CONTROLLED MECHANICAL VENTILATION LEADS TO REMODELING OF THE RAT DIAPHRAGM

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1 Online Supplement for: CONTROLLED MECHANICAL VENTILATION LEADS TO REMODELING OF THE RAT DIAPHRAGM Pharmacologic and Surgical Preparation All surgical procedures in the controlled mechanical ventilation (CMV) and spontaneously breathing anesthetized (ANE) groups were performed under sterile conditions. After induction of anesthesia by intraperitoneal administration of sodium pentobarbital (50 mg/kg), rats in the CMV and ANE groups were tracheostomized. The femoral artery was cannulated to allow monitoring of heart rate and blood pressure, as well as withdrawal of arterial blood samples. The internal jugular and femoral veins were also cannulated to permit administration of the following medications: (1) diazepam 1 mg/kg/hour by continuous infusion as well as buprenorphine 0.05 mg/kg every 8 hours, with supplemental doses of intraperitoneal sodium pentobarbital given as needed to achieve optimal sedation and analgesia; (2) prophylactic antibiotics (ceftriaxone 100 mg/kg/day and ticarcillin-clavulanate 300 mg/kg/day); (3) doxacurium 0.6 mg/kg/hour in the CMV group only, with supplemental doses given as needed to completely suppress spontaneous respiratory efforts. Adequate analgesia was verified by the absence of heart rate or blood pressure responses to painful stimuli. In the CMV group, an endotracheal tube was connected to a pediatric ventilator (Babylog 8000; Drager, Lubeck, Germany) equipped with clinical-grade sterile tubing and filters. Ventilator settings were as follows: respiratory frequency = 90/minute, tidal volume = 5 ml/kg, fractional inspired oxygen (FIO 2 ) = , and positive end-expiratory pressure (PEEP) = 4 cm H 2 O. The ventilator was placed in the assist-control mode with a trigger threshold of cm H 2 O. Absence of respiratory muscle effort was confirmed by the lack of triggering of the E1

2 ventilator as well as the stable and reproducible shape of the tracheal pressure waveform. The FIO 2 and level of ventilation were adjusted in CMV group rats to ensure an arterial PO 2 > 100 mm Hg and P CO2 = mm Hg. Supplemental oxygen was also provided to rats in the ANE group using a flow-by system. Serum electrolytes were monitored and remained within normal limits throughout the study. Tracheal pressure, arterial blood pressure, and central venous pressure were also monitored. Nutrition consisted of a rodent liquid diet (LD 101; PMI Feeds, Inc., St. Louis, MO) providing 1 kcal (16% protein, 15% fat) per gram. This was administered via an esophageal tube at a dose that provided approximately 50 kcal/kg body weight per day. Body temperature was monitored and maintained within the normal range with a heating blanket. Other aspects of general care included regular turning of the animals, airway suctioning, eye lubrication, and periodic expressing of the bladder. All animals were continuously monitored by two physicians trained in critical care medicine, who performed alternating 12-hour shifts throughout the duration of each experiment. The decision to terminate an experiment was based on the occurrence of technical complications such as loss of intravenous or intraarterial access needed for drug delivery and monitoring, difficulty in maintaining adequate analgesia without unacceptable hemodynamic side effects, or an inability to continue monitoring the animals due to excessive fatigue of the research personnel. Immunohistochemical and Morphometric Analysis Frozen costal diaphragm sections were treated with antibodies specific for slow type I and all fast type II myosin heavy chain (MHC) isoforms (Sigma Chemical Co., St. Louis, MO) as previously described (E1). The diaphragm strip was embedded in mounting medium and serial sections (6 μm thick) midway between central tendon and rib cage were cut with a cryostat at -20ºC. After E2

3 air-drying, the sections were reacted with antibodies (E2, E3) specific for slow type I (NOQ7.5.4D, 1:100) and fast type II (MY-32, 1:200) isoforms (the latter reacts with all type II MHCs: IIa, IIx and IIb). The primary antibodies (raised in mouse) were diluted in PBS and incubated with tissue sections at 4ºC overnight, rinsed and then incubated for an additional hour with a biotinylated anti-mouse secondary antibody, and subsequently visualized by peroxidase staining using the Vectastain ABC kit (Vector Labs, Burlingame, CA). By reacting serial sections with the above antibodies, it was possible to identify hybrid fibers (i.e., fibers co-expressing both type I and II MHC isoforms) that were in transition from a type I-to-type II MHC phenotype. The same approaches were used to evaluate MHC isoform expression in the soleus and extensor digitorum longus (EDL), which are prototypical slow- and fast-twitch hindlimb muscles, respectively. Images of serially stained muscle sections were captured to computer, and the fibers were categorized as type I, type II, or hybrid fibers. The number of fibers in each category was determined from randomly selected fields (minimum of 200 fibers per muscle); individual diaphragm fiber cross-sectional areas were also measured from the calibrated computer image. Muscle Contractility Analysis In a subset of rats, costal diaphragm muscle strips were obtained for in vitro contractility measurements under isometric conditions as previously described in detail (E1). Freshly dissected diaphragm strips were briefly transferred to chilled Ringer s solution (composition: 119 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl 2, 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 20 mm NaHCO 3, and 12 μm d-tubocurarine chloride) perfused with 95% O 2 :5% CO 2 (ph 7.4). The muscles were then mounted vertically in a jacketed tissue bath chamber filled with continuously perfused Ringer s solution maintained at 25ºC, and a thermoequilibration period of 10 minutes was observed before initiating contractile measurements. One end of each muscle was securely E3

4 anchored to a platform near the base of the chamber, while the opposite tendon was tied to the lever arm of a force transducer/length servomotor system (Model 300B dual mode; Cambridge Technology, Watertown, MA) (E4, E5). The latter was mounted on a mobile micrometer stage (Newport Instruments, Toronto, Canada) to allow incremental adjustments of muscle length. Electrical field stimulation was induced via platinum plate electrodes placed into the bath on both sides of the muscle. Supramaximal stimuli with a monophasic pulse duration of 2 ms were delivered using a computer-controlled electrical stimulator (Model S44; Grass Instruments, Quincy, MA) connected in series to a power amplifier (Model 6824A; Hewlett Packard, Palo Alto, CA). Muscle force was displayed on a storage oscilloscope (Tektronix, Beaverton, OR), and the data were simultaneously acquired to computer (Labdat/Anadat software; RHT-InfoData, Inc., Montreal, Canada) via an analog-to-digital converter at a sampling rate of 1,000 Hz for later analysis. After adjusting each muscle to optimal length (Lo, the length at which maximal twitch force is achieved), five twitch stimulations were recorded and the mean value was used to determine the following: twitch contraction time (time to peak force), twitch half-relaxation time (time for force to decrease to one-half of its peak value), and maximal isometric twitch force. Maximal isometric tetanic force was then measured by stimulating the muscle at 120 Hz for 1 second, allowing a clear plateau in force to be attained. Fatiguability of the diaphragm was assessed by measuring the loss of force in response to repeated stimulations (30 Hz, 0.33 duty cycle, 90 trains/minute) for 2 minutes. The muscle was then removed from the bath and Lo was directly measured under a dissecting microscope with microcalipers accurate to 0.1 mm. Total muscle strip cross-sectional area was determined by dividing muscle weight by its length and tissue density (1.06 g/cm 3 ). This allowed specific force (force/cross-sectional area) to be calculated, which was expressed as N/cm 2. E4

5 Northern Blot Analysis Total cellular RNA was isolated from frozen hemidiaphragm samples using the guanidinium thiocyanate-phenol-chloroform extraction method (E2). Total RNA was quantitated and purity assessed by measuring spectrophotometric absorption at 260 and 280 nm. Aliquots (10 μg) of RNA were size-fractionated by electrophoresis on a 1% agarose/6.6% formaldehyde gel, transferred to nylon membranes (GeneScreen, Biotechnology Systems; NEN Research Products, Boston, MA) and immobilized by UV cross-linking. The membranes were prehybridized at 56ºC overnight in a solution containing 50% formamide, 5x SSC, 0.5% SDS, 2.5x Denhardt s, 50 μg/ml trna, and 100 μg/ml salmon sperm DNA. The membranes were then hybridized overnight at the same temperature to 32 P-labelled crna probes (riboprobes) transcribed from MHC isoform-specific cdna sequences corresponding to portions of the 3'-untranslated region of each major MHC isoform found in rat diaphragm; the sequences of the probes used in this study have been reported earlier (E6). The membranes were washed in 2x SSC/1% SDS at 65ºC followed by 0.1x SSC/0.1% SDS, and then exposed on a storage phosphor screen (Fuji Photo Film Co., Stamford, CT) as well as on X-ray film at -80ºC with two intensifying screens. The same blots were alternately stripped (confirmed by phosphorimager) and reprobed at separate times. Relative amounts of MHC isoform mrna were determined by phosphorimager (Fujix Bio-imaging Analyzer System; Bas 1,000, Fuji Photo Film Co.); the MHC mrna phosphorimager scores were normalized to 18S ribosomal RNA using a random-primer-labelled cdna probe as previously described (E7). E5

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7 References E1. Yang L, Bourdon J, Gottfried SB, Zin WA, Petrof BJ. Regulation of myosin heavy chain gene expression after short-term diaphragam inactivation. Am J Physiol 1998;274:L980- L989. E2. Petrof BJ, Kelly AM, Rubinstein NA, Pack AI. Effect of hypothyroidism on myosin heavy chain expression in rat pharyngeal dilator muscles. J Appl Physiol 1992;73: E3. Petrof BJ, Gottfried SB, Eby J, LaManca J, Levine S. Growth hormone does not prevent corticosteroid-induced changes in rat diaphragm structure and function. J Appl Physiol 1995;79: E4. Petrof BJ, Stedman HH, Shrager JB, Eby J, Sweeney HL, Kelly AM. Adaptations in myosin heavy chain expression and contractile function in the dystrophic (MDX) mouse diaphragm. Am J Physiol 1993;265:C834-C841. E5. Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci USA 1993;90: E6. DeNardi C, Ausoni S, Moretti P, Gorza L, Velleca M, Buckingham M, Schiaffino S. Type 2X-myosin heavy chain is coded by a muscle fiber type-specific and developmentally regulated gene. J Cell Biol 1993;123: E7. Szyf M, Milstone DS, Schimmer BP, Parker KL, Seidman JG. Cis modification of the steroid 21-hydroxylase gene prevents its expression in the Y1 mouse adrenocortical tumor cell line. Mol Endocrinol 1990;90: E7