Transcriptomic characterization of the aberrant alternative splicing in skeletal muscles of sarcopenia patients

Figures

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  Fig. 1. Transcriptome analysis of DEGs in muscle biopsies from sarcopenic subjects (Sar), subjects with only low muscle mass (Low_mass) and subjects with only low muscle strength (Low_str) compared to healthy subjects (Ctrl). (A) Principal component analysis (PCA) of muscle biopsies from healthy subjects (Ctrl) and sarcopenic subjects (Sar), subjects with only low muscle mass (Low_mass), and subjects with only low muscle strength (Low_str) based on the FPKM value of all DEGs. The ellipse for each group is the confidence ellipse. (B). Bar plot of DEGs in three groups (Low_mass, Low_str samples, and Sar samples) compared to Ctrl samples, respectively. (C). Venn diagram for up-regulated DEGs and down-regulated DEGs of Low_mass samples, Low_str samples, and Sar samples compared to Ctrl samples, respectively. (D). Bubble plot of the top ten enriched GO BP terms of upregulated and downregulated DEGs from the Sar group compared to Ctrl samples.

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  Fig. 2. Transcriptome analysis of alternative splicing events in muscle biopsies from Sar, Low_mass, and Low_str samples compared to Ctrl samples. (A). Classification of all regulated alternative splicing events (DASEs). X-axis: the number of DASEs. (B). Principal component analysis (PCA) of Low_mass samples, Low_str samples, and Sar samples compared to Ctrl samples based on the PSI (Percent Spliced In) values of different NIR (non-intron retention) events. The ellipse for each group is the confidence ellipse. (C). PSI heatmap of all NIR DASEs in Low_mass samples, Low_str samples, and Sar samples compared to Ctrl samples. AS filter had detectable splice junctions in all samples and at least 80% of samples had > = 10 splice junction reads supporting AS. (D). Bubble plot showing the GO term and KEGG pathway analysis of differential alternative splicing genes (DASGs) in Sar samples compared to Ctrl samples. (E). Box plot showing alternative splicing ratio change in DASE of FOXO3. The ratio of alternative splice junction reads and model splice junction reads were calculated and presented. F. Box plots showing alternative splicing ratio changes in DASEs of CD38, PARP2, SIRT2, SIRT3 and SIRT5…

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  Fig. 3. Overlap analysis of DASEs in Low_mass samples, Low_str samples, and Sar samples. Venn diagram of DASEs in Low_mass samples, Low_str samples and Sar samples. Venn diagram of DASGs in Low_mass samples, Low_str samples and Sar samples. Bubble plot showing the GO term and KEGG pathway analysis of DASGs in Low_mass samples overlap Sar samples. Bubble plot showing GO terms and KEGG pathway analysis of DASGs in Low_str samples overlapping Sar samples.

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  Fig. 4. Splicing events regulated important genes in Low_mass samples, Low_str samples, and Sar samples compared to Ctrl samples. Box plots showing PSI changes in four DASEs in Low_mass samples, Low_str samples, and Sar samples compared to Ctrl samples. Visualization of read distribution of A3SS of PPARGC1A in Low_mass samples, Low_str samples, and Sar samples compared to Ctrl samples.

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  Fig. 5. Differential alternative splicing in a sarcopenic mouse model. Box plots showing grip strength, fall times and running duration between DM-treated and negative control (NC) mice. Student’s t-test was used for comparisons between two groups. Error bars represent mean ± SEM. **** P-value < 0.0001. Volcano plot showing all differentially expressed genes (DEGs) between DM versus NC samples. Bar plot showing the differential alternative splicing events (DASEs) between DM-treated sarcopenic and control mice. Bubble plot exhibiting the enriched top 10 GO biological process of mouse sarcopenic differentially alternatively spliced genes (DASGs). Venn diagram showing the shared differentially alternatively spliced genes (DASGs) in sarcopenic mand patients. Bubble plot exhibiting the most enriched top 10 GO biological process of the overlaped DASGs between Sar versus Ctrl (patients) and DM versus NC (mice) groups.

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  Fig. 6. Verification of differential alternative splicing events. Bar plot showing the splic ratio of four DASEs between Sar samples and Ctrl samples. They are located in genes AKT2, FMNL2, TCF7L2 and USP40. The ratio of alternative splice junction reads and model splice junction reads were calculated and presented. Student’s t-test was used for comparisons between two groups. Error bars represent mean ± SEM. * P-value < 0.05; ** P-value < 0.01. RT-qPCR verification of the differential alternative splicing events in the same genes as in (A) using the muscle tissue of sarcopenic mice. The primers amplifying the alternative splice junction and model splice junction reads were designed. The ratio of the corresponding PCR products were quantified. Group comparisons were performed with the two-way ANOVA. Error bars represent mean ± SEM. * P-value < 0.05; **** P-value < 0.0001. The PSI changes of the four DASEs between the DM model and control mice were ploted on the left of their corresponding RT-qPCR results.