Diaphragm weakness and proteomics (global and redox) modifications in heart failure with reduced ejection fraction in rats

Abstract

Inspiratory dysfunction occurs in patients with heart failure with reduced ejection fraction (HFrEF) in a manner that depends on disease severity and by mechanisms that are not fully understood. In the current study, we tested whether HFrEF effects on diaphragm (inspiratory muscle) depend on disease severity and examined putative mechanisms for diaphragm abnormalities via global and redox proteomics. We allocated male rats into Sham, moderate (mHFrEF), or severe HFrEF (sHFrEF) induced by myocardial infarction and examined the diaphragm muscle. Both mHFrEF and sHFrEF caused atrophy in type IIa and IIb/x fibers. Maximal and twitch specific forces (N/cm 2 ) were decreased by 19±10% and 28±13%, respectively, in sHFrEF (p < 0.05), but not in mHFrEF. Global proteomics revealed upregulation of sarcomeric proteins and downregulation of ribosomal and glucose metabolism proteins in sHFrEF. Redox proteomics showed that sHFrEF increased reversibly oxidized cysteine in cytoskeletal and thin filament proteins and methionine in skeletal muscle α-actin (range 0.5 to 3.3-fold; p < 0.05).In conclusion, fiber atrophy plus contractile dysfunction caused diaphragm weakness in HFrEF. Decreased ribosomal proteins and heighted reversible oxidation of protein thiols are candidate mechanisms for atrophy or anabolic resistance as well as loss of specific force in sHFrEF.

Skeletal muscle Nox4 knockout prevents and Nox2 knockout blunts loss of maximal diaphragm force in mice with heart failure with reduced ejection fraction

“…Ventricles were separated, weighed, and the left ventricle was cut down the interventricular septum from the base to the apex to determine infarct size by planimetry (Finsen et al ., 2005; Ahn et al ., 2015). Inspiratory dysfunction worsens with the severity of HFrEF in patients (Kelley & Ferreira, 2017) and diaphragm dysfunction is prevalent in rodents with severe HFrEF (Kelley et al ., 2020). For this reason, we restricted our HFrEF experimental groups to mice displaying severe HFrEF, defined as infarcted left ventricle area greater than 30% (Bayat et al ., 2002) and right ventricle weight/body weight ratio two standard deviations greater than sham group average (indicative of right ventricular hypertrophy secondary to pulmonary arterial hypertension) (Lindsey et al ., 2018; Kelley et al ., 2020).…”

“…The diaphragm is the primary inspiratory muscle, and dysfunction in this skeletal muscle contributes to the inspiratory dysfunction seen in HFrEF. Muscle biopsies from patients and extensive work in animal models suggest contractile dysfunction underpins diaphragm abnormalities in HFrEF, along with reports of muscle atrophy, fiber-type shifts, and altered mitochondrial function (Howell et al, 1995;Tikunov et al, 1997;Ahn et al, 2015;Laitano et al, 2016;Adams et al, 2019;Coblentz et al, 2019;Kelley et al, 2020;Mangner et al, 2021).…”

“…Reactive oxygen species (ROS) are key mediators of diaphragm dysfunction in HFrEF: increased markers of protein oxidation and intracellular redox shifts have been observed in the diaphragm of patients and animal models of HFrEF (Supinski & Callahan, 2005;Ahn et al, 2016;Laitano et al, 2016;Kelley et al, 2020;Mangner et al, 2021) but the source of excess ROS remains unknown. NAD(P)H oxidases (Nox), specifically the Nox2 and 4 isoforms, are important sources of ROS in skeletal muscle (Sakellariou et al, 2013;Ferreira & Laitano, 2016;Henríquez-Olguín et al, 2019).…”

“…The diaphragm is the primary inspiratory muscle, and dysfunction in this skeletal muscle contributes to the inspiratory dysfunction seen in HFrEF. Muscle biopsies from patients and extensive work in animal models suggest contractile dysfunction underpins diaphragm abnormalities in HFrEF, along with reports of muscle atrophy, fiber-type shifts, and altered mitochondrial function (Howell et al ., 1995; Tikunov et al ., 1997; Ahn et al ., 2015; Laitano et al ., 2016; Adams et al ., 2019; Coblentz et al ., 2019; Kelley et al ., 2020; Mangner et al ., 2021). Reactive oxygen species (ROS) are key mediators of diaphragm dysfunction in HFrEF: increased markers of protein oxidation and intracellular redox shifts have been observed in the diaphragm of patients and animal models of HFrEF (Supinski & Callahan, 2005; Ahn et al ., 2016; Laitano et al ., 2016; Kelley et al ., 2020; Mangner et al ., 2021) but the source of excess ROS remains unknown.…”

Cardiac and respiratory muscle responses to dietary N-acetylcysteine in rats consuming a high-saturated fat, high-sucrose diet

“…Diaphragm bundles were processed for immunohistochemical analyses of fiber cross-sectional area and myosin heavy chain isoforms as described previously. 57,61 Bundles embedded in Tissue-Tek OCT freezing medium were sliced into sections of 10 μm thickness at approximately -20ºC using a cryostat (Leica, CM 3050S We used semi-automatic muscle analysis using segmentation of histology (SMASH) code, run in MATLAB software, to quantify fiber type distribution and fiber crosssectional area for multiple images per rat. 62 We analyzed 269-737 diaphragm fibers per rat.…”