A, Left panel: ACE-2 mRNA in diaphragm specimens determined by quantitative polymerase chain reaction (qPCR) and normalized to housekeeping gene TBP. Right panel: α-ACE-2 antibody localization with fluoresceine microscopy on diaphragm cross-sections; the arrowheads show membrane and cytosolic localization (bar = 50 μm). B, Left panel: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA, determined by qPCR and normalized to housekeeping gene TBP, is detected in the diaphragm of 4 coronavirus disease 2019 (COVID-19)–intensive care unit (ICU) patients (patients 7, 9, 33, and 36). Right panel: in situ hybridization using RNAscope on patient #7 shows intramyofiber SARS-CoV-2 virus particles (red dots, indicated with arrowheads); a myofiber edge is highlighted with dashed line (bar = 30 μm). C, Left panels: representative images of picrosirius red-stained diaphragm cross-sections to highlight fibrosis; patients #22 and 3 are shown (bar = 100 μm). Right panel: quantification of the amount of fibrosis.
eTable 1. The demographic and clinical characteristics per patient
eTable 2. List of antibodies
eTable 3. List of primers
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Shi Z, de Vries HJ, Vlaar APJ, et al. Diaphragm Pathology in Critically Ill Patients With COVID-19 and Postmortem Findings From 3 Medical Centers. JAMA Intern Med. Published online November 16, 2020. doi:10.1001/jamainternmed.2020.6278
Extrapulmonary manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are now widely recognized and have important clinical implications.1,2 To our knowledge, the association of SARS-CoV-2 with the respiratory muscles has not been studied. This is surprising, as the respiratory muscles drive alveolar ventilation and their weakness results in acute respiratory failure. In critically ill patients undergoing ventilation, respiratory muscle weakness prolongs mechanical ventilation and increases mortality.3 The aim of this study was to investigate the association of severe coronavirus disease 2019 (COVID-19) with the respiratory muscles in critically ill patients and compare the findings with those obtained from non-COVID-19 critically ill patients.
Our study focused on the diaphragm, the main muscle of respiration. Consecutive diaphragm muscle specimens were collected during autopsy from the corpses of 26 patients who had been critically ill with COVID-19 in 3 academic medical centers in the Netherlands (referred to as COVID-19–intensive care unit [ICU]) in April and May 2020. As a control group, autopsy diaphragm specimens were collected from corpses of 8 patients who had been critically ill without COVID-19 (referred to as control-ICU). Specimens from the left midcostal diaphragm were used for analyses. Methodological details are described in the eMethods and eTables 2 and 3 in the Supplement. This study was approved by the medical ethical committee at Amsterdam UMC, and written informed consent was provided by the decedents’ next of kin. Data were analyzed using SPSS, version 22 (IBM), and visualized with GraphPad Prism, version 7.0 (GraphPad). Statistical significance was set at P < .05.
The median age of COVID-19–ICU patients was 71 years (interquartile range, 61-74 years), and 21 (81%) were men. Twenty-four patients (92.3%) received invasive mechanical ventilation for a median of 12 days (interquartile range, 6-25 days). The number of days receiving invasive mechanical ventilation and ICU length of stay were comparable between COVID-19–ICU and control-ICU patients. COVID-19–ICU patients had higher body mass index (calculated as weight in kilograms divided by height in meters squared) and were less likely to be treated with steroids (Table). No patients in either group had preexisting neuromuscular disease.
We report angiotensin-converting enzyme 2 (ACE-2) in the diaphragm of COVID-19–ICU and control-ICU patients (Figure, A). The ACE-2 predominantly localizes at the myofiber membrane (Figure, A), providing an entry point for SARS-CoV-2 to infect diaphragm myofibers. Evidence for SARS-CoV-2 viral RNA in the diaphragm was found in 4 patients (15.4%; Figure, B). Further analyses, for which we applied RNA in situ hybridization, indicated that viral RNA localized inside diaphragm myofibers (Figure, B). The RNA sequencing analyses showed that 315 genes were upregulated and 281 were downregulated in the diaphragm of COVID-19–ICU patients compared with control-ICU patients. Subsequent analyses of all upregulated and downregulated genes revealed activation of fibrosis pathways (fibroblast growth factor signaling). In line with these findings, epimysial and perimysial fibrosis was more than 2-fold higher in the diaphragms of COVID-19–ICU patients compared with control-ICU patients (Figure, C).
In this study, we provide unique evidence for ACE-2 expression in the human diaphragm and SARS-CoV-2 viral infiltration in the diaphragm of a subset of COVID-19–ICU patients. In COVID-19–ICU patients, we report increased expression of genes involved in fibrosis and histological evidence for the development of fibrosis in the diaphragm. This myopathic phenotype was distinctly different from that of control-ICU patients, with comparable duration of mechanical ventilation and ICU length of stay.4,5 It remains to be established whether diaphragm myopathy is a direct effect of SARS-CoV-2. Only 3 patients in the control-ICU group (37.5%) had viral lung disease, and the association of viral pneumonia with diaphragm muscles is unknown. We hypothesize that severe diaphragm myopathy associated with COVID-19, as described in this study, may lead to diaphragm weakness and might contribute to ventilator weaning failure, persistent dyspnea, and fatigue in patients with COVID-19 who survive their ICU stay.6
Accepted for Publication: September 10, 2020.
Published Online: November 16, 2020. doi:10.1001/jamainternmed.2020.6278
Corresponding Author: Coen Ottenheijm, PhD, Department of Physiology, Amsterdam UMC, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands (firstname.lastname@example.org).
Author Contributions: Dr Ottenheijm had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Concept and design: Shi, Boon, Heunks, Ottenheijm.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Shi, Heunks, Ottenheijm.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Shi, de Vries, Ottenheijm.
Obtained funding: Ottenheijm.
Administrative, technical, or material support: de Vries, Heunks, Ottenheijm.
Supervision: Vlaar, Boon, Heunks, Ottenheijm.
Conflict of Interest Disclosures: Dr de Vries reported grants from Amsterdam Cardiovascular Sciences during the conduct of the study and personal fees from a Dutch ultrasound center outside the submitted work. Dr Heunks reported personal fees from Getinge and grants from Liberate Medical outside the submitted work. No other disclosures were reported.
Dutch COVID-19 Diaphragm Investigators: Bernadette Schurink, MD, PhD, Eva Roos, MD, PhD, Hans W.M. Niessen, MD, PhD, Sylvia Bogaards, BSc, Stefan Conijn, BSc, Yeszamin L. Onderwater, MSc, Pedro Espinosa, MSc, Anke van Bergen, BSc, Diewertje I. Bink, MSc, Marloes van den Berg, MD (Amsterdam UMC, location VUMC),and Benno Kusters, MD, PhD (Radboudumc).
Funding/Support: The research reported in this work was supported by a grant from the National Institutes of Health/ Heart Lung and Blood Institute (R01HL121500 to Dr Ottenheijm).
Role of the Funder/Sponsor: The National Institutes of Health/ Heart Lung and Blood Institute had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
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