MAPK/PKR Modulation of Diaphragm Weakness

The respiratory muscles of critically ill patients requiring mechanical ventilation are profoundly weak and infections are a major factor contributing to the development of this debility. The cellular mechanisms by which systemic infection induces respiratory muscle weakness, however, are poorly understood. In other tissues, the effects of infection are mediated by a variety of signaling pathways but virtually nothing is known about the signaling pathways activated in respiratory skeletal muscle in response to infection. The purpose of the experiments in this proposal is to examine this issue. These experiments will test our central hypothesis: p38, JNK, and PKR kinase pathways interact to regulate diaphragm muscle death receptor activation, death receptor active caspase 8 formation, and caspase mediated reductions in diaphragm force generation. Aim I studies will test the hypothesis that p38 and JNK modulate inflammation induced skeletal muscle caspase 8 activation and contractile dysfunction. Studies will be done using mice given endotoxin and C2C12 muscle cells exposed to cytokines. We will examine the time course of activation of p38 and JNK and will also determine if chemical or genetic inhibition of p38 and/or JNK prevents inflammation induced caspase 8 formation and diaphragm weakness. We will also determine if NADPH oxidase mediated superoxide generation causes skeletal muscle p38 and JNK activation in response to inflammation. Aim \I studies will test the hypothesis that PKR also contributes to inflammation induced skeletal muscle caspase 8 activation and weakness. We will examine the time time course of skeletal muscle PKR activation and will determine if chemical or genetic inhibition of PKR prevents inflammation induced skeletal muscle caspase 8 formation and weakness. We will also determine if PACT/RAX activation is responsible for activating PKR in skeletal muscle following inflammatory stimuli. Aim III studies will test the hypothesis that inflammation induces specific changes in skeletal muscle death receptor complex (DISC) characteristics that are linked to activation of the p38, JNK and PKR signaling pathways. We will both characterize the effects of inflammation on DISC formation, localization, and autocatalytic activity and determine if these alterations can be blocked by inhibition of p38, JNK, and/or PKR signaling pathways.