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 dexamethasoneinduced 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. K E Y W O R D Sgene regulation, insulin-like growth factor 1, muscle atrophy 556 |

The Effects of Muscle Cell Aging on Myogenesis

“…These findings further support the notion of a differential regulation and role of IGF-1 isoforms in muscle biology and possibly in the myogenic program of aged muscle cells. More specifically, the IGF-1Eb isoform is known to produce the Eb peptide and this post-translational processing product, as well as its human counterpart (Ec peptide), has been documented to act as an IGF-1R independent competence growth factor in several biological systems [ 37 , 38 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 ]. Previous studies have examined the potential impact and the possible mechanisms of action of IGF-1 isoform over-expression on muscle aging in vivo [ 62 , 69 , 71 ]; however, more studies are needed to mechanistically investigate the potentially distinct roles of the IGF-1 isoforms and particularly of IGF-1Eb in the aging of myoblasts.…”

LncRNA CASC2 is involved in the development of chronic obstructive pulmonary disease via targeting miR-18a-5p/IGF1 axis

“… 33 IGF1 is also known to be a key regulator of muscle mass, and muscle dysfunction is one of the common systemic manifestations of COPD. 34 In addition, IGF1 is also reported to play an important role in the regulation of inflammation in the immune system. 25 The evidence all supported our present results about the potential role of IGF1 in CSE-induced cell apoptosis and inflammation.…”

Role of Alternatively Spliced Messenger RNA (mRNA) Isoforms of the Insulin-Like Growth Factor 1 (IGF1) in Selected Human Tumors

“…IGF1 is an important positive regulator of muscle mass, playing a mainly autocrine/paracrine role in this tissue [ 24 , 128 ]. Additionally, proper levels of IGF1 in muscles is not only implicated in their growth, but also in the maintenance of the glucose homeostasis.…”

“…Recent studies, using CRISPR-Cas9 gene activation approach, note an upregulation of different IGF1 variants in cell lines derived from mouse and human skeletal muscle myoblast, as well as a selective upregulation of class I/II IGF1 transcripts through the change of the target location of a single-guide RNA. These isoforms promoted myotube differentiation and prevented dexamethasone-induced atrophy in myotubes in vitro [ 128 ].…”

“…Biochemical basics and biological relevance of different IGF1 mRNA isoform and post-translational processing products (E peptides) formation are only partially discovered, both in the context of physiology and pathophysiology [ 88 , 103 , 128 ].…”