Effect of Diaphragm Stimulation During Surgery

University of Florida25 enrolled

Overview

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.

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.

Eligibility

Sex: ALLMin age: 18 YearsMax age: 85 Years

Medical Language ↔ Plain English

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,

Outcomes

Primary Outcomes

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

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

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

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

Single Diaphragm Fiber, Specific Force

Specific force of single diaphragm fibers represents the force generated per unit area.

Time frame: 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).

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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).

Time frame: 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

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: Up to eight hours

Effect of Diaphragm Stimulation During Surgery

Overview

Conditions

Interventions

Eligibility

Locations (1)

Outcomes

Primary Outcomes