7. SUMMARY.The long-term goal of this proposal is to gain detailed understanding of how the diaphragm the main muscleof respiration rapidly weakens in response to mechanical unloading and of the mechanisms whereby thegiant elastic protein titin influences this response.The diaphragm is a unique muscle in that it is constantly subjected to mechanical loading. Recent worksuggests that diaphragm strength is remarkably sensitive to mechanical unloading as occurs duringmechanical ventilation in the ICU. How unloading affects diaphragm strength is poorly understood. Increasingthis understanding is critically important: within hours diaphragm unloading during mechanical ventilationcauses diaphragm weakness in critically ill patients contributing to weaning failure. The search for themolecular triggers for the development of diaphragm weakness is ongoing. The potential role ofmechanosensor proteins that link unloading to protein turnover is under-explored but an exciting conceptthat needs to be studied. A candidate mechanosensor is titin a giant elastic protein that has been suggestedto sense mechanical stress and link this to trophic signalling pathways. This proposals aims to understandmechanosensing in the diaphragm in health and disease and the role of titin therein.Aim 1 will critically test how titin affects muscle trophicity. We will use unilateral diaphragm denervation(UDD). A property that can be observed during UDD is an initial hypertrophy response and we have shown thatthis hypertrophy of the denervated hemidiaphragm is caused by cyclic passive stretch of diaphragm fibers andthat titins elastic properties dictate the magnitude of the response. In this Aim we will identify the titin-basedsignalling pathways involved. Aim 2 determines the role of titins elasticity in PEEP ventilation-inducedlongitudinal diaphragm atrophy. This work builds on our recent finding that mechanical ventilation withPEEP which unloads the diaphragm at a shortened length causes longitudinal atrophy of fibers. Pilot datasuggest that titin-based mechanosensing modulates this response. To critically test the role of titin we willstudy the effect of PEEP ventilation on longitudinal atrophy in two titin KO mouse models: one with increasedtitin stiffness and one with lowered. In Aim 3 we will use unique diaphragm biopsies of critically illpatients to validate the findings from aim 1&2 and study whether titin-based mechanosensing contributes todiaphragm weakness. Up/downregulated titin binding proteins will be determined the significance of which istested in mouse models through genetic deletion.The innovation of this proposal lies in the novel research foci with innovative guiding hypotheses its innovativemouse models unique diaphragm biopsies from mechanically ventilated critically ill patients and its novelexperimental tools. The proposals integrative approach is expected to lead to a significant step forward inour understanding of diaphragm function and the role of titin therein.