Diaphragm weakness and proteomics (global and redox) modifications in heart failure with reduced ejection fraction in rats
Introduction
As the main inspiratory muscle, the diaphragm is essential in ventilatory and non-ventilatory behaviors [1]. Patients with heart failure who have reduced ejection fraction (HFrEF) show a decline in maximal inspiratory pressure, which is an indirect marker of diaphragm force/function [2,3]. Maximal inspiratory pressure is an independent predictor of prognosis in patients with HFrEF [4,5]. Patient data and direct measurement of diaphragm abnormalities in rodent models of HFrEF suggest that dysfunction of this respiratory muscle contributes to the pathophysiology of HFrEF [3]. In general, diaphragm dysfunction leads to impaired airway clearance and predisposition to pneumonia, inability to sustain ventilation and shallow breathing that limits gas exchange during physical activity, and reflex sympathetic activation, which vasoconstricts the periphery as well as exacerbates pathological cardiac remodeling [3,6]. These physiological changes worsen patient symptoms of dyspnea and discomfort, and can accelerate disease progression. Therefore, it is important to resolve the underlying mechanisms of diaphragm dysfunction and develop new therapies for patients with HFrEF.
Assuming a maximal voluntary effort and sufficient neuromuscular transmission, the decrease in maximal inspiratory pressure in patients is the result of diaphragm fiber atrophy and contractile dysfunction. In animal models of HFrEF, some studies support and others refute the existence of diaphragm atrophy or contractile dysfunction [[7], [8], [9], [10], [11], [12]]. The decline in maximal inspiratory pressure in patients depends on disease severity [3,13]. Thus, we have recently postulated that diaphragm atrophy or loss of force in animal models of HFrEF depends on disease severity [3].
The systemic factors leading to diaphragm atrophy and contractile dysfunction in HFrEF appear to involve neurohumoral and inflammatory signaling [3]. These signaling events culminate in accumulation of reactive oxygen species and an oxidized shift in the intracellular redox balance [14,15]. Knockout of oxidant-producing enzymes or systemic antioxidant interventions prevent diaphragm weakness in HFrEF [8,9,16], suggesting that oxidants alter abundance or function of proteins to cause loss of force. Oxidant-signaling activates proteolytic pathways and increases protein degradation [17]. One of the main mechanisms of oxidant-mediated cellular signaling is through reversible oxidation of cysteine residues or other thiol groups in proteins [18]. For instance, reversible cysteine oxidation of myofibrillar and calcium-handling proteins lowers skeletal muscle shortening and force [[19], [20], [21], [22]]. It is important to note, however, that the specific changes in protein abundance and thiol redox modifications of proteins associated with diaphragm abnormalities in HFrEF remain unknown.
The main goal of our study was to understand the physiological and molecular basis of diaphragm weakness in HFrEF. We tested the hypothesis that diaphragm atrophy and loss of force depend on severity of HFrEF. Overall, diaphragm abnormalities were more pronounced in severe HFrEF. Therefore, we performed global label free proteomics and differential cysteine labeling focused on diaphragm of rats with severe HFrEF and healthy controls to better understand the protein abundance and redox modifications associated with diaphragm weakness. Our general hypotheses were that severe HFrEF increased proteins of catabolic pathways, decreased abundance of myofibrillar proteins, and heightened reversibly oxidized proteins in the diaphragm. Our data partially supported our hypotheses and reveal novel candidate mechanisms of diaphragm atrophy and contractile dysfunction, which include decreased abundance of key components of anabolic pathways and increased reversibly oxidized thiol groups in thin-filament proteins.
Section snippets
Animals and surgical intervention
All procedures conformed to the guiding principles for use and care of laboratory animals of the American Physiological Society and were approved by the University of Florida Institutional Animal Care and Use Committee (IACUC 201607964). Adult male Sprague-Dawley rats (initially 8–10 weeks old) were used in the present study. Rats were housed at the University of Florida under 12 h:12 h light-dark cycle and had access to standard chow and water ad libitum. Rats were randomly assigned to undergo
Animals
The characteristics of Sham, mHFrEF, and sHFrEF rats used for measurements of diaphragm isometric function, fiber cross-sectional area, and proteomic analysis are shown in Table 1. Rats with mHFrEF and sHFrEF demonstrated reduced fractional shortening, which is a defining characteristic of HFrEF. As expected by design, infarcted area of the left ventricle and right ventricle hypertrophy were higher in sHFrEF compared to mHFrEF. Serum BNP, a biomarker of heart failure, was significantly elevated
Discussion
The main findings of our study were: 1) moderate and severe HFrEF caused diaphragm fiber atrophy; 2) maximal diaphragm specific force was diminished in severe HFrEF, but not moderate HFrEF; 3) severe HFrEF decreased abundance of proteins involved in ribosomal function and glucose and glycolytic metabolism; 4) redox proteomics in severe HFrEF showed heightened reversible cysteine oxidation of thin-filament proteins, filamin C, and SERCA1, and increased methionine oxidation in sarcomeric actin.
Summary and conclusions
Our study shows that diaphragm fiber atrophy occurs in moderate and severe HFrEF, and loss of diaphragm fiber force occurs in severe HFrEF. These factors are the most likely culprits in the progressive decline in inspiratory function in patients. Our global proteomic analysis revealed downregulation of ribosomal and glucose metabolism/glycolysis proteins that will contribute to fiber atrophy or anabolic resistance and further accelerate fatigue in severe HFrEF. The redox proteomics
Funding
This study was funded by National Institutes of Health (NIH) grants R01 HL130318-01 (L.F. Ferreira) and BREATHE T32 HL134621 (trainee: R.C. Kelley, principal investigator: Gordon S. Mitchell, University of Florida).
Disclosures
None.
Author contributions
Conception and design of the experiments: R.C.K., B.M., and L.F.F. Collection and analysis of data: R.C.K., B.M., B.B., G.A.W., R.V., and L.F.F. Interpretation of data: R.C.K., B.M., B.B., G.A.W., R.V., and L.F.F. Drafting the article: R.C.K., B.M., and L.F.F. Critical revision of article for intellectual content: R.C.K., B.M., B.B., G.A.W., R.V., and L.F.F. All authors read and approved the manuscript before submission. All persons designated as authors qualify for authorship, and all those
Acknowledgements
We would like to thank Philip Coblentz, Nikhil Patel, and Ravi Patel for technical assistance. A portion of this work was performed in the McKnight Brain Institute at the National High Magnetic Field Laboratory's AMRIS Facility, which is supported by National Science Foundation Cooperative Agreement No. DMR-1644779* and the State of Florida.
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