Sanford Levine

Rapid Disuse Atrophy of Diaphragm Fibers in Mechanically Ventilated Humans

BACKGROUND The combination of complete diaphragm inactivity and mechanical ventilation (for more than 18 hours) elicits disuse atrophy of myofibers in animals. We hypothesized that the same may also occur in the human diaphragm. METHODS We obtained biopsy specimens from the costal diaphragms of 14 brain-dead organ donors before organ harvest (case subjects) and compared them with intraoperative biopsy specimens from the diaphragms of 8 patients who were undergoing surgery for either benign lesions or localized lung cancer (control subjects). Case subjects had diaphragmatic inactivity and underwent mechanical ventilation for 18 to 69 hours; among control subjects diaphragmatic inactivity and mechanical ventilation were limited to 2 to 3 hours. We carried out histologic, biochemical, and gene-expression studies on these specimens. RESULTS As compared with diaphragm-biopsy specimens from controls, specimens from case subjects showed decreased cross-sectional areas of slow-twitch and fast-twitch fibers of 57% (P=0.001) and 53% (P=0.01), respectively, decreased glutathione concentration of 23% (P=0.01), increased active caspase-3 expression of 100% (P=0.05), a 200% higher ratio of atrogin-1 messenger RNA (mRNA) transcripts to MBD4 (a housekeeping gene) (P=0.002), and a 590% higher ratio of MuRF-1 mRNA transcripts to MBD4 (P=0.001). CONCLUSIONS The combination of 18 to 69 hours of complete diaphragmatic inactivity and mechanical ventilation results in marked atrophy of human diaphragm myofibers. These findings are consistent with increased diaphragmatic proteolysis during inactivity.

Prolonged mechanical ventilation alters diaphragmatic structure and function

Objective:To review current knowledge about the impact of prolonged mechanical ventilation on diaphragmatic function and biology. Measurements:Systematic literature review. Conclusions:Prolonged mechanical ventilation can promote diaphragmatic atrophy and contractile dysfunction. As few as 18 hrs of mechanical ventilation results in diaphragmatic atrophy in both laboratory animals and humans. Prolonged mechanical ventilation is also associated with diaphragmatic contractile dysfunction. Studies using animal models revealed that mechanical ventilation-induced diaphragmatic atrophy is due to increased diaphragmatic protein breakdown and decreased protein synthesis. Recent investigations have identified calpain, caspase-3, and the ubiquitin-proteasome system as key proteases that contribute to mechanical ventilation-induced diaphragmatic proteolysis. The scientific challenge for the future is to delineate the mechanical ventilation-induced signaling pathways that activate these proteases and depress protein synthesis in the diaphragm. Future investigations that define the signaling mechanisms responsible for mechanical ventilation-induced diaphragmatic weakness will provide the knowledge required for the development of new medicines that can maintain diaphragmatic mass and function during prolonged mechanical ventilation.

Both High Level Pressure Support Ventilation and Controlled Mechanical Ventilation Induce Diaphragm Dysfunction and Atrophy

Objectives:Previous workers have demonstrated that controlled mechanical ventilation results in diaphragm inactivity and elicits a rapid development of diaphragm weakness as a result of both contractile dysfunction and fiber atrophy. Limited data exist regarding the impact of pressure support ventilation, a commonly used mode of mechanical ventilation—that permits partial mechanical activity of the diaphragm—on diaphragm structure and function. We carried out the present study to test the hypothesis that high-level pressure support ventilation decreases the diaphragm pathology associated with CMV. Methods:Sprague-Dawley rats were randomly assigned to one of the following five groups:1) control (no mechanical ventilation); 2) 12 hrs of controlled mechanical ventilation (12CMV); 3) 18 hrs of controlled mechanical ventilation (18CMV); 4) 12 hrs of pressure support ventilation (12PSV); or 5) 18 hrs of pressure support ventilation (18PSV). Measurements and Main Results:We carried out the following measurements on diaphragm specimens: 4-hydroxynonenal—a marker of oxidative stress, active caspase-3 (casp-3), active calpain-1 (calp-1), fiber type cross-sectional area, and specific force (sp F). Compared with the control, both 12PSV and 18PSV promoted a significant decrement in diaphragmatic specific force production, but to a lesser degree than 12CMV and 18CMV. Furthermore, 12CMV, 18PSV, and 18CMV resulted in significant atrophy in all diaphragm fiber types as well as significant increases in a biomarker of oxidative stress (4-hydroxynonenal) and increased proteolytic activity (20S proteasome, calpain-1, and caspase-3). Furthermore, although no inspiratory effort occurs during controlled mechanical ventilation, it was observed that pressure support ventilation resulted in large decrement, approximately 96%, in inspiratory effort compared with spontaneously breathing animals. Conclusions:High levels of prolonged pressure support ventilation promote diaphragmatic atrophy and contractile dysfunction. Furthermore, similar to controlled mechanical ventilation, pressure support ventilation-induced diaphragmatic atrophy and weakness are associated with both diaphragmatic oxidative stress and protease activation. (Crit Care Med 2012; 40:–1260)

Increased proteolysis, myosin depletion, and atrophic AKT-FOXO signaling in human diaphragm disuse

RATIONALE Patients on mechanical ventilation who exhibit diaphragm inactivity for a prolonged time (case subjects) develop decreases in diaphragm force-generating capacity accompanied by diaphragm myofiber atrophy. OBJECTIVES Our objectives were to test the hypotheses that increased proteolysis by the ubiquitin-proteasome pathway, decreases in myosin heavy chain (MyHC) levels, and atrophic AKT-FOXO signaling play major roles in eliciting these pathological changes associated with diaphragm disuse. METHODS Biopsy specimens were obtained from the costal diaphragms of 18 case subjects before harvest (cases) and compared with intraoperative specimens from the diaphragms of 11 patients undergoing surgery for benign lesions or localized lung cancer (control subjects). Case subjects had diaphragm inactivity and underwent mechanical ventilation for 18 to 72 hours, whereas this state in controls was limited to 2 to 4 hours. MEASUREMENTS AND MAIN RESULTS With respect to proteolysis in cytoplasm fractions, case diaphragms exhibited greater levels of ubiquitinated-protein conjugates, increased activity of the 26S proteasome, and decreased levels of MyHCs and α-actin. With respect to atrophic signaling in nuclear fractions, case diaphragms exhibited decreases in phosphorylated AKT, phosphorylated FOXO1, increased binding to consensus DNA sequence for Atrogin-1 and MuRF-1, and increased supershift of DNA-FOXO1 complexes with specific antibodies against FOXO1, as well as increased Atrogin-1 and MuRF-1 transcripts in whole myofiber lysates. CONCLUSIONS Our findings suggest that increased activity of the ubiquitin-proteasome pathway, marked decreases in MyHCs, and atrophic AKT-FOXO signaling play important roles in eliciting the myofiber atrophy and decreases in diaphragm force generation associated with prolonged human diaphragm disuse.

Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems

Glycolysis is the initial step of glucose catabolism and is up‐regulated in cancer cells (the Warburg Effect). Such shifts toward a glycolytic phenotype have not been explored widely in other biological systems, and the molecular mechanisms underlying the shifts remain unknown. With proteomics, we observed increased glycolysis in disused human diaphragm muscle. In disused muscle, lung cancer, and H2O2‐treated myotubes, we show up‐regulation of the rate‐limiting glycolytic enzyme muscle‐type phosphofructokinase (PFKm, >2 fold, P<0.05) and accumulation of lactate (>150%, P< 0.05). Using microRNA profiling, we identify miR‐320a as a regulator of PFKm expression. Reduced miR‐320a levels (to ~50% of control, P<0.05) are associated with the increased PFKm in each of these diverse systems. Manipulation of miR‐320a levels both in vitro and in vivo alters PFKm and lactate levels in the expected directions. Further, miR‐320a appears to regulate oxidative stress‐induced PFKm expression, and reduced miR‐320a allows greater induction of glycolysis in response to H2O2 treatment. We show that this microRNA‐mediated regulation occurs through PFKms 3′ untranslated region and that Ets proteins are involved in the regulation of PFKm via miR‐320a. These findings suggest that oxidative stress‐responsive microRNA‐320a may regulate glycolysis broadly within nature.—Tang, H., Lee, M., Sharpe, O., Salamone, L., Noonan, E. J., Hoang, C. D., Levine, S., Robinson, W. H., Shrager, J. B. Oxidative stress‐responsive microRNA‐320 regulates glycolysis in diverse biological systems. FASEB J. 26, 4710–4721 (2012). www.fasebj.org

Respiratory muscle fiber remodeling in chronic hyperinflation: dysfunction or adaptation?

The diaphragm and other respiratory muscles undergo extensive remodeling in both animal models of emphysema and in human chronic obstructive pulmonary disease, but the nature of the remodeling is different in many respects. One common feature is a shift toward improved endurance characteristics and increased oxidative capacity. Furthermore, both animals and humans respond to chronic hyperinflation by diaphragm shortening. Although in rodent models this clearly arises by deletion of sarcomeres in series, the mechanism has not been proven conclusively in human chronic obstructive pulmonary disease. Unique characteristics of the adaptation in human diaphragms include shifts to more predominant slow, type I fibers, expressing slower myosin heavy chain isoforms, and type I and type II fiber atrophy. Although some laboratories report reductions in specific force, this may be accounted for by decreases in myosin heavy chain content as the muscles become more oxidative and more efficient. More recent findings have reported reductions in Ca(2+) sensitivity and reduced myofibrillar elastic recoil. In contrast, in rodent models of disease, there is no consistent evidence for loss of specific force, no consistent shift in fiber populations, and atrophy is predominantly seen only in fast, type IIX fibers. This review challenges the hypothesis that the adaptations in human diaphragm represent a form of dysfunction, secondary to systemic disease, and suggest that most findings can as well be attributed to adaptive processes of a complex muscle responding to unique alterations in its working environment.

Intrinsic apoptosis in mechanically ventilated human diaphragm: linkage to a novel Fos/FoxO1/Stat3-Bim axis

Mechanical ventilation (MV) is a life‐saving measure in many critically ill patients. However, prolonged MV results in diaphragm dysfunction that contributes to the frequent difficulty in weaning patients from the ventilator. The molecular mechanisms underlying ventilator‐induced diaphragm dysfunction (VIDD) remain poorly understood. We report here that MV induces myonuclear DNA fragmentation (3‐fold increase; P<0.01) and selective activation of caspase 9 (P<0.05) and Bcl2‐interacting mediator of cell death (Bim; 2‐ to 7‐fold increase; P<0.05) in human diaphragm. MV also statistically significantly down‐regulates mitochondrial gene expression and induces oxidative stress. In cultured muscle cells, we show that oxidative stress activates each of the catabolic pathways thought to underlie VIDD: apoptotic (P<0.05), proteasomal (P<0.05), and autophagic (P<0.01). Further, silencing Bim expression blocks (P<0.05) oxidative stress‐induced apoptosis. Overlapping the gene expression profiles of MV human diaphragm and H2O2‐treated muscle cells, we identify Fos, FoxO1, and Stat3 as regulators of Bim expression as well as of expression of the catabolic markers atrogin and LC3. We thus identify a novel Fos/FoxO1/Stat3‐Bim intrinsic apoptotic pathway and establish the centrality of oxidative stress in the development of VIDD. This information may help in the design of specific drugs to prevent this condition.—Tang, H., Lee, M., Budak, M. T., Pietras, N., Hittinger, S., Vu, M., Khuong, A., Hoang, C. D., Hussain, S. N. A., Levine, S., Shrager, J. B. Intrinsic apoptosis in mechanically ventilated human diaphragm: linkage to a novel Fos/FoxO1/Stat3‐Bim axis. FASEB J. 25, 2921–2936 (2011). www.fasebj.org

Myosin heavy chain and physiological adaptation of the rat diaphragm in elastase-induced emphysema

BackgroundSeveral physiological adaptations occur in the respiratory muscles in rodent models of elastase-induced emphysema. Although the contractile properties of the diaphragm are altered in a way that suggests expression of slower isoforms of myosin heavy chain (MHC), it has been difficult to demonstrate a shift in MHCs in an animal model that corresponds to the shift toward slower MHCs seen in human emphysema.MethodsWe sought to identify MHC and corresponding physiological changes in the diaphragms of rats with elastase-induced emphysema. Nine rats with emphysema and 11 control rats were studied 10 months after instillation with elastase. MHC isoform composition was determined by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry by using specific probes able to identify all known adult isoforms. Physiological adaptation was studied on diaphragm strips stimulated in vitro.ResultsIn addition to confirming that emphysematous diaphragm has a decreased fatigability, we identified a significantly longer time-to-peak-tension (63.9 ± 2.7 ms versus 53.9 ± 2.4 ms). At both the RNA (RT-PCR) and protein (immunocytochemistry) levels, we found a significant decrease in the fastest, MHC isoform (IIb) in emphysema.ConclusionThis is the first demonstration of MHC shifts and corresponding physiological changes in the diaphragm in an animal model of emphysema. It is established that rodent emphysema, like human emphysema, does result in a physiologically significant shift toward slower diaphragmatic MHC isoforms. In the rat, this occurs at the faster end of the MHC spectrum than in humans.

Diaphragm adaptations elicited by severe chronic obstructive pulmonary disease: lessons for sports science.

LEVINE, S., NGUYEN, T., SHRAGER, J., KAISER, L., CAMASAMUDRAM, V., and RUBINSTEIN, N. Diaphragm adaptations elicited by severe chronic obstructive pulmonary disease: lessons for sports science. Exerc. Sport Sci. Rev., Vol. 29, No. 1, pp 71-75, 2001. In humans, the diaphragm adapts to severe chronic obstructive pulmonary disease with (a) fast-to-slow transformations of the fiber types and myofibrillar proteins and (b) increases in the activity of mitochondrial oxidative enzymes. We suggest that progressive endurance training over several decades accounts for these adaptations.

CrossTalk proposal: Mechanical ventilation-induced diaphragm atrophy is primarily due to inactivity

Mechanical ventilation (MV) is used clinically to maintain adequate alveolar ventilation in patients that are incapable of doing so on their own. Although MV can be a life-saving intervention for patients in respiratory failure, prolonged MV promotes the rapid development of diaphragmatic atrophy and contractile dysfunction. This unfortunate consequence of prolonged MV is referred to as ventilator-induced diaphragm dysfunction (VIDD). Thus, the question arises, ‘what factors are responsible for the speedy onset of diaphragmatic atrophy that occurs during MV’? Although a definitive answer to this question does not exist, at least four different physiological mechanisms could make a primary contribution to MV-induced diaphragm atrophy. These