Effect of Diaphragm Stimulation During Surgery

The Effect of Intermittent Hemidiaphragm Stimulation During Surgery on Mitochondrial Function, Single Fiber Contractile Force and Catabolic Pathways in Humans

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.

Study Overview

• Status: Completed
• Conditions: Mechanical Ventilation Complication; Diaphragm Injury
• Intervention / Treatment: Other: Electrical stimulation of hemidiaphragm

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.

• Study Type: Interventional
• Enrollment (Actual): 25
• Phase: Not Applicable

Contacts and Locations

Study Locations

• United States
  • Florida
    • Gainesville, Florida, United States, 32610
      • University of Florida

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 85 years (Adult, Older Adult)
• Accepts Healthy Volunteers: No

Description

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,

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Prevention
• Allocation: Non-Randomized
• Interventional Model: Parallel Assignment
• Masking: None (Open Label)

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Stimulation; Electrical stimulation of hemidiaphragm	Other: Electrical stimulation of hemidiaphragm; Electrical impulses
No Intervention: Control; No stimulation of hemidiaphragm

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
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.	Up to eight hours
Aconitase Activity	In order to evaluate mitochondrial damage, actonitase activity will be measured spectrophotometrically. It will be quantified as units/mg protein.	Up to eight hours
Lipid Peroxidation	Lipid peroxidation will be assessed by measuring 4-hydroxy-2-nonenal-modified proteins. It will be quantified as arbitrary optical density units.	Up to eight hours
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.	Up to eight hours
Single Diaphragm Fiber, Specific Force	Specific force of single diaphragm fibers represents the force generated per unit area.	Up to eight hours
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).	Up to eight hours
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.	Up to eight hours
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.	Up to eight hours
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).	Up to eight hours
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	Up to eight hours
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.	Up to eight hours
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.	Up to eight hours
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.	Up to eight hours
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.	Up to eight hours
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.	Up to eight hours

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Caspase-9	Caspase-9 will be measured with Western Blot anaylsis and will be presented as percent difference in expression.	Up to eight hours
MurF1	MurF1 will be measured with Western Blot anaylsis and will be presented as percent difference in expression.	Up to eight hours
Foxo-3	Foxo-3 will be measured with Western Blot anaylsis and will be presented as percent difference in expression.	Up to eight hours
28SrRNA	28SrRNA will be measured with Western Blot anaylsis and will be presented as percent difference in expression.	Up to eight hours
18SrRNA	18SrRNA will be measured with Western Blot anaylsis and will be presented as percent difference in expression.	Up to eight hours
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.	Up to eight hours
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.	Up to eight hours
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.	Up to eight hours
Titin Size	Titin integrity will be assessed. A relative titin size will be quantified in nm.	Up to eight hours
20S Proteasome	20S proteasome will be measured with Western Blot anaylsis and will be presented as percent difference in expression.	Up to eight hours
26S Proteasome	26S proteasome will be measured with Western Blot anaylsis and will be presented as percent difference in expression.	Up to eight hours
45S Pre-rRNA	45S pre-rRNA will be measured with Western Blot anaylsis and will be presented as percent difference in expression.	Up to eight hours
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.	Up to eight hours

Collaborators and Investigators

• Sponsor: University of Florida
• Collaborators: National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS); National Institutes of Health (NIH); University of Arizona
• Investigators: Principal Investigator: Anatole D Martin, PhD, University of Florida; Principal Investigator: Thomas M Beaver, MD, University of Florida; Principal Investigator: Barbara Smith, PhD, PT, University of Florida

Study record dates

Study Major Dates

• Study Start (Actual): February 14, 2018
• Primary Completion (Actual): May 31, 2022
• Study Completion (Actual): December 31, 2023

Study Registration Dates

• First Submitted: September 26, 2017
• First Submitted That Met QC Criteria: October 4, 2017
• First Posted (Actual): October 5, 2017

Study Record Updates

• Last Update Posted (Actual): June 26, 2024
• Last Update Submitted That Met QC Criteria: June 13, 2024
• Last Verified: June 1, 2024

More Information

Terms related to this study

• Keywords: Mechanical ventilation; Diaphragm weakness; Difficulty weaning from mechanical ventilation; Diaphragm stimulation
• Other Study ID Numbers: IRB201602186-N; R01AR072328 (U.S. NIH Grant/Contract)

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No