Fellowship: Leonardo Ferreira: Intracellular Signaling of Cytokine Stimulated Protein Degradation in Skeletal Muscle

Patients with chronic heart failure (CHF) display elevated levels of circulating cytokines (TNF-alpha and interleukins) that trigger intracellular events resulting in cachexia, a debilitating loss of respiratory and limb muscle mass. Cardiac cachexia makes patients more susceptible to fatigue lessening the quality of life and is associated with increased mortality in CHF patients. Cardiac cachexia is thought to be caused, in part, by TNF-alpha and interleukins but the cellular mechanisms, post-receptor signaling and cytokine-sensitive genes, are poorly understood in skeletal muscle. Activation of the ubiquitin/proteasome pathway is the primary mechanism for protein degradation. In catabolic states, general activity of the ubiquitin/proteasome pathway is regulated at the transcriptional level by upregulation of ubiquitin, subunits of the proteasome complex, and muscle-specific E2 and E3 proteins. Importantly, circulating cytokines stimulate reactive oxygen species, nuclear factor kappa-B (NF-kappaB), and muscle atrophy. The current project is based on the general concept that specific catabolic cytokines (increased in CHF patients) act via muscle-derived reactive oxygen species to upregulate muscle-specific E2 and E3 proteins, thereby stimulating ubiquitin conjugation and protein loss. Specifically, our project uses C2C12 cell lines and primary mouse myotubes to address two specific aims: 1) To evaluate putative signaling events in cytokine-stimulated catabolic myocytes and 2) To evaluate MuRF1 (a muscle-specific E3 protein) as a target of cytokine-stimulated ROS/NF-kappaB signaling. The hypotheses being tested under specific aim 1 are: 1A) IL-1Beta stimulates TNF-alpha-sensitive signaling, ubiquitin conjugation, and protein loss; 1B) Mitochondrial ROS production is increased by IL-1Beta and TNF-alpha; 1C) IL-1Beta and TNF-alpha stimulate oxidant activity via iPLA2 (IL-1Beta) and cPLA2 (TNF-alpha)]. For specific aim 2 we hypothesize that: 2A) IL-1Beta and TNF-alpha stimulate MuRF1 expression via ROS-dependent signaling; 2B) NF-kappaB mediates cytokine and ROS effects on MuRF1; and 2C) MuRF1 contributes to cytokine-stimulated protein loss. Thus our research is expected to define signaling events and muscle-specific gene products by which cytokines induce skeletal muscle atrophy. This information is essential to expand our understanding of cytokine biology and will facilitate development of therapeutic strategies to treat cachexia in CHF patients.