Summary Our 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 in mechanical loading rapidly affect diaphragm contractility, e.g., within hours of diaphragm unloading during mechanical ventilation (MV) in ICU patients, severe diaphragm weakness ensues, contributing to difficult ventilator weaning. The pathophysiology of diaphragm weakness is incompletely understood. In the proposal at hand, we will study the role of depressed interactions between myosin and actin, the two key contractile proteins in myofibers, and the role of titin in force depression. Our pilot data suggest that unloading of the diaphragm causes the myosin motors to adopt the so-called ‘super-relaxed state’ (SRX). Once in the SRX state, less myosin motors are available for binding to actin, and less force can be generated. We will also study the role of the giant protein titin in releasing myosin from the SRX state and investigate whether during MV-induced unloading of the diaphragm, this mechanism is perturbed. Aim 1 will determine the SRX state of myosin in the diaphragm of ventilated ICU patients. We will use our diaphragm biopsies of ICU patients to study the contractile force of diaphragm myofibers and determine the number of myosin motors that attach to actin during activation. We will combine mechanics with X-ray diffraction and biochemical assays to resolve with nanometer resolution the position of myosin motors relative to actin during activation and the proportion of myosin in the SRX state. In Aim 2 we will determine the role of posttranslational 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 determine the phospho-proteome, and we will establish whether there is a cause-and-effect relation between RLC phosphorylation and SRX by performing RLC exchange experiments into patients’ myofibers. To critically test whether changes in RLC are caused by diaphragm inactivity, we will ventilated healthy rats, and study RLC phosphorylation. Aim 3 will determine whether, in addition to RLC phosphorylation, direct mechanical effects of titin on myosin induce SRX. Titin-based passive tension strains the myosin filament which regulates the SRX state. We will study whether the reduction in titin-based passive tension, due to diaphragm unloading during MV, reduces the strain in the myosin filament and increases SRX. We will use mouse models with genetically engineered increased or decreased titin-based passive tension and study the effect of MV on the SRX state of myosin. The innovation of this proposal lies in the novel research foci and guiding hypotheses, unique diaphragm biopsies from patients, and its novel tools and mouse models, The proposal’s integrative approach is expected to lead to a m...