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DCRL RESEARCH: Molecular Mechanisms of Diaphragm Myopathy in Humans With Severe Heart Failure

Norman Mangner , Jens Garbade , Estelle Heyne , Marloes van den Berg, Ephraim B. Winzer , Jennifer Hommel ,
Marcus Sandri, Joanna Jozwiak-Nozdrzykowska, Anna L. Meyer, Sven Lehmann, Clara Schmitz, Edoardo Malfatti,
Michael Schwarzer, Coen A.C. Ottenheijm, T. Scott Bowen , Axel Linke,* Volker Adams*
RATIONALE: Diaphragm weakness impairs quality of life, exercise capacity, and survival in patients with chronic heart failure
(CHF) and reduced left ventricular ejection fraction. However, the underlying cellular mechanisms responsible in humans
remain poorly resolved.
OBJECTIVES: We prospectively evaluated clinical, functional (in vivo/in vitro), histological/ultrastructural, and molecular
alterations of the diaphragm from patients with CHF receiving a left ventricular assist device compared with patients without
CHF undergoing elective coronary bypass grafting (control) in the observational LIPAMUS-HF (Lipsia Diaphragm And
Muscle Heart Failure).
METHODS AND RESULTS: Participants (controls=21, CHF=18) underwent cardiopulmonary exercise and spirometry/respiratory
muscle testing alongside diaphragm and cardiac imaging. Diaphragm biopsies were phenotyped for mitochondrial respiration,
muscle fiber function, histology/ultrastructure, and protein expression. In vivo respiratory muscle function and diaphragm
thickness were reduced in CHF by 38% and 23%. Diaphragm biopsies revealed a fiber-type shift and severe fiber atrophy in
CHF alongside elevated proteasome-dependent proteolysis (ie, MuRF1 [muscle-specific RING finger protein 1] expression,
ubiquitination, ubiquitin-proteasome activity) and myofibrillar protein oxidation, which corresponded to upregulated Nox
(NADPH [nicotinamide adenine dinucleotide phosphate oxidase] oxidase; Nox2/Nox4) signaling. Mitochondria demonstrated
severe intrinsic functional and ultrastructural abnormalities in CHF characterized by accumulation of small mitochondria and
inhibited autophagy/mitophagy. Single muscle fiber contractile function revealed reduced Ca2+ sensitivity in CHF and there
was evidence of RyR1 (ryanodine receptor 1) dysfunction indicating Ca2+ leak from the sarcoplasmic reticulum. Mitochondrial
and Ca2+ measures corresponded to upregulated Nox4 isoform NADPH oxidase expression. Molecular markers correlated to
whole-body exercise intolerance and diaphragm dysfunction/wasting.
CONCLUSIONS: Patients with CHF demonstrate an obvious diaphragm myopathy independent of disuse or other confounding
factors, such as aging, obesity, or hypertension. Diaphragm weakness in CHF was associated with intracellular abnormalities
characterized by fiber atrophy, oxidative stress, mitochondrial dysfunction, impaired Ca2+ homeostasis, elevated proteasomedependent
proteolysis, but inhibited autophagy/mitophagy, which we speculate offers a novel therapeutic molecular target
regulated by a Nox-MuRF1/ubiquitin-proteasome-mitochondria-RyR1/Ca2+ signaling axis.
REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02663115.
GRAPHIC ABSTRACT: A graphic abstract is available for this article.
Key Words: diaphragm ◼ heart failure ◼ mitochondria ◼ mitophagy ◼ reactive oxygen species