CRISPR-Cas9-induced IGF1 gene activation as a tool for enhancing muscle differentiation via multiple isoform expression

Abstract

Muscle wasting, or muscle atrophy, can occur with age, injury, and disease; it affects the quality of life and complicates treatment. Insulin-like growth factor 1 (IGF1) is a key positive regulator of muscle mass. The IGF1/Igf1 gene encodes multiple protein isoforms that differ in tissue expression, potency, and function, particularly in cellular proliferation and differentiation, as well as in systemic versus localized signaling. Genome engineering is a novel strategy for increasing gene expression and has the potential to recapitulate the diverse biology seen in IGF1 signaling through the overexpression of multiple IGF1 isoforms. Using a CRISPR-Cas9 gene activation approach, we showed that the expression of multiple IGF1 or Igf1 mRNA variants can be increased in human and mouse skeletal muscle myoblast cell lines using a single-guide RNA (sgRNA). We found increased IGF1 protein levels in the cell culture media and increased cellular phosphorylation of AKT1, the main effector of IGF1 signaling. We also showed that the expression of Class 1 or Class 2 mRNA variants can be selectively increased by changing the sgRNA target location. The expression of multiple IGF1 or Igf1 mRNA transcript variants in human and mouse skeletal muscle myoblasts promoted myotube differentiation and prevented dexamethasone-induced atrophy in myotubes in vitro. Our findings suggest that this novel approach for enhancing IGF1 signaling has potential therapeutic applications for treating skeletal muscle atrophy.

Keywords: gene regulation; insulin‐like growth factor 1; muscle atrophy.

Figure-1.

Use of CRISPR-Cas9–mediated gene activation to increase IGF1 or Igf1 expression in human or mouse myoblasts and myotubes. (A) Schematic representation of the alignment of human and mouse genomic DNA showing the conservation of human sgRNA-targeting sites in mice. The sgRNA-targeting sequences are highlighted in gray, and the start of the first exon is underlined. (B) A schematic illustrating the transcriptional activation of IGF1 (or Igf1) by the CRISPR-Cas9–based three-component SAM gene activation system. (C) A schematic of the semi-quantitative PCR strategy for determining the relative quantities of total human IGF1 or mouse Igf1 mRNA transcripts and the relative quantities of their respective mRNA transcript variants. Arrows indicate the binding sites for the semi-quantitative PCR primers used in expression profiling.

Figure-2.

Expression of human IGF1 or mouse Igf1 mRNA and their respective transcript variants due to CRISPR-Cas9-mediated gene activation. (A) The relative quantity (RQ) of total IGF1 mRNA in HEK 293T cell lines measured by semi-quantitative PCR. Data are shown for different sgRNAs. Naïve cells are uninfected HEK 293T cells and empty cells are HEK 293T cell lines that have the gene activation system but express a sgRNA without a targeting sequence. (B) The RQ of total IGF1 mRNA in undifferentiated and differentiated (day-8) human skeletal muscle myoblast (HSMM) cell lines measured by semi-quantitative PCR. (C) The RQ of total Igf1 mRNA in undifferentiated and differentiated (day-3) C2C12 cell lines measured by semi-quantitative PCR. (D) RQ of IGF1 mRNA or Igf1 mRNA transcript variants in undifferentiated and differentiated human skeletal muscle myoblast (HSMM) and C2C12 cell lines measured by semi-quantitative PCR. (E) Time course of the RQ of Igf1 mRNA or Igf1 mRNA transcript variants determined by semi-quantitative PCR. (F) Analysis of semi-quantitative PCR data showing total Igf1 mRNA or Igf1 mRNA variant expression in NIH/3T3 cell lines expressing the SAM-mediated gene activation system and various Igf1 sgRNAs. (G) Analysis of semi-quantitative PCR data from undifferentiated C2C12 cell lines expressing different sgRNAs. The RQ of samples was normalized to that of Igf1 mRNA transcript variants (Class 1 or Class 2) in C2C12 cells expressing ms.sgRNA2. Samples in A-D and F were normalized to the corresponding empty vector sample to determine the RQ of total IGF1 mRNA transcript expression. Samples in E were normalized to empty vector C2C12 cells on day-0, and samples in G are normalized to that of Igf1 mRNA transcript variants (Class 1 or Class 2) in C2C12 cells expressing ms.sgRNA2. Three biological replicates were performed in all experiments. In A-D and F, * indicates a statistically significant difference when compared with the corresponding empty vector sample (P<0.05). In E, * indicates a significant difference (P<0.05) between the RQ of mRNA in cells expressing ms.sgRNA2 and the RQ of mRNA in cells expressing empty vector at the same time point. In G, * indicates a statistical significance between the mRNA transcript and the ms.sgRNA2 sample. Error bars represent the standard deviation.

Figure-3.

Inhibition of dexamethasone (DEX)-induced atrophy in human and mouse myotubes by CRISPR-Cas9–mediated gene activation to increase the expression of IGF1 or Igf1 mRNA variants. (A) Anti-myosin (MF20) staining of human skeletal muscle myoblast (HSMM)-derived myotubes expressing empty vector or human sgRNA2 after 6 days of differentiation and 2 days of treatment or no treatment (control) with 50 μM DEX. (B) Quantification of myotube diameter and fusion index (nuclei per myotube) of MF20-positive HSMM-derived myotubes after DEX treatment. (C) MF20 staining of C2C12-derived myotubes expressing empty vector or mouse ms.sgRNA2 after 2 days of differentiation and 1 day of treatment or no treatment with 50 μM DEX. (D) Quantification of myotube diameter and fusion index of MF20-positive C2C12-derived myotubes after DEX treatment. (E) Enzyme-linked immunosorbent assay (ELISA) results showing the concentration of IGF1 in the conditioned media of C2C12 cells before and after differentiation for 3 days. (F) Western blot analysis showing the levels of phosphorylated AKT (p-AKT) in C2C12 cells expressing empty vector or mouse ms.sgRNA2 after 3 days of differentiation. Scale bars, 50 μm. DAPI-positive nuclei are blue and MF20-positive myotubes are red. In all DEX-treated cells, ethanol was used as a carrier control. * P<0.01; n/s, no statistically significant difference between samples; braces show the statistical comparisons performed. Experiments were performed in biological triplicates in panels (A), (B), (E), and (F), and four biological replicates were performed in panels (C) and (D). Error bars in panel (E) represent 95% confidence intervals.

Figure-4.

Enhanced muscle cell differentiation and reduced response to glucocorticoids in C2C12 myotubes expressing multiple Igf1 mRNA transcript variants. (A) Heatmap of the Z-scores for mRNA transcripts that showed a significant change in expression (at least a 1.5-fold change) between untreated C2C12 myotubes expressing ms.sgRNA2 and untreated C2C12 myotubes expressing empty vector. mRNA variant data were determined by RNA-seq. (B) From the RNA-seq data, P-values are shown for representative gene ontology (GO) terms associated with muscle development, the response to dexamethasone (DEX), and cell death. (C) Representative heatmaps of genes that are differentially expressed in untreated C2C12-derived myotubes expressing ms.sgRNA2, DEX-treated C2C12-derived myotubes, and DEX-treated C2C12-derived myotubes expressing empty vector when compared to untreated C2C12-derived myotubes expressing empty vector. Genes shown are associated with the GO terms Response to Oxidative Stress and Striated Muscle Cell Development. Four biological replicates were performed per condition.

Figure-5.

Differential regulation of a subset of genes by IGF1 signaling in dexamethasone (DEX)-treated C2C12 cells. (A) A Venn diagram summarizing data from the RNA-seq analysis showing the number of differentially regulated genes that were in common between the comparison of untreated C2C12 cells expressing empty vector versus DEX-treated C2C12 cells expressing empty vector (Empty vs Empty DEX) and the comparison of DEX-treated C2C12 cells expressing empty vector versus DEX-treated C2C12 cells expressing ms.sgRNA2 (ms.sgRNA2 DEX vs Empty DEX). (B) A heatmap of the Z-scores for the 33 genes that were identified in (A). Four biological replicates were performed.