Electronic Supplementary Material (ESM).
VENTILATOR-INDUCED DIAPHRAGM DYSFUNCTION:
The clinical relevance of animal models
THEODOROS VASSILAKOPOULOS MD
DEPARTMENT OF CRITICAL CARE AND PULMONARY SERVICES
UNIVERSITY OF ATHENSMEDICALSCHOOL
EVANGELISMOSHOSPITAL,
ATHENS, GREECE.
ADDRESS: Critical Care Department
Evangelismos Hospital
45-47 Ipsilandou Str
Athens, Greece
10675
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Force-velocity relationship of the diaphragm secondary to CMV
During spontaneous respiration, thediaphragmusually contracts against loads that can be overcome, resulting in diaphragmatic shortening and thus thoracic excursions. This can be “simulated” to some extent in vitro by the isotonic contractile properties i.e. diaphragmatic strip contractions against different loads and thus different velocities of shortening, allowing for the establishment of the force-velocity relationship. The force-velocity relationship of the diaphragm changes secondary to CMV, the maximum shortening velocity increasing after 1, 2, and 3 days of CMV, respectively (figure ESM 2)(1). The increased maximum shortening velocity correlated with the volume density of abnormal myofibrilsand not to changes inmyosin isoform composition(1). The increase in maximum shortening velocity is a compensatorymechanism to preserve the power output of the diaphragm in the face of the force deficit in the low force-high velocity region of the force-velocity relationship (power is the product of force and velocity of contraction). Thus, the functional consequences of VIDD would be minimized when the diaphragm would contract against normal (small) loads i.e. when the lungs and the thoracic wall are normal (e.g. in the postoperative setting). It should be noted however, that the reduction in maximal isometric force is so profound that it functionally truncates the force-velocity relationship by about 40% and that the power output of the diaphragm subjected to CMV achieves that of the normal diaphragm at loads (i.e. isotonic forces of contraction) less than 5 % of the maximal isometric force(1).
Diaphragm fatiguability secondary to CMV
Increased in vitro fatiguability of the diaphragmatic strips has been documented after 51 hours of CMV in rabbits (tidal volume= 8ml/kgr, frequency = 60/min, PEEP = 2 cmH2O)(2). In contrast, Sassoon et al did not report changes in fatiguability after 24 or 72 hours of CMV in rabbits (tidal volume= 6-8ml/kgr, frequency = 40-50/min, PEEP = 0 cmH2O)(3). Given the similar duration of CMV between the 2 studies it could be speculated that the use of PEEP rendered the diaphragm more susceptible to fatigue in the study of Capdevila et al(2). In rats at an early stage (18 hours) of CMV (tidal volume= 10ml/kgr, frequency = 80/min, PEEP = 1 cmH2O) fatigue resistance is improved (4). However, after longer duration (44-93 hours) CMV (tidal volume= 5ml/kgr, frequency = 90/min, PEEP = 4 cmH2O) in vitro fatiguability was unchanged (5). These differences could be attributed to the different ventilatory settings (with PEEP again associated with increased fatiguability), or to a time effect, with its attendant different fiber type transformations (see below Muscle Fiber Remodeling).
Antioxidant enzymes in the diaphragm after CMV
The response of antioxidant enzymes in the diaphragm to CMV is controversial and might be both time- and species-dependent. In rats, 12 hours of CMV led to decreased protein levels of the cytosolic copper-zinc superoxide dismutase (Cu-Zn-SOD) and glutathione peroxidase (but no change in the respective mRNA levels), increased mRNA levels of thioredoxin reductase-1 and the mitochondrial manganese superoxide dismutase (Mn-SOD) (but no change in the respective protein levels), no change in catalase and significant upregulation of the mRNA and protein levels of heme oxygenase-1 (6). The same group reported that 18 hours of CMV led to augmented SOD activity of both isoforms in rats (4). In piglets, 3 days of CMV (10.5-14 ml / kg, frequency = 21 min-1) resulted in reduced SOD activity and a tendency for reduced glutathione peroxidase activity compared to 2-4 hours of CMV (7), whereas 5 days of CMV (tidal volume 12-15 ml/ kg, PEEP = 3-5 cmH2O) did not change the SOD activity (8). It could be speculated that the early increase in SOD activity might be a defense mechanism against the increased oxidative stress, but as oxidative stress persists antioxidant defenses begin to be exhausted (7), to be restored to normal levels as CMV persists (8).
CMV in rats led to reductions in the total antioxidant capacity, (an indication of non-enzymatic antioxidants-scavengers)(6), and both in the total (6)and in the reduced (9)glutathione levels (the primary non-enzymatic intracellular antioxidant). In contrast, neither the reduced nor the oxidized glutathione were altered after 5 days of CMV in piglets (8). Further studies are needed to elucidate the response of the antioxidant defenses in the diaphragm secondary to CMV.
Potential mechanisms of oxidative stress generation secondary to CMV
The mechanisms of oxidative stress generation remain elusive. Immobilization models in limb muscles show up-regulation of the superoxide-generating enzyme xanthine oxidase and elevated levels of transition metals such as iron, calcium, copper and manganese (10). The increase in [Fe2+] would facilitate the generation of hydroxyl radicals from superoxide and hydrogen peroxide and both copper and manganese could catalyze the oxidation of glutathione thus reducing the overall antioxidant capacity. Whether such a mechanism is operative in the diaphragm needs experimental confirmation. The nitric oxide synthases do not contribute to oxidative stress generation at least up to 18 hours of CMV in rats, since neither their protein expression nor the various end-products of nitric oxide reaction in tissues (nitrite, nitrate, S-nitrosothiols, 3-nitrotyrosine protein levels) are altered (9). The mitochondria can also produce superoxide at complexes I and III along the electron transport chain. After 48 hours of CMV in rabbits, increased oxygen consumption by unstimulated diaphragmatic mitochondria was detected with pyruvate and malate as substrates but not with succinate and retinone(11). Since succinate and retinone inhibit complex I of the mitochondria, these results suggest that the increased mitochondrial oxygen consumption was due to increased complex I activity. Whether other possible sources of ROS such as the NADPH oxidases (12) are operative during CMV awaits experimental confirmation.
Changes in the optimal length of the diaphragm (Lo) secondary to CMV
The remodeling of the diaphragm is not limited to fiber type transformations. Whereas 24 hours of CMV do not affect the optimal length of the diaphragm (Lo), (the muscle length where actin-myosin interposition in sarcomeres is optimum resulting in the greatest force generation) (13), more than 48 hours result in significant decreases in Lo (14), (5), suggesting that the diaphragm is dropping out sarcomeres in series, (15)in the face of increased lung volume due to the use of PEEP during CMV.
Metabolic enzymes and mitochondrial function in the diaphragm secondary to CMV
Enzymes implicated in the metabolic pathways of glycolysis (lactate dehydrogenase) or the Krebs cycle (citrate synthase, succinate dehydrogenase) do not change secondary to CMV (14), (3), (7), (8) although quite early (18 hours) there is transiently increased citrate synthase activity (4), which does not remain elevated when the duration of CMV is prolonged. This initially observed increased activity of citrate synthase (the first enzyme of the Krebs cycle) which is a marker of the oxidative capacity of the diaphragm is in keeping with the improvement in fatigue resistance at this early time point (4).
Mitochondria isolated from the diaphragm of rabbits after 48 hours of CMV were smaller, with focal disruption of their outer membrane, and exhibited increased oxygen consumption in vitro in the unstimulated-resting (basal) state (state 2) which was due to augmented complex I (NADH reductase) activity of the respiratory chain(11). This might be attributable to proton leakage through the inner mitochondrial membrane leading to decreased ATP production, which would be compensated for by increased activity of respiratory chain complexes upstream from the site of ATP production(8).
Decreased complex IV activity (i.e. cytochrome c oxidase, which mediates the final step in the electron transport chain leading to ATP production) was documented in piglet intermyofibrillar (but not subsarcolemmal) mitochondria after 5 days of CMV(8), which would compromise energy production at the site where it is used for muscle contraction, and thus contribute to VIDD.
FIGURE LEGENDS
Figure ESM 1. Transdiaphragmatic pressure (Pdi) development upon electrical stimulation of the phrenic nerves in vivo with various frequencies (force-frequency curve) in adult baboons before (continuous lines) and after (interrupted lines) 11 days of controlled mechanical ventilation. Each trace corresponds to a representative animal. From reference (3) with permission. Please note the decline in the transdiaphragmatic pressure following CMV, which is the functional consequence of VIDD.
Figure ESM 2. Force-velocity and force-power relationships of the diaphragm in control and 1-day (1d), 2-day (2d), and 3-day (3d) controlled mechanical ventilation (CMV) groups. Note that force is expressed as N/cm2 in the top and as percentage of maximum tetanic force [i.e., relative to maximal isometric tension (Po)] in the bottom. P < 0.02, for maximum shortening velocity after 3 day of CMV vs. control. Power was not significantly different among groups. From reference (11) with permission.Please note that the increase in maximum shortening velocity is a compensatorymechanism to preserve the power output of the diaphragm in the face of the force deficit in the low force-high velocity region of the force-velocity relationship. Thus, the functional consequences of VIDD would be minimized when the diaphragm would contract against normal (small) loads i.e. when the lungs and the thoracic wall are normal. However, the reduction in maximal isometric force is so profound that it functionally truncates the force-velocity relationship by about 40% and the power output of the diaphragm subjected to CMV achieves that of the normal diaphragm at loads (i.e. isotonic forces of contraction) less than 5 % of the maximal isometric force (11).
Figure ESM 3. Illustration of the calpain-mediated release of myofilaments during mechanical ventilation (MV)-induced diaphragmatic atrophy. Because the proteasome cannot degrade intact myofilbrillar proteins, it is possible that calpain activation is a required first step to disassemble the sarcomere. This calpain-mediated release of myofibrils would permit the degradation of these sarcomeric proteins by the proteasome system. From reference (21)with permission.
Figure ESM 4. Degradation of oxidized proteins by the 20S proteasome. Following an oxidant attack, most proteins are partially unfolded exposing their inner hydrophobic residues. The 20S core proteasome can recognize such proteins with increased hydrophobicity and degrade them into peptides and amino acids. Such recognition and degradation of oxidatively modified proteins does not require ATP hydrolysis or substrate ubiquitinylation. The peptides may be further broken down into amino acids by other cellular peptidases, and the undamaged amino acids are recycled for protein synthesis. From reference (29) with permission.
Figure ESM 5.Transverse cross section of diaphragm myofibrils in control, 1-day, 2-day, and 3-day CMV groups (x 24.000). Lipid droplets (short red arrow), vacuoles (curved yellow arrow) abnormal myofibrils (long yellow arrow) and mitochondria are shown. After 2 days of CMV, demarcation among myofibril bundles is less tight, with disintegration of the myofibrils between the existing myofibril bundles, and after 3 days of CMV the myofibrils’ “moth-eaten” appearance becomes more distinct. From reference (11) with permission.
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14. Le Bourdelles G, Viires N, Boczkowski J, Seta N, Pavlovic D, Aubier M (1994). Effects of mechanical ventilation on diaphragmatic contractile properties in rats. Am J Respir Crit Care Med 149:1539-1544.
15. Farkas GA and Roussos C (1983). Diaphragm in emphysematous hamsters: sarcomere adaptability. J Appl Physiol 54:1635-1640.
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