SPIF promotes myoblast differentiation and utrophin expression while inhibiting fibrosis in Duchenne muscular dystrophy via the H19/miR-675/let-7 and miR-21 pathways

Daria Morgoulis,Chaya Brodie,Simona Cazacu,Amir Dori,Peter Berenstein,Gila Kazimirsky,Eytan R Barnea

doi:10.1038/s41419-019-1307-9

Abstract

Duchenne muscular dystrophy (DMD) is a progressive, lethal, X-linked disease of skeletal and cardiac muscles caused by mutations in the dystrophin gene. Loss of dystrophin leads to muscle fiber damage and impairment of satellite cell asymmetric division, which are essential for muscle regeneration. These processes ultimately result in muscle wasting and the replacement of the degenerating muscles by fibrogenic cells, a process that leads to the generation of fibrotic tissues. Preimplantation factor (PIF) is an evolutionary conserved 15-amino acid peptide secreted by viable mammalian embryos. Synthetic PIF (sPIF) reproduces the protective/regenerative effects of the endogenous peptide in immune disorders and transplantation models. In this study, we demonstrated that sPIF treatment promoted mouse and human myoblast differentiation and inhibited the expression of collagen 1A1, collagen 1A2, and TGF-β in DMD patient-derived myoblasts. Additionally, sPIF increased the expression of utrophin, a homolog of dystrophin protein. sPIF effects were mediated via the upregulation of lncRNA H19 and miR-675 and downregulation of let-7. sPIF also inhibited the expression of miR-21, a major fibrosis regulator. The administration of sPIF in mdx mice significantly decreased serum creatine kinase and collagen I and collagen IV expression in the diaphragm, whereas it increased utrophin expression in the diaphragm, heart and quadriceps muscles. In conclusion, sPIF promoted the differentiation of DMD myoblasts, increased utrophin expression via the H19/miRNA-675/let-7 pathway, and reduced muscle fibrosis possibly via the upregulation of miR-675 and inhibition of miR-21 expression. These findings strongly support pursuing sPIF as a potential therapeutic agent for DMD. Moreover, the completion of an sPIF phase I safety trial will further promote the use of sPIF for the treatment of muscular dystrophies.

Highlights

Duchenne muscular dystrophy (DMD) is a recessive, fatal, X-linked disease and the most common form of Official journal of the Cell Death Differentiation AssociationMorgoulis et al Cell Death and Disease (2019)10:82 dystrophin is essential for the asymmetric division of satellite cells, and the lack of functional protein interferes with the regenerative capacity of these cells[7]
We first examined the effects of synthetic PIF analog (sPIF) on myoblast differentiation by analyzing the expression of the myogenic factors, MyoD and myogenin, which are required for the proper differentiation of myogenic cells during the differentiation and repair of myosin heavy chain (MyHC), which is expressed in differentiated muscle cells
Myoblasts were cultured in a medium containing 2% horse serum for 10 days to allow cell differentiation. sPIF was added to the cells at the beginning of the culture and every 2 days thereafter

Summary

Introduction

Duchenne muscular dystrophy (DMD) is a recessive, fatal, X-linked disease and the most common form of Official journal of the Cell Death Differentiation AssociationMorgoulis et al Cell Death and Disease (2019)10:82 dystrophin is essential for the asymmetric division of satellite cells, and the lack of functional protein interferes with the regenerative capacity of these cells[7]. Various therapeutic approaches have been explored for treating DMD, including promotion of muscle regeneration[9], anti-inflammatory and anti-fibrotic agents[2,10,11], and exon-skipping with antisense oligonucleotides[12,13]. Despite these efforts, there is currently no cure for DMD, and treatments focus mainly on relieving the symptoms and minimizing complications[14]. The synthetic PIF analog (sPIF) replicates the native peptide actions and has comprehensive immune protective and regenerative properties. SPIF was the subject of a successful Universityinitiated FDA Fast-Track-awarded Phase I clinical trial for autoimmune hepatitis and obtained Orphan Drug Designation (www.clinicaltrials.gov, NCT 02239562)

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Results

Conclusion