Effects of reactive oxygen species in skeletal muscle growth and development

Reactive oxygen species (ROS) generation is a ubiquitous phenomenon in eukaryotic cells. Increase of ROS inside the cells due to various internal and external stimuli such as disease, ionizing radiation, smoking, inflammatory cytokines, etc. leads to increased intracellular concentrations of free radicals. This leads to oxidative stress in the cells and is associated with changes in physiological conditions of the cell and can result in cell death. This effect can be observed in all cell types. In skeletal muscle cells generation of ROS is regulated primarily by NADPH oxidase-4 (Nox4 ) and partially by mitochondrial pathways and xanthine oxidase. Upregulation of these enzymes and pathways leads to increased concentrations of free radicals in muscle. Oxidative stress in skeletal muscle tissue can even lead to skeletal muscle atrophy. These consequences can result in reduced energy generation and misbalanced thermoregulation. The effects can result in imbalance of cellular homeostatis. Thus, dysregulation of ROS is a threat to normal health. However, recent research has revealed the importance of ROS in maintaining homeostasis of the body. Apart from their importance in host-pathogen interactions, their unique properties as second messengers in cell signaling pathways have attracted substantial research interest. It has been observed that free radicals generated inside the body trigger various cell cycle pathways such as the mitogen-activated protein kinase (MAPK) pathway that plays a major role in proliferation and differentiation of skeletal myoblasts. We evaluated the role of Nox4 generated ROS in a cell culture model of skeletal muscle, the mouse C2C12 cell line. Increased expression of Nox4 resulted in increased generation of ROS and vice versa. In induction of both increased and reduced expression of Nox4, the expression of myogenic markers (Myogenin, MyoD and MyfS) was reduced suggesting a reduced rate of differentiation. This regulation was found to be associated with reduced expression of phosphorylated ERK1/2; a signaling protein of the MAPK pathway. The effects were eminent on day 2 and day 4. However, by day 6, the induced changes in expression patterns of Nox4 were ameliorated. No difference in phosphorylated ERK1/2 was observed between treated and untreated conditions. These data suggest that there is a slow temporal cellular recovery from induced overexpression or knock-down of Nox4. Thus, we revealed the unique property of Nox4 in regulation of ROS and its importance in myogenesis.;We evaluated antioxidative properties of manniflavanone, a new member of the flavonoid group of antioxidants using a skeletal muscle cell culture model. Manniflavanone showed unique properties as an efficient scavenger of ROS with reduced expression of Nox4 in the C2C12 cell culture model. Also, manniflavanone was less cytotoxic compared to ascorbic acid and myricetin. Our study demonstrates the possible use of manniflavanone as a new scavenger of ROS and opens up doorways for its use as an effective antioxidant.;We also established a novel Nox4 siRNA delivery platform using gold nanoparticles in differentiated myotubes with high cellular uptake as compared to lipofectamine mediated transfection. Transfection of siRNA in differentiated myotubes has been problematic and more efficient approaches are needed. We constructed a nanoconjugate with an endoplasmic reticulum (ER) targeting peptide, containing a C-terminal KDEL motif and siRNA. In our experiments, we have demonstrated successful uptake of siRNA in differentiated myotubes with significant gene knock-down. This suggests KDEL gold nanoconjugates as a new platform for efficient delivery of siRNA inside differentiated myotubes.;In conclusion, these studies have identified the importance of Nox4 generated ROS in skeletal muscle cell culture, the pathway involved and developed novel techniques to manipulate Nox4 expression in vitro. Our studies implicate the importance in balance of ROS in the cellular environment. When cells are in stressed conditions due to environmental, physiological or metabolic imbalances, ROS concentration inside the cells fluctuates. There could be either increases or decreases in total ROS concentration. We demonstrated that in both cases, cellular homeostasis is disturbed resulting in inhibition of important myogenic regulatory factors. Therefore, care should be taken while using enzymatic or non-enzymatic antioxidants to study myogenic cellular homeostasis to avoid the negative effects on cellular physiology.