Measuring neuromuscular junction functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle. Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1)

A DIC based technique to measure the contraction of a skeletal muscle engineered tissue

Tissue engineering is a multidisciplinary science based on the application of engineering approaches to biologic tissue formation. Engineered tissue internal organization represents a key aspect to increase biofunctionality before transplant and, as regarding skeletal muscles, the potential of generating contractile forces is dependent on the internal fiber organization and is reflected by some macroscopic parameters, such as the spontaneous contraction. Here we propose the application of digital image correlation (DIC) as an independent tool for an accurate and noninvasive measurement of engineered muscle tissue spontaneous contraction. To validate the proposed technique we referred to the X-MET, a promising 3-dimensional model of skeletal muscle. The images acquired through a high speed camera were correlated with a custom-made algorithm and the longitudinal strain predictions were employed for measuring the spontaneous contraction. The spontaneous contraction reference values were obtained by studying the force response.The relative error between the spontaneous contraction frequencies computed in both ways was always lower than 0.15%. In conclusion, the use of a DIC based systemallows for an accurate and noninvasive measurement of biological tissues’ spontaneous contraction, in addition to the measurement of tissue strain field on any desired region of interest during electrical stimulation

Counteracting muscle wasting in aging and neuromuscular diseases: the critical role of IGF-1

Most muscle pathologies are characterized by the progressive loss of muscle tissue due to chronic degeneration combined with the inability of regeneration machinery to replace the damaged muscle. These pathological changes, known as muscle wasting, can be attributed to the activation of several proteolytic systems, such as calpain, ubiquitin-proteasome and caspases, and to the alteration in muscle growth factors. Among them, insulin-like growth factor-1 (IGF-1) has been implicated in the control of skeletal muscle growth, differentiation, survival, and regeneration and has been considered a promising therapeutic agent in staving off the advance of muscle weakness. Here we review the molecular basis of muscle wasting associated with diseases, such as sarcopenia, muscular dystrophy and Amyotrophic Lateral Sclerosis, and discuss the potential therapeutic role of local IGF-1 isoforms in muscle aging and diseases

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

A Principal component analysis to detect cancer cell line aggressiveness

In this paper, we propose the use of Principal Component Analysis (PCA) as a new post-processing method for the detection of breast and bone cancer cell lines cultured in vitro using a microwave biosensor. MDA-MB-231 and MCF7 breast cancer cell lines and SaOS-2 and 143B osteosarcoma cell lines were characterized using a circular patch resonator in the 1 MHz – 3 GHz frequency range. The return loss of each cancer cell line was analyzed, and the differences among each other were determined through Principal Component Analysis according to a protocol previously proposed mainly for electrocardiogram processing and X-ray photoelectron spectroscopy. Our results showed that the four cancer cell lines analyzed exhibited peculiar dielectric properties when compared to each other and the growth medium, confirming that PCA could be employed as an alternative methodology to analyze microwave characterization of cancer cell lines which, in turn, may be deeply exploited as a tool for the detection of cancer cells in healthy tissues

Development and validation of a device for in vitro uniaxial cell substrate deformation with real-time strain control

Substrate deformation affects the behavior of many cell types, as for example bone, skeletal muscle and endothelial cells. Nowadays, in vitro tests are widely employed to study the mechanotransduction induced by substrate deformation. The aim of in vitro systems is to properly reproduce the mechanical stimuli sensed by the tissue in the cellular microenvironment. An accurate strain measurement and control is therefore necessary to ensure the cell sensing the proper strain for the entire treatment. Different types of in vitro systems are commercially available or custom made designed; however, none of these devices performs a real-time measurement of the induced strains. In this study, we proposed a uniaxial strain device for in vitro cell stimulation with an innovative real-time strain control. The system was designed to induce sinusoidal waveform stimulation in a huge range of amplitude and frequency, to three silicone chambers stretched by a linear actuator. The real-time strain measurement and control algorithm is based on an optical tracking method implemented in LabView 2015, and it is able adapting the input amplitude to the linear motor, if necessary, hanging the stimulation signal for about 120 ms. A validation of the strain values measured during the real-time tracking algorithm was carried out through a comparison with digital image correlation (DIC) technique. We investigated the influence of number of reference points and image size on the algorithm accuracy. Experimental results showed that the tracking algorithm allowed for a real-time measurement of the membrane longitudinal strains with a relative error of 0.3%, on average, in comparison to the strains measured with DIC in post-processing analysis. We showed a high homogeneity of the strain pattern on the entire chamber base for different stimulation conditions. Finally, as proof of concept, we employed the uniaxial strain device to induce substrate deformation on human Osteosarcoma cell line (SaOS-2). Experimental results showed a consistent cells’ change in shape in response to the mechanical strain

Measuring the maximum power of an ex vivo engineered muscle tissue with isovelocity shortening technique

The final aim of muscle tissue engineering (TE) is to create a new tissue able to restore the functionality of impaired muscles once transplanted in the site of injury. Therefore, functional contractile properties close to that of healthy muscles are desirable to allow for a good compatibility and a proper functional contribution. Since skeletal muscles deal with locomotion during their normal activity, an accurate measurement of ex vivo muscle engineered tissues' isotonic properties is crucial. In this paper, we devised an experimental system to measure the mechanical power generated by an ex vivo muscle engineered tissue, the X-MET, based on the isovelocity contraction technique. The X-MET is developed without the use of any scaffolds, so that its mechanical properties are not affected by endogenous components. Our experiments allowed for delimiting the ranges of shortening and shortening velocity for which the tissue is able to generate and maintain power for the entire stimulation, which is the condition that better reproduces muscle physiological activity. Then, we measured the power generated by the X-MET and fit the experimental results to the Hill's equation usually employed for modeling the force-velocity relationship of skeletal muscles. The use of this model yielded to the measurement of maximum power and maximum shortening velocity. Results revealed that most of the isotonic properties were consistent with that proposed in the literature for slow-twitch muscles; in particular, the X-METs were able to generate a maximum power of 2.08± 0.78 W/kg and had a maximum shortening velocity of 1.84 ± 0.57 L₀/s, on average

Increased levels of interleukin-6 exacerbate the dystrophic phenotype in mdx mice

Duchenne muscular dystrophy (DMD) is characterized by progressive lethal muscle degeneration and chronic inflammatory response. The mdx mouse strain has served as the animal model for human DMD. However, while DMD patients undergo extensive necrosis, the affected muscles of adult mdx mice rapidly regenerates and regains structural and functional integrity. The basis for the mild effects observed in mice compared with the lethal consequences in humans remains unknown. In this study, we provide evidence that interleukin-6 (IL-6) is causally linked to the pathogenesis of muscular dystrophy. We report that forced expression of IL-6, in the adult mdx mice, recapitulates the severe phenotypic characteristics of DMD in humans. Increased levels of IL-6 exacerbate the dystrophic muscle phenotype, sustaining inflammatory response and repeated cycles of muscle degeneration and regeneration, leading to exhaustion of satellite cells. The mdx/IL6 mouse closely approximates the human disease and more faithfully recapitulates the disease progression in humans. This study promises to significantly advance our understanding of the pathogenic mechanisms that lead to DMD

Skeletal muscle myopenia in mice model of bile duct ligation and carbon tetrachloride-induced liver cirrhosis

Skeletal muscle myopathy is universal in cirrhotic patients, however, little is known about the main mechanisms involved. The study aims to investigate skeletal muscle morphological, histological, and functional modifications in experimental models of cirrhosis and the principal molecular pathways responsible for skeletal muscle myopathy. Cirrhosis was induced by bile duct ligation (BDL) and carbon tetrachloride (CCl4) administration in mice. Control animals (CTR) underwent bile duct exposure or vehicle administration only. At sacrifice, peripheral muscles were dissected and weighed. Contractile properties of extensor digitorum longus (EDL) were studied in vitro. Muscle samples were used for histological and molecular analysis. Quadriceps muscle histology revealed a significant reduction in cross-sectional area of muscle and muscle fibers in cirrhotic mice with respect to CTR. Kinetic properties of EDL in both BDL and CCl4 were reduced with respect to CTR; BDL mice also showed a reduction in muscle force and a decrease in the resistance to fatigue. Increase in myostatin expression associated with a decrease in AKT-mTOR expressions was observed in BDL mice, together with an increase in LC3 protein levels. Upregulation of the proinflammatory citochines TNF-a and IL6 and an increased expression of NF-kB and MuRF-1 were observed in CCl4 mice. In conclusion, skeletal muscle myopenia was present in experimental models of BDL and CCl4-induced cirrhosis. Moreover, reduction in protein synthesis and activation of protein degradation were the main mechanisms responsible for myopenia in BDL mice, while activation of ubiquitin-pathway through inflammatory cytokines seems to be the main potential mechanism involved in CCl4 mice

Modulation of Caspase Activity in Muscle Stem Cells Regulates Muscle Regeneration and Function

Muscle homeostasis involves de novo myogenesis, as observed in conditions of acute or chronic muscle damage. Tumor Necrosis Factor (TNF) triggers skeletal muscle wasting and inhibits muscle regeneration. We show that intramuscular treatment with the myogenic factor Arg8-vasopressin (AVP) enhanced skeletal muscle regeneration and rescued the inhibitory effects of TNF on regeneration. The functional analysis of regenerating muscle performance following TNF or AVP treatments revealed that the two factors had opposite effects on muscle force and fatigue, and that AVP rescued TNF negative effects on muscle performance. Muscle regeneration is, at least in part, regulated by caspase activation in PW1 Interstitial Cells (PICs). The participation of these CD34+ Sca-1+ PW1+ cells to muscle regeneration is hampered by TNF and rescued by AVP. The contrasting effects of AVP and TNF in vivo are recapitulated in cultured myogenic cells, which express both PW1, a caspase activator, and Hsp70, a caspase inhibitor. Hsp70 and PW1 co-immunoprecipitated and co-localized in muscle cells. In vivo Hsp70 expression was upregulated by AVP, and Hsp70 overexpression per se counteracted the TNF block of muscle regeneration. In summary, AVP counteracts TNF effects through a cross-talk at the level of Hsp70, a pivotal regulator of caspase activity in myogenic cells. Diminishing caspase activity is important for a prompt morphological and functional recovery following injury