Intraoperative hemidiaphragm electrical stimulation reduces oxidative stress and upregulates autophagy in surgery patients undergoing mechanical ventilation: exploratory study
© The Author(s) 2016
Received: 28 July 2016
Accepted: 11 October 2016
Published: 26 October 2016
Mechanical ventilation (MV) during a cardio-thoracic surgery contributes to diaphragm muscle dysfunction that impairs weaning and can lead to the ventilator- induced diaphragm dysfunction. Especially, it is critical in older adults who have lower muscle reparative capacity following MV. Reports have shown that the intraoperative intermittent hemidiaphragm electrical stimulation can maintain and/or improve post-surgery diaphragm function. In particular, from a molecular point of view, intermittent electrical stimulation (ES) may reduce oxidative stress and increase regulatory autophagy levels, and therefore improve diaphragm function in animal studies. We have recently shown in humans that intraoperative ES attenuates mitochondrial dysfunction and force decline in single diaphragm muscle fibers. The aim of this study was to investigate an effect of ES on oxidative stress, antioxidant status and autophagy biomarker levels in the human diaphragm during surgery.
One phrenic nerve was simulated with an external cardiac pacer in operated older subjects (62.4 ± 12.9 years) (n = 8) during the surgery. The patients received 30 pulses per min every 30 min. The muscle biopsy was collected from both hemidiaphragms and frozen for further analyses. 4-hydroxynonenal (4-HNE), an oxidative stress marker, and autophagy marker levels (Beclin-1 and the ratio of microtubule-associated protein light chain 3, I and II-LC3 II/I) protein concentrations were detected by the Western Blot technique. Antioxidant enzymatic activity copper-zinc (CuZnSOD) and manganese (MnSOD) superoxide dismutase were analyzed.
Levels of lipid peroxidation (4-HNE) were significantly lower in the stimulated side (p < 0.05). The antioxidant enzyme activities (CuZnSOD and MnSOD) in the stimulated side of the diaphragm were not different than in the unstimulated side (p > 0.05). Additionally, the protein concentrations of Beclin-1 and the LC3 II/I ratio were higher in the stimulated side (p < 0.05).
These results suggest that the intraoperative electrical stimulation decreases oxidative stress levels and upregulates autophagy levels in the stimulated hemidiaphragm. These results may contribute future studies and clinical applications on reducing post-operative diaphragm dysfunction.
Mechanical ventilation (MV) is a life-saving component of modern intensive care and surgery. However, diaphragmatic unloading induced by MV may result in deleterious changes on the cellular level even within the first few hours from the intubation . Diaphragm unloading may contribute to diaphragm muscle dysfunction that impairs weaning and can lead to the ventilator- induced diaphragm dysfunction (VIDD) . Studies have shown that the intermittent intraoperative hemidiaphragm electrical stimulation may maintain or improve post-surgery diaphragm function [3–6]. For example, recently, we were the first to report that intraoperative hemidiaphragm electrical stimulation improved state III (25%) and state IV (42%) mitochondrial respiration in comparison with the unstimulated hemidiaphragm in older adults who underwent cardio-thoracic surgeries . Moreover, single fiber force was improved by 30% in the stimulated side of the diaphragm muscle in the same group of patients . These results warranted further biochemical analyses investigating molecular pathways of these beneficial effects of electrical stimulation on diaphragm function.
Animal studies showed that diaphragmatic inactivity promotes reactive oxygen species (ROS) formation and mitochondrial dysfunction [7, 8] and may contribute to VIDD . Potentially, excessive production of reactive oxygen species may be caused by mechanical inactivity of the respiratory muscles during MV—a state of metabolic oversupply [9, 10]. On the other hand, MV induces oxidative stress that may also reduce mitochondrial oxygen phosphorylation and may lead to reduced overall energy supply to muscle and to cell apoptosis (showed in cultured human diaphragm muscle cells) . However, these molecular pathways have not been well studied in humans undergoing MV.
It has also been shown that autophagy is an important process in maintaining cell homeostasis by disposing cytotoxic elements of malfunctioned cell components e.g. mitochondrial turnover—mitophagy and thereby reducing pathophysiological ROS formation . Importantly, levels of autophagy diminish with age, which may be an additive factor to diaphragm mitochondrial dysfunction in post-operative weaning from MV in older adults .
We hypothesized that intermittent electrical stimulation may reduce deleterious oxidative stress levels and increase basal regulatory autophagy levels, which maybe one of the key factors to improve diaphragm function during cardio-thoracic surgeries in mechanically ventilated patients [5, 6].
Patient demographics and surgery description
MV start to biopsy (min)
Aortic valve replacement
Ascending aortic aneurysm
Aortic graft, aortic valve replacement, CABG
Aortic valve replacement, double CABG
Aortic valve replacement
62.4 ± 12.9
172.6 ± 6.7
92.5 ± 10.6
284.1 ± 53.8
Equal amounts of protein (50 lg/condition) were resolved by 12% sodium dodecyl sulfate (SDS)–gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes, according to a partially modified conventional protocol . The immunodetection included the transfer (20 V for 25 min, per each membrane) and blocking of the membrane with Western Blot (WB) blocking solution (2 h at room temperature). After washing the membranes two times with TBST 1, the blots were incubated with the corresponding primary antibodies (Cell Signaling, USA) against 4-Hydroxynonenal (4-HNE), Peroxysome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), Beclin-1, microtubule-associated protein 1 light chain 3 I and II (LC3) (1:000) and (incubated at 4 °C overnight. The LC3-II/I ratio was calculated based on densitometry analysis of both bands. The ratio is a widely used indicator of autophagy flux . The membranes were washed two times with TBST 1 and subsequently incubated with their respective HRP-conjugated secondary antibodies (1:10,000) (1 h at room temperature). The detection of bound antibodies was visualized by chemiluminescence with enhanced chemiluminescence (ECL) substrate. Finally, a quantification analysis was performed with Image J software (NIH), using Ponceau stain.
Antioxidant enzymatic activity
Superoxide dismutase (SOD) activity was measured as previously described .
Two-tailed t-tests for matched pairs were used to compare distributions and statistical significance was set at p < 0.05. Data are shown as mean ± standard deviation.
Stimulation of a hemidiaphragm was well tolerated and the biopsies were obtained without complication. All subjects were extubated between 6.7 and 67.1 h after surgery (27.5 ± 22.8 h). The first stimulation was initialized when the phrenic nerve and diaphragm were visualized by a surgeon (95.9 ± 12 min after intubation). The patients received an average of 6.4 ± 1.8 stimulation bouts with the mean amplitude of 19.6 ± 5.8 mA. Muscle biopsies were harvested 28.3 ± 1.8 min after the last stimulation bout.
We are the first to report potentially beneficial effects of electrical stimulation on oxidative stress and autophagy levels in human diaphragm in mechanically ventilated older patients during cardio-thoracic surgeries. The main finding of this exploratory study was that levels of oxidative stress were lower in the stimulated hemidiaphragm. Additionally, higher levels of autophagy levels markers i.e. Beclin-1 and the LC3-II/I ratio in the stimulated hemidiaphragm suggest upregulation of macroautophagy levels.
Previous reports suggested that higher levels of oxidative stress may be a key factor of the majority of pathways leading to VIDD in the mechanically ventilated patients, with mitochondrial dysfunction being an essential part [11, 17]. We reported that electrical stimulation improved mitochondrial function (i.e. state III and IV mitochondrial respiration) in comparison with the unstimulated hemidiaphragm . These results suggest improved mitochondrial function in the stimulated hemidiaphragm, which maybe a contributing factor to reducing risk of VIDD in surgery patients. Mitochondrial function can be improved by generating new mitochondrial and/or removing the dysfunctional mitochondria (mitophagy) that are the source of oxidative stress . In the current analysis we showed no difference in the protein concentration of PGC-1α, a biomarker of mitochondrial biogenesis levels. This suggests that autophagy-related mechanisms and reduced oxidative stress levels may have improved mitochondrial function in the stimulated hemidiaphragm  and that our observation is not due to an altered level of mitochondrial biogenesis.
The link between increased oxidative stress and MV-related diaphragm dysfunction has also been shown in animal models [17, 20]. Indeed, we found increases in 4-HNE protein concentration, a biomarker of lipid peroxidation, and thus an indication of increased oxidative stress levels in the unstimulated hemidiaphragm. Additionally, the activity levels of endogenous antioxidant defense system (CuZnSOD and MnSOD) were not different between the hemidiaphragms that may be associated with a limited samples size. The future studies may include measurements of other biomarkers of oxidative stress levels e.g. protein carbonylation and 8-isoprostane levels to better interpret the increased 4-HNE levels in the current analysis.
Taken together, we can speculate that decreased oxidative stress levels maybe a product of increased cell-protective autophagy levels (Fig. 3). Although, these results may support our previous report of improved mitochondrial function in the stimulated hemidiaphragms, these exploratory results warrant future studies involving confocal microscopy methods in order to study the autophagy flux and support the clinical value of the diaphragm electrical stimulation.
Although these results suggest electrical stimulation (ES) has a potential protective effect against VIDD, this study has a few limitations. A larger sample size would increase the statistical power and the ability to detect more differences between stimulated and control tissue. Additionally, studying the biological pathways more comprehensively would have been informative, but was limited by small muscle specimens (~20 mg). However, despite the relatively small number of subjects and dependent variables examined, this study presents novel and important findings regarding the effect of electrical stimulation on the human diaphragm during surgery and MV.
Intraoperative hemidiaphragm electrical stimulation may decrease oxidative damage and upregulate autophagy levels. Further studies on molecular bases of the diaphragm electrical stimulation are warranted on a larger population and using confocal technologies. These results may contribute future studies and clinical applications on reducing post-operative diaphragm dysfunction.
ventilator induced diaphragm dysfunction
New York Heart Association
forced expiratory volume 1
reactive oxygen species
Institutional Review Board
sodium dodecyl sulfate
microtubule-associated protein light chain 3
copper-zinc superoxide dismutase
manganese superoxide dismutase
RT, CL, and DM participated in the conception, drafting, and revision of the manuscript. TB, PH, BS and TM performed the surgeries, harvested diaphragm muscle biopsies, gave final approval of the version to be published and were involved in revising the manuscripts for important intellectual content. RT, SA, MD, CH and SS were involved in the tissues processing, biochemical analyses, data analysis and revising the manuscript. All authors read and approved the final manuscript.
We are grateful to research nurses for assisting in the recruitment of the subjects. We would also like to thank the subjects for their participation.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets analyzed during the current study could be available from the corresponding author on reasonable request.
Consent to publish
Written informed consent was obtained from the patients for publication of their individual details in this manuscript.
Ethics approval and consent to participate
The University of Florida IRB approved this study protocol, and all subjects gave their consent for participation.
This study was supported by the University of Florida Clinical Translational Science Institute and Pilot Grant (UL1 RR029890), the Persistent Inflammation, Immunosuppression and Catabolism Syndrome (PICS): A New Horizon for Surgical Critical Care, P50 Grant (P50GM111152-01), the Claude D. Pepper Older Americans Independence Center (OAIC) Metabolism and Translational Science Core (CL) and K12-HD055929 (BKS) and Eli Lilly company. The OAIC is supported by a Grant from the National Institutes of Health/National Institute on Aging (1P30AG028740).
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