Augmenting PGE2 as a Novel Strategy to Treat Ventilator-Induced Diaphragmatic Dysfunction (VIDD)

Background and Rationale: Though the ongoing COVID-19 pandemic affects all age groups, elderly patients (over 75) are much more likely to require mechanical ventilation (MV) upon contracting COVID-19. MV support can lead to progressive and often deadly diaphragmatic muscle atrophy and contractile dysfunction referred to as ventilator-induced diaphragmatic dysfunction (VIDD). VIDD risk increases proportionally to time spent on MV, and COVID-19 patients, who spend weeks to months on a ventilator, are therefore at particularly high risk. Currently there are no effective treatments for COVID-19-associated VIDD. During my SNF Early Postdoc.mobility fellowship, I discovered that short term treatment of skeletal muscle stem cells with the eicosanoid PGE2 enhances their long-term regenerative potential via epigenetic mechanisms. Normally, upon injury, elevated PGE2 signals via the EP4 receptor to augment the function of muscle stem cells (MuSCs) to repair injured myofibers. PGE2 is degraded by 15-hydroxyprostaglandin dehydrogenase (15-PGDH), can be inhibited by the small molecule noncompetitive inhibitor SW033291 (SW). Aged muscle expresses higher concentrations of 15-PGDH and consequently significantly lower levels of PGE2 than young muscle. We found that a systemic decrease in 15-PGDH activity via SW treatment increases PGE2 levels, attenuates skeletal muscle atrophy, and augments limb muscle strength in aged mice (Palla, Ravichandran, et al., Science, Accepted for publication with minor revisions). Diaphragm muscle is replete with MuSCs and express the PGE2 receptor, EP4, and the PGE2 degrading enzyme,15-PGDH, the target of SW. These data motivate my hypothesis that using SW to boost PGE2 levels in the diaphragm will augment the regenerative function of diaphragm MuSCs, rescue atrophy, and rebuild strength allowing COVID-19 survivors to surmount VIDD. Our preliminary data suggest that this approach will be particularly effective for the elderly.Objectives and Specific Aims: Although limb muscle MuSCs have been extensively characterized, studies of diaphragm MuSCs in mouse and human are lacking. My objective is to establish 15-PGDH inhibition as a therapeutic strategy to stimulate diaphragm regeneration and strength. My specific aims are to 1) demonstrate that PGE2 augments the proliferation and regenerative function of human diaphragm-derived muscle stem cells; 2) show that increasing PGE2 levels via 15-PGDH inhibition enhances strength and reduces diaphragm muscle atrophy via (i) augmenting MuSC proliferation and function and (ii) promoting hypertrophy and improving strength of mature myofibers; and 3) characterize the downstream mechanisms of PG2/EP4 mediated of atrophy and strength.Methods: This proposal represents an ongoing collaboration between the labs of Helen Blau, PhD, and Joseph Shrager, MD, both faculty at Stanford University. For my studies, I will obtain human diaphragm biopsies from our collaborator, Dr. Shrager, a cardiothoracic surgeon. To establish if PGE2 augments the regeneration and function of diaphragm MuSCs, I will transiently expose human diaphragm MuSCs isolated from biopsies from young (under 40) and elderly (over 70) patients to PGE2 and carry out assays of proliferation and function using (1) time-lapse imaging to track muscle stem cell proliferation, death and changes in cell fate, (2) non-invasive bioluminescence imaging assays to monitor the engraftment of these human diaphragm MuSCs in vivo into immunodeficient mice (3) non-invasive in vivo force measurements to assess muscle strength post-transplant. In addition to human diaphragm studies, I will employ a well-established 'Rodent ICU' model of VIDD using Fischer F344/BN hybrid rats (male and female; aged 4-6 months for young and >2 years for aged). The effect of SW on rescuing diaphragm atrophy will be measured by quantifying the diaphragm isometric specific contractile force in conjunction with histology. Molecular biology and biochemical assays (qPCR, immunohistochemistry, western blotting) will be employed to characterize the PGE2 mediated signaling mechanisms that counter diaphragm atrophy, and to assess age-associated differences in outcomes.Expected Results and Impact: This proposal aims to characterize a novel therapeutic strategy that will strengthen the diaphragm muscles and therefore reduce the duration of mechanical ventilation. This research has the potential to have a major clinical impact and benefit elderly patients most affected by prolonged ventilation due to pneumonia or COVID-19, aiding in weaning patients from respiratory support and substantially decreasing their mortality rates.