Evaluation of pro-regenerative and anti-inflammatory effects of isolecanoric acid in the muscle: Potential treatment of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating degenerative disease of skeletal muscles caused by loss of dystrophin, a key protein that maintains muscle integrity, which leads to progressive muscle degeneration aggravated by chronic inflammation, muscle stem cells' (MuSCs) reduced regenerative capacity and replacement of muscle with fibroadipose tissue. Previous research has shown that pharmacological GSK-3ß inhibition favors myogenic differentiation and plays an important role in modulating inflammatory processes. Isolecanoric acid (ILA) is a natural product isolated from a fungal culture displaying GSK-3ß inhibitory properties. The present study aimed to investigate the proregenerative and anti-inflammatory properties of this natural compound in the DMD context. Our results showed that ILA markedly promotes myogenic differentiation of myoblasts by increasing ß-Catenin signaling and boosting the myogenic potential of mouse and human stem cells. One important finding was that the GSK-3ß/ß-Catenin pathway is altered in dystrophic mice muscle and ILA enhances the myofiber formation of dystrophic MuSCs. Treatment with this natural compound improves muscle regeneration of dystrophic mice by, in turn, improving functional performance. Moreover, ILA ameliorates the inflammatory response in both muscle explants and the macrophages isolated from dystrophic mice to, thus, mitigate fibrosis after muscle damage. Overall, we show that ILA modulates both inflammation and muscle regeneration to, thus, contribute to improve the dystrophic phenotype.

Understanding Epicardial Cell Heterogeneity during Cardiogenesis and Heart Regeneration.

The outermost layer of the heart, the epicardium, is an essential cell population that contributes, through epithelial-to-mesenchymal transition (EMT), to the formation of different cell types and provides paracrine signals to the developing heart. Despite its quiescent state during adulthood, the adult epicardium reactivates and recapitulates many aspects of embryonic cardiogenesis in response to cardiac injury, thereby supporting cardiac tissue remodeling. Thus, the epicardium has been considered a crucial source of cell progenitors that offers an important contribution to cardiac development and injured hearts. Although several studies have provided evidence regarding cell fate determination in the epicardium, to date, it is unclear whether epicardium-derived cells (EPDCs) come from specific, and predetermined, epicardial cell subpopulations or if they are derived from a common progenitor. In recent years, different approaches have been used to study cell heterogeneity within the epicardial layer using different experimental models. However, the data generated are still insufficient with respect to revealing the complexity of this epithelial layer. In this review, we summarize the previous works documenting the cellular composition, molecular signatures, and diversity within the developing and adult epicardium.

miR-106b is a novel target to promote muscle regeneration and restore satellite stem cell function in injured Duchenne dystrophic muscle.

Satellite cells (SCs), muscle stem cells, display functional heterogeneity, and dramatic changes linked to their regenerative capabilities are associated with muscle-wasting diseases. SC behavior is related to endogenous expression of the myogenic transcription factor MYF5 and the propensity to enter into the cell cycle. Here, we report a role for miR-106b reinforcing MYF5 inhibition and blocking cell proliferation in a subset of highly quiescent SC population. miR-106b down-regulation occurs during SC activation and is required for proper muscle repair. In addition, miR-106b is increased in dystrophic mice, and intramuscular injection of antimiR in injured mdx mice enhances muscle regeneration promoting transcriptional changes involved in skeletal muscle differentiation. miR-106b inhibition promotes the engraftment of human muscle stem cells. Furthermore, miR-106b is also high in human dystrophic muscle stem cells and its inhibition improves intrinsic proliferative defects and increases their myogenic potential. This study demonstrates that miR-106b is an important modulator of SC quiescence, and that miR-106b may be a new target to develop therapeutic strategies to promote muscle regeneration improving the regenerative capabilities of injured dystrophic muscle.

Pitx2 Differentially Regulates the Distinct Phases of Myogenic Program and Delineates Satellite Cell Lineages During Muscle Development.

The knowledge of the molecular mechanisms that regulate embryonic myogenesis from early myogenic progenitors to myoblasts, as well as the emergence of adult satellite stem cells (SCs) during development, are key concepts to understanding the genesis and regenerative abilities of the skeletal muscle. Several previous pieces of evidence have revealed that the transcription factor Pitx2 might be a player within the molecular pathways controlling somite-derived muscle progenitors' fate and SC behavior. However, the role exerted by Pitx2 in the progression from myogenic progenitors to myoblasts including SC precursors remains unsolved. Here, we show that Pitx2 inactivation in uncommitted early myogenic precursors diminished cell proliferation and migration leading to muscle hypotrophy and a low number of SCs with decreased myogenic differentiation potential. However, the loss of Pitx2 in committed myogenic precursors gave rise to normal muscles with standard amounts of SCs exhibiting high levels of Pax7 expression. This SC population includes few MYF5+ SC-primed but increased amount of less proliferative miR-106b+cells, and display myogenic differentiation defects failing to undergo proper muscle regeneration. Overall our results demonstrate that Pitx2 is required in uncommitted myogenic progenitors but it is dispensable in committed precursors for proper myogenesis and reveal a role for this transcription factor in the generation of diverse SC subpopulations.

Regulation of Epicardial Cell Fate during Cardiac Development and Disease: An Overview.

The epicardium is the outermost cell layer in the vertebrate heart that originates during development from mesothelial precursors located in the proepicardium and septum transversum. The epicardial layer plays a key role during cardiogenesis since a subset of epicardial-derived cells (EPDCs) undergo an epithelial-mesenchymal transition (EMT); migrate into the myocardium; and differentiate into distinct cell types, such as coronary vascular smooth muscle cells, cardiac fibroblasts, endothelial cells, and presumably a subpopulation of cardiomyocytes, thus contributing to complete heart formation. Furthermore, the epicardium is a source of paracrine factors that support cardiac growth at the last stages of cardiogenesis. Although several lineage trace studies have provided some evidence about epicardial cell fate determination, the molecular mechanisms underlying epicardial cell heterogeneity remain not fully understood. Interestingly, seminal works during the last decade have pointed out that the adult epicardium is reactivated after heart damage, re-expressing some embryonic genes and contributing to cardiac remodeling. Therefore, the epicardium has been proposed as a potential target in the treatment of cardiovascular disease. In this review, we summarize the previous knowledge regarding the regulation of epicardial cell contribution during development and the control of epicardial reactivation in cardiac repair after damage.

Muscle Satellite Cell Heterogeneity: Does Embryonic Origin Matter?

Muscle regeneration is an important homeostatic process of adult skeletal muscle that recapitulates many aspects of embryonic myogenesis. Satellite cells (SCs) are the main muscle stem cells responsible for skeletal muscle regeneration. SCs reside between the myofiber basal lamina and the sarcolemma of the muscle fiber in a quiescent state. However, in response to physiological stimuli or muscle trauma, activated SCs transiently re-enter the cell cycle to proliferate and subsequently exit the cell cycle to differentiate or self-renew. Recent evidence has stated that SCs display functional heterogeneity linked to regenerative capability with an undifferentiated subgroup that is more prone to self-renewal, as well as committed progenitor cells ready for myogenic differentiation. Several lineage tracing studies suggest that such SC heterogeneity could be associated with different embryonic origins. Although it has been established that SCs are derived from the central dermomyotome, how a small subpopulation of the SCs progeny maintain their stem cell identity while most progress through the myogenic program to construct myofibers is not well understood. In this review, we synthesize the works supporting the different developmental origins of SCs as the genesis of their functional heterogeneity.

MiRNAs and Muscle Regeneration: Therapeutic Targets in Duchenne Muscular Dystrophy.

microRNAs (miRNAs) are small non-coding RNAs required for the post-transcriptional control of gene expression. MicroRNAs play a critical role in modulating muscle regeneration and stem cell behavior. Muscle regeneration is affected in muscular dystrophies, and a critical point for the development of effective strategies for treating muscle disorders is optimizing approaches to target muscle stem cells in order to increase the ability to regenerate lost tissue. Within this framework, miRNAs are emerging as implicated in muscle stem cell response in neuromuscular disorders and new methodologies to regulate the expression of key microRNAs are coming up. In this review, we summarize recent advances highlighting the potential of miRNAs to be used in conjunction with gene replacement therapies, in order to improve muscle regeneration in the context of Duchenne Muscular Dystrophy (DMD).

Isolation and Culture of Quiescent Skeletal Muscle Satellite Cells.

It has been shown that freshly isolated satellite cells from adult muscle constitute a stem cell-like population that exhibits more efficient engraftment and self-renewal activity in regenerating muscle than myoblast. Thus, purification of pure populations of quiescent satellite cells from adult skeletal muscle is highly necessary, not only for understanding the biology of satellite cells and myoblasts but also for improving cell-based therapies for muscle regeneration. This chapter describes a basic protocol used in our laboratory to isolate quiescent muscle satellite cells from adult skeletal muscle by enzymatic dissociation followed by a sequential magnetic-activated cell sorting (MACS). This method is cheap and fast providing and alternative procedure to other purification methods that require fluorescence-activated cell sorting (FACS) machines. Freshly isolated quiescent satellite cells purified by this method can be used in a broad range of experiments including cell transplantation for satellite cell self-renewal experiments or cell therapies.

PITX2 Enhances the Regenerative Potential of Dystrophic Skeletal Muscle Stem Cells.

Duchenne muscular dystrophy (DMD), one of the most lethal genetic disorders, involves progressive muscle degeneration resulting from the absence of DYSTROPHIN. Lack of DYSTROPHIN expression in DMD has critical consequences in muscle satellite stem cells including a reduced capacity to generate myogenic precursors. Here, we demonstrate that the c-isoform of PITX2 transcription factor modifies the myogenic potential of dystrophic-deficient satellite cells. We further show that PITX2c enhances the regenerative capability of mouse DYSTROPHIN-deficient satellite cells by increasing cell proliferation and the number of myogenic committed cells, but importantly also increasing dystrophin-positive (revertant) myofibers by regulating miR-31. These PITX2-mediated effects finally lead to improved muscle function in dystrophic (DMD/mdx) mice. Our studies reveal a critical role for PITX2 in skeletal muscle repair and may help to develop therapeutic strategies for muscular disorders.

Pitx2 in Embryonic and Adult Myogenesis.

Skeletal muscle is a heterogeneous tissue that represents between 30 and 38% of the human body mass and has important functions in the organism, such as maintaining posture, locomotor impulse, or pulmonary ventilation. The genesis of skeletal muscle during embryonic development is a process controlled by an elaborate regulatory network combining the interplay of extrinsic and intrinsic regulatory mechanisms that transform myogenic precursor cells into functional muscle fibers through a finely tuned differentiation program. However, the capacity of generating muscle still remains once these fibers have matured. Adult myogenesis resembles many of the embryonic morphogenetic episodes and depends on the activation of satellite cells that have the potential to differentiate into new muscle fibers. Pitx2 is a member of the bicoid family of homeodomain transcription factors that play an important role in morphogenesis. In the last decade, Pitx2 has emerged as a key element involved in the fine-tuning mechanism that regulates skeletal-muscle development as well as the differentiation and cell fate of satellite cells in adult muscle. Here we present an integrative view of all aspects of embryonic and adult myogenesis in which Pitx2 is involved, from embryonic development to satellite-cell proliferation, fate specification, and differentiation. Those new Pitx2 functions on satellite-cell biology might open new perspectives to develop therapeutic strategies for muscular disorders.