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

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 CONTEXT: Duchenne 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.

Autophagy regulates satellite cell ability to regenerate normal and dystrophic muscles.

Autophagy is emerging as a key regulatory process during skeletal muscle development, regeneration and homeostasis, and deregulated autophagy has been implicated in muscular disorders and age-related muscle decline. We have monitored autophagy in muscles of mdx mice and human Duchenne muscular dystrophy (DMD) patients at different stages of disease. Our data show that autophagy is activated during the early, compensatory regenerative stages of DMD. A progressive reduction was observed during mdx disease progression, in coincidence with the functional exhaustion of satellite cell-mediated regeneration and accumulation of fibrosis. Moreover, pharmacological manipulation of autophagy can influence disease progression in mdx mice. Of note, studies performed in regenerating muscles of wild-type mice revealed an essential role of autophagy in the activation of satellite cells upon muscle injury. These results support the notion that regeneration-associated autophagy contributes to the early compensatory stage of DMD progression, and interventions that extend activation of autophagy might be beneficial in the treatment of DMD. Thus, autophagy could be a 'disease modifier' targeted by interventions aimed to promote regeneration and delay disease progression in DMD.

Interactions between muscle stem cells, mesenchymal-derived cells and immune cells in muscle homeostasis, regeneration and disease.

Recent evidence has revealed the importance of reciprocal functional interactions between different types of mononuclear cells in coordinating the repair of injured muscles. In particular, signals released from the inflammatory infiltrate and from mesenchymal interstitial cells (also known as fibro-adipogenic progenitors (FAPs)) appear to instruct muscle stem cells (satellite cells) to break quiescence, proliferate and differentiate. Interestingly, conditions that compromise the functional integrity of this network can bias muscle repair toward pathological outcomes that are typically observed in chronic muscular disorders, that is, fibrotic and fatty muscle degeneration as well as myofiber atrophy. In this review, we will summarize the current knowledge on the regulation of this network in physiological and pathological conditions, and anticipate the potential contribution of its cellular components to relatively unexplored conditions, such as aging and physical exercise.

Protein kinase Cθ is required for cardiomyocyte survival and cardiac remodeling.

Protein kinase Cs (PKCs) constitute a family of serine/threonine kinases, which has distinguished and specific roles in regulating cardiac responses, including those associated with heart failure. We found that the PKCθ isoform is expressed at considerable levels in the cardiac muscle in mouse, and that it is rapidly activated after pressure overload. To investigate the role of PKCθ in cardiac remodeling, we used PKCθ(-/-) mice. In vivo analyses of PKCθ(-/-) hearts showed that the lack of PKCθ expression leads to left ventricular dilation and reduced function. Histological analyses showed a reduction in the number of cardiomyocytes, combined with hypertrophy of the remaining cardiomyocytes, cardiac fibrosis, myofibroblast hyper-proliferation and matrix deposition. We also observed p38 and JunK activation, known to promote cell death in response to stress, combined with upregulation of the fetal pattern of gene expression, considered to be a feature of the hemodynamically or metabolically stressed heart. In keeping with these observations, cultured PKCθ(-/-) cardiomyocytes were less viable than wild-type cardiomyocytes, and, unlike wild-type cardiomyocytes, underwent programmed cell death upon stimulation with α1-adrenergic agonists and hypoxia. Taken together, these results show that PKCθ maintains the correct structure and function of the heart by preventing cardiomyocyte cell death in response to work demand and to neuro-hormonal signals, to which heart cells are continuously exposed.