Fetal bovine serum contains biologically available ATP.

ATP is a ubiquitous extracellular messenger released in a wide number of pathophysiological conditions. ATP is known to be present in minute amounts in the extracellular space in healthy tissues and in the blood, and to modulate a multiplicity of cell responses. Cell culture systems are widely used to explore purinergic signaling. We show here that currently used fetal bovine sera contain ATP in the 300-1300 pmol/L range. Serum ATP is associated with albumin as well as with microparticle/microvesicle fraction. Serum microparticles/microvesicles affect in vitro cell responses due to their content of miRNAs, growth factors, and other bioactive molecules. ATP is likely to be one of these bioactive factors found in a variable amount in sera of different commercial sources. ATP in serum supports ATP-dependent biochemical reactions such as the hexokinase-dependent phosphorylation of glucose to glucose 6-phosphate, and affects purinergic signaling. These findings show that cells growing in vitro in serum-supplemented media are exposed to varying levels of extracellular ATP, and thus to varying degrees of purinergic stimulation.

Modeling Sarcoglycanopathy in Danio rerio.

Sarcoglycanopathies, also known as limb girdle muscular dystrophy 3-6, are rare muscular dystrophies characterized, although heterogeneous, by high disability, with patients often wheelchair-bound by late adolescence and frequently developing respiratory and cardiac problems. These diseases are currently incurable, emphasizing the importance of effective treatment strategies and the necessity of animal models for drug screening and therapeutic verification. Using the CRISPR/Cas9 genome editing technique, we generated and characterized δ-sarcoglycan and ß-sarcoglycan knockout zebrafish lines, which presented a progressive disease phenotype that worsened from a mild larval stage to distinct myopathic features in adulthood. By subjecting the knockout larvae to a viscous swimming medium, we were able to anticipate disease onset. The δ-SG knockout line was further exploited to demonstrate that a δ-SG missense mutant is a substrate for endoplasmic reticulum-associated degradation (ERAD), indicating premature degradation due to protein folding defects. In conclusion, our study underscores the utility of zebrafish in modeling sarcoglycanopathies through either gene knockout or future knock-in techniques. These novel zebrafish lines will not only enhance our understanding of the disease's pathogenic mechanisms, but will also serve as powerful tools for phenotype-based drug screening, ultimately contributing to the development of a cure for sarcoglycanopathies.

Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation.

Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling.