Adam W. Beharry

HDAC1 activates FoxO and is both sufficient and required for skeletal muscle atrophy

ABSTRACT The Forkhead box O (FoxO) transcription factors are activated, and necessary for the muscle atrophy, in several pathophysiological conditions, including muscle disuse and cancer cachexia. However, the mechanisms that lead to FoxO activation are not well defined. Recent data from our laboratory and others indicate that the activity of FoxO is repressed under basal conditions via reversible lysine acetylation, which becomes compromised during catabolic conditions. Therefore, we aimed to determine how histone deacetylase (HDAC) proteins contribute to activation of FoxO and induction of the muscle atrophy program. Through the use of various pharmacological inhibitors to block HDAC activity, we demonstrate that class I HDACs are key regulators of FoxO and the muscle-atrophy program during both nutrient deprivation and skeletal muscle disuse. Furthermore, we demonstrate, through the use of wild-type and dominant-negative HDAC1 expression plasmids, that HDAC1 is sufficient to activate FoxO and induce muscle fiber atrophy in vivo and is necessary for the atrophy of muscle fibers that is associated with muscle disuse. The ability of HDAC1 to cause muscle atrophy required its deacetylase activity and was linked to the induction of several atrophy genes by HDAC1, including atrogin-1, which required deacetylation of FoxO3a. Moreover, pharmacological inhibition of class I HDACs during muscle disuse, using MS-275, significantly attenuated both disuse muscle fiber atrophy and contractile dysfunction. Together, these data solidify the importance of class I HDACs in the muscle atrophy program and indicate that class I HDAC inhibitors are feasible countermeasures to impede muscle atrophy and weakness.

Diaphragm and ventilatory dysfunction during cancer cachexia

Cancer cachexia is characterized by a continuous loss of locomotor skeletal muscle mass, which causes profound muscle weakness. If this atrophy and weakness also occurs in diaphragm muscle, it could lead to respiratory failure, which is a major cause of death in patients with cancer. Thus, the purpose of the current study was to determine whether colon‐26 (C‐26) cancer cachexia causes diaphragm muscle fiber atrophy and weakness and compromises ventilation. All diaphragm muscle fiber types were significantly atrophied in C‐26 mice compared to controls, and the atrophy‐related genes, atrogin‐1 and MuRF1, significantly increased. Maximum isometric specific force of diaphragm strips, absolute maximal calcium activated force, and maximal specific calcium‐activated force of permeabilized diaphragm fibers were all significantly decreased in C‐26 mice compared to controls. Further, isotonic contractile properties of the diaphragm were affected to an even greater extent than isometric function. Ventilation measurements demonstrated that C‐26 mice have a significantly lower tidal volume compared to controls under basal conditions and, unlike control mice, an inability to increase breathing frequency, tidal volume, and, thus, minute ventilation in response to a respiratory challenge. These data demonstrate that C‐26 cancer cachexia causes profound respiratory muscle atrophy and weakness and ventilatory dysfunction.—Roberts, B. M., Ahn, B., Smuder, A. J., Al‐Rajhi, M., Gill, L. C., Beharry, A. W., Powers, S. K., Fuller, D. D., Ferreira, L. F., Judge, A. R. Diaphragm and ventilatory dysfunction during cancer cachexia. FASEB J. 27, 2600‐2610 (2013). www.fasebj.org

NAD(P)H oxidase subunit p47phox is elevated, and p47phox knockout prevents diaphragm contractile dysfunction in heart failure

Patients with chronic heart failure (CHF) have dyspnea and exercise intolerance, which are caused in part by diaphragm abnormalities. Oxidants impair diaphragm contractile function, and CHF increases diaphragm oxidants. However, the specific source of oxidants and its relevance to diaphragm abnormalities in CHF is unclear. The p47(phox)-dependent Nox2 isoform of NAD(P)H oxidase is a putative source of diaphragm oxidants. Thus, we conducted our study with the goal of determining the effects of CHF on the diaphragm levels of Nox2 complex subunits and test the hypothesis that p47(phox) knockout prevents diaphragm contractile dysfunction elicited by CHF. CHF caused a two- to sixfold increase (P < 0.05) in diaphragm mRNA and protein levels of several Nox2 subunits, with p47(phox) being upregulated and hyperphosphorylated. CHF increased diaphragm extracellular oxidant emission in wild-type but not p47(phox) knockout mice. Diaphragm isometric force, shortening velocity, and peak power were decreased by 20-50% in CHF wild-type mice (P < 0.05), whereas p47(phox) knockout mice were protected from impairments in diaphragm contractile function elicited by CHF. Our experiments show that p47(phox) is upregulated and involved in the increased oxidants and contractile dysfunction in CHF diaphragm. These findings suggest that a p47(phox)-dependent NAD(P)H oxidase mediates the increase in diaphragm oxidants and contractile dysfunction in CHF.

Transcriptional regulation of myotrophic actions by testosterone and trenbolone on androgen-responsive muscle

Androgens regulate body composition and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. Recently, we demonstrated that trenbolone (a potent synthetic testosterone analogue that is not a substrate for 5-alpha reductase or for aromatase) induces myotrophic effects in skeletal muscle without causing prostate enlargement, which is in contrast to the known prostate enlarging effects of testosterone. These previous results suggest that the 5α-reduction of testosterone is not required for myotrophic action. We now report differential gene expression in response to testosterone versus trenbolone in the highly androgen-sensitive levator ani/bulbocavernosus (LABC) muscle complex of the adult rat after 6weeks of orchiectomy (ORX), using real time PCR. The ORX-induced expression of atrogenes (Muscle RING-finger protein-1 [MuRF1] and atrogin-1) was suppressed by both androgens, with trenbolone producing a greater suppression of atrogin-1 mRNA compared to testosterone. Both androgens elevated expression of anabolic genes (insulin-like growth factor-1 and mechano-growth factor) after ORX. ORX-induced increases in expression of glucocorticoid receptor (GR) mRNA were suppressed by trenbolone treatment, but not testosterone. In ORX animals, testosterone promoted WNT1-inducible-signaling pathway protein 2 (WISP-2) gene expression while trenbolone did not. Testosterone and trenbolone equally enhanced muscle regeneration as shown by increases in LABC mass and in protein expression of embryonic myosin by western blotting. In addition, testosterone increased WISP-2 protein levels. Together, these findings identify specific mechanisms by which testosterone and trenbolone may regulate skeletal muscle maintenance and growth.

Identification of the Acetylation and Ubiquitin-Modified Proteome during the Progression of Skeletal Muscle Atrophy

Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the acetylation and ubiquitination status of these identified proteins regulates the muscle atrophy phenotype.

Differential expression of HDAC and HAT genes in atrophying skeletal muscle.

Introduction: Histone deacetylase (HDAC) proteins, which counter the activity of histone acetyltransferases (HATs), are necessary for normal muscle atrophy in response to several pathophysiological conditions. Despite this, it remains unknown whether a common or unique transcriptional profile of HDAC and HAT genes exist during the progression of muscle atrophy. Methods: Muscles were harvested from cast immobilized, denervated, or nutrient deprived animals for quantitative reverse transcriptase‐polymerase chain reaction analysis of HDAC and HAT gene expression. Results: The mRNA levels of Hdac2, Hdac4, Hdac6, Sirt1, p300, Cbp, and Pcaf increased, and Hdac7 decreased in skeletal muscle in each experimental model of muscle atrophy. Hdac1 and Hdac3 were increased only in cast immobilized and denervated muscles. Conclusions: While specific HDACs and HATs are increased in multiple models of muscle atrophy, increased expression of class I HDACs was unique to muscle disuse, reinforcing that specific HDAC inhibitors may be more effective than pan‐HDAC inhibitors at countering muscle atrophy. Muscle Nerve 52: 1098–1101, 2015

Diaphragm Abnormalities in Patients with End-Stage Heart Failure: NADPH Oxidase Upregulation and Protein Oxidation

Patients with heart failure (HF) have diaphragm abnormalities that contribute to disease morbidity and mortality. Studies in animals suggest that reactive oxygen species (ROS) cause diaphragm abnormalities in HF. However, the effects of HF on ROS sources, antioxidant enzymes, and protein oxidation in the diaphragm of humans is unknown. NAD(P)H oxidase, especially the Nox2 isoform, is an important source of ROS in the diaphragm. Our main hypothesis was that diaphragm from patients with HF have heightened Nox2 expression and p47phox phosphorylation (marker of enzyme activation) that is associated with elevated protein oxidation. We collected diaphragm biopsies from patients with HF and brain-dead organ donors (controls). Diaphragm mRNA levels of Nox2 subunits were increased 2.5–4.6-fold over controls (p < 0.05). Patients also had increased protein levels of Nox2 subunits (p47phox, p22phox, and p67phox) and total p47phox phosphorylation, while phospho-to-total p47phox levels were unchanged. The antioxidant enzyme catalase was increased in patients, whereas glutathione peroxidase and superoxide dismutases were unchanged. Among markers of protein oxidation, carbonyls were increased by ~40% (p < 0.05) and 4-hydroxynonenal and 3-nitrotyrosines were unchanged in patients with HF. Overall, our findings suggest that Nox2 is an important source of ROS in the diaphragm of patients with HF and increases in levels of antioxidant enzymes are not sufficient to maintain normal redox homeostasis. The net outcome is elevated diaphragm protein oxidation that has been shown to cause weakness in animals.

Janus kinase inhibition prevents cancer- and myocardial infarction-mediated diaphragm muscle weakness in mice

Respiratory dysfunction is prevalent in critically ill patients and can lead to adverse clinical outcomes, including respiratory failure and increased mortality. Respiratory muscles, which normally sustain respiration through inspiratory muscle contractions, become weakened during critical illness, and recent studies suggest that respiratory muscle weakness is related to systemic inflammation. Here, we investigate the pathophysiological role of the inflammatory JAK1/3 signaling pathway in diaphragm weakness in two distinct experimental models of critical illness. In the first experiment, mice received subcutaneous injections of PBS or C26 cancer cells and were fed chow formulated with or without the JAK1/3 inhibitor R548 for 26 days. Diaphragm specific force was significantly reduced in tumor-bearing mice receiving standard chow; however, treatment with the JAK1/3 inhibitor completely prevented diaphragm weakness. Diaphragm cross-sectional area was diminished by ∼25% in tumor-bearing mice but was similar to healthy mice in tumor-bearing animals treated with R548. In the second study, mice received sham surgery or coronary artery ligation, leading to myocardial infarction (MI), and were treated with R548 or vehicle 1 h postsurgery, and once daily for 3 days. Diaphragm specific force was comparable between sham surgery/vehicle, sham surgery/R548 and MI/R548 groups, but significantly decreased in the MI/vehicle group. Markers of oxidative damage and activated caspase-3, mechanisms previously identified to reduce muscle contractility, were not elevated in diaphragm extracts. These experiments implicate JAK1/3 signaling in cancer- and MI-mediated diaphragm weakness in mice, and provide a compelling case for further investigation.