The Effect of Intermittent Hemidiaphragm Stimulation During Surgery on Mitochondrial Function, Single Fiber Contractile Force and Catabolic Pathways in Humans
Phase: NA
Conditions: Mechanical Ventilation Complication, Diaphragm Injury
Sponsor: University of Florida

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Protocol 
 
1. 
Project Title: 
The effect of hemidiaphragm stimulation during surgery on gene expression and single 
fiber contractile properties in humans 
 
2. 
Investigator(s): 
Barbara Smith, PhD, PT;  
Thomas Beaver, MD, MPH  
 
3. 
Abstract: 
Approximately 10% of the patients who receive mechanical ventilation (MV) experience 
difficulty weaning, and these patients account for approximately 40-50% of the total MV 
days.  Numerous animal studies have documented that MV induced muscle inactivity 
leads to diaphragm atrophy, oxidative stress, altered diaphragm gene expression and 
reduced muscle strength in as little as 6-24 hours.  This phenomenon has been termed 
ventilator induced diaphragm dysfunction (VIDD).  Spontaneous breathing for 5 minutes 
every 6 hours over a 24 hour period of MV support can attenuate VIDD by 
approximately 50% in a rodent model, indicating that very brief periods of normal 
diaphragm activity can be protective.  Recent human work has documented VIDD 
(diaphragm muscle fibers atrophying ~ 55%) following approximately 39 hours of 
controlled MV.  Clinical human studies have shown that patients experiencing difficulty 
weaning from MV have impaired diaphragm performance, consistent with changes seen 
in VIDD models. Therefore, while there is growing evidence that MV rapidly leads to 
VIDD in animals and humans, and brief periods of diaphragm activity during MV support 
can lessen VIDD in a rodent model, there are no data examining strategies to attenuate 
this problem in humans.  We have data examining human diaphragm gene expression 
(within subjects design) at the beginning and end of ~5 hours of surgery/MV support.  
The data revealed up regulation of several diaphragm genes controlling ubiquitin 
mediated catabolic activity and down regulation of genes critical to normal muscle 
contractile function.  We propose to study the effects of periodically stimulating one 
hemidiaphragm and using the unstimulated hemidiaphragm as a control in a human 
MV/surgical model.  The right or left hemidiaphragm will be stimulated via the 
corresponding phrenic nerve during surgery depending upon the ease of access. Costal 
diaphragm muscle samples will be obtained from each hemidiaphragm at the end of 
surgery.  Gene expression, mitochondrial function and single muscle fiber contractile 
properties in the stimulated and unstimulated hemidiaphragms will be contrasted. This 
study will provide the first data examining the molecular and contractile effects of 
increased diaphragm activity during periods of MV/surgery in humans.  The results from 
this research study will help direct efforts to develop rehabilitation interventions in the 
preoperative and postoperative period aimed at preventing VIDD in humans. 
 
 
 
 

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4.  
Background: 
Failure to wean from mechanical ventilation (MV) is a growing clinical and economic 
problem in the United States health care system. The vast majority of patients 
supported by MV are quickly weaned, but a minority (~10%) of these patients 
experience difficult weaning and require prolonged MV support, often for months.  
Approximately 250,000 patients annually in the US experience difficult weaning and the 
estimated annual cost of providing care during these prolonged MV support episodes is 
16 billion dollars [1]. 
Several articles have documented that patients experiencing difficult weaning have 
inactivity-induced diaphragm muscle dysfunction [2-6].  It is not surprising that the use 
of MV leads to muscle atrophy and dysfunction.  Mechanical ventilators are in essence, 
artificial breathing “muscles” that pump air into the patients’ lungs, facilitating gas 
exchange. While MV support is life-saving for patients with respiratory failure, MV 
support can have deleterious effects on the respiratory muscles. Numerous [7-17] 
animal studies have shown that even short periods of controlled MV support (6-24 
hours) can lead to physiologically significant diaphragm atrophy, decreased muscle 
force production, oxidative stress and gene expression changes promoting muscle 
dysfunction which has been termed ventilator induced diaphragm dysfunction (VIDD).   
Furthermore, animal work has shown that the diaphragm atrophies approximately 8 
times faster than limb muscle, indicating that the diaphragm is uniquely sensitive to 
inactivity-induced atrophy and dysfunction [10].  While rodent diaphragm function is 
extraordinarily sensitive to reduced activity, it takes very little activity to maintain 
function. Gayan-Ramirez [18] has shown that allowing rats to breathe spontaneously for 
5 minutes every 6 hours reduces the VIDD effect by 50% when animals receive 24 
hours of MV support.  Sassoon [19] found that ventilating rats with assist–control 
ventilation, which requires the animals to generate some diaphragmatic activity, also 
attenuated VIDD when compared to controlled MV, which eliminates all active diagram 
activity.  Increased activity is not the only possible means of attenuating VIDD.  Maes et 
al. [20] found that leupeptin, an inhibitor of lysosomal proteases and calpain completely 
prevented VIDD and atrophy over a 24 our period in a rat MV model. Trolox can also 
partially protect the diaphragm from VIDD by decreasing myofilament protein substrate 
availability to proteasomes in rats [7].  Data examining the human diaphragm following 
periods of MV are sparse, but largely consistent with the animal data. 
Recent work by Levine, et al. in humans has shown that controlled MV for ~ 36 hours 
led to 55% atrophy of diaphragm muscle fibers, increased markers of oxidative stress 
and upregulation of genes controlling pathways leading to muscle atrophy [21].  Like 
animals, the rate of diaphragm atrophy in humans is much greater than seen in limb 
muscles.  For example, 5 weeks of bedrest inactivity led to 12% atrophy in the thigh and 
calf muscles in healthy young subjects [22]. Ohira reported that 120 days of bedrest in 
healthy young subjects resulted in a 32% reduction in the soleus muscle cross sectional 
area [23].  Buckey found that 10 days of bedrest atrophied calf muscles by 7% [24]. 
Ogawa found that 20 days of inactivity reduced quadriceps muscle cross sectional area 
by 6% [25].  Clearly, the human diaphragm atrophies much faster than human limb 
muscle. Despite the importance of VIDD’s probable contribution to weaning difficulties, 
the vast majority of work examining the effects of MV and VIDD has been conducted in 

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animal models and has focused on the dysfunction side of the muscle plasticity 
continuum, with sparse attention given to measures to reduce VIDD [9-12, 15, 17, 26].  
Only two studies have directly studied the impact of MV on human diaphragm structure 
and none have directly measured muscle contractile performance following periods of 
MV.  Levine’s recent report on the effects of 36 hours of MV in humans was discussed 
earlier.  The only other human study used a case control design that qualitatively 
showed profound diaphragm muscle fiber atrophy in a young child ventilated for 47 days 
compared to a child ventilated for 3 days [27].  The current evidence indicates that short 
periods of MV leads to VIDD in animal models and difficult to wean humans clinically 
demonstrate VIDD.  Given the evidence of VIDD, it is clear that mechanistic human data 
are needed to design strategies to prevent or treat the problem.  
In this study, we propose to stimulate either the right or left human hemidiaphragm 
hourly during routine cardiothoracic (CT) surgery and to obtain diaphragm muscle 
biopsies from the stimulated and unstimulated hemidiaphragms at the end of surgery for 
analysis with micro gene arrays, mitochondrial function and single muscle fiber 
contractile properties.  To our knowledge, this project would provide the first 
simultaneous data on the human diaphragm’s gene expression, mitochondrial function 
and contractile responses to activity during surgery and MV support.  This knowledge 
will be invaluable to developing strategies to protect the diaphragm from the deleterious 
effects of MV and surgery.   
This project will help establish our human diaphragm surgery/MV model, facilitating 
applications for support from the NIH to study preoperative diaphragm training 
paradigms and intraoperative pharmacologic interventions. 
 
5. 
Specific Aims: 
Aim 1:  Compare the expression of the human diaphragm genes that control energy 
metabolism, excitation–contraction coupling, contractile proteins and oxidative stress in 
stimulated and unstimulated diaphragm muscle samples. 
Aim 2:  Compare the single muscle fiber contractile characteristics (subdivided by 
muscle fiber type) of stimulated and control diaphragm muscle fibers. 
Aim 3: Compare the mitochondrial function of stimulated and control diaphragm muscle 
fibers.  
We hypothesize that intermittent stimulation of either the right or left human 
hemidiaphragm during thoracic surgical procedures and lung transplant lasting 
approximately 5 hours or longer will prevent or attenuate inactivity-induced changes in 
gene expression, mitochondrial function and single fiber contractile properties as 
compared to the unstimulated hemidiaphragm. 
 
 

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6. 
Research Plan: 
Subjects: 
 
Patients undergoing regularly scheduled cardiothoracic surgery, including lung 
transplants, at Shands Hospital at the University of Florida will be recruited to enroll in 
this prospective study. From each subject an informed consent will be obtained to 
donate two diaphragm muscle biopsy samples during the surgery.  
After recruitment, an initial evaluation consisting of basic demographic information as 
well as diagnostic information pertaining to the surgery will be performed before the 
scheduled surgery to search for exclusion factors. In addition, we will track medications, 
ventilator use, complications, and length of stay during the patient’s hospitalization for 
surgery.  Up to 100 subjects will be studied. 
Exclusion Criteria: 
1. Age < 18 or > 80, 
2. history of prior surgery to the diaphragm or pleura; 
3. a diagnosis of COPD will be determined from a clinical history consistent with 
chronic bronchitis and/or emphysema, a long history of cigarette smoking, and 
pulmonary function tests consistent with irreversible airflow obstruction (FEV1 < 
40% predicted, according to European Respiratory Society criteria (1).  
4. a diagnosis of chronic heart failure (NYHA class IV) (2) 
5. clinical diagnosis of other lung disease (cystic fibrosis, bronchiectasis, lung 
cancer; etc.); 
6. renal insufficiency (serum creatinine > 1.6 mg/dl);  
7. severe hepatic disease (any liver function tests > 1.5 times the upper limit of 
normal);   
8. undernourishment (body mass index < 20 kg/m2), 
9. chronic uncontrolled or poorly controlled metabolic diseases (e.g., diabetes, 
hypo- or hyperthyroidism) 
10. orthopedic diseases, suspected paraneoplastic or myopathic syndromes,  
11. if in the surgeons’ judgment the patients’ clinical status warrants, diaphragm 
stimulation will be stopped and biopsies will not be obtained, 
12. items #3 and #5 above will not apply to transplant patients. 
Experimental Procedure: 
Before the surgery: 
A pulmonary function test will be performed to determine whether the subject qualifies 
for the study.   
PFT: Subjects will complete a forced expiratory maneuver to determine eligibility for 
training.  Tests will be completed according to American Thoracic Society guidelines,(3) 
and 3 to 5 trials administered, separated by 2 minutes of rest.  If an individual 
demonstrates FEV1<40% of predicted, he or she will be removed from any further 

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participation in the study. An FEV1.0 of 40% or less of predicted as an exlcusionary 
factor will not apply to transplant patients. 
In addition, the qualified subjects will undergo maximum inspiratory pressure test before 
the scheduled surgery. 
Maximal Inspiratory Pressure:  Subjects will perform a maximal inspiratory pressure 
maneuver from residual volume, in accordance with American Thoracic Society testing 
guidelines.(3)  Trials will be administered until 5% variability is achieved between three 
trials (typically achieved within 5 trials), and separated by a two-minute rest. A short rest 
between maneuvers will be provided to prevent muscle fatigue. 
In conjunction with respiratory testing, we will monitor baseline and exertional blood 
pressure, heart and respiratory rate, and pulse oximetry. The following tests will be 
completed on all subjects.   
During t surgery following procedures will be performed, 
Diaphragm stimulation: Either the right or the left hemidiaphragm will be stimulated 
during routine cardiothoracic surgical procedures. This will be decided by the surgeon 
on the basis of ease of access to the corresponding phrenic nerve. Rodent models of 
MV have shown that different neuromuscular blockers can exert varied effects on the 
diaphragm contractile properties (4-7). The right or the left hemidiaphragm will be 
stimulated via the corresponding phrenic nerve with a temporary cardiac pacer 
(Medtronic 5388) with wire electrodes.  The surgeon will identify the phrenic nerve on 
the pericardium and the pacing electrodes will be sutured to the pericardium 
immediately adjacent (~8 mm) to either side of the phrenic nerve (8). It is not necessary 
for the pacing electrodes to touch the phrenic nerve to stimulate the phrenic nerve and 
generate a hemidiaphragm contraction.   
Dr. Beaver has observed vigorous left hemidiaphragm contractions on many occasions 
during temporary heart pacing when electrodes were applied to the pericardium close to 
the left phrenic nerve. Great care will be taken to ensure that the stimulating electrodes 
do not contact the phrenic nerve. The left phrenic nerve will be stimulated near the 
pulmonary artery to prevent pacing the heart.  The right phrenic nerve will be stimulated 
on the right side of the pericardium.  
Patients receive non-depolarizing neuromuscular blockers during surgery to prevent 
limb muscle contractions.  The diaphragm is more resistant to neuromuscular blockade 
than limb muscles, and remains responsive to stimulation despite neuromuscular 
blockade sufficient to temporarily paralyze limb muscles (9, 10). The surgeon will select 
the hemidiaphragm for stimulation depending upon the ease of access to the 
corresponding phrenic nerve. There is no evidence that the muscle fiber contractile 
characteristics or gene expression differ between the right and left human 
hemidiaphragms.  
Once the electrodes are sutured in place, the stimulator will be activated with the lowest 
possible current. The stimulator current will then be systematically increased until a 
vigorous contraction of the corresponding hemidiaphragm is observed, which will be 

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termed the threshold stimulus. The stimulus intensity will be adjusted to three times the 
threshold level for subsequent stimulation. The hemidiaphragm on the unstimulated side 
will be observed to verify that it is not entrained with the hemidiaphragm contractions on 
the stimulated side. Stimulation will be conducted for one minute (30 pulses per minute, 
15 msec duration) either 30 or 60 minutes following initiation of MV and every 30 or 60 
minutes thereafter. The frequency of stimulation (every 30 or 60 minutes) will be 
determined by the estimated duration of surgery. In order for both human 
hemidiaphragms to generate contractions, stimulation must be applied to both phrenic 
nerves or hemidiaphragms (11).  The temporary wire electrodes that are used for the 
electrical stimulation will be removed at the end of the surgery. 
Our model is not a pure MV inactivity paradigm with the addition of surgery, but it is not 
possible to ventilate humans and obtain diaphragm biopsies for experimental purposes, 
as is possible with animal models.  Our surgery/MV model, however, is the actual 
clinical situation that often leads to VIDD and difficult weaning in humans, and thus, our 
results will be highly relevant to the clinical management of these patients.  
Data safety monitoring board: 
A data safety monitoring board comprised of three physicians will be impaneled before 
any additional subjects are studied.  Prior to collecting additional data, the panel will be 
convened to review the study protocol, safety measures and establish operating 
policies. The data safety monitoring board will meet at least annually and will be given 
immediate reports of any serious and unexpected adverse events that may occur.  
DSMB findings and reports the will be included in the annual renewal of this protocol to 
the IRB. 
Diaphragm Biopsies: 
The method selected to obtained diaphragm biopsies was designed to minimize the risk 
of bleeding, phrenic nerve injury, or diaphragmatic impairment.  The surgeon will obtain 
a full-thickness diaphragm sample, approximately 6 mm X 20 mm, from the antero-
lateral coastal regions of the stimulated and unstimulated hemidiaphragms, shortly 
before the surgery is completed. By harvesting the diaphragm biopsy from the anterior 
costal diaphragm, in a region opposite of the phrenic nerve insertion, we will minimize 
the proximity to phrenic insertion. The wounds will be closed with sutures. The surgeon 
will place the biopsy samples on ice and hand them to study staff. Tissue samples will 
be divided using the procedures described below. One part of the sample will be sent to 
Christiaan Leeuwenburgh's  laboratory for mitochondrial respiration and gene analysis. 
One sample will be sent to Dr. Karan Esser’s laboratory (University of Florida, College 
of Medicine) for RNA analysis.  Samples to be analyzed with micro gene array and RNA 
analysis will be immediately placed in RNAlater solution and stored at –80° C until 
analysis.  Another  part of the sample will be sent to Dr Leo Ferreira's laboratory ( 
College of Health and Human Performance, University of Florida) for contractile and 
fiber type measures. Samples destined for single fiber contractile properties will be 
placed in a relaxing solution containing 50% glycerol (vol/vol) and stored at 20°C for no 
more than 4 wk before subsequent analysis. (12) A portion of the tissue samples will be 
sent to a non-UF investigator, Dr. Coen Ottenheijm at the University of Arizona for 

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analysis of muscle titan integrity.  After harvest, tissue samples will be coded to protect 
subject confidentiality and tissue samples will not contain identifying information such as 
the subject name, medical record are other identifying information. All of the tissue will 
be consumed during the analysis of the various variables and will not be stored or bank 
for use outside of this protocol. 
In addition we will request the subject’s permission to be videotaped for this research 
project. The process of stimulation of the phrenic nerve and the contraction or 
movement of the diaphragm muscle will be videotaped during the surgery. A separate 
consent [Consent to be videotaped and to different uses of the videotape(s)] will be 
obtained from the subjects.  
C.  After Surgery: 
Data Analysis: 
Microarray Analysis: 
The gene micro array analysis will be performed in Dr Christians Leeuwenburgh's  
laboratory (Department of Aging at the University of Florida, College of Medicine).  
In brief, a U133 plus 2.0 Affymetrix GeneChip or similar product will be used to query 
the entire human genome (~ 54,000 probes). Total RNA will be harvested for cDNA 
preparation using protocols and reagents recommended by Qiagen Inc. cDNA will be 
prepared using protocols recommended by Affymetrix and reagents obtained from 
Invitrogen. Biotin labeled-cRNA preparation will be accomplished using protocols and 
reagents recommended by Affymetrix. The labeled-cRNA will be used to interrogate 
DNA microarrays prepared by Affymetrix. cRNA will be synthesized from cellular RNA 
from specimens based on protocols and recommendations from the GeneChip 
manufacturer, Affymetrix. 
Single fiber contractile and fiber type measures:   
Single fiber contractile and fiber type measures will be done in Dr Ferreira’s laboratory 
(Physiology laboratory at the Center for Exercise Science, University of Florida).  Dr 
Ferraria’s laboratory has extensive experience with these techniques (13). 
Muscle fibers treatment:  
Following collection, muscle fiber bundles will be stretched ~20%, pinned on cork, and 
placed in a relaxing solution consisting of 59.0 mM potassium acetate, 6.7 mM 
magnesium acetate, 5.6 mM NaATP, 10 mM EGTA, 2.0 mM dithiothreitol, 15.0 mM 
creatine phosphate, 1 mg/ml phosphocreatine kinase, and 50 mM imidazole for a total 
ionic strength of 200 mM at a pH of 7.0 at 5°C and then shipped to Dr Mantilla’s 
laboratory for analysis. The fiber bundles can be stored in relaxing solution for up to 
3 weeks before analysis.   
Myosin heavy chain concentration and single-fiber gel electrophoresis:   
Single fiber segments (~1.5-2.5 mm in length) will be fixed in 4% paraformaldehyde for 
30 s and placed on a microscope stage with a CCD camera. The fiber image will be 

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projected onto a video screen, and the total number of sarcomeres will be counted. 
Fibers will be placed in 25 µl of SDS sample buffer containing 62.5 mM Tris-HCl, 2% 
(wt/vol) SDS, 10% (vol/vol) glycerol, 5% 2-mercaptoethanol, and 0.001% (wt/vol) 
bromophenol blue (pH 6.8). Boiling for 2 min will denature the samples. Samples will be 
separated electrophoretically in gradient gels (stacking gel: 3.5% acrylamide -pH 6.8, 
and separating gel: 5-8% acrylamide -pH 8.8), and gels will be silver stained. Control 
samples at known concentrations of myosin heavy chain will be run for quantification. 
Myosin heavy chain concentration is determined by dividing the myosin heavy chain 
content determined from the myosin standard curve by the fiber volume. This 
concentration will then multiplied by the half-sarcomere volume of the fiber, normalized 
to optimal length (2.5 µm/sarcomere). Space limitations prevent complete descriptions 
of the procedures for measuring myosin heavy chain concentrations and gel 
electrophoresis for fiber classification, but more details on these methods can be found 
in articles from Dr. Mantilla’s laboratory.(14, 15) 
Single-fiber mechanical measurements:  
Fibers will be retrieved from the relaxing solution and fiber bundles will be placed in a 
solution containing 1% Triton X-100 to permeabilize the plasma membrane for ~20 min, 
and dissected under a dissecting microscope. Single fibers will be transferred from the 
skinning solution to a relaxing solution (pCa 9.0) for force measurements. Force 
measurements will be performed in solutions containing the following (in mM): 
10.0 EGTA, 1.0 free Mg2+, 5.0 MgATP, 15.0 creatine phosphate, 50.0 imidazole, 
2.0 DTT, and PCK at 1 mg/ml with a total ionic strength of 150 mM. The relaxing 
solution will have pCa of 9.0, and the activating solution a pCa of 4.0.  Fiber ends will be 
fixed by exposing them to a 5% glutaraldehyde solution to maintain noncompliant 
attachments of the fibers to a force transducer and servo-controlled motor. Fibers will be 
mounted horizontally on two small stainless steel hooks in a temperature-controlled 
flow-through acrylic chamber located on the stage of an inverted microscope. Fibers will 
be attached at one end to a force transducer (resonant frequency of 5 kHz), and the 
other end to a servo-motor (step time: 800 µs). Sarcomere length will be set at 2.5 µm 
and monitored by first-order laser diffraction. Signals will be recorded with a custom-
designed LabView-based software and a data acquisition board. Fiber dimensions will 
be measured to calculate fiber cross-sectional area. A baseline force measurement will 
be obtained while fibers are perfused with a pCa 9.0 solution. The perfusate will be 
switched to a pCa 4.0 solution (in the same flow-through chamber) to maximally activate 
the fibers, and after maximal activation is achieved, the fiber will again be perfused with 
a pCa 9.0 solution to verify that force returns to its original baseline level. Maximum 
specific force (N/cm2) will be calculated by dividing the maximum isometric force by 
fiber cross-sectional area. Approximately 4 fibers of each fiber type will be analyzed. 
This technique has been previously reported in greater detail. (15) 
Mitochondrial function measurements in diaphragm muscle: 
Elevated levels of oxidative stress produced during mechanical ventilation (MV) directly 
contribute to diaphragmatic atrophy and contractile dysfunction, thus preventing the 
successful removal of patients from the ventilator (i.e. “weaning”) (16, 17). Numerous 
lines of evidence (18, 19) have surfaced implicating reactive oxygen species (ROS) and 

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specifically mitochondrial-mediated signaling pathways in mitochondrial ventilation 
(MV)-induced diaphragmatic dysfunction. 
Human biopsy pickups will be performed by specific personnel from Dr. 
Leeuwenburgh’s lab. Diaphragm muscle tissue (~20-30 mg) will be immediately placed 
on ice in Buffer. The sample will be taken to Dr. Leeuwenburgh’s laboratory, and 
samples will be blotted and dissected free of blood, fat, and connective tissue.  
The muscle sample will then be separated into different portions and processed as 
follows: 
~8 mg will be placed on ice in isolation medium (10 mM Ca-EGTA, 0.1 µM free calcium, 
20 mM imidazole, 20 mM taurine, 49 mM K-MES, 3 mM K2HPO4, 9.5 mM MgCl2, 5.7 
mM ATP, 15 mM phosphocreatine, 1 µM leupeptin, pH 7.1) and be utilized for 
respirometry; ~30 mg of muscle tissue will be frozen in liquid nitrogen, stored at -80°C 
(in Room P1-09), and used for BN-PAGE; remaining tissue will be frozen immediately in 
liquid nitrogen and stored at −80°C (Room P1-09) for further analysis (i.e. biochemical, 
immunoblotting, PCR).  
Procedure  
The parameters of mitochondrial function will be assessed in these patients using 
conventional biochemical (i.e., electrophoresis and immunoblotting) and histological 
(light and electron microscopy) methods, as well as several novel technologies listed 
below; 
High-resolution respirometry will be utilized in freshly permeabilized human muscle 
samples to assess mitochondrial respiration, and electron transport chain and capacity 
of oxidative phosphorylation (OXPHOS) in situ. This methodology is advantageous over 
measurements on isolated mitochondria since it: A) substantially reduces the overall 
tissue necessary, B) avoids selective isolation and assessment of mitochondrial 
subpopulations and, C) comprehensively examines function of all mitochondria within a 
muscle sample, independent of size, location, functional status, and in their natural 
intracellular position and assembly. 
Blue native polyacrylamide gel electrophoresis (BN-PAGE) will be used to determine 
content and enzymatic activity of individual mitochondrial respiratory complexes. 
Specific Techniques: 
Determination of mitochondrial function using high-resolution respirometry of 
permeabilized muscle fibers.  
Permeabilized skeletal muscle samples will be prepared for respirometry as described 
in (20). Briefly, 2-10 mg of freshly collected muscle sample will be washed in 1 mL ice-
cold isolation medium (10 mM Ca-EGTA, 0.1 µM free calcium, 20 mM imidazole, 20 mM 
taurine, 49 mM K-MES, 3 mM K2HPO4, 9.5 mM MgCl2, 5.7 mM ATP, 15 mM 
phosphocreatine, 1 µM leupeptin, pH 7.1). Skeletal muscle strips will be separated to 
form thin muscle-fiber bundles, permeabilized in isolation medium containing 50 µg mL-
1 saponin, at 4 °C, and washed several times in respiration medium (0.5 mM EGTA, 3 

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mM MgCl2, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 1 g L-1 essentially fatty-
acid free BSA, 60 mM K-lactobionate, 110 mM mannitol, 0.3 mM DTT, pH 7.1). 
Respiration medium will be added to the high-resolution Oxygraph 2k (OROBOROS 
INSTRUMENTS, Innsbruck, Austria), equilibrated to the required experimental 
temperature (37 °C) and oxygenated with atmospheric oxygen to ~600 µM O2. The 
permeabilized muscle fiber bundle will be blotted dry, weighed and added to the 
chamber. Respiratory function of mitochondria and flux ratios will be determined using 
the following titration protocol (final concentrations and assessment in parenthesis). 
After assessment of resting respiration, the following reagents will be added: 
glutamate/malate (10mM/2mM; leak rate with complex I supported respiration); ADP (1-
5 mM; state 3, complex I supported respiration; OXPHOS capacity C-I); cytochrome c 
(10 µM; integrity of the outer mitochondrial membrane); succinate (10 mM; state 3, 
complex II supported respiration; OXPHOS capacity C-I+II); oligomycin (2 µg mL-1; 
inhibition of ATP synthase); stepwise titration with the uncoupler FCCP (0.5-5 µM; ETC 
capacity and limitation of OXPHOS relative to ETC by the phosphorylation system); 
rotenone (0.5µM; complex II-only supported respiration); antimycin A (2.5 µM; non-
respiratory residual oxygen consumption). The respiratory control ratio (RCR), or 
OXPHOS capacity, will be determined by calculating the ratio of state 3 (with complex I 
+ II substrates, and ADP) to state 4 (oligomycin-inhibited respiration); the ETC capacity 
will be determined using the ratio of uncoupled respiration to state 3 respiration. In 
addition to the calculation of flux control ratios, respiration will be related to 
mitochondrial DNA content (21) as well as citrate synthase activity (22). 
Determination of respiratory complex content and activities using Blue Native 
Polyacrylamide Gel Electrophoresis (BN-PAGE).  
For determination of the content and activity of the ETC complexes we will follow the 
protocol as described by Schagger et al. with some modification (23, 24) . Briefly, ~30 
mg of muscle tissue will be homogenized in buffer 1 (20 mM MOPS, 440 mM sucrose, 1 
mM EDTA and 0.5 mM PMSF, pH 7.2,) at 4°C. The homogenates will be centrifuged at 
20,000 x g for 20 min. The pellet will be re-suspended in 80 µl of buffer 2 (1 M 
aminocaproic acid, 50 mM Bis-tris and 0.5 mM PMSF, pH 7.0). The membranes will be 
solubilized by the addition of 30 µl 10% n-dodecylmaltoside. Mitochondrial suspensions 
will be incubated on ice for 30 min and vortexed every 5 min, followed by 
ultracentrifugation for 25 min at 100,000 x g (Beckman, Optima LE-80K). The 
solubilized mitochondrial membrane proteins (in 5% w/v Coomassie brilliant blue G-250 
and 1M aminocaproic acid) will be separated by BN-PAGE using a 3-12 % gradient gel. 
The anode buffer is comprised of 50 mM Bis-Tris, pH 7.0. The cathode buffer is 
comprised of 50 mM tricine, 15 mM Bis-Tris, and Coomassie brilliant blue G-250 (0.02% 
w/v), pH 7.0. Samples will be electrophoresed at 90 V for 20 min, and thereafter at 170 
V for 2 h, at 4°C. For determination of the content of all of the respiratory complexes, 
gels will be blotted onto polyvinylidene fluoride membranes, which will then be probed 
with the MitoProfile Human Total OXPHOS Detection Kit (Mitosciences). For 
determination of enzymatic activity of respiratory complexes I, II, IV and V (complex III 
activity cannot be assessed using this technique), in-gel enzymatic colorimetric 
reactions will be performed. Gels will be incubated overnight at room temperature with 
the following solutions: Complex I: 2 mM Tris-HCl, pH 7.4, 0.1 mg/ml NADH, and 2.5 

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mg/ml NTB (nitrotetrazolium blue). Complex II: 4.5 mM EDTA, 10 mM KCN, 0.2 mM 
phenazine methasulfate, 84 mM succinic acid and 50 mM NTB in 1.5mM phosphate 
buffer, pH 7.4. Complex IV: 5 mg 3:30-Diamidobenzidine tetrahydrochloride (DAB) 
dissolved in 9 ml phosphate buffer (0.05 M, pH 7.4), 1 ml catalase (20 µg/ml), 10 mg 
cytochrome c, and 750 mg sucrose. Complex V: 35 mM Tris, 270 mM glycine, 14mM 
MgSO4, 0.2% Pb(NO3)2, and 8 mM ATP, pH 7.8. Gels will be washed in distilled water 
and the bands quantified by digital imaging using a CCD camera. Activity and content 
will be normalized to muscle weight, mitochondrial DNA content (21) as well as citrate 
synthase activity (22). 
3.   Titan analysis:   The integrity and phosphorylation of Titan subunits of the 
sarcomere will be analyzed in Dr. Ottenheijm’s laboratory at the University of Arizona. 
Dr. Ottenheijm’s group has extensive experience making this unique measurement  and 
will follow procedures as they have previously published.(25, 26) 
Statistical Analysis: 
 
We will use a mixed effects model with random intercept for each subject to evaluate 
the fixed effects of stimulation and fiber type on single-fiber force mechanics. For other 
dependent variables (i.e. mitochondrial respiration, RNA, protein quantification), we will 
test differences between the stimulated and unstimulated tissue samples using paired 
parametric T tests when the data meet the assumptions of parametric analysis, or non-
parametric tests if the data are not normally distributed.  
 
7. 
Possible Discomforts and Risks: 
The risks of diaphragm biopsy include failure of the biopsy site to close, bleeding, 
phrenic nerve injury, and diaphragm dysfunction.  The portion of the diaphragm from 
which the biopsy is obtained (6 mm by 20 mm section of tissue) will be isolated from the 
remaining muscle by surgical staples, prior to excision of the full-thickness biopsy 
sample.  The site will also be reinforced with sutures, minimizing the risks of bleeding or 
failure of the biopsy site to close. 
While human diaphragm biopsies are a relatively new model to study muscle, numerous 
workers have obtained non-therapeutic, experimental human diaphragm tissue samples 
during routine surgeries, as proposed in this work (27-35) and no complications were 
reported.  We have recently completed a study in which two diaphragm samples were 
obtained from 5 subjects at the beginning and end of CT surgery, and there were no 
intra or postoperative complications associated with the diaphragm biopsies. The 
addition of indirect phrenic nerve stimulation to the protocol poses no significant risk to 
the subjects. 
Respiratory testing (pulmonary function testing & maximum inspiratory pressure testing) 
may elicit transient fatigue or shortness of breath that resolves within several seconds of 
completion.  Each training set typically lasts less than 15 seconds, and at least two to 
three minutes of rest will be provided between trials to minimize feelings of exertion, and 
perceived exertion will be monitored during training sessions.   

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This study may include risks that are unknown at this time. 
 
8. 
Possible Benefits: 
There are no direct benefits to the subjects for participating in this study, but our results 
will lead to a better understanding of how MV affects human diaphragm and may lead to 
improved clinical care for MV patients. The potential importance of optimizing 
diaphragmatic function prior to surgery was show by Hulzebos (2006). These 
researchers showed that 2 weeks of specific diaphragm training prior to coronary 
bypass surgery significantly reduced the incidence of post operative pneumonia and 
duration of hospitalization. The molecular basis of the improved post-operative 
diaphragmatic function following preoperative conditioning is unknown. 
 
9. 
Conflict of Interest: 
No real or potential conflict of interest exists for any investigator with regard to this 
research project. 
     
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