Reactive Oxygen Species and Respiratory Muscle Dysfunction in Heart Failure

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Abstract Diaphragm weakness is a significant health problem in patients with chronic heart failure and reduced ejection fraction (CHF) because it compromises ventilation and airway clearance and contributes to cardiac arrhythmias, peripheral vasoconstriction, and limb muscle fatigue. A mismatch in production and scavenging of reactive oxygen species (ROS), accompanied by oxidative modifications of myofibrillar proteins, is a critical determinant of loss of specific force (i.e., contractile dysfunction) in CHF. In this proposal, our goal is to identify the mechanisms which are causative in diaphragm redox imbalance and ROS-mediated contractile dysfunction in CHF. Based on our data collected during the previous funding cycle and preliminary studies, coupled with knowledge from the existing literature, we propose three specific aims to achievel our goal. In Aim 1, we will determine whether downregulation of a transcription factor, which is known to regulate several redox genes, in the diaphragm is sufficient and required for excess ROS and weakness in CHF. In Aim 2, we will define the role and mechanisms of protein oxidation on diaphragm weakness in CHF. Our preliminary data suggests that overexpression of enzymes that reverse oxidation of specific thiol groups prevents CHF-induced contractile dysfunction. We will expand on these studies to define the protective effects of these enzymes in CHF-induced diaphragm weakness and the cellular and molecular mechanisms involved in this process. Aim 3 will test oxidation of diaphragm α-actin (skeletal) as an essential molecular event in CHF-induced contractile dysfunction. Actin is particularly relevant because it contains amino acid residues that are highly sensitive to oxidation in regions critical for protein structure and function. We will use viral-mediated plasmid transduction to elicit overexpression and knockdown of the relevant genes. The general main dependent variables are diaphragm contractile properties in intact bundles and permeabilized single fibers, muscle ultrastructre, RNA- Sequencing, ROS emission and markers of redox balance, and methionine redox proteomics. The aims and experiments in our proposal will build an in-depth understanding of respiratory muscle biology, redox-mediated contractile dysfunction, and identify novel mechanisms and targets to treat diaphragm weakness in CHF.