# Role of titin in the pathophysiology of diaphragm weakness during mechanical ventilation

> **NIH NIH R01** · UNIVERSITY OF ARIZONA · 2024 · $587,842

## Abstract

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...

## Key facts

- **NIH application ID:** 10898757
- **Project number:** 5R01HL121500-11
- **Recipient organization:** UNIVERSITY OF ARIZONA
- **Principal Investigator:** Coen Ottenheijm
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $587,842
- **Award type:** 5
- **Project period:** 2014-01-01 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10898757

## Citation

> US National Institutes of Health, RePORTER application 10898757, Role of titin in the pathophysiology of diaphragm weakness during mechanical ventilation (5R01HL121500-11). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10898757. Licensed CC0.

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