Inhibiting EZH2 complements steroid effects in Duchenne muscular dystrophy [mouse]

Duchenne muscular dystrophy (DMD) is a devastating X-linked disorder caused by mutations in the dystrophin gene. Despite recent advances in understanding the disease etiology and applying emerging treatment methodologies, glucocorticoid derivatives remain the only general therapeutic option that can slow disease development. However, the precise molecular mechanism of glucocorticoid action remains unclear, and there is still need for additional remedies to complement the treatment. Here, using single-nucleus RNA-sequencing and spatial transcriptome analyses of human and mouse muscles, we investigated pathogenic features in DMD patients and palliative effects of glucocorticoids. Our approach further illuminated the importance of proliferating satellite cells, and revealed increased activity of a signal transduction pathway involving EZH2 in the patient cells. Subsequent administration of EZH2 inhibitors to Dmd mutant mice resulted in improved muscle phenotype through maintaining the immune-suppressing effect but overriding the muscle weakness and fibrogenic effects exerted by glucocorticoids. Our analysis reveals pathogenic mechanisms that can be readily targeted by extant therapeutic options for DMD. DBA/2J and DBA/2J-mdx mice (D2.B10-Dmdmdx/J), hereafter denoted as D2-mdx mice, were purchased from the Jackson Laboratory (JAX ID: 013141; Bar Harbor, ME). All experiments were approved by the Institutional Animal Care and Use Committee in Seoul National University Hospital (#20-0216-S1A0) and animals were maintained in a facility accredited by AAALAC International (#001169) in accordance with the Guide for the Care and Use of Laboratory Animals 8th edition, NRC. Muscle samples were taken from the hindlimb and quadriceps and frozen with liquid nitrogen. snRNA-seq was performed on fresh frozen muscle cells from two male wild-type DBA/2J mice, two male D2-mdx mice, and two male D2-mdx mice treated with deflazacort. Each genotype group included one 7-week-old and one 28-week-old mouse. Deflazacort (SML0123-10MG; Sigma, St. Louis, MO) was formulated as a 0.2 mg/ml suspension in a solution comprised of DMSO (10%), PEG300 (40%), Tween-80 (5%), and saline (45%). This formulation was administered at a dose of 1 mg/kg by intraperitoneal injection once a week for 28 days to four week-old mice. GSK126 (S7061; Selleck chemicals, Houston, TX) was administered to four-week-old mice by intraperitoneal injection once every two days for 28 days at a dose of 50 mg/kg in 20% Captisol.