Staff member

Fibroadipogenic precursor cells (FAPs) are key contributors to the fibrotic and adipogenic remodeling observed in Duchenne muscular dystrophy (DMD), yet their precise role in muscle degeneration remains unclear. In this study, we investigated how FAPs from DMD muscle influence myogenesis by conducting co-culture experiments using healthy myoblasts with FAPs derived from either healthy or DMD biopsies, in both direct and indirect contact conditions. We observed that healthy FAPs enhance myogenic differentiation, increasing myotube area, while DMD FAPs significantly inhibit it. To identify underlying molecular mechanisms, we performed mass spectrometry of the FAP secretome and found that 368 of 760 detected proteins were upregulated in DMD FAPs, including C4b-binding protein alpha chain (C4BPA), which showed a notable increase. In vitro treatment of myoblasts with recombinant C4BPA led to severe impairment of myotube formation, evidenced by reduced myotube area, fewer nuclei per myotube, and smaller myotube size. C4BPA exposure also downregulated myogenic markers and upregulated genes associated with muscle atrophy. These findings were further validated in a 3D engineered muscle model, where C4BPA significantly reduced contractile function. Importantly, silencing C4BPA in DMD-derived cultures partially restored myogenic capacity, improving both the differentiation index and nuclear content per myotube. Together, our data demonstrate that DMD FAPs exert anti-myogenic effects, at least in part, through elevated secretion of C4BPA, which interferes with muscle differentiation and function. This highlights C4BPA as a novel effector of muscle degeneration and a promising therapeutic target for modulating the fibrotic environment in DMD. Targeting specific secreted factors from pathological FAPs may help preserve muscle regeneration and mitigate disease progression in dystrophic muscles.

JTD

The degeneration of the muscle in muscle dystrophies involves complex interactions among the different cell types. Here, we have used datasets from single-nuclei RNA sequencing (snRNAseq) of Duchenne Muscular Dystrophy (DMD) muscle samples to study the dysregulation of molecular pathways compared to healthy control muscle. We have observed that the epidermal growth factor (EGF) signaling is upregulated in DMD by an increase of the ligands EGF and EGF containing fibulin extracellular matrix protein 1 (EFEMP1). This study explores the role of EGF and EFEMP1 in FAPs and myoblasts in vitro. We provide evidence that EFEMP1 is secreted by FAPs in DMD and is mainly involved with increased myotube size but without enhancing muscle strength. Conversely, EGF enhances fibrotic differentiation in FAPs and promote smaller, proliferative myotubes in myoblasts, aligning with a fibrotic and dysfunctional muscle phenotype in DMD. The cellular differences from both ligands can be explained by the interactions with the receptor type, with EGF activating both EGFR and ErbB2, while EFEMP1 selectively maintained ErbB4 in an inactive state but promoting EGFR-ErbB2 and ErbB2-ErbB4 heterodimerization, potentially enhancing EGF signaling. Consequently, this study examinates the alteration of the EGF signalling in DMD and provides new molecular interactions in muscle that can be useful for targeted therapies of muscle degeneration.

JTD Keywords: Deposits, Efemp1, Migration, Regeneration, Satellite cells

Electrical pulse stimulation (EPS) is used to replicate motor neuron activation in muscle tissues, enabling in vitro studies of muscle contraction. However, both custom-built and commercial existing EPS systems often suffer from significant limitations, including limited scalability, high cost, and lack of flexibility for experimental adaptation. This work presents the Myo-MOVES platform, a practical solution for stimulating 3D skeletal muscle tissues. The device has been designed as an intuitive EPS system consisting of two main components: a selector and a stimulator that adapts to commercial 24-well culture plates. The Myo-MOVES selector enables targeted stimulation of single or multiple wells, while the stimulator delivers electrical signals via graphite electrodes to the plate containing 3D skeletal muscle samples. The Myo-MOVES platform was technically validated and employed as a proof of concept to investigate sarcolemmal damage induced by muscle contraction in Duchenne muscular dystrophy (DMD) 3D skeletal muscle tissues. Taking advantage of the versatility of the device, we validated Myo-MOVES through the assessment of force generation in DMD engineered muscle tissues and the detection of contraction-induced sarcolemmal damage via Evans blue dye uptake and the release of creatine kinase (CK), the gold standard marker of muscle damage. These findings demonstrate the feasibility of using Myo-MOVES to induce and study functionally relevant disease phenotypes in DMD 3D skeletal muscle tissues. These results highlight the system's potential as a valuable tool for future applications in the field of 3D skeletal muscle tissue engineering, including drug screening and the study of DMD therapies and other muscular diseases.

JTD

Ramón-Azcón, J.

JTD

Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disease diagnosed in childhood. It is a progressive and wasting disease, characterized by a degeneration of skeletal and cardiac muscles caused by the lack of dystrophin protein. The absence of this crucial structural protein leads to sarcolemmal fragility, resulting in muscle fiber damage during contraction. Despite ongoing efforts, there is no cure available for DMD patients. One of the primary challenges is the limited efficacy of current preclinical tools, which fail in modeling the biological complexity of the disease. Human-based three-dimensional (3D) cell culture methods appear as a novel approach to accelerate preclinical research by enhancing the reproduction of pathophysiological processes in skeletal muscle. In this work, we developed a patient-derived functional 3D skeletal muscle model of DMD that reproduces the sarcolemmal damage found in the native DMD muscle. These bioengineered skeletal muscle tissues exhibit contractile functionality, as they responded to electrical pulse stimulation. Sustained contractile regimes induced the loss of myotube integrity, mirroring the pathological myotube breakdown inherent in DMD due to sarcolemmal instability. Moreover, damaged DMD tissues showed disease functional phenotypes, such as tetanic fatigue. We also evaluated the therapeutic effect of utrophin upregulator drug candidates on the functionality of the skeletal muscle tissues, thus providing deeper insight into the real impact of these treatments. Overall, our findings underscore the potential of bioengineered 3D skeletal muscle technology to advance DMD research and facilitate the development of novel therapies for DMD and related neuromuscular disorders.

JTD Keywords: 3d cell culture, disease modeling, drug testing, duchenne muscular dystrophy, sarcolemmal damage, skeletal muscle, 3d cell culture, Animal-models, Disease modeling, Dmso, Drug testing, Duchenne muscular dystrophy, Gene, Humans, Image, Mechanisms, Muscle fibers, skeletal, Muscle, skeletal, Muscular dystrophy, duchenne, Myocardium, Sarcolemmal damage, Skeletal muscle, Tissue engineering, Utrophin

JTD Keywords: extracellular vesicles, microfluidic platforms, micrornas, obesity, plasma, rnas, Biomarkers, Mirnas

JTD Keywords: beta-cell function, organ-on-a-chip, pancreatic islets, plasmonic biosensors, responses, skeletal muscle, tissue engineering, Microfluidics, Type-2 diabetic-patients

JTD Keywords: Good health and well-being

Research into the development of therapeutic strategies is basedmainly on animal models and cell cultures. The ability to extrapolatedata from them is limited, and research on new drugs cannot beperformed efficiently. This is especially dramatic in rare diseases,which are intrinsically very heterogeneous. The generation of ad-vanced models using tissue engineering and patient-derived cellsallows fabricating new platforms for studying pathological processesand discovering new potential drugs. Here, we developed a patient-derived 3D functional skeletal muscle for Duchenne muscular dys-trophy (DMD). DMD is the most prevalent neuromuscular diseasediagnosed during childhood. The disease is characterized by pro-gressive degeneration of skeletal and cardiac muscle caused by thelack of dystrophin protein. Although there are several molecules indrug development for DMD, there is no treatment available for pa-tients to date. By using a 3D-printed casting mold, we encapsulatedpatient-derived myogenic precursor cells in a fibrin-composite ma-trix. This platform incorporated two flexible T-shaped pillars thatprovided continuous tension to the tissue, thus allowing the orien-tation of the muscle fibers. Our 3D muscle model expressed maturemuscle markers and responded to electric pulse stimulation (EPS).Besides, contraction dynamics between DMD and control tissueswere shown to be different. Moreover, an increase of damagemarkers after EPS was observed in DMD but not in healthy tissues.Finally, the tissues will be integrated into a microfluidic device tomonitor drug administration. Eventually, the microfluidic systemwill be connected to a biosensors system for the real-time detectionof biomarkers.

JTD Keywords: Casting, Contraction dynamics, Muscular dystrophy