Pharmacological Inhibition of PKCθ Counteracts Muscle Disease in a Mouse Model of Duchenne Muscular Dystrophy

Abstract

Inflammation plays a considerable role in the progression of Duchenne Muscular Dystrophy (DMD), a severe muscle disease caused by a mutation in the dystrophin gene. We previously showed that genetic ablation of Protein Kinase C θ (PKCθ) in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease.Research in contextDuchenne muscular dystrophy (DMD) is a severe muscle disease affecting 1:3500 male births. DMD is caused by a mutation in dystrophin gene, coding for a protein required for skeletal and cardiac muscle integrity. Lack of a functional dystrophin is primarily responsible for the muscle eccentric contraction-induced muscle damage, observed in dystrophic muscle. However, inflammation plays a considerable role in the progression of DMD. Glucocorticoids, which have anti-inflammatory properties, are being used to treat DMD with some success; however, long term treatment with these drugs induces muscle atrophy and wasting, outweighing their benefit. The identification of specific targets for anti-inflammatory therapies is one of the ongoing therapeutic options. Although blunting inflammation would not be a “cure” for the disease, the emerging clue is that multiple strategies, addressing different aspects of the pathology, which may eventually converge, may be successful. In this context, we previously showed that genetic ablation of Protein Kinase C θ (PKCθ), an enzyme known to be involved in immune response, in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease.

Targeting PKCθ Promotes Satellite Cell Self-Renewal

“…We previously showed that knock-out of PKCθ or its inhibition in a mouse model of Duchenne Muscular Dystrophy (mdx mouse) reduces muscle damage, by modulating the immune cell infiltration. This beneficial effect was mainly due to the inhibition of PKCθ in T cells, which leads to reduced inflammation [10][11][12]. Interestingly, we also found that lack of PKCθ promotes muscle repair in dystrophic mice, even at advanced stages of the disease, supporting SCs survival and maintenance [13].…”

“…We next sought to validate our results from the PKCθ-/-mouse model, using the C20specific inhibitor of PKCθ [11,12,18,19]. To analyze the effects of C20 throughout the different phases of myogenic progression, we isolated SC from WT and PKCθ-/-mice and cultured them for 4 days in growth medium (GM), followed by 2 days in differentiation medium (DM).C20 was used at the concentration range (0.5 µM, 1 µM and 2 µM) we previously found not to be toxic for in vitro treatment [11]. Control cultures were treated with 0.1% DMSO, the same concentration used for C20 dilution.…”

PKC and PKN in heart disease

“…Other studies investigating cardiac transplant rejection and cardiac muscle structure in dystrophic mice identified and characterized novel derivatives of 2,4-diaminopyrimidine and 2,4-diamino-5-nitropyrimidine as selective inhibitors against PKCθ [149][150][151]. Treatment with these compounds reduced inflammation in dystrophic mice, and prolonged graft survival in an in vivo rat heterotopic cardiac transplant model.…”

Lack of PKCθ Promotes Regenerative Ability of Muscle Stem Cells in Chronic Muscle Injury

“…We previously showed that lack or pharmacological inhibition of PKCθ reduced muscle loss and inflammation, and increased muscle regeneration and performance in mdx mice. The observed phenotype was primarily due to lack of PKCθ in hematopoietic cells [19,20], and in particular inhibition of early T cells infiltration in dystrophic muscle [21]. However, PKCθ is also expressed in muscle, where it modulates several signalling pathways involved in foetal and early post-natal tissue growth and maturation [22][23][24].…”