SummaryOur long-term goal is to understand diaphragm contractility in health and disease and the role of titin therein.The diaphragm muscle drives respiration and is constantly subjected to mechanical loading. Changes inmechanical loading rapidly affect diaphragm contractility e.g. within hours of diaphragm unloading duringmechanical ventilation (MV) in ICU patients severe diaphragm weakness ensues contributing to difficultventilator weaning. The pathophysiology of diaphragm weakness is incompletely understood. In the proposal athand we will study the role of depressed interactions between myosin and actin the two key contractile proteinsin myofibers and the role of titin in force depression. Our pilot data suggest that unloading of the diaphragmcauses the myosin motors to adopt the so-called super-relaxed state (SRX). Once in the SRX state less myosinmotors are available for binding to actin and less force can be generated. We will also study the role of the giantprotein titin in releasing myosin from the SRX state and investigate whether during MV-induced unloading of thediaphragm this mechanism is perturbed.Aim 1 will determine the SRX state of myosin in the diaphragm of ventilated ICU patients. We will use ourdiaphragm biopsies of ICU patients to study the contractile force of diaphragm myofibers and determine thenumber of myosin motors that attach to actin during activation. We will combine mechanics with X-ray diffractionand biochemical assays to resolve with nanometer resolution the position of myosin motors relative to actinduring activation and the proportion of myosin in the SRX state. In Aim 2 we will determine the role ofposttranslational modifications in the SRX of myosin. Phosphorylation of regulatory light chains (RLC)regulates the SRX state of myosin and we will apply mass-spectrometry on the diaphragm biopsies to determinethe phospho-proteome and we will establish whether there is a cause-and-effect relation between RLCphosphorylation and SRX by performing RLC exchange experiments into patients myofibers. To critically testwhether changes in RLC are caused by diaphragm inactivity we will ventilated healthy rats and study RLCphosphorylation. Aim 3 will determine whether in addition to RLC phosphorylation direct mechanicaleffects of titin on myosin induce SRX. Titin-based passive tension strains the myosin filament which regulatesthe SRX state. We will study whether the reduction in titin-based passive tension due to diaphragm unloadingduring MV reduces the strain in the myosin filament and increases SRX. We will use mouse models withgenetically engineered increased or decreased titin-based passive tension and study the effect of MV on theSRX state of myosin.The innovation of this proposal lies in the novel research foci and guiding hypotheses unique diaphragm biopsiesfrom patients and its novel tools and mouse models The proposals integrative approach is expected to leadto a major step forward in our understanding of diaphragm function and titins role therein.