Rat Diaphragm Research Articles

Anti-inflammatory and metabolic effects of remote ischemic preconditioning and postconditioning on the contractile performance of the diaphragm in endotoxemic rats

BackgroundRespiratory failure is a major cause of mortality during septic shock and is due in part to the decreased ventilatory muscles contraction. These muscles depend mainly on fatty acid oxidation as an important source of ATP. We investigated the effects of remote ischemic preconditioning and postconditioning (RpostC) on the contractile functions of the diaphragm in relation to metabolic functions during systemic inflammation in lipopolysaccharide (LPS)-induced endotoxemia model.Materials and methodsA total of 24 adult male albino rats were divided equally into four groups: (i) the control group (group 1); (ii) LPS group (group 2), in which rats were treated with a single intraperitoneal injection of LPS (0.8 mg/kg intraperitoneal single dose); (iii) preconditioned group (group 3), in which remote ischemic preconditioning was induced with three ischemia/reperfusion cycles of the right hind limbs just before LPS injection; and (iv) postconditioned group (group 4), in which remote ischemic postconditioning was induced as in group 3 just after LPS injection. The animals were killed 5 days after the treatment. Among all groups, the contractility of the diaphragm was examined and the gene expressions of the carnitine palmitoyl transferase 1β (CPT-10β), the nuclear receptor peroxisome proliferator-activated receptor-α (PPARα), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and the nuclear factor erythroid 2-related factor 2 ( Nrf2) - the master of antioxidant response element - by real-time reverse transcription-PCR in the rat diaphragm tissue were determined; in addition, the serum levels of inflammatory markers, tumor necrosis factor α (TNFα) and interleukin-6, and lipids were measured.ResultsBoth types of remote ischemic, preconditioning and postconditioning, significantly improved the contractile performance of the diaphragm ( P < 0.001) compared with rats in the LPS group. These improvements were accompanied with significant elevation in the diaphragmatic expression of PPARα, PGC-1α, and CPT-1β, together with decreased serum lipids. In addition, there was a significant improvement in Nrf2 with a reduction in serum TNFα and interleukin-6.ConclusionOur results showed that the improvement of contractile performance of the diaphragm during sepsis was related to the improvement in the expression of nuclear hormone receptors involved in fat oxidation and modulation in oxidative stress and its consequences on inflammatory response. On the basis of these findings, we can suggest that early rational postconditioning interventions may be one of the promising strategies in the management of sepsis.

Therapeutic Approaches in Mitochondrial Dysfunction, Proteolysis, and Structural Alterations of Diaphragm and Gastrocnemius in Rats With Chronic Heart Failure.

Patients with chronic heart failure (CHF) experience exercise intolerance, fatigue and muscle wasting, which negatively influence their survival. We hypothesized that treatment with either the antioxidant N-acetyl cysteine (NAC) or the proteasome inhibitor bortezomib of rats with monocrotaline-induced CHF may restore inspiratory and limb muscle mass, function, and structure through several molecular mechanisms involved in protein breakdown and metabolism in the diaphragm and gastrocnemius. In these muscles of CHF-cachectic rats with and without treatment with NAC or bortezomib (N = 10/group) and non-cachectic controls, proteolysis (tyrosine release, proteasome activities, ubiquitin-proteasome markers), oxidative stress, inflammation, mitochondrial function, myosin, NF-κB transcriptional activity, muscle structural abnormalities, and fiber morphometry were analyzed together with muscle and cardiac functions. In diaphragm and gastrocnemius of CHF-cachectic rats, tyrosine release, proteasome activity, protein ubiquitination, atrogin-1, MURF-1, NF-κB activity, oxidative stress, inflammation, and structural abnormalities were increased, while muscle and cardiac functions, myosin content, slow- and fast-twitch fiber sizes, and mitochondrial activity were decreased. Concomitant treatment of CHF-cachectic rats with NAC or bortezomib improved protein catabolism, oxidative stress, inflammation, muscle fiber sizes, function and damage, superoxide dismutase and myosin levels, mitochondrial function (complex I, gastrocnemius), cardiac function and decreased NF-κB transcriptional activity in both muscles. Treatment of CHF-cachectic animals with NAC or bortezomib attenuated the functional (heart, muscles), biological, and structural alterations in muscles. Nonetheless, future studies conducted in actual clinical settings are warranted in order to assess the potential beneficial effects and safety concerns of these pharmacological agents on muscle mass loss and wasting in CHF-cachectic patients.

Orthotopic transplantation of a tissue engineered diaphragm in rats

The currently available surgical options to repair the diaphragm are associated with significant risks of defect recurrence, lack of growth potential and restored functionality. A tissue engineered diaphragm has the potential to improve surgical outcomes for patients with congenital or acquired disorders. Here we show that decellularized diaphragmatic tissue reseeded with bone marrow mesenchymal stromal cells (BM-MSCs) facilitates in situ regeneration of functional tissue. A novel bioreactor, using simultaneous perfusion and agitation, was used to rapidly decellularize rat diaphragms. The scaffolds retained architecture and mechanical properties and supported cell adhesion, proliferation and differentiation. Biocompatibility was further confirmed in vitro and in vivo. We replaced 80% of the left hemidiaphragm with reseeded diaphragmatic scaffolds. After three weeks, transplanted animals gained 32% weight, showed myography, spirometry parameters, and histological evaluations similar to native rats. In conclusion, our study suggested that reseeded decellularized diaphragmatic tissue appears to be a promising option for patients in need of diaphragmatic reconstruction.

Age-dependent action of reactive oxygen species on transmitter release in mammalian neuromuscular junctions

Reactive oxygen species (ROS) are implicated in aging, but the neurobiological mechanisms of ROS action are not fully understood. Using electrophysiological techniques and biochemical assays, we studied the age-dependent effect of hydrogen peroxide (H2O2) on acetylcholine release in rat diaphragm neuromuscular junctions. H2O2 significantly inhibited both spontaneous (measured as frequency of miniature end-plate potentials) and evoked (amplitude of end-plate potentials) transmitter release in adult rats. The inhibitory effect of H2O2 was much stronger in old rats, whereas in newborns tested during the first postnatal week, H2O2 did not affect spontaneous release from nerve endings and potentiated end-plate potentials. Proteinkinase C activation or intracellular Ca2+ elevation restored redox sensitivity of miniature end-plate potentials in newborns. The resistance of neonates to H2O2 inhibition was associated with higher catalase and glutathione peroxidase activities in skeletal muscle. In contrast, the activities of these enzymes were downregulated in old rats. Our data indicate that the vulnerability of transmitter release to oxidative damage strongly correlates with aging and might be used as an early indicator of senescence.

Mesenchymal expression of the FRAS1/FREM2 gene unit is decreased in the developing fetal diaphragm of nitrofen-induced congenital diaphragmatic hernia.

Developmental mutations that inhibit normal formation of extracellular matrix (ECM) in fetal diaphragms have been identified in congenital diaphragmatic hernia (CDH). FRAS1 and FRAS1-related extracellular matrix 2 (FREM2), which encode important ECM proteins, are secreted by mesenchymal cells during diaphragmatic development. The FRAS1/FREM2 gene unit has been shown to form a ternary complex with FREM1, which plays a crucial role during formation of human and rodent diaphragms. Furthermore, it has been demonstrated that the diaphragmatic expression of FREM1 is decreased in the nitrofen-induced CDH model. We hypothesized that FRAS1 and FREM2 expression is decreased in the developing diaphragms of fetal rats with nitrofen-induced CDH. Pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on D13, D15 and D18. Microdissected diaphragms were divided into nitrofen-exposed/CDH and control samples (n=12 per time-point and experimental group, respectively). Diaphragmatic gene expression levels of FRAS1 and FREM2 were analyzed by qRT-PCR. Immunofluorescence double staining for FRAS1 and FREM2 was combined with the mesenchymal marker GATA4 in order to evaluate protein expression and localization in pleuroperitoneal folds (PPFs) and fetal diaphragmatic tissue. Relative mRNA expression of FRAS1 and FREM2 were significantly reduced in PPFs of nitrofen-exposed fetuses on D13 (1.76±0.86 vs. 3.09±1.15; p<0.05 and 0.47±0.26 vs. 0.82±0.36; p<0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (1.45±0.80 vs. 2.63±0.84; p<0.05 and 0.41±0.16 vs. 1.02±0.49; p<0.05) and fully muscularized diaphragms of CDH fetuses on D18 (1.35±0.75 vs. 2.32±0.92; p<0.05 and 0.37±0.24 vs. 0.70±0.32; p<0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished FRAS1 and FREM2 immunofluorescence in diaphragmatic mesenchyme, which was associated with reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal CDH diaphragms on D13, D15 and D18 compared to controls. Decreased mesenchymal expression of FRAS1 and FREM2 in the nitrofen-induced CDH model may cause failure of the FRAS1/FREM2 gene unit to activate FREM1 signaling, disturbing the formation of diaphragmatic ECM and thus contributing to the development of diaphragmatic defects in CDH.

Beneficial effects of dantrolene on sepsis-induced diaphragmatic dysfunction are associated with downregulation of high-mobility group box 1 and calpain-caspase-3 proteolytic pathway

BackgroundIntracellular calcium overload is a major contributing factor to diaphragmatic dysfunction triggered by sepsis. In this study, the possible role of dantrolene, a ryanodine receptor inhibitor, in preventing the release of calcium from the sarcoplasmic reticulum in diaphragmatic dysfunction and weakness was explored. MethodsA middle-grade severity sepsis rat model was established for the effects of treatment with dantrolene, on diaphragm harvested 24 h after cecal ligation and puncture (CLP), and analyzed using functional, histologic, and biomarker assays. ResultsIt was found that in septic rats, treatment with dantrolene significantly improved the contractility, relaxation, and fatigue index of the diaphragm in a dose-dependent manner. The benefits are associated with improvement in ultrastructural changes of Z band integrity and myofilament arrangements along with increases both in the ratio of slow-twitch type composition. Moreover, dantrolene effectively inhibits the overexpression of high-mobility group box 1 and reduces the calpain-1-caspase-3 proteolytic activity. ConclusionsDantrolene can effectively attenuate the dysfunction of diaphragm in septic rats; Furthermore, the beneficial effects were associated with downregulation of high-mobility group box 1 and calpain-1-caspase-3 proteolytic activity.

Effects of bispyridinium non-oximes on nerve agent blocked neuromuscular transmission in rat diaphragm muscle and neuronal nicotinic receptors

Reactivation by bispyridinium mono-oximes (Hagedorn-oximes) and some classical oximes (0.03 or 1 mM) was studied in vitro of rat, bovine and human erythrocyte acetylcholinesterase and of electric eel acetylcholinesterase inhibited by soman. Relative reactivating potencies of the oximes are similar for the three inhibited erythrocyte enzymes. In general, Hagedorn-oximes are more potent than the classical oximes. Amont the Hagedorn-oximes, HI-6 is the most potent reactivator for the three inhibited enzymes. Relative reactivating potencies for the inhibited erythrocyte acetylcholinesterases and electric eel acetylcholinesterase, however, clearly differ.Since the reactivation experiments were carried out with racemic soman, a mixture of the two inhibited enzymes may be formed, which may cause additional problems in the comparison of various results. In order to get more detailed information on differences between human erythrocyte and electric eel acetylcholinesterase, reactivation of these enzymes inhibited with the P(−)-isomers of C(+)- and C(−)-soman were studied separately. Reactivation appeared to be dependent on the chirality of the α-carbon atom in the pinacolyl group. HI-6 is by far the most potent reactivator for the human enzyme inhibited by the two P(−)-isomers. It is suggested that electric eel acetylcholinesterase is not a reliable model for in vitro testing of therapeutic potencies of oximes against soman intoxication in mammals.Rate constants of aging of the four acetylcholinesterases inhibited with racemic soman and of the human and eel enzyme inhibited by the P(−)-isomers of C(+)- and C(−)-soman were also determined. The aging of the inhibited rat enzymes proceeds remarkably slowly (t12 = 21 min). The rate of aging is not affected by the chirality on the α-carbon atom in the pinacolyl group.Consequences of the present results are discussed in view of extrapolation of reactivation data of a series of reactivators to their relative therapeutic effect, ultimately in man. It is speculated that the more rapid aging of the human inhibited enzyme may hamper oxime-therapy in man more seriously than in rat.

Gene Expression of FRAS1-Related Extracellular Matrix 1 Is Decreased in Nitrofen-Induced Congenital Diaphragmatic Hernia.

The origin of congenital diaphragmatic hernia (CDH) is considered to lie in a malformation of the nonmuscular primordial diaphragm. It is known that fetal diaphragmatic development requires the structural integrity of its underlying mesenchymal tissue. Developmental mutations that inhibit the formation of normal diaphragmatic mesenchyme have been shown to cause CDH. FRAS1-related extracellular matrix 1 (FREM1) plays a critical role in the development of the fetal diaphragm. It has been demonstrated that a deficiency of FREM1 can lead to CDH both in humans and mice. Furthermore, FREM1-deficient fetuses exhibit a decreased level of mesenchymal cell proliferation in their developing diaphragms. We hypothesized that FREM1 expression is decreased in developing diaphragms of fetal rats with nitrofen-induced CDH. Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time-points D13, D15, and D18. Dissected diaphragms (n = 72) were divided into control and nitrofen-exposed samples (n = 12 per time-point and experimental group). Diaphragmatic gene expression levels of FREM1 were analyzed by quantitative polymerase chain reaction. Immunofluorescence staining for FREM1 was combined with the mesenchymal marker GATA4 to localize FREM1 protein expression and tissue distribution in fetal diaphragms. In nitrofen-exposed fetuses, relative mRNA expression of FREM1 was significantly reduced in pleuroperitoneal folds on D13 (0.30 ± 0.23 vs. 0.83 ± 0.19; p < 0.05), developing diaphragms on D15 (0.54 ± 0.22 vs. 1.19 ± 0.28; p < .05) and fully muscularized diaphragms on D18 (0.49 ± 0.37 vs. 0.97 ± 0.53; p < 0.05) in comparison with controls. Confocal laser scanning microscopy revealed markedly diminished diaphragmatic FREM1 immunofluorescence, which was associated with reduced proliferation of diaphragmatic mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared to controls. Decreased expression of FREM1 in the nitrofen-induced CDH model may disturb the formation of the diaphragmatic mesenchyme, thus contributing to the development of diaphragmatic defects.

Partial Support Ventilation and Mitochondrial-Targeted Antioxidants Protect against Ventilator-Induced Decreases in Diaphragm Muscle Protein Synthesis.

Mechanical ventilation (MV) is a life-saving intervention in patients in respiratory failure. Unfortunately, prolonged MV results in the rapid development of diaphragm atrophy and weakness. MV-induced diaphragmatic weakness is significant because inspiratory muscle dysfunction is a risk factor for problematic weaning from MV. Therefore, developing a clinical intervention to prevent MV-induced diaphragm atrophy is important. In this regard, MV-induced diaphragmatic atrophy occurs due to both increased proteolysis and decreased protein synthesis. While efforts to impede MV-induced increased proteolysis in the diaphragm are well-documented, only one study has investigated methods of preserving diaphragmatic protein synthesis during prolonged MV. Therefore, we evaluated the efficacy of two therapeutic interventions that, conceptually, have the potential to sustain protein synthesis in the rat diaphragm during prolonged MV. Specifically, these experiments were designed to: 1) determine if partial-support MV will protect against the decrease in diaphragmatic protein synthesis that occurs during prolonged full-support MV; and 2) establish if treatment with a mitochondrial-targeted antioxidant will maintain diaphragm protein synthesis during full-support MV. Compared to spontaneously breathing animals, full support MV resulted in a significant decline in diaphragmatic protein synthesis during 12 hours of MV. In contrast, diaphragm protein synthesis rates were maintained during partial support MV at levels comparable to spontaneous breathing animals. Further, treatment of animals with a mitochondrial-targeted antioxidant prevented oxidative stress during full support MV and maintained diaphragm protein synthesis at the level of spontaneous breathing animals. We conclude that treatment with mitochondrial-targeted antioxidants or the use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during prolonged MV.

Role of quinidine therapy after dimethoate acute intoxication in the rat diaphragm

Background Accidental and occupational acute intoxication by dimethoate (an organophosphorous compound) has gained attention in developing countries. The antiarrhythmic drug quinidine is known to decrease the conductivity and irritability of the myocardium. Aim of the work The current work studied the role of quinidine therapy in the biochemical and ultrastructural changes on the diaphragm, at the neuromuscular junction, of male albino rats following acute dimethoate intoxication. Materials and methods Fifty adult male Wistar albino rats were divided equally into five groups. Groups I and II served as the control group; group III received 200 mg/kg body weight (BW) of quinidine; group IV received oral dimethoate (300 mg/kg BW); and in group V the dimethoate-intoxicated animals were treated with 200 mg/kg BW of quinidine. Serum creatine kinase and lactate dehydrogenase were measured and assessed statistically. The left hemidiaphragms were processed for transmission electron microscopic study. Results The study of the diaphragm after treatment of the dimethoate-intoxicated group with quinidine showed residual foci of subsarcolemmal aggregation of the mitochondria. The sarcomere banding, sarcoplasmic reticulum, T tubules, and Z lines were nearly similar to control sections. In addition, mitochondria were observed around the Z lines, compared with untreated intoxicated rats. The electron density of the postsynaptic membrane at the junctional folds had an uneven appearance. The increased serum creatine kinase and lactate dehydrogenase of the intoxicated group decreased significantly after quinidine therapy. Conclusion The current results suggest that quinidine had a partial protective effect on the acute toxicity from dimethoate on the rat diaphragmatic ultrastructure. Thus, early administration of quinidine is beneficial for guarding against the respiratory failure following acute organophosphorus insecticide poisoning.

The effects of buthionine sulfoximine treatment on diaphragm contractility and SERCA pump function in adult and middle aged rats.

This study examined the effects of 10 days of buthionine sulfoximine (BSO) treatment on in vitro contractility and sarcoplasmic reticulum calcium pump (SERCA) expression and function in adult (AD; 6–8 months old) and middle aged (MA; 14–17 months old) rat diaphragm in both the basal state and following fatiguing stimulation. BSO treatment reduced the cellular concentrations of free glutathione (GSH) by >95% and oxidized glutathione (GSSG) by >80% in both age cohorts. GSH content in AD Control diaphragm was 32% higher (P < 0.01) than in MA Control, with no differences in GSSG. The ratio of GSH:GSSG, an indicator of cellular oxidative state, was 34.6 ± 7.4 in MA Control, 52.5 ± 10.1 in AD Control, 10.6 ± 1.7 in MA BSO, and 9.5 ± 1.1 in AD BSO (BSO vs. Control, P < 0.05). Several findings suggest that the effects of BSO treatment are age dependent. AD BSO diaphragm had 26% higher twitch and 28% higher tetanic force (both P < 0.05) than AD Controls, whereas no significant difference existed between the two MA groups. In contrast to our previous work on BSO-treated AD rats, BSO treatment did not influence maximal SERCA ATPase activity in MA rat diaphragm, nor did SERCA2a expression increase in BSO-treated MA diaphragm. Biotinylated iodoacetamide binding to SERCA1a, a specific marker of free cysteine residues, was reduced by 35% (P < 0.05) in AD Control diaphragm following fatiguing stimulation, but was not reduced in any other group. Collectively, these results suggest an important role for redox regulation in both contractility and SERCA function which is influenced by aging.

High levels of positive end-expiratory pressure preserve diaphragmatic contractility during acute respiratory distress syndrome in rats.

What is the central question of this study? Higher levels of positive end-expiratory pressure (PEEP) have recently been used in patients with acute respiratory distress syndrome (ARDS). In normal physiological conditions, the ability of the diaphragm to generate pressure is reduced when the lung volume is elevated beyond its functional residual capacity. It is unknown whether higher levels of PEEP will have a negative impact on diaphragmatic contraction in the presence of the pathophysiology of ARDS. What is the main finding and its importance? Mechanical ventilation with higher levels of PEEP reduced lung injury, improved diaphragmatic contractility and increased the expression of both dihydropyridine receptor and ryanodine receptor in the diaphragms of rats with ARDS. Higher levels of positive end-expiratory pressure (PEEP) have recently been used in patients with acute respiratory distress syndrome (ARDS). In normal physiological conditions, the ability of the diaphragm to generate pressure is reduced when the lung volume is elevated beyond its functional residual capacity. Thus, it is critical to understand whether higher levels of PEEP will have a negative impact on diaphragmatic contraction in the presence of the pathophysiology of ARDS. This study was designed to determine whether higher levels of PEEP reduce diaphragmatic contractility in a rat model of ARDS generated using i.p. lipopolysaccharide. Forty rats were randomly assigned to the following five groups: a control group with no special treatment; an ARDS group with no mechanical ventilation; and three ARDS groups with mechanical ventilation with PEEP at 0, 5 or 10 cmH2 O, respectively. We found that mechanical ventilation with PEEP reduced lung injury, improved diaphragmatic contractility and increased the expression of both dihydropyridine receptor and ryanodine receptor in the diaphragms of rats with ARDS. These changes were most significant at a PEEP of 10 cmH2 O among all applied levels of PEEP. In conclusion, using a rat ARDS model, this study confirmed that diaphragmatic contractility was preserved by mechanical ventilation with high levels of PEEP.

Exercise training causes differential changes in gene expression in diaphragm arteries and 2A arterioles of obese rats.

We employed next-generation, transcriptome-wide RNA sequencing (RNA-Seq) technology to assess the effects of two different exercise training protocols on transcriptional profiles in diaphragm second-order arterioles (D2a) and in the diaphragm feed artery (DFA) from Otsuka Long Evans Tokushima Fatty (OLETF) rats. Arterioles were isolated from the diaphragm of OLETF rats that underwent an endurance exercise training program (EX; n = 13), interval sprint training program (SPRINT; n = 14), or remained sedentary (Sed; n = 12). Our hypothesis was that exercise training would have similar effects on gene expression in the diaphragm and soleus muscle arterioles because diaphragm blood flow increases during exercise to a similar extent as in soleus. Results reveal that several canonical pathways that were significantly altered by exercise in limb skeletal muscles were not among the pathways significantly changed in the diaphragm arterioles including actin cytoskeleton signaling, role of NFAT in regulation of immune response, protein kinase A signaling, and protein ubiquitination pathway. EX training altered the expression of a smaller number of genes than did SPRINT in the DFA but induced a larger number of genes with altered expression in the D2a than did SPRINT. In fact, FDR differential expression analysis (FDR, 10%) indicated that only two genes exhibited altered expression in D2a of SPRINT rats. Very few of the genes that exhibited altered expression in the DFA or D2a were also altered in limb muscle arterioles. Finally, results indicate that the 2a arterioles of soleus muscle (S2a) from endurance-trained animals and the DFA of SPRINT animals exhibited the largest number of genes with altered expression.

Inhibition of forkhead boxO-specific transcription prevents mechanical ventilation-induced diaphragm dysfunction.

Mechanical ventilation is a lifesaving measure for patients with respiratory failure. However, prolonged mechanical ventilation results in diaphragm weakness, which contributes to problems in weaning from the ventilator. Therefore, identifying the signaling pathways responsible for mechanical ventilation-induced diaphragm weakness is essential to developing effective countermeasures to combat this important problem. In this regard, the forkhead boxO family of transcription factors is activated in the diaphragm during mechanical ventilation, and forkhead boxO-specific transcription can lead to enhanced proteolysis and muscle protein breakdown. Currently, the role that forkhead boxO activation plays in the development of mechanical ventilation-induced diaphragm weakness remains unknown. This study tested the hypothesis that mechanical ventilation-induced increases in forkhead boxO signaling contribute to ventilator-induced diaphragm weakness. University research laboratory. Young adult female Sprague-Dawley rats. Cause and effect was determined by inhibiting the activation of forkhead boxO in the rat diaphragm through the use of a dominant-negative forkhead boxO adeno-associated virus vector delivered directly to the diaphragm. Our results demonstrate that prolonged (12 hr) mechanical ventilation results in a significant decrease in both diaphragm muscle fiber size and diaphragm-specific force production. However, mechanically ventilated animals treated with dominant-negative forkhead boxO showed a significant attenuation of both diaphragm atrophy and contractile dysfunction. In addition, inhibiting forkhead boxO transcription attenuated the mechanical ventilation-induced activation of the ubiquitin-proteasome system, the autophagy/lysosomal system, and caspase-3. Forkhead boxO is necessary for the activation of key proteolytic systems essential for mechanical ventilation-induced diaphragm atrophy and contractile dysfunction. Collectively, these results suggest that targeting forkhead boxO transcription could be a key therapeutic target to combat ventilator-induced diaphragm dysfunction.

Adaptation of the Rat Diaphragm Proteome in Response to Endurance Exercise Training

Mechanical ventilation is a lifesaving intervention for patients in respiratory failure. However, this leads to diaphragmatic atrophy and contractile dysfunction, termed ventilator-induced diaphragm dysfunction (VIDD). We recently discovered that endurance exercise training (ET) results in a diaphragmatic phenotype that resists VIDD. However, the cellular mechanisms responsible for ET-induced protection remain unknown. Therefore, ET provides a unique experimental tool to investigate novel biological targets to prevent VIDD. PURPOSE: Using a proteomics analysis, we examined the global protein expression changes in the diaphragm muscle following ET. METHODS: Female Sprague Dawley rats (∼3 months of age) were assigned to control (CON; N=8) or 2 weeks of endurance exercise-training (60 min/day at ∼ 70% VO2) (ET; N=8). Soluble proteins were extracted from diaphragm muscle, digested with trypsin, and analyzed using label-free liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: LC-MS/MS analysis of soluble proteins isolated from the diaphragm of CON and ET rats identified 1397 protein groups and label-free profiling was performed on 813 protein groups that had 3 or more unique peptides. The normalized abundance of 70 proteins was statistically (P<0.05) different between CON and ET samples. Importantly, our analysis revealed that ET leads to significant changes in the protein abundance of several potential cytoprotective proteins, which fall into the following categories, based upon biological functions: aerobic/fatty acid metabolism, redox regulation, stress adaptation, and calcium handling. Examples of proteins in each of these categories include long-chain-fatty-acid-CoA ligase 1 (+14%), microsomal glutathione S-transferase 3 (+18%), heat shock protein 70 kDa protein 1A/1B (+22%), and calsequestrin (-15%), respectively. CONCLUSION: These results uncover novel proteins that are differentially regulated with ET and highlight new areas of investigation to identify potential cytoprotective proteins that may serve as biological targets to prevent VIDD. Supported by the NIH R01 AR064189 awarded to SKP and alumni fellowship awarded to KJS.

Effects of treadmill running on diaphragm and hind-limb muscles in rats with emphysema

Effects of treadmill running on diaphragm and hind-limb muscles in rats with emphysema

Differential Effects Of Flow Stasis On Flow-mediated Dilation In Diaphragm And Soleus Skeletal Muscle Arterioles

Unlike limb muscle, the diaphragm is active during the entire lifespan of an individual. However, with inactivity from mechanical ventilation (MV), there is a time-dependent reduction in diaphragm blood flow and inability to augment flow with contractions which is not evident in hindlimb locomotory muscle. One potential mechanism for the reduced hyperemia after MV is a diminished vasodilatory capacity due to the prolonged reduction in blood flow in the diaphragm. PURPOSE: The purpose of the present investigation was to test the hypothesis that prolonged flow stasis (i.e., absence of flow through vascular lumen) will elicit a rapid reduction in flow-mediated dilation (FMD) in diaphragm resistance vessels, whereas those from locomotory muscle (soleus) muscle will exhibit a preserved vasodilation. METHODS: Using the isolated microvessel technique, resistance arterioles were harvested from the diaphragm and soleus muscles of female Sprague-Dawley rats (n=8, 4 mo., ∼275 g). After the vessels were cannulated, FMD was measured after 1, 3, and 6 hours of flow stasis in the vessels. RESULTS: Despite no difference in initial diameter after cannulation between muscles, the maximal arteriolar diameter (at the end of the experiment) of the arterioles in the diaphragm was smaller than those of the soleus (165 ± 16 vs. 195 ± 22 μm, respectively; p≤0.05). After 1 hour of stasis, maximal FMD was not different between groups (diaphragm, 49 ± 4, Sol, 44 ± 7%, p>0.1). However, in the diaphragm at 3 and 6 hours of flow stasis, there was a 46 ± 6% and 83 ± 4% reduction in maximal FMD, respectively (both p<0.05 vs. 1 hour). There was no difference in maximal FMD over time in the soleus muscle arterioles. After 6 hours of flow stasis maximal FMD was lower in the diaphragm (8 ± 2%) compared to the soleus (42 ± 9%; p<0.05) muscle arterioles. CONCLUSION: There are several unique vascular properties of the diaphragm in relation to other skeletal muscle which, when coupled with its chronic activation, may expedite the time-course of vasomotor dysfunction with disuse in this muscle. The severely blunted FMD observed herein may predispose patients subjected to MV to weaning difficulties due, in part, to a diminished capacity to increase oxygen delivery to the diaphragm during contractions. Supported by NIH AG-31317 (BJB) and R21 AG044858 (JMD).

Skeletal muscle alterations in chronic heart failure: differential effects on quadriceps and diaphragm.

BackgroundChronic heart failure (CHF) results in limb and respiratory muscle weakness, which contributes to exercise intolerance and increased morbidity and mortality, yet the molecular mechanisms remain poorly understood.Therefore, we aimed to compare parameters of antioxidative capacity, energy metabolism, and catabolic/anabolic balance in diaphragm and quadriceps muscle in an animal model of CHF.MethodsLigation of the left anterior descending coronary artery (n = 13) or sham operation (n = 11) was performed on Wistar Kyoto rats. After 12 weeks, echocardiography and invasive determination of maximal rates of left ventricular (LV) pressure change were performed. Antioxidative and metabolic enzyme activities and expression of catabolic/anabolic markers were assessed in quadriceps and diaphragm muscle.ResultsLigated rats developed CHF (i.e. severe LV dilatation, reduced LV ejection fraction, and impaired maximal rates of LV pressure change; P < 0.001). There was a divergent response for antioxidant enzymes between the diaphragm and quadriceps in CHF rats, with glutathione peroxidase and manganese superoxide dismutase activity increased in the diaphragm but reduced in the quadriceps relative to shams (P < 0.01). Metabolic enzymes were unaltered in the diaphragm, but cytochrome c oxidase activity (P < 0.01) decreased and lactate dehydrogenase activity (P < 0.05) increased in the quadriceps of CHF animals. Protein expression of the E3 ligase muscle ring finger 1 and proteasome activity were increased (P < 0.05) in both the diaphragm and quadriceps in CHF rats compared with shams.ConclusionChronic heart failure induced divergent antioxidative and metabolic but similar catabolic responses between the diaphragm and quadriceps. Despite the quadriceps demonstrating significant impairments in CHF, apparent beneficial adaptations of an increased antioxidative capacity were induced in the diaphragm. Nevertheless, muscle ring finger 1 and proteasome activity (markers of protein degradation) were elevated and oxidative enzyme activity failed to increase in the diaphragm of CHF rats, which suggest that a myopathy is likely present in respiratory muscle in CHF, despite its constant activation.

Effects of Acute Respiratory and Metabolic Acidosis on Diaphragm Muscle Obtained from Rats

Acute respiratory acidosis is associated with alterations in diaphragm performance. The authors compared the effects of respiratory acidosis and metabolic acidosis in the rat diaphragm in vitro. Diaphragmatic strips were stimulated in vitro, and mechanical and energetic variables were measured, cross-bridge kinetics calculated, and the effects of fatigue evaluated. An extracellular pH of 7.00 was obtained by increasing carbon dioxide tension (from 25 to 104 mmHg) in the respiratory acidosis group (n = 12) or lowering bicarbonate concentration (from 24.5 to 5.5 mM) in the metabolic acidosis group (n = 12) and the results compared with a control group (n = 12, pH = 7.40) after 20-min exposure. Respiratory acidosis induced a significant decrease in maximum shortening velocity (-33%, P < 0.001), active isometric force (-36%, P < 0.001), and peak power output (-59%, P < 0.001), slowed relaxation, and decreased the number of cross-bridges (-35%, P < 0.001) but not the force per cross-bridge, and impaired recovery from fatigue. Respiratory acidosis impaired more relaxation than contraction, as shown by impairment in contraction-relaxation coupling under isotonic (-26%, P < 0.001) or isometric (-44%, P < 0.001) conditions. In contrast, no significant differences in diaphragmatic contraction, relaxation, or contraction-relaxation coupling were observed in the metabolic acidosis group. In rat diaphragm, acute (20 min) respiratory acidosis induced a marked decrease in the diaphragm contractility, which was not observed in metabolic acidosis.

Effects of Mechanical Ventilation and Autophagy on Diaphragm Oxidative Stress and Proteolysis

Mechanical ventilation (MV) is a life-saving measure for patients in respiratory failure. However, prolonged MV results in diaphragm weakness due to muscle fiber atrophy and contractile dysfunction. Therefore, identifying the signaling pathways responsible for ventilator-induced diaphragm dysfunction (VIDD) is important. While previous reports demonstrate an increase in the autophagy/lysosomal system during MV, the contribution of autophagy to VIDD is unknown. Thus, these experiments were designed to determine the effects of accelerated autophagy on the diaphragm during MV. Cause and effect was determined by inhibiting MV-induced autophagy via adeno-associated virus overexpression of mutated autophagy-related protein 5 (ATG5) in the diaphragm of rats. Our results reveal that inhibiting autophagy prevented the MV-induced reduction in both diaphragm muscle fiber size and force generation. In addition, overexpression of mutated ATG5 prior to MV caused a significant reduction in MV-induced diaphragm lipid peroxidation and mitochondrial ROS production. Also, transduction of mutated ATG5 resulted in increased diaphragm cytosolic catalase content compared to mechanically ventilated control animals. Finally, our results show a regulatory cross-talk may exist between autophagy, calpain and caspase-3. Specifically, inhibition of autophagy resulted in a reduction of the MV-induced increase in proteolytic activity of calpain and caspase-3. Therefore, our data indicate that MV-induced autophagy is detrimental to the diaphragm and inhibition of autophagic signaling protects against VIDD by reducing diaphragmatic oxidative stress and proteolysis.