Diaphragm Injury, Mechanical Ventilation Complication
Conditions
Keywords
Mechanical ventilation, Diaphragm weakness, Difficulty weaning from mechanical ventilation, Diaphragm stimulation
Brief summary
During major surgical procedures general anesthesia is used to make the patient unconscious. General anesthesia insures that the patient is unaware of any pain caused by surgery. General anesthesia also prevents the patient from moving to prevent any potential surgical error. At the same time general anesthesia makes it impossible for the patient to breathe. To help the patient breathe a breathing tube is placed into the patient's airway and connected to the mechanical ventilator. A mechanical ventilator is an artificial breathing pump, which delivers gas into a patient's airways. The purpose of this research study is to determine if brief periods of diaphragm stimulation can prevent diaphragm problems caused by the use of mechanical ventilators and surgery. To answer this question the changes in the genes responsible for maintaining diaphragm function will be studied. A gene is the code present in each cell in your body and controls the behavior of that cell. In addition, the changes in the contractile properties of muscle fibers will be studied. The results from this study may help develop new treatments to prevent diaphragm weakness resulting from mechanical ventilation use.
Detailed description
Although mechanical ventilation (MV) is life-sustaining, it comes with a cost. MV dramatically reduces diaphragm contractility, induces ventilator-induced diaphragm dysfunction (VIDD) and sometimes leads to weaning failure. VIDD includes reduced mitochondrial respiration and increased oxidative stress, muscle fiber damage and decreased diaphragm force production. In animal models, intermittent diaphragm contraction during MV support attenuates VIDD. However, there are only limited data addressing this problem in humans. Here, the study team propose to directly test the hypothesis that intermittent electrical stimulation (ES) of the human hemidiaphragm during prolonged cardiac surgeries with MV support prevents/attenuates VIDD in the active hemidiaphragm. Mitochondrial function is central to energy metabolism and skeletal muscle function in a chronically active muscle, such as the diaphragm. Although abnormal mitochondrial function is thought to precipitate VIDD in animal models, limited data are available concerning mitochondrial contributions to VIDD in humans. Of even greater importance, there are no interventions available to attenuate these defects in humans. Here, the study team will test the impact of an innovative experimental treatment, intermittent electrical stimulation (ES) of the hemidiaphragm during prolonged surgeries with MV, on mitochondrial function, single fiber contractile properties and catabolic muscle pathways in human diaphragm. Using a within-subjects experimental design, muscle samples from a stimulated hemidiaphragms will be compared with samples from the unstimulated hemidiaphragm. The study team will investigate mitochondrial dysfunction and oxidative stress during prolonged CTS/MV, and the potential of ES to attenuate or prevent VIDD. Next, the study team will investigate the effects of ES on single fiber contractile properties and Titin integrity. Finally, the study team will study the effect of ES on proteolytic pathways (caspase, calpain and ubiquitin-proteasome) and ribosomal RNA markers of decreased protein synthesis implicated in VIDD.
Interventions
OTHERElectrical stimulation of hemidiaphragm
Electrical impulses
Sponsors
National Institutes of Health (NIH)
University of Arizona
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
University of Florida
Study design
Allocation
NON_RANDOMIZED
Intervention model
PARALLEL
Primary purpose
PREVENTION
Masking
NONE
Intervention model description
This is a within subjects design. One side of each subject's diaphragm will be stimulated. The other side of the subject's diaphragm will not be stimulated and will therefore serve as the control. Biopsies will be taken from both sides and compared.
Eligibility
Sex/Gender
ALL
Age
18 Years to 85 Years
Healthy volunteers
No
Inclusion criteria
* Patients undergoing complex, elective prolonged surgeries, usually lasting 5-8 hours or longer, including lung transplants (e.g. valveoplasty, coronary artery bypass and/or aortic repairs)
Exclusion criteria
* history of prior surgery to the diaphragm or pleura; * 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 \[will not apply to transplant patients\] * a diagnosis of chronic heart failure (NYHA class IV) * clinical diagnosis of other lung disease (cystic fibrosis, bronchiectasis, lung cancer; etc.) \[will not apply to transplant patients\] * renal insufficiency (serum creatinine \> 1.6 mg/dl); * severe hepatic disease (any liver function tests \> 1.5 times the upper limit of normal); * undernourishment (body mass index \< 20 kg/m2), * chronic uncontrolled or poorly controlled metabolic diseases (e.g., diabetes, hypo- or hyperthyroidism) * orthopedic diseases, suspected paraneoplastic or myopathic syndromes, * if in the surgeons' judgment the patients' clinical status warrants, diaphragm stimulation will be stopped and biopsies will not be obtained,
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Atrogin 1 | Up to eight hours | Atrogin 1 will be measured with Jess protein immunoassay analysis, normalized to total protein, and will be presented as the corrected peak area (AU) in stimulated and unstimulated hemidiaphragm muscle fibers. |
| Mitochondrial Respiration | Up to eight hours | High-resolution respirometry will be used to assess mitochondrial respiration of permeablilized diaphragm bundles. Addition of substrate medium to the Oroboros O2K respirometry instrument enables quantification of leak respiration and peak uncoupled respiration, expressed as pmol oxygen/sec/mg wet weight. |
| Aconitase Activity | Up to eight hours | In order to evaluate mitochondrial damage, actonitase activity will be measured spectrophotometrically. It will be quantified as units/mg protein. |
| Lipid Peroxidation | Up to eight hours | Lipid peroxidation will be assessed by measuring 4-hydroxy-2-nonenal-modified proteins. It will be quantified as arbitrary optical density units. |
| Citrate Cynthase Activity | Up to eight hours | Changes in electron transport chain will be assessed by measuring citrate cynthase activity. It will be quantified as nmol/mg protein/min. |
| Single Diaphragm Fiber, Specific Force | Up to eight hours | Specific force of single diaphragm fibers represents the force generated per unit area. |
| Single Diaphragm Fiber, Rate of Tension Redevelopment | Up to eight hours | Single diaphragm fiber mechanical force properties will be measured. The rate of tension redevelopment is quantified as s\^(-1). |
| Calcium Sensitivity (pCa50) | Up to eight hours | The pCa50 value is the logarithmic scale of pCa (sensitivity of Ca+2) at which half-maximal force generation was obtained. The pCa value is calculated as the -log10\[Ca (nm)\]; the pCa50 is the -log10\[Ca (nm)\] at which half-maximal force is generated. |
| Difference in Total Titin to Myosin Heavy Chain Ratio | Up to eight hours | The quantities of total titin protein and myosin heavy chain protein content in homogenized diaphragm fiber specimens were measured and then calculated as a ratio of total titin to myosin heavy chain content (unitless value). The statistical approach was selected apriori as the difference of the ratio between the stimulated and unstimulated sides. |
| Difference in Titin Exon Composition | Up to eight hours | The composition of titin exons will be assessed and quantified via real-time polymerase chain reaction (qPCR). The N2A and tT2 will be calculated as a percentage of total titin. |
| Difference in Calpain 1 Protein Content | Up to eight hours | Calpain 1 (mu-calpain) will be measured with Western Blot analysis and will be presented as percent of total intensity in stimulated and unstimulated hemidiaphragms |
| Difference in Calpain 2 Protein Content | Up to eight hours | Calpain 2 will be measured with automated, capillary-based immunoassay using a Jess System, normalized to total protein, and will be presented as an area of corrected peak (AU) in stimulated and unstimulated hemidiaphragms. |
| Difference in Calpain 3 Protein Content | Up to eight hours | Calpain 3 will be measured with Western Blot analysis and will be presented as a ratio of cleaved to total calpain 3 (unitless value) in stimulated and unstimulated hemidiaphragms. |
| Difference in Caspase-3 Protein Content | Up to eight hours | Caspase-3 will be measured with Western Blot analysis, normalized to total protein loaded in each lane, and will be presented as an area of corrected peak (AU) in stimulated and unstimulated hemidiaphragm muscle fibers. |
| Difference in Titin Binding Protein Content | Up to eight hours | The content of titin binding proteins will be quantified via Western blot. It will be normalized to a reference protein (GAPDH) and presented as optical intensity (AU). |
Other
| Measure | Time frame | Description |
|---|---|---|
| Cytochrome c Oxidase (COX) Activity | Up to eight hours | Changes in electron transport chain will be assessed by measuring cytochrome c oxidase (COX) activity. It will be quantifed as Units/mcg protein. |
| Nuclear DNA Mutation Frequency | Up to eight hours | Long-Amplicon quantitative PCR will be used to measure the frequency of nuclear DNA mutations. It will be quantified as number of lesions/10 kilobases. |
| Titin Size | Up to eight hours | Titin integrity will be assessed. A relative titin size will be quantified in nm. |
| Caspase-9 | Up to eight hours | Caspase-9 will be measured with Western Blot anaylsis and will be presented as percent difference in expression. |
| 20S Proteasome | Up to eight hours | 20S proteasome will be measured with Western Blot anaylsis and will be presented as percent difference in expression. |
| 26S Proteasome | Up to eight hours | 26S proteasome will be measured with Western Blot anaylsis and will be presented as percent difference in expression. |
| 28SrRNA | Up to eight hours | 28SrRNA will be measured with Western Blot anaylsis and will be presented as percent difference in expression. |
| 18SrRNA | Up to eight hours | 18SrRNA will be measured with Western Blot anaylsis and will be presented as percent difference in expression. |
| Foxo-3 | Up to eight hours | Foxo-3 will be measured with Western Blot anaylsis and will be presented as percent difference in expression. |
| 45S Pre-rRNA | Up to eight hours | 45S pre-rRNA will be measured with Western Blot anaylsis and will be presented as percent difference in expression. |
| Mitochondrial DNA Mutation Frequency | Up to eight hours | Long-Amplicon quantitative PCR will be used to measure the frequency of mitochondrial DNA mutations. It will be quantified as number of lesions/10 kilobases. |
| MurF1 | Up to eight hours | MurF1 will be measured with Western Blot anaylsis and will be presented as percent difference in expression. |
| Mitochondrial Reactive Oxygen Species Production | Up to eight hours | Mitochondrial reactive oxygen species (ROS) production will be assessed using an in situ approach to measure hydrogen peroxide production in permeabilized diaphragm skeletal muscle fiber bundles. It will be quantified as pmol/min/mg dry weight. |
Countries
United States
Participant flow
Participants by arm
| Arm | Count |
|---|---|
| Participants Undergoing Elective, Open Cardiothoracic Surgical Procedures Lasting 4 Hours or Greater Patients undergoing complex, elective prolonged surgeries, usually lasting 5-8 hours or longer, including lung transplants (e.g. valvuloplasty, coronary artery bypass and/or aortic repairs) | 21 |
| Total | 21 |
Baseline characteristics
| Characteristic | Participants Undergoing Elective, Open Cardiothoracic Surgical Procedures Lasting 4 Hours or Greater |
|---|---|
| Age, Categorical <=18 years | 0 Participants |
| Age, Categorical >=65 years | 6 Participants |
| Age, Categorical Between 18 and 65 years | 15 Participants |
| Age, Continuous | 59 Number STANDARD_DEVIATION 11.4 |
| Body Mass Index | 29.8 Kg/m^2 STANDARD_DEVIATION 3.96 |
| Cardiopulmonary Bypass | 137 Minutes STANDARD_DEVIATION 65 |
| Core Temperature at Biopsy | 36.5 Degrees Celsius STANDARD_DEVIATION 0.8 |
| Ethnicity (NIH/OMB) Hispanic or Latino | 2 Participants |
| Ethnicity (NIH/OMB) Not Hispanic or Latino | 19 Participants |
| Ethnicity (NIH/OMB) Unknown or Not Reported | 0 Participants |
| % Forced Expiratory Volume | 83.2 Percentage of predicted STANDARD_DEVIATION 15 |
| %Force Vital Capacity | 86.6 Percentage of predicted value STANDARD_DEVIATION 14.4 |
| Intubation to Biopsy | 278 Minutes STANDARD_DEVIATION 65 |
| Intubation to Frist Stimulation | 98 Minute STANDARD_DEVIATION 33 |
| Maximal Inspiratory Pressure | 90.6 cm H2O STANDARD_DEVIATION 23 |
| Minimum Core Temperature | 33.5 Degrees Celsius STANDARD_DEVIATION 2.4 |
| Number of stimulations | 6.2 Number of Stimulations STANDARD_DEVIATION 1.9 |
| Race (NIH/OMB) American Indian or Alaska Native | 0 Participants |
| Race (NIH/OMB) Asian | 1 Participants |
| Race (NIH/OMB) Black or African American | 2 Participants |
| Race (NIH/OMB) More than one race | 0 Participants |
| Race (NIH/OMB) Native Hawaiian or Other Pacific Islander | 0 Participants |
| Race (NIH/OMB) Unknown or Not Reported | 0 Participants |
| Race (NIH/OMB) White | 18 Participants |
| Region of Enrollment United States | 21 participants |
| Sex: Female, Male Female | 9 Participants |
| Sex: Female, Male Male | 12 Participants |
| Stimulation Current Intensity | 18.0 mA STANDARD_DEVIATION 5.4 |
Adverse events
| Event type | EG000 affected / at risk |
|---|---|
| deaths Total, all-cause mortality | 0 / 21 |
| other Total, other adverse events | 21 / 21 |
| serious Total, serious adverse events | 14 / 21 |
Outcome results
Primary
Aconitase Activity
In order to evaluate mitochondrial damage, actonitase activity will be measured spectrophotometrically. It will be quantified as units/mg protein.
Time frame: Up to eight hours
Population: Aconitase activity was measured in the stimulated (n=20) and unstimulated (n=18) hemidiaphragms from subjects who completed the entire intra-operative stimulation protocol. Insufficient tissue was available in two unstimulated hemidiaphragms. Tissue obtained from one participant contained large proportions of intramuscular fat that prevented a measurement.
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Control | Aconitase Activity | 0.175 mU/mg protein | Standard Deviation 0.01 |
| Stimulation | Aconitase Activity | 0.196 mU/mg protein | Standard Deviation 0.01 |
Primary
Atrogin 1
Atrogin 1 will be measured with Jess protein immunoassay analysis, normalized to total protein, and will be presented as the corrected peak area (AU) in stimulated and unstimulated hemidiaphragm muscle fibers.
Time frame: Up to eight hours
Population: Protein content of AKT, p-AKT, calpain-2, calpain-3, caspase-3, atrogin-1 was measured by automated, capillary-based immunoassay using a Jess System. Samples were analyzed from the stimulated and unstimulated hemidiaphragms of the first 9 participants and normalized to total protein.
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Control | Atrogin 1 | 634046 AU | Standard Deviation 377885 |
| Stimulation | Atrogin 1 | 675965 AU | Standard Deviation 379914 |
Primary
Calcium Sensitivity (pCa50)
The pCa50 value is the logarithmic scale of pCa (sensitivity of Ca+2) at which half-maximal force generation was obtained. The pCa value is calculated as the -log10\[Ca (nm)\]; the pCa50 is the -log10\[Ca (nm)\] at which half-maximal force is generated.
Time frame: Up to eight hours
Population: pCa50 represents the sensitivity of pCa (concentration of Ca+2: -log10\[Ca+2\]) at which half-maximal force generation was obtained. It was calculated from an average of 10 slow and 10 fast single fibers obtained from each hemidiaphragm, per subject. Of the 21 patients who completed intraoperative stimulation procedures, single fiber measurements were not obtained in two participants due to unavailability of study personnel (n=2) and non-cardiac surgery (n=1).
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Control | Calcium Sensitivity (pCa50) | Slow Muscle Fibers | 5.805 -log10[Ca2+] | Standard Error 0.158 |
| Control | Calcium Sensitivity (pCa50) | Fast Muscle Fibers | 5.888 -log10[Ca2+] | Standard Error 0.115 |
| Stimulation | Calcium Sensitivity (pCa50) | Slow Muscle Fibers | 5.797 -log10[Ca2+] | Standard Error 0.161 |
| Stimulation | Calcium Sensitivity (pCa50) | Fast Muscle Fibers | 5.887 -log10[Ca2+] | Standard Error 0.121 |
Primary
Citrate Cynthase Activity
Changes in electron transport chain will be assessed by measuring citrate cynthase activity. It will be quantified as nmol/mg protein/min.
Time frame: Up to eight hours
Population: Citrate synthase activity was measured in the stimulated (n=20) and unstimulated (n=18) hemidiaphragms from subjects who completed the entire intra-operative stimulation protocol. Insufficient tissue was available in two unstimulated hemidiaphragms. Tissue obtained from one participant contained large proportions of intramuscular fat that prevented a measurement.
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Control | Citrate Cynthase Activity | 0.084 nmol/mg protein/min | Standard Deviation 0.006 |
| Stimulation | Citrate Cynthase Activity | 0.082 nmol/mg protein/min | Standard Deviation 0.004 |
Primary
Difference in Calpain 1 Protein Content
Calpain 1 (mu-calpain) will be measured with Western Blot analysis and will be presented as percent of total intensity in stimulated and unstimulated hemidiaphragms
Time frame: Up to eight hours
Population: Calpain-1 (µ-calpain) protein content was measured by traditional Western blot. Samples were analyzed from the stimulated and unstimulated hemidiaphragms of the first 9 participants and normalized to total protein. The statistical approach selected to test the effect of stimulation on oxidative stress-related protein content was selected apriori as the difference between the stimulated and unstimulated sides. Full-length (80kDa) and truncated (76 kDa) isoforms are reported.
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Control | Difference in Calpain 1 Protein Content | Full length (80 kDa) CLP1 isoform | 74 Percent of total Intensity | Standard Deviation 5.4 |
| Control | Difference in Calpain 1 Protein Content | Truncated (76 kDa) CLP1 isoform | 14.1 Percent of total Intensity | Standard Deviation 2.6 |
| Stimulation | Difference in Calpain 1 Protein Content | Full length (80 kDa) CLP1 isoform | 72.4 Percent of total Intensity | Standard Deviation 5.4 |
| Stimulation | Difference in Calpain 1 Protein Content | Truncated (76 kDa) CLP1 isoform | 15 Percent of total Intensity | Standard Deviation 3 |
Primary
Difference in Calpain 2 Protein Content
Calpain 2 will be measured with automated, capillary-based immunoassay using a Jess System, normalized to total protein, and will be presented as an area of corrected peak (AU) in stimulated and unstimulated hemidiaphragms.
Time frame: Up to eight hours
Population: Protein content of AKT, p-AKT, calpain-2, calpain-3, caspase-3, atrogin-1 was measured by automated, capillary-based immunoassay using a Jess System. Samples were analyzed from the stimulated and unstimulated hemidiaphragms of the first 9 participants and normalized to total protein.
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Control | Difference in Calpain 2 Protein Content | 274938 AU | Standard Deviation 118636 |
| Stimulation | Difference in Calpain 2 Protein Content | 357182 AU | Standard Deviation 219400 |
Primary
Difference in Calpain 3 Protein Content
Calpain 3 will be measured with Western Blot analysis and will be presented as a ratio of cleaved to total calpain 3 (unitless value) in stimulated and unstimulated hemidiaphragms.
Time frame: Up to eight hours
Population: Protein content of AKT, p-AKT, calpain-2, calpain-3, caspase-3, atrogin-1 was measured by automated, capillary-based immunoassay using a Jess System. Samples were analyzed from the stimulated and unstimulated hemidiaphragms of the first 9 participants and normalized to total protein. The statistical approach selected to test the effect of stimulation on oxidative stress-related protein was selected apriori as the difference between the stimulated and unstimulated sides.
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Control | Difference in Calpain 3 Protein Content | 2.98 ratio of cleaved/total calpain 3 | Standard Deviation 1.35 |
| Stimulation | Difference in Calpain 3 Protein Content | 3.21 ratio of cleaved/total calpain 3 | Standard Deviation 1.9 |
Primary
Difference in Caspase-3 Protein Content
Caspase-3 will be measured with Western Blot analysis, normalized to total protein loaded in each lane, and will be presented as an area of corrected peak (AU) in stimulated and unstimulated hemidiaphragm muscle fibers.
Time frame: Up to eight hours
Population: Protein content of calpain-2, calpain-3, caspase-3, atrogin-1 was measured by automated, capillary-based immunoassay using a Jess System. Samples were analyzed from the stimulated and unstimulated hemidiaphragms of the first 9 participants and normalized to total protein.
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Control | Difference in Caspase-3 Protein Content | 274328 AU | Standard Deviation 70600 |
| Stimulation | Difference in Caspase-3 Protein Content | 317726 AU | Standard Deviation 164524 |
Primary
Difference in Titin Binding Protein Content
The content of titin binding proteins will be quantified via Western blot. It will be normalized to a reference protein (GAPDH) and presented as optical intensity (AU).
Time frame: Up to eight hours
Population: Content of titin binding proteins including MARP1 (ANKRD-1) and MARP2 (ANKRD-2) were measured in homogenized tissue obtained from the stimulated and unstimulated hemidiaphragms of the first 11 subjects and quantified using western blot. The results were normalized to the optical intensity of GAPDH (AU).
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Control | Difference in Titin Binding Protein Content | ANKRD1 | 0.0165 AU | Standard Deviation 0.0279 |
| Control | Difference in Titin Binding Protein Content | ANKRD2 | 1.39 AU | Standard Deviation 0.44 |
| Stimulation | Difference in Titin Binding Protein Content | ANKRD1 | 0.0167 AU | Standard Deviation 0.025 |
| Stimulation | Difference in Titin Binding Protein Content | ANKRD2 | 1.28 AU | Standard Deviation 0.52 |
Primary
Difference in Titin Exon Composition
The composition of titin exons will be assessed and quantified via real-time polymerase chain reaction (qPCR). The N2A and tT2 will be calculated as a percentage of total titin.
Time frame: Up to eight hours
Population: The composition of the N2A and tT2 exons of the titin protein was obtained from homogenized tissue of the unstimulated and stimulated hemidiaphragms of the first 11 subjects. The relative abundance of each exon was calculated as a percentage of total titin.
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Control | Difference in Titin Exon Composition | Percentage N2A | 96.1 Percent of total titin | Standard Deviation 9.3 |
| Control | Difference in Titin Exon Composition | Percentage tT2 | 3.9 Percent of total titin | Standard Deviation 9.3 |
| Stimulation | Difference in Titin Exon Composition | Percentage N2A | 96.3 Percent of total titin | Standard Deviation 6.9 |
| Stimulation | Difference in Titin Exon Composition | Percentage tT2 | 3.7 Percent of total titin | Standard Deviation 6.9 |
Primary
Difference in Total Titin to Myosin Heavy Chain Ratio
The quantities of total titin protein and myosin heavy chain protein content in homogenized diaphragm fiber specimens were measured and then calculated as a ratio of total titin to myosin heavy chain content (unitless value). The statistical approach was selected apriori as the difference of the ratio between the stimulated and unstimulated sides.
Time frame: Up to eight hours
Population: The quantities of total titin protein and myosin heavy chain protein content in homogenized diaphragm fiber specimens were measured and then calculated as a ratio of total titin to myosin heavy chain content. The statistical approach selected to test the effect of stimulation on titin:myosin heavy chain ratio was selected apriori as the difference between the stimulated and unstimulated sides.
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Control | Difference in Total Titin to Myosin Heavy Chain Ratio | 0.111 Total titan/MHC Ratio | Standard Deviation 0.042 |
| Stimulation | Difference in Total Titin to Myosin Heavy Chain Ratio | 0.103 Total titan/MHC Ratio | Standard Deviation 0.044 |
Primary
Lipid Peroxidation
Lipid peroxidation will be assessed by measuring 4-hydroxy-2-nonenal-modified proteins. It will be quantified as arbitrary optical density units.
Time frame: Up to eight hours
Population: Protein content was measured by traditional Western blot. Samples were analyzed from the stimulated and unstimulated hemidiaphragms of the first 11 participants. Protein quantification was normalized to total protein and optical density units of the stimulated and unstimulated hemidiaphragm were reported.
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Control | Lipid Peroxidation | 1.037 Arbitrary optical density units (U) | Standard Deviation 0.145 |
| Stimulation | Lipid Peroxidation | 1.061 Arbitrary optical density units (U) | Standard Deviation 0.134 |
Primary
Mitochondrial Respiration
High-resolution respirometry will be used to assess mitochondrial respiration of permeablilized diaphragm bundles. Addition of substrate medium to the Oroboros O2K respirometry instrument enables quantification of leak respiration and peak uncoupled respiration, expressed as pmol oxygen/sec/mg wet weight.
Time frame: Up to eight hours
Population: Mitochondrial respiration was measured in the stimulated (n=19) and unstimulated (n=19) hemidiaphragms from subjects who completed the entire intra-operative stimulation protocol. Biopsy tissue was specifically reserved for analysis. From the full patient sample, insufficient contractile tissue was available to measure mitochondrial respiration in two unstimulated and two stimulated hemidiaphragms.
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Control | Mitochondrial Respiration | Leak Respiration | 3.5 pmol/s/mg wwt | Standard Deviation 0.7 |
| Control | Mitochondrial Respiration | ECI+II | 31.1 pmol/s/mg wwt | Standard Deviation 3.5 |
| Stimulation | Mitochondrial Respiration | Leak Respiration | 2.9 pmol/s/mg wwt | Standard Deviation 0.6 |
| Stimulation | Mitochondrial Respiration | ECI+II | 27.1 pmol/s/mg wwt | Standard Deviation 2.1 |
Primary
Single Diaphragm Fiber, Rate of Tension Redevelopment
Single diaphragm fiber mechanical force properties will be measured. The rate of tension redevelopment is quantified as s\^(-1).
Time frame: Up to eight hours
Population: Rate constant of tension development (ktr) represents the half-time of force recovery after release of a standardized stretch. ktr was calculated from an average of 10 slow and 10 fast single fibers obtained from each hemidiaphragm, per subject. Of the 21 patients who completed intraoperative stimulation procedures, single fiber measurements were not obtained in two participants due to unavailability of study personnel (n=2) and non-cardiac surgery (n=1).
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Control | Single Diaphragm Fiber, Rate of Tension Redevelopment | Fast Fibers | 4.744 per second (force recovery rate) | Standard Error 1.322 |
| Control | Single Diaphragm Fiber, Rate of Tension Redevelopment | Slow Fibers | 1.102 per second (force recovery rate) | Standard Error 0.269 |
| Stimulation | Single Diaphragm Fiber, Rate of Tension Redevelopment | Slow Fibers | 1.143 per second (force recovery rate) | Standard Error 0.236 |
| Stimulation | Single Diaphragm Fiber, Rate of Tension Redevelopment | Fast Fibers | 4.777 per second (force recovery rate) | Standard Error 1.369 |
Primary
Single Diaphragm Fiber, Specific Force
Specific force of single diaphragm fibers represents the force generated per unit area.
Time frame: Up to eight hours
Population: Specific force represents force generation per cross sectional area of single diaphragm fibers and was calculated from an average of 10 slow and 10 fast single fibers obtained from each hemidiaphragm, per subject. Of the 21 patients who completed intraoperative stimulation procedures, single fiber measurements were not obtained in two participants due to unavailability of study personnel (n=2) and non-cardiac surgery (n=1).
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Control | Single Diaphragm Fiber, Specific Force | Slow Fibers | 102.64 kN/m2 | Standard Error 2.38 |
| Control | Single Diaphragm Fiber, Specific Force | Fast Fibers | 124.5 kN/m2 | Standard Error 2.39 |
| Stimulation | Single Diaphragm Fiber, Specific Force | Slow Fibers | 95.85 kN/m2 | Standard Error 4.62 |
| Stimulation | Single Diaphragm Fiber, Specific Force | Fast Fibers | 126.93 kN/m2 | Standard Error 4.3 |
Other Pre-specified
18SrRNA
18SrRNA will be measured with Western Blot anaylsis and will be presented as percent difference in expression.
Time frame: Up to eight hours
Population: Differences in 18SrRNA were not completed due to challenges with cross-species validation of reagents and reference values.
Other Pre-specified
20S Proteasome
20S proteasome will be measured with Western Blot anaylsis and will be presented as percent difference in expression.
Time frame: Up to eight hours
Population: Changes in 20S proteasome protein were not evaluated due to lack of a reliable antibody with preparatory validation studies.
Other Pre-specified
26S Proteasome
26S proteasome will be measured with Western Blot anaylsis and will be presented as percent difference in expression.
Time frame: Up to eight hours
Population: Changes in 26S Proteasome protein were not evaluated due to lack of a reliable antibody with preparatory validation studies.
Other Pre-specified
28SrRNA
28SrRNA will be measured with Western Blot anaylsis and will be presented as percent difference in expression.
Time frame: Up to eight hours
Population: Differences in 28SrRNA were not completed due to challenges with cross-species validation of reagents and reference values.
Other Pre-specified
45S Pre-rRNA
45S pre-rRNA will be measured with Western Blot anaylsis and will be presented as percent difference in expression.
Time frame: Up to eight hours
Population: Changes in 45S pre-rRNA protein were not evaluated due to lack of a reliable antibody with preparatory validation studies.
Other Pre-specified
Caspase-9
Caspase-9 will be measured with Western Blot anaylsis and will be presented as percent difference in expression.
Time frame: Up to eight hours
Population: Changes in Caspase-9 protein were not evaluated due to lack of a reliable antibody with preparatory validation studies.
Other Pre-specified
Cytochrome c Oxidase (COX) Activity
Changes in electron transport chain will be assessed by measuring cytochrome c oxidase (COX) activity. It will be quantifed as Units/mcg protein.
Time frame: Up to eight hours
Population: Limited tissue quantity did not permit analysis.
Other Pre-specified
Foxo-3
Foxo-3 will be measured with Western Blot anaylsis and will be presented as percent difference in expression.
Time frame: Up to eight hours
Population: Changes in Foxo-3 protein were not evaluated due to lack of a reliable antibody with preparatory validation studies.
Other Pre-specified
Mitochondrial DNA Mutation Frequency
Long-Amplicon quantitative PCR will be used to measure the frequency of mitochondrial DNA mutations. It will be quantified as number of lesions/10 kilobases.
Time frame: Up to eight hours
Population: Limited tissue quantity did not permit analysis.
Other Pre-specified
Mitochondrial Reactive Oxygen Species Production
Mitochondrial reactive oxygen species (ROS) production will be assessed using an in situ approach to measure hydrogen peroxide production in permeabilized diaphragm skeletal muscle fiber bundles. It will be quantified as pmol/min/mg dry weight.
Time frame: Up to eight hours
Population: Limited tissue quantity did not permit analysis.
Other Pre-specified
MurF1
MurF1 will be measured with Western Blot anaylsis and will be presented as percent difference in expression.
Time frame: Up to eight hours
Population: Changes in MuRF1 protein were not evaluated due to lack of a reliable antibody with preparatory validation studies.
Other Pre-specified
Nuclear DNA Mutation Frequency
Long-Amplicon quantitative PCR will be used to measure the frequency of nuclear DNA mutations. It will be quantified as number of lesions/10 kilobases.
Time frame: Up to eight hours
Population: Limited tissue quantity did not permit analysis.
Other Pre-specified
Titin Size
Titin integrity will be assessed. A relative titin size will be quantified in nm.
Time frame: Up to eight hours
Population: Limited tissue quantity did not permit analysis.