The research activity of the group is focused on the identification of new strategies to improve skeletal muscle regeneration. In more detail, the team is studying the role of electrical activity and local trophic factors in the microenvironment of satellite cell niche and in the post-mitotic myogenesis. Through the recombinant synthesis of extracellular matrix proteins, the roles of immobilized adhesion, proliferative and differentiative cues are evaluated. Skeletal muscle progenitors (satellite cells), murine cell line and immortalized human myoblasts are studied in vitro to identify, in a controlled environment, the pathways regulating the regenerative potential of the skeletal muscle. The experimental planning includes the use of electrical field stimulation, electrophysiological recordings, biochemical techniques, recombinant synthesis of matrix proteins, immunofluorescence and videoimaging.

The final aim is to discover new strategies to counteract the impaired functionality of the neuromuscular system due to ageing and diseases.   

Currently, four are the main research projects.

1) Interplay between adenosine and acetylcholine receptors

Adenosine is a well-known modulator of metabotropic and ionotropic neuroreceptors at the central and peripheral synapses. Adenosine receptors (ARs) control the cAMP/PKA cascade and cause the functional modulation of neuronal acetylcholine receptor (AChR) through its phosphorylation. Several types of adenosine receptors have been detected in developing and differentiated skeletal muscle cells, but little is known about the functional outcome of AR modulation on muscle AChRs.

In this project, the group intends to investigate the nature and the role of adenosine and AR signalling pathways on the two isoforms of muscle AChRs: the embryonic (g-AChR) regulating synaptogenesis and muscle development and the adult (e-AChR) with a role in nerve-muscle communication.

The results of the proposed research will advance the understanding of important aspects of the neuromuscular physiology such as the modulation of the neuromuscular transmission and the skeletal muscle plasticity. In this light, they will be useful for identification of new pharmacological tools to control the activity of muscle AChRs and, thus, for finding novel strategies for neuromuscular diseases associated with altered neuromuscular transmission.

Distribution of the AChR isoforms in skeletal muscle fibres

2) Role of neural agrin in skeletal muscle ageing

With ageing, skeletal muscle undergoes a severe reduction in tissue mass, leading to a decrease in strength (sarcopenia). A gradual decrease in the number of muscle fibres begins in the fifth decade, such that about 50% of skeletal muscle mass is lost by the ninth decade. Recent experimental evidences suggest that altered systemic and/or local trophic factors are crucial in the development of sarcopenia. The contribution of nerve-derived factors remains the less characterised. One of the trophic factors released by the nerve terminals is agrin. The group already discovered that neural agrin improves the differentiation of human muscle satellite cells. However, the successful outcome of muscle regeneration also depends on the satellite cell proliferation. Currently, the goal of the project is to explore the mitogenic potential of neural agrin. 

Demographic trend suggests that sarcopenia will soon reach epidemic proportions. The aged-related musculoskeletal impairment and the consequent reduction of independence and quality of life pose an outstanding social burden. The project fits within this frame as it aims at identifying molecular mechanisms able to foster the intrinsic regenerative potential of satellite cells, to prevent sarcopenia and/or counteract it in the elderly.

Figure modified from Lorenzon et al, 2004, Exp Gerontol 39:1545-1554

3) Electrical stimulation to improve skeletal muscle regeneration

Skeletal muscle produces reactive oxygen species (ROS) in an activity-dependent way. Interestingly, muscles undergoing massive exercise are characterized by an imbalance between the production of ROS and the antioxidant defenses.

The resulting oxidative stress is one of the proposed reasons for the well-known impaired activity of satellite cells upon extreme exercise. On the other hand, ROS are also important physiological signaling molecules. According to the hormetic effects, low concentrations of ROS are protective versus skeletal muscle progenitors causing muscle regeneration and maintaining muscle mass, whereas high concentrations cause damage.

Nowadays, field electrical stimulation provides an excellent new tool to study the role of ROS in skeletal muscle progenitors at the single cell level. The research team has recently developed a bioreactor producing electrical stimulation of muscle cells. Combining electrophysiological and biochemical techniques, the working hypothesis is to identify electrical activity patterns able to control and maintain ROS at “permissive” levels to preserve and/or foster the regenerative capacity of skeletal muscle.

The project aim at contributing to the future design of more efficient stimulation protocols and innovative electrical devices for rehabilitation of patients suffering from weakened or injured muscles.

Figure modified from Sciancalepore et al, 2012, Free Radic Biol Med 53:1392-1398

4) Extracellular matrix control of muscle formation: Human Elastin-Like Polypeptides as biomimetic substrates for regenerative myogenesis

The functional development of skeletal muscle is critically dependent upon the extracellular matrix (ECM) for spatial organization, structural support, and mechanical function. The linkage between ECM and the cell cytoskeleton, mediated by cell adhesion complexes, is essential to stabilize the muscle cell membrane under force transmission. The evidence that mutations altering the interplay between cells and ECM invariably lead to functional impairment and muscle waste, dramatically highlights the crucial role of these interactions.

Aiming at skeletal muscle regeneration strategies, the goal of mimicking the structure of ECM and its biological functions requires the design of artificial substrates that reproduce the properties and functionalities of natural tissues. A new generation of synthetic ECM proteins, the Human Elastin-Like Polypeptides (HELPs), have been developed in our lab. They consist of recombinant artificial polypeptides based on repetitive sequences found in human elastin. A key feature of the HELPs is their modular structure that allows the tailoring of the protein: the fusion of a bioactive domain of choice to the elastin-like backbone confers specific function to the recombinant protein.

When employed as adhesion substrates for C2C12 myoblasts, HELPs stimulate cell adhesion, spreading, proliferation, myogenic differentiation and the development of the excitation-contraction coupling mechanism. Intriguingly, all these processes are finely tuned by the polypeptide sequence.

On the whole, HELP polypeptides revealed suitable tools for investigating structure-functions relationships in skeletal muscle biology and prototypes of new biomaterials for skeletal muscle regeneration. The long-term goal of the project is to produce a synthetic ECM scaffold mimicking the biochemical and mechanical properties of the satellite cell microenvironment ultimately improving cell survival by counteracting the degenerative signals of aging and diseases.

Calcium imaging of C2C12 myoblasts during in vitro differentiation. Cells adhering to one of the HELP polypeptides show calcium transients induced by depolarization (K⁺ 60mM) and by caffeine (20mM). modified from D’Andrea et al, 2015, Biomaterials, 67:240-253