In a few weeks, a post-mortem collection of diaphragm and abdominal muscle samples will begin at the GHU Pitié-Salpêtrière-Charles Foix as part of the DIAVITAL protocol. The team coordinating the collection of samples (B. Riou, V. Justice) will organise the muscle biopsies, which will immediately be managed by the Institute of Myology’s 
MYOBANK-AFM tissue bank (M. Chapart*, S. Vasseur*) for researchers at the Myology Centre for Research (C. Coirault **, V. Mouly ***). The aim is to study diaphragmatic dysfunction related to mechanical ventilation (MV) during ageing.
The context
The consequences of mechanical ventilation on the diaphragm
Mechanical ventilation leads to rapid atrophy and diaphragm dysfunction, which makes its weaning difficult and increases the morbidity/mortality during reanimation and post-hospitalisation. This diaphragmatic dysfunction related to MV is frequent and therefore represents a major public health problem. In older patients, and particularly frail individuals such as those with myopathies, the impact is probably even greater.
A pilot study on the effect of maintaining diaphragmatic contraction during MV
A pilot study was conducted by T. Similowski (Head of the Department of Pneumology, Intensive Care and Reanimation at the Pitié-Salpêtrière) and C. Coirault, to determine whether maintenance of diaphragmatic contraction during MV made it possible to prevent diaphragm dysfunction. The study showed that electrical stimulation of the phrenic nerve (which innervates the diaphragm and is responsible for its contraction) prevented its atrophy. Diaphragm dysfunction is therefore directly related to its resting state.
The effects of ageing on the diaphragm
It is not known whether sarcopenia affects the diaphragm and other muscles in the same way. Indeed, most sarcopenia is related to a decrease in physical activity. Since the diaphragm is a muscle that contracts throughout life, one can hypothesise that it may be protected against sarcopenia. This very particular muscle has yet to be well studied, but embryologically, it has been shown to be derived from cell lines of the muscles of the head and neck, and not those of the trunk and legs. Furthermore, it benefits from a double vascularization – a network from the thorax and another from the abdomen.
The DIAVITAL protocol
A close collaboration between 3 teams: sampling, Myobank and researchers
The team from the Pitié-Salpêtrière will sample the various organs of individuals deceased while under MV. In the context of organ donation, the team will have obtained the families’ consent and will be able to collect samples of the diaphragm and abdominal muscles. For its part, the Myobank team will be on call 24/7 for this project, over 18 months to collect the tissues as soon as they have been sampled, and prepare them according to different protocols: cryopreservation, mounting in isopentane and rapid freezing in liquid nitrogen. The samples will then be stored at the Myobank within the Pitié-Salpêtrière Charles Foix biological resource platform. Samples will be available to researchers at the Institute of Myology.
Comparing the diaphragm and another muscle under sustained contraction
The idea behind this new DIAVITAL protocol is therefore to collect human diaphragmatic tissue from 7 young people (18-65 years old) and 7 elderly people (over 65 years old) to highlight specific mechanisms of sarcopenia in this muscle. A sample of the upper right abdominal muscle is also taken to determine the state of a non-rhythmic muscle, on which one would expect to observe markers of sarcopenia in the elderly.
Addressing key questions specific to the senescent diaphragm
There is very little data on this muscle. It is not known whether it is able to regenerate, if the number of satellite cells evolves with age, and how. Researchers have therefore planned to prepare cultures of diaphragm stem cells and observe their behaviour in vitro: this may allow a more precise interpretation of the myogenic capacity and the presence of epigenetic markers. It will be possible to follow muscle fibre atrophy in the diaphragm at the tissue level via histological studies, but also at the molecular level by analysing modifications and deregulation of its genes, methylation of epigenetic markers, etc.
Even though the researchers know for a fact that they will not be able to answer all of their questions, they plan to identify trends and specific research avenues for further studies.
*Maud Chapart and Stéphane Vasseur work at the Institute of Myology’s MYOBANK-AFM tissue bank
**Catherine Coirault leads the Physiopathology of Contractile Dysfunction group, Team 1 – Genetics, Physiopathology & Therapeutic Approaches to Muscle Diseases, at the Myology Centre for Research, Institute of Myology
***Vincent Mouly leads Team 3 – Regeneration, Physiopathology & Therapeutic Approaches: Cellular Models, at the Myology Centre for Research , Institute of Myology
