paired analysis of PABPN1 and SIRT1 fold change. Line connects values with a mouse. Mice with the smallest and highest PABPN1 fold changes are depicted with green and blue lines, respectively. (C) A representative western blot in muscle cell culture. GAPDH was used as loading control. GAPDH-normalized values are plotted in the bar chart. (D) A schematic presentation of alternative PAS utilization in the 3′ UTR in conditions with reduced PABPN1 levels. The positions of distal (D) and proximal (P) primer sets in Sirt1 mRNA are depicted. (E and F) Distal to proximal ratio in Sirt1 3′ UTR (E) and Sirt1 fold change (F) in scram or shPab muscle cell cultures. Fold change was calculated after normalization to Hprt housekeeping gene and scram control. Statistical difference was assessed with the Student's t test. p < 0.05. (G) A schematic presentation showing the position of Sirt1 AONs. AONs are designed to mask proximal PAS in Sirt1 mRNA. (H and I) Bar charts show the distal to proximal ratio in Sirt1 3′ UTR (H) and the Sirt1 fold change (I). Scrambled AON is depicted in the black bar, and with Sirt1 AONs are depicted in dashed bars. Fold change (I) was calculated after normalization to Hprt housekeeping gene and scram control. Averages and standard deviations of all the experiments here are from three biological replicates. Statistical difference was assessed with the Student's t test. p < 0.05.

Figure 6

Sirtinol Treatment Reverses Myogenic Defects in shPab Muscle Cell Culture (A) Mitochondrial membrane potential in shPab vehicle and sirtinol-treated muscle cell cultures. Images show an overlay between monomers (green) and J-aggregates (red) and the nuclei (blue). Scale bar, 10 μm. The ratio is measured from >30,000 cells. (B) Muscle cell fusion in shPab vehicle and sirtinol-treated cell cultures. Images show an overlay between MyHC staining (green) and the nuclei (blue). Scale bar, 20 μm. The fraction of nuclei within MyHC regions is from >50,000 nuclei. (C) Western blot analysis of PABPN1 levels in sirtinol-treated scram and shPab cell cultures. Tubulin is used as loading control and for normalization. (D) Accumulation of nuclear PABPN1. Images show an overlay between PABPN1 (red) and nuclei (blue) in shPab vehicle and sirtinol-treated cell cultures. The segmented nuclei are depicted with a blue line. Scale bar, 10 μm. Bar chart shows the average MFI of nuclear PABPN1 from >50,000 nuclei. (E and F) Distal to proximal ratio in Sirt1 3′ UTR (E) or mRNA fold change (F) in mock and sirtinol-treated shPab cell cultures. Fold change was calculated after normalization to Hprt and scram mock. Averages and standard deviations are from three biological replicates (A, B, D, E, F, and C, respectively). Statistical difference was assessed with the Student's t test. *p < 0.05.

Figure 7

Sirtinol Treatment in shPab Muscles Reverses PABPN1-Induced Muscle Pathology (A) An overview of sirtinol treatment in shPab muscles. Muscles were injected with AAV9 shPab in both right and left tibialis anterior muscles in three mice. After 3 weeks muscles were injected twice, with a 10-day interval, with empty vehicle or sirtinol. (B) A representative western blot shows PABPN1 protein levels in shPab vehicle and sirtinol-injected muscles. Equal loading is assessed with Coomassie blue (CB). The mouse with the lowest PABPN1 fold change is depicted with a light gray line. (C) Dot plot shows normalized PABPN1 expression levels. Lines connect paired muscles. Means and standard deviations are depicted with a black line. (D) Gomori trichrome staining in muscle cryosections. Extracellular thickening is depicted with a white arrow, examples of central myonuclei and split myofibers are indicated with red and black arrows, respectively. Scale bar, 20 μm. (E) Dot plot shows the fraction of central nuclei in mock and sirtinol-treated shPab muscles; >1,500 myofibers were counted per condition per mouse. Lines connect between paired muscles. Control AAV9 scrambled shRNA was injected into tibialis anterior TA (N = 4 muscles). Means and standard deviations are depicted with a black line. (F) Cumulative distribution plot of myofiber cross-sectional area (CSA) in vehicle and sirtinol-treated muscles. (G) Myofiber typing: Images show an overlay of MyHC-2b (green), MyHC-2a (red), and DAPI (blue) staining in vehicle or sirtinol-treated shPab muscles. Scale bar, 20 μm. Cumulative distribution plot of MyHC-2a and MyHC-2b MFI in single myofibers from mock or sirtinol-treated muscles. Statistical difference was determined with the Kolmogorov-Smirnov test; *p < 0.0005. (H) Representative images of MyHC-2b foci in vehicle and sirtinol-treated shPab muscles. Scale bar, 50 μm. The upper right box shows segmented MyHC-2b foci. Chart bars show mean MyHC-2b foci per myofiber (left panel) and mean foci area (right panel). Statistical difference was assessed with the Student's t test. (F–H) Pooled myofibers from all muscles, number of single myofibers > 1,000.