Development of antisense-mediated myostatin knockdown for the treatment of insulin resistance

Keith Foster,Wouter Eilers,Mark Cleasby

doi:10.1038/s41598-021-81222-7

Abstract

Myostatin is a negative regulator of muscle mass and its inhibition represents a promising strategy for the treatment of muscle disorders and type 2 diabetes. However, there is currently no clinically effective myostatin inhibitor, and therefore novel methods are required. We evaluated the use of antisense phosphorodiamidate morpholino oligomers (PMO) to reduce myostatin expression in skeletal muscle and measured their effects on muscle mass and glucose uptake. C57/Bl6 mice received intramuscular or intravenous injections of anti-myostatin PMOs. Repeated intramuscular administration lead to a reduction in myostatin transcript levels (~ 20–40%), and an increase in muscle mass in chow and high-fat diet (HFD)-fed mice, but insulin-stimulated glucose uptake was reduced in PMO-treated muscles of HFD-fed mice. Five weekly intravenous administrations of 100 nmol PMO did not reduce myostatin expression, and therefore had no significant physiological effects. Unexpectedly, exon skipping levels were higher after intramuscular administration of PMO in HFD- than chow-fed mice. These results suggest that a modest PMO-induced reduction in myostatin transcript levels is sufficient to induce an increase in muscle mass, but that a greater degree of inhibition may be required to improve muscle glucose uptake.

Highlights

Myostatin is a negative regulator of muscle mass and its inhibition represents a promising strategy for the treatment of muscle disorders and type 2 diabetes
We evaluated a number of phosphorodiamidate morpholino oligomers (PMO) complementary to exon 2 or overlapping the splice donor site of intron 2 of the mouse myostatin transcript, on the basis of the location of binding sites for serine/arginine-rich splicing factors predicted using ESEfinder v3.029,30 by administering a single local injection into the cranial compartment of the lower leg, which includes the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles, followed by RT-PCR analysis of myostatin exon 2 skipping 1 week post-injection
We found that a mixture of two PMO 30-mers (Fig. 1a) resulted in the highest level of exon skipping in the TA muscle when compared to single PMOs at an equimolar total PMO concentration

Summary

Introduction

Myostatin is a negative regulator of muscle mass and its inhibition represents a promising strategy for the treatment of muscle disorders and type 2 diabetes. We evaluated the use of antisense phosphorodiamidate morpholino oligomers (PMO) to reduce myostatin expression in skeletal muscle and measured their effects on muscle mass and glucose uptake. Exon skipping levels were higher after intramuscular administration of PMO in HFDthan chow-fed mice These results suggest that a modest PMO-induced reduction in myostatin transcript levels is sufficient to induce an increase in muscle mass, but that a greater degree of inhibition may be required to improve muscle glucose uptake. Hybridisation of AOs to specific complementary sequences on the (pre-)mRNA allows the recruitment of RNAse, which induces degradation of the target transcript or the steric blockade of RNA regulatory proteins, to alter RNA processing Examples of how this could modify disease phenotypes include the manipulation of specific signalling pathways or the correction of aberrant RNA splicing caused by genetic mutations. Therapeutic MSTN silencing has the potential to ameliorate IR via dual mechanisms: an augmentation of muscle mass and a reduction in adipose mass

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