Bumsoo Ahn

Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration.

Skeletal muscle regeneration following injury is a highly coordinated process that involves transient muscle inflammation, removal of necrotic cellular debris and subsequent replacement of damaged myofibers through secondary myogenesis. However, the molecular mechanisms which coordinate these events are only beginning to be defined. In the current study we demonstrate that Heat shock protein 70 (Hsp70) is increased following muscle injury, and is necessary for the normal sequence of events following severe injury induced by cardiotoxin, and physiological injury induced by modified muscle use. Indeed, Hsp70 ablated mice showed a significantly delayed inflammatory response to muscle injury induced by cardiotoxin, with nearly undetected levels of both neutrophil and macrophage markers 24 hours post-injury. At later time points, Hsp70 ablated mice showed sustained muscle inflammation and necrosis, calcium deposition and impaired fiber regeneration that persisted several weeks post-injury. Through rescue experiments reintroducing Hsp70 intracellular expression plasmids into muscles of Hsp70 ablated mice either prior to injury or post-injury, we confirm that Hsp70 optimally promotes muscle regeneration when expressed during both the inflammatory phase that predominates in the first four days following severe injury and the regenerative phase that predominates thereafter. Additional rescue experiments reintroducing Hsp70 protein into the extracellular microenvironment of injured muscles at the onset of injury provides further evidence that Hsp70 released from damaged muscle may drive the early inflammatory response to injury. Importantly, following induction of physiological injury through muscle reloading following a period of muscle disuse, reduced inflammation in 3-day reloaded muscles of Hsp70 ablated mice was associated with preservation of myofibers, and increased muscle force production at later time points compared to WT. Collectively our findings indicate that depending on the nature and severity of muscle injury, therapeutics which differentially target both intracellular and extracellular localized Hsp70 may optimally preserve muscle tissue and promote muscle functional recovery.

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

Redox control of skeletal muscle atrophy.

Skeletal muscles comprise the largest organ system in the body and play an essential role in body movement, breathing, and glucose homeostasis. Skeletal muscle is also an important endocrine organ that contributes to the health of numerous body organs. Therefore, maintaining healthy skeletal muscles is important to support overall health of the body. Prolonged periods of muscle inactivity (e.g., bed rest or limb immobilization) or chronic inflammatory diseases (i.e., cancer, kidney failure, etc.) result in skeletal muscle atrophy. An excessive loss of muscle mass is associated with a poor prognosis in several diseases and significant muscle weakness impairs the quality of life. The skeletal muscle atrophy that occurs in response to inflammatory diseases or prolonged inactivity is often associated with both oxidative and nitrosative stress. In this report, we critically review the experimental evidence that provides support for a causative link between oxidants and muscle atrophy. More specifically, this review will debate the sources of oxidant production in skeletal muscle undergoing atrophy as well as provide a detailed discussion on how reactive oxygen species and reactive nitrogen species modulate the signaling pathways that regulate both protein synthesis and protein breakdown.

Cancer cachexia decreases specific force and accelerates fatigue in limb muscle

Cancer cachexia is a complex metabolic syndrome that is characterized by the loss of skeletal muscle mass and weakness, which compromises physical function, reduces quality of life, and ultimately can lead to mortality. Experimental models of cancer cachexia have recapitulated this skeletal muscle atrophy and consequent decline in muscle force generating capacity. However, more recently, we provided evidence that during severe cancer cachexia muscle weakness in the diaphragm muscle cannot be entirely accounted for by the muscle atrophy. This indicates that muscle weakness is not just a consequence of muscle atrophy but that there is also significant contractile dysfunction. The current study aimed to determine whether contractile dysfunction is also present in limb muscles during severe Colon-26 (C26) carcinoma cachexia by studying the glycolytic extensor digitorum longus (EDL) muscle and the oxidative soleus muscle, which has an activity pattern that more closely resembles the diaphragm. Severe C-26 cancer cachexia caused significant muscle fiber atrophy and a reduction in maximum absolute force in both the EDL and soleus muscles. However, normalization to muscle cross sectional area further demonstrated a 13% decrease in maximum isometric specific force in the EDL and an even greater decrease (17%) in maximum isometric specific force in the soleus. Time to peak tension and half relaxation time were also significantly slowed in both the EDL and the solei from C-26 mice compared to controls. Since, in addition to postural control, the oxidative soleus is also important for normal locomotion, we further performed a fatigue trial in the soleus and found that the decrease in relative force was greater and more rapid in solei from C-26 mice compared to controls. These data demonstrate that severe cancer cachexia causes profound muscle weakness that is not entirely explained by the muscle atrophy. In addition, cancer cachexia decreases the fatigue resistance of the soleus muscle, a postural muscle typically resistant to fatigue. Thus, specifically targeting contractile dysfunction represents an additional means to counter muscle weakness in cancer cachexia, in addition to targeting the prevention of muscle atrophy.

Diaphragm Atrophy and Contractile Dysfunction in a Murine Model of Pulmonary Hypertension

Pulmonary hypertension (PH) causes loss of body weight and inspiratory (diaphragm) muscle dysfunction. A model of PH induced by drug (monocrotaline, MCT) has been extensively used in mice to examine the etiology of PH. However, it is unclear if PH induced by MCT in mice reproduces the loss of body weight and diaphragm muscle dysfunction seen in patients. This is a pre-requisite for widespread use of mice to examine mechanisms of cachexia and diaphragm abnormalities in PH. Thus, we measured body and soleus muscle weight, food intake, and diaphragm contractile properties in mice after 6–8 weeks of saline (control) or MCT (600 mg/kg) injections. Body weight progressively decreased in PH mice, while food intake was similar in both groups. PH decreased (P<0.05) diaphragm maximal isometric specific force, maximal shortening velocity, and peak power. Protein carbonyls in whole-diaphragm lysates and the abundance of select myofibrillar proteins were unchanged by PH. Our findings show diaphragm isometric and isotonic contractile abnormalities in a murine model of PH induced by MCT. Overall, the murine model of PH elicited by MCT mimics loss of body weight and diaphragm muscle weakness reported in PH patients.

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.

Phrenic Nerve Stimulation Increases Human Diaphragm Fiber Force after Cardiothoracic Surgery

To the Editor: Mechanical ventilation (MV) causes diaphragm inactivity and unloading, and this quiescence results in ventilator-induced diaphragm dysfunction (VIDD). VIDD is characterized by oxidative stress, muscle atrophy, and decreases in diaphragm fiber force, as documented in both animal models and humans (1). VIDD is a major clinical problem because the ensuing decreased ability to develop inspiratory pressure will contribute to the difficulties in weaning patients from MV. During open-chest cardiothoracic surgery in humans, a rapid form of VIDD has been observed, with 20–30% depression of diaphragm force occurring within 2 hours of commencing MV (2). Extensive experiments establishing a cause-and-effect relationship for the mechanisms leading to VIDD in animals are available (reviewed in Reference 3), but interventions in humans are scant. Recently, intermittent phrenic nerve stimulation during MV has emerged as a potential VIDD countermeasure (4, 5). Specifically, hemidiaphragm stimulation during MV resulted in greater type 2 muscle fiber area in sheep (4), improved contractile properties in rats (6), and higher mitochondrial respiration rates in humans undergoing cardiothoracic surgery (5). In this preliminary study, we examined whether intermittent phrenic nerve stimulation during cardiothoracic surgery prevents diaphragm fiber contractile function impairment. We obtained diaphragm biopsies from four patients undergoing sternotomy for elective cardiothoracic surgical procedures (Table 1). The University of Florida Institutional Review Board approved the protocol, and participants gave written consent. Patients received nondepolarizing neuromuscular blockers during intubation, but none during surgery. The experimental intervention consisted of unilateral of phrenic nerve stimulation using an external cardiac pacemaker (Medtronic 5388; Medtronic, Minneapolis, MN) with temporary wire electrodes (AE Medical Corp., Farmingdale, NJ). The stimulation parameters and protocol were reported previously (5) and are given in Table 1. Briefly, stimulations were conducted every 30 minutes as soon as the phrenic nerve and diaphragm were exposed. The contralateral hemidiaphragm served as intrasubject control. There were no complications from the stimulations or biopsies. All participants were successfully extubated on the first attempt. Table 1. Patient Characteristics and Stimulation Parameters Muscle biopsies from each hemidiaphragm were either frozen immediately and later prepared for protein immunoblotting (see online supplement) or placed in an ice-cold solution containing low calcium and high ATP concentration (“relaxing” solution) (7), processed for chemical permeabilization (7), and stored at −20°C in relaxing solution (50% glycerol) until measurements of contractile properties (≤3 wk of surgery). We carefully isolated single fibers from diaphragm bundles in ice-cold relaxing solution and clamped the fibers to a force transducer and length controller for mechanical testing at 15°C, using calcium activation (7). We discarded fibers with sarcomere length greater than 2.65 μm under slack conditions on final mounting on the mechanics apparatus. This was the only exclusion criterion we adopted to avoid selectively studying “healthy” fibers. In our hands, slack sarcomere length greater than 2.65 μm is a sign of damage imposed during isolation of the fiber. We studied fibers from each hemidiaphragm (stimulated or control) in all subjects and determined fiber types using myosin heavy chain gel electrophoresis (8). Incidentally, the vast majority of fibers were type 2 (38 fibers); only four fibers were type 1 (three control, one stimulated). Hence, we report here only findings for type 2 fibers. These fibers account for approximately half of the diaphragm fiber composition (2). We analyzed the data from four patients with a varying number of fibers in each of the stimulated and control muscles, using a mixed effects model with random intercept for each individual and a fixed effect for group (stimulated or control), using the parametric bootstrap to assess statistical significance, as described by Faraway (9). This statistical approach takes into consideration the within-subject design in which different numbers of fibers are analyzed per subject in each condition. Specific force averaged 77 kN/m2 in control/inactive fibers compared with 100–150 kN/m2 reported in healthy active type 2 fibers [rodents (7), humans (2)]. Stimulated fibers elicited an approximately 30% greater specific force compared with the inactive fibers (P = 0.028; Figure 1). We found no differences between stimulated and control hemidiaphragms in other measures of isometric contractile properties (Figure 1). We also measured the abundance of protein carbonyls and ubiquitin conjugates (n = 3 patients), which, respectively, are general markers of oxidative damage and targeting for degradation by the ubiquitin-proteasome system. There was no difference between stimulated and control hemidiaphragms for protein carbonyls (control, 8.5 ± 2.8; stimulated, 9.2 ± 3.1) or ubiquitin conjugates (control, 2.3 ± 1.1; stimulated, 2.5 ± 1.0; Figures E1–E2 in the online supplement). Figure 1. Phrenic nerve stimulation increases diaphragm fiber force after cardiothoracic surgery. (A) Specific force versus pCa relationship for all fibers from control and stimulated hemidiaphragms. Specific force is absolute force (in kilonewtons) normalized ... Several mechanisms are involved in VIDD, including activation of proteolytic pathways, oxidative stress, impaired excitation–contraction coupling, and posttranslational modification of sarcomeric proteins. Our preliminary findings suggest that intermittent phrenic nerve stimulation preserves human diaphragm fiber contractile function, possibly by inhibiting proteolysis and/or posttranslational modification of sarcomeric proteins seen after MV (3, 10). However, the protection does not appear to be mediated by changes in protein carbonyls or ubiquitination. The resolution of specific cellular and molecular mechanisms will require a larger number of subjects and different analysis techniques. It is possible that indirect stretch imposed onto the control hemidiaphragm by stimulation of the treated side conferred some protection against VIDD (11). Therefore, our data may underestimate the benefits of intermittent phrenic nerve stimulation to diaphragm fiber function. However, our intrasubject design was sufficiently sensitive to detect significant differences in function that may be obscured by the large variability in an intersubject design resulting from factors such as age, sex, genetics, duration of surgery, and so on. We cannot determine whether a 30% increase in diaphragm fiber force is clinically relevant. Of note, an increase in inspiratory/diaphragm muscle strength elicited by inspiratory strength training is associated with greater weaning rate than standard care in chronic failure-to-wean patients (12). Phrenic nerve stimulation has been shown to increase the type 2 fiber cross-sectional area in sheep (4), mitochondrial respiration rates (5), and specific force in rats (6) and humans, as shown for the first time to our knowledge in the present study. These observations suggest that intermittent diaphragm activity during MV offers some protection against VIDD. Thus, intermittent diaphragm activity may ultimately prove to be a useful clinical strategy to prevent or attenuate VIDD in a subset of patients that has yet to be identified in larger trials.

Pharmacological targeting of mitochondrial reactive oxygen species counteracts diaphragm weakness in chronic heart failure

Diaphragm muscle weakness in chronic heart failure (CHF) is caused by elevated oxidants and exacerbates breathing abnormalities, exercise intolerance, and dyspnea. However, the specific source of oxidants that cause diaphragm weakness is unknown. We examined whether mitochondrial reactive oxygen species (ROS) cause diaphragm weakness in CHF by testing the hypothesis that CHF animals treated with a mitochondria-targeted antioxidant have normal diaphragm function. Rats underwent CHF or sham surgery. Eight weeks after surgeries, we administered a mitochondrial-targeted antioxidant (MitoTEMPO; 1 mg·kg(-1)·day(-1)) or sterile saline (Vehicle). Left ventricular dysfunction (echocardiography) pre- and posttreatment and morphological abnormalities were consistent with the presence of CHF. CHF elicited a threefold (P < 0.05) increase in diaphragm mitochondrial H2O2 emission, decreased diaphragm glutathione content by 23%, and also depressed twitch and maximal tetanic force by ∼20% in Vehicle-treated animals compared with Sham (P < 0.05 for all comparisons). Diaphragm mitochondrial H2O2 emission, glutathione content, and twitch and maximal tetanic force were normal in CHF animals receiving MitoTEMPO. Neither CHF nor MitoTEMPO altered the diaphragm protein levels of antioxidant enzymes: superoxide dismutases (CuZn-SOD or MnSOD), glutathione peroxidase, and catalase. In both Vehicle and MitoTEMPO groups, CHF elicited a ∼30% increase in cytochrome c oxidase activity, whereas there were no changes in citrate synthase activity. Our data suggest that elevated mitochondrial H2O2 emission causes diaphragm weakness in CHF. Moreover, changes in protein levels of antioxidant enzymes or mitochondrial content do not seem to mediate the increase in mitochondria H2O2 emission in CHF and protective effects of MitoTEMPO.

Diaphragm dysfunction caused by sphingomyelinase requires the p47phox subunit of NADPH oxidase

Sphingomyelinase (SMase) activity is elevated in inflammatory states and may contribute to muscle weakness in these conditions. Exogenous SMase depresses muscle force in an oxidant-dependent manner. However, the pathway stimulated by SMase that leads to muscle weakness is unclear. In non-muscle cells, SMase activates the Nox2 isoform of NADPH oxidase, which requires the p47(phox) subunit for enzyme function. We targeted p47(phox) genetically and pharmacologically (apocynin) to examine the role of NADPH oxidase on SMase-induced increase in oxidants and diaphragm weakness. SMase increased cytosolic oxidants (arbitrary units: control 203±15, SMase 276±22; P<0.05) and depressed maximal force in wild type mice (N/cm(2): control 20±1, SMase 16±0.6; P<0.05). However, p47(phox) deficient mice were protected from increased oxidants (arbitrary units: control 217±27, SMase 224±17) and loss of force elicited by SMase (N/cm(2): control 20±1, SMase 19±1). Apocynin appeared to partially prevent the decrease in force caused by SMase (n=3 mice/group). Thus, our study suggests that NADPH oxidase plays an important role on oxidant-mediated diaphragm weakness triggered by SMase. These observations provide further evidence that NADPH oxidase modulates skeletal muscle function.

Global proteome changes in the rat diaphragm induced by endurance exercise training

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in the rapid development of diaphragmatic atrophy and weakness. Importantly, endurance exercise training results in a diaphragmatic phenotype that is protected against ventilator-induced diaphragmatic atrophy and weakness. The mechanisms responsible for this exercise-induced protection against ventilator-induced diaphragmatic atrophy remain unknown. Therefore, to investigate exercise-induced changes in diaphragm muscle proteins, we compared the diaphragmatic proteome from sedentary and exercise-trained rats. Specifically, using label-free liquid chromatography-mass spectrometry, we performed a proteomics analysis of both soluble proteins and mitochondrial proteins isolated from diaphragm muscle. The total number of diaphragm proteins profiled in the soluble protein fraction and mitochondrial protein fraction were 813 and 732, respectively. Endurance exercise training significantly (P<0.05, FDR <10%) altered the abundance of 70 proteins in the soluble diaphragm proteome and 25 proteins of the mitochondrial proteome. In particular, key cytoprotective proteins that increased in relative abundance following exercise training included mitochondrial fission process 1 (Mtfp1; MTP18), 3-mercaptopyruvate sulfurtransferase (3MPST), microsomal glutathione S-transferase 3 (Mgst3; GST-III), and heat shock protein 70 kDa protein 1A/1B (HSP70). While these proteins are known to be cytoprotective in several cell types, the cyto-protective roles of these proteins have yet to be fully elucidated in diaphragm muscle fibers. Based upon these important findings, future experiments can now determine which of these diaphragmatic proteins are sufficient and/or required to promote exercise-induced protection against inactivity-induced muscle atrophy.