Abstract
Introduction
Global ischemia reperfusion (I/R) stimulation induced by cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) triggers multiple forms of programmed cell death including pyroptosis and necroptosis, and further results in post-resuscitation myocardial damage. Recently, a specific inhibitor of histone deacetylase 6 activity, tubastatin A (TubA) was preliminarily shown to protect the heart against global and regional I/R stimulation. The present study was designed to investigate the effect of TubA on post-resuscitation myocardial pyroptosis and necroptosis in a porcine model of CA and resuscitation.
Methods
A total of 18 pigs were randomly assigned to one of the following three groups (n = 6 each): Sham group, CA/CPR group, and CA/CPR + TubA group. The setting of 9 min of CA and 6 min of CPR was used to establish the porcine model of CA and resuscitation. A dose of 4.5 mg/kg of TubA was intravenously infused within 1 h after successful resuscitation. Myocardial function including stroke volume and global ejection fraction, and cardiac injury biomarkers including cardiac troponin I and creatine kinase-MB were regularly evaluated for 24 h after resuscitation. Thereafter, the pigs were euthanized, and myocardial tissues were harvested to evaluate the ratio of cell apoptosis, the contents of high mobility group box 1, IL-1β, and IL-18, and the expression levels of caspase 3, gasdermin E (GSDME), GSDME N-terminal (GSDME-N), receptor-interacting protein 1 (RIP1), RIP3, mixed lineage kinase domain-like protein (MLKL), and phosphorylated MLKL (p-MLKL).
Results
After resuscitation, stroke volume and global ejection fraction were significantly decreased while serum cardiac troponin I and creatine kinase-MB were significantly increased in the two groups experiencing the CA/CPR procedure compared with the Sham group. However, myocardial dysfunction and cardiac injury were significantly milder in the CA/CPR + TubA group than in the CA/CPR group. At 24 h after resuscitation, apoptosis ratio, pyroptosis-related proteins (caspase 3, GSDME, GSDME-N), necroptosis-related proteins (RIP1, RIP3, MLKL, p-MLKL), and proinflammatory cytokines (high mobility group box 1, IL-1β, IL-18) in myocardium were significantly increased in the CA/CPR and CA/CPR + TubA groups compared with the Sham group. Nevertheless, all of them were significantly decreased in those pigs treated with the TubA compared to the CA/CPR group.
Conclusions
TubA could effectively alleviate post-resuscitation myocardial damage in a porcine model of CA and resuscitation, in which the protective role was possibly related to the inhibition of GSDME-mediated pyroptosis and MLKL-mediated necroptosis.
Keywords: Tubastatin A, Cardiac arrest, Cardiopulmonary resuscitation, Myocardial damage, Pyroptosis, Necroptosis
Introduction
Cardiac arrest (CA) is an unpredictable whole-body ischemic insult and results in significant morbidity and mortality in the world.1, 2, 3 After achieving successful cardiopulmonary resuscitation (CPR) from CA events, the following post-cardiac arrest syndrome, including myocardial dysfunction, brain injury, and systemic inflammation, became the main contributors to death during hospital stay.4 Among these, post-resuscitation myocardial dysfunction can lead to low cardiac output, malignant arrhythmia, and even recurrent CA, and is proven to be the key contributing factor to early death.5, 6 However, no intervention is effective enough to improve post-resuscitation myocardial dysfunction in the clinical setting. Currently, it is needed to explore the key pathogenesis of myocardial dysfunction after resuscitation and further its potential therapeutic target.
Nowadays, studies have manifested that multiple forms of programmed cell death were involved in the pathophysiologic process of myocardial damage induced by global and regional ischemia reperfusion (I/R) stimulation, such as CA and myocardial infarction.7, 8 Especially, cell pyroptosis and necroptosis, known as the lytic types of programmed cell death, can amplify tissue inflammation and therefore promote the development of myocardial damage after resuscitation.9, 10 However, the upstream regulators for governing the processes of cell pyroptosis and necroptosis in post-resuscitation myocardium remain to be investigated. Recently, the activation of histone deacetylases (HDACs) was confirmed to drive those classical pathological injuries including inflammation and oxidative stress in a series of I/R stimulation.11, 12 Currently, the inhibition of HDAC6 by tubastatin A (TubA) treatment has been preliminarily demonstrated to alleviate post-resuscitation myocardial damage via the inhibition of cardiomyocyte pyroptosis.13 Nevertheless, the regulatory mechanism by which TubA treatment inhibits post-resuscitation myocardial pyroptosis remains unclear. In addition, whether TubA treatment provides post-resuscitation myocardial protection by targeting cell necroptosis and its potential mechanism requires further investigation. Thus, this study was designed to investigate the effects of HDAC6 inhibition by TubA treatment on myocardial pyroptosis and necroptosis in a clinically relevant, pig model of CA and resuscitation. Our results showed that TubA treatment simultaneously inhibited cell pyroptosis mediated by gasdermin E (GSDME) activation and cell necroptosis mediated by mixed lineage kinase domain-like protein (MLKL) activation, and thereby alleviated myocardial damage after resuscitation in pigs.
Materials and methods
All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals. A total of 21 healthy male white domestic pigs, aged 4–6 months, weighing between 33 kg and 40 kg, were supplied by Shanghai Jiagan Biotechnology Inc. (Shanghai, China). The number of animal qualification certification was SCXK (Shanghai) 2020-0006. After the pigs arrived, they were fed in standard conditions, such as standard atmospheric pressure, 20–25 °C of room temperature, 60–80 % of humidity, 12 h/12 h of light/dark cycle, free water intake, regular feeding, cleaning, and disinfection. This study was approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine (approval number: 2021-103). Study protocol was prepared in advance, including research question, key design features, primary outcome measure, and analysis plan. Currently, the protocol was not registered in a public repository. However, our protocol and study data were available from the corresponding author upon reasonable request. All experimental procedures were performed according to the requirements of the ARRIVE guidelines (Supplementary Table S1). Those pigs who were successfully resuscitated were included for data analysis, and other pigs who died during animal preparation and model establishment were excluded.
Animal preparation
All pigs were fasted for a duration of 12 h. After that, the pigs were sedated with intramuscular injection of tiletamine/zolazepam (5 mg/kg) and xylazine (1 mg/kg), and then intravenous injection of propofol (2 mg/kg) followed by its continuous infusion (4 mg/kg/h). In addition, the analgesia was performed with intravenous injection of butorphanol (8 μg/kg) followed by its continuous infusion (3 μg/kg/h). Subsequently, the animals were rapidly intubated and ventilated with a Monnal T75 ventilator (Air Liquide Medical Systems, Antony Cedex, France). The working parameter of the ventilator was set with a volume-controlled mode, tidal volume (10 ml/kg), respiratory frequency (12 breaths/min), peak flow (40 L/min), and FiO2 (0.21). The conventional lead II electrocardiogram and peripheral oxygen saturation were continuously monitored with an iM60 patient monitoring system (Edan, Shenzhen, China).
For the measurements of aortic and right atrial pressures, two 7Fr pressure-monitoring catheters were inserted from the right femoral artery and vein into the thoracic aorta and right atrium, respectively. Both of them were connected to the patient monitoring system mentioned above. For the measurements of myocardial function indicators, including stroke volume and global ejection fraction, a 4 Fr thermistor-tipped arterial catheter was inserted into the left femoral artery, and another 7 Fr central venous catheter was inserted into the right internal jugular vein. Both of them were connected to a PiCCO monitor system (Pulsion Medical Systems, Munich, Germany). For CA induction, a 5-F pacing catheter was inserted from the right external jugular vein into the right ventricle and then connected to an electrical stimulation device (Weil Institute of Critical Care Medicine, Rancho Mirage, United States). All the catheters were regularly flushed with normal saline containing 5 IU/ml heparin. The animals’ temperature was maintained at a normal temperature of 37.5 ± 0.5 °C using the Blanketrol III Hyper-Hypothermia System (Cincinnati Sub-Zero, Cincinnati, United States) throughout the experiment.
Experimental procedures
After animal preparation was finished, the animals were stabilized for 15 min, and then baseline measurements were obtained, and experimental randomization was performed. The animals were randomized with the sealed envelope method into the following three groups (n = 6 each): Sham group, CA/CPR group, and CA/CPR + TubA group. During the experiment, one nurse who didn’t participate in the experimental procedures was invited to be responsible for performing animal randomization and then providing the corresponding medication according to the treatment assignment. The nurse was aware of the group allocation at the different stages of the experiment. Sham animals only experienced animal preparation, but didn’t establish the experimental model. In the CA/CPR and CA/CPR + TubA groups, the experimental model was established by 9 min of CA and then 6 min of CPR. Once the animal was successfully resuscitated, a dose of 4.5 mg/kg of TubA (APExBIO, Houston, United States) dissolved in 50 ml of normal saline was intravenously infused for 30 min in the CA/CPR + TubA group. The dose of TubA administration was chosen according to several previous studies and the principle of dose conversion between the animals.13, 14, 15 In addition, the same volume of saline was similarly administered in the other two groups.
For the establishment of an experimental model, 1 mA of alternating current was initially delivered via the electrical stimulation device into the right ventricle until CA was successfully induced. Subsequently, the pacing electrode was removed out and mechanical ventilation was stopped. After a duration of 9 min of untreated CA, a duration of 6 min of manual CPR was performed at a ratio of 30:2 of compression to ventilation according to the CPR guideline.16 Chest compression was implemented by two professional providers, and its quality was monitored by a PlamCPR feedback device (Sunlife, Suzhou, China) to guarantee 50–60 mm of compression depth and 100–120/min of compression rate. The ventilation was implemented using a bag respirator connected to the endotracheal tube with room air. At 2 min after CPR, a dose of 20 μg/kg of epinephrine was intravenously injected, and followed by the same dose every 3 min if needed. At 6 min after CPR, a single 150-J biphasic electrical shock was delivered by a M Series defibrillator (ZOLL Medical Corporation, Chelmsford, United States). If an organized rhythm was observed, accompanied by a mean arterial pressure >50 mm Hg for 5 min or more, the animal was regarded as successful CPR. Thereafter, the animals were ventilated and monitored for 4 h, and then sent back to be observed in their cages for an additional 20 h. At 24 h after resuscitation, the animals were euthanized with intravenous injection of propofol (3 mg/kg) and then potassium chloride (10 %, 10 ml). Subsequently, the thoracotomy was rapidly performed to expose and remove the whole heart. After that, tissue samples were obtained from the left ventricular apex, and then myocardial tissues in the subendocardium were further harvested and stored for the following measurements.
Measurements
Physiological indexes and CPR outcomes
Before the model establishment, baseline body weight, heart rate, and mean arterial pressure were recorded. In addition, arterial pH and lactate concentrations were measured with a 2 ml of arterial blood sample using an i15 blood gas analyzer (Edan, Shenzhen, China). During CPR, coronary perfusion pressure was regularly calculated according to the difference between decompression diastolic aortic and time-coincident right atrial pressures. Other CPR indexes including duration of CPR, dosage of epinephrine, and number of defibrillations were recorded.
Myocardial function indicators and cardiac injury biomarkers
The indicators of myocardial function including stroke volume and global ejection fraction were measured using the PiCCO monitor system at baseline, and at 1 h, 2 h, and 4 h after resuscitation. At each time point, the PiCCO monitor system was regularly calibrated and then operated continuously for three times’ measurements of stroke volume and global ejection fraction, and the latter was calculated by (4 × stroke volume)/global end-diastolic volume. The average of three measurements was used to reflect the status of myocardial function. The biomarkers of cardiac injury including cardiac troponin I and creatine kinase-MB were measured with 2 ml of venous blood sample using their enzyme-linked immunosorbent assay kits (Meixuan Biotechnology, Shanghai, China) at baseline, and at 1 h, 2 h, 4 h, and 24 h after resuscitation.
TUNEL assay
Myocardial apoptosis was evaluated by the TdT-mediated dUTP nick end labeling (TUNEL) staining at 24 h after resuscitation. Those tissue samples obtained from the subendocardium of the left ventricular apex were fixed in 4 % paraformaldehyde for 24 h, then embedded in paraffin, and finally sliced into 5-μm-thick sections. The sections were stained with the TUNEL assay kit (Boster Biological Technology, Wuhan, China) according to the manufacturer’s instructions, and then six fields were randomly photographed at 200× magnification using a CX31 optical microscope (Olympus, Tokyo, Japan) to count the number of TUNEL-positive cells and total cells. Apoptosis ratio was calculated as the percentage of TUNEL-positive cells/total cells.
Immunohistochemistry
The expression of GSDME N-terminal (GSDME-N) and phosphorylated-MLKL (p-MLKL) in the myocardium was measured by immunohistochemistry at 24 h after resuscitation. Those tissue samples obtained from the subendocardium of the left ventricular apex were similarly fixed, then embedded, and finally sliced into 5-μm-thick sections. The sections were incubated with primary anti-GSDME-N (1:200, Cell Signaling Technology, Danvers, United States) and anti-p-MLKL (1:200, ZenBio, Chengdu, China), then treated with the secondary antibody, and finally reacted with diaminobenzidine (Boster Biological Technology, Wuhan, China). Six fields were randomly photographed at 200× magnification with the CX31 optical microscope. The semiquantitative analysis of the intensity of GSDMD-N and p-MLKL positive staining was performed through integrated optical density (IOD) using the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, United States).
Proinflammatory cytokines
The contents of proinflammatory cytokines including high mobility group box 1 (HMGB1), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in the myocardium were measured with an enzyme-linked immunosorbent assay at 24 h after resuscitation. Those tissue samples obtained from the subendocardium of the left ventricular apex were homogenized with normal saline on ice and centrifuged at 2500g at 4 °C for 15 min. The supernatants were collected to measure the contents of HMGB1, IL-1β, and IL-18 with their enzyme-linked immunosorbent assay kits (Meixuan Biotechnology, Shanghai, China) according to the manufacturer’s instructions.
Immunoblotting analysis
The expression of pyroptosis and necroptosis-related proteins in the myocardium was evaluated by western blotting at 24 h after resuscitation. Those tissue samples obtained from the subendocardium of the left ventricular apex were lysed with the RIPA lysate to extract tissue proteins. The lysates were then centrifuged at 12,000 rpm at 4 °C for 20 min, and then the total protein concentrations were quantified using a BCA protein quantitation kit (Beyotime Biotechnology, Shanghai, China). Subsequently, the protein samples were denatured and electrophoresed in 10 % SDS-PAGE, then transferred onto a PVDF membrane, and finally blocked with 5 % non-fat milk. All the PVDF membranes were incubated with primary anti-Caspase 3 (1:1000, ZenBio, Chengdu, China), anti-GSDME (1:1,000, Proteintech, Rosemount, United States), receptor-interacting protein 1 (RIP1, 1:1,000, Proteintech, Rosemount, United States), RIP3(1:1,000, ZenBio, Chengdu, China), MLKL (1:1,000, Proteintech, Rosemount, United States), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1,000, BBI Life Science Corporation, Shanghai, China) at 4 °C for 24 h. Thereafter, they were incubated with a secondary antibody (1:5,000, BBI Life Science Corporation, Shanghai, China) at room temperature for 1 h. The proteins were then visualized with ECL luminescent substrates and analyzed by Image J software (NIH, Bethesda, United States). The expression levels of Caspase 3, GSDME, RIP1, RIP3, and MLKL were standardized by GAPDH.
Statistical analysis
Statistical analysis was performed using the SPSS 20.0 software (IBM, Armonk, United States). Sample size was calculated based on our previous study to detect a difference with respect to global ejection fraction (primary outcome) of ≥40 %,17 in which the number of 6 animals in each group was needed to get an α of 0.05 and a power of 80 %. Those potential confounders related to the order of experimental procedures were minimized through randomization across the experimental days. In addition, the animals’ cage location was not systematically controlled and might represent a potential limitation in this study. Continuous variables were analyzed with the Kolmogorov-Smirnov test to confirm their normal distribution, and then their data were shown as mean ± SD. The comparisons between two groups were performed using the Student t test, and three groups were performed by 1-way ANOVA. For the time-based measurements within each group, the comparisons were performed using repeated-measurement ANOVA. If a significant difference was observed in the overall comparison of groups, the comparisons between any other two groups were performed using the Bonferroni test. A value of P < 0.05 was considered statistically significant.
Results
Baseline and CPR characteristics
Twenty-one pigs were used in this study, in which 1 pig in the CA/CPR group and 2 pigs in the CA/CPR + TubA group were not resuscitated. Finally, the data of those unresuscitated pigs were excluded, and the remaining 18 pigs were included for data analysis. At baseline, no significant differences were observed in body weight, heart rate, mean arterial pressure, and arterial pH and lactate among the three groups (Fig. 1A–E). During CPR, the same levels of coronary perfusion pressure were achieved in the CA/CPR and CA/CPR + TubA groups. Consequently, coronary perfusion pressure and other CPR characteristics including duration of CPR, dosage of epinephrine, and number of defibrillations were not significantly different between the CA/CPR and CA/CPR + TubA groups (Fig. 1F–I).
Fig. 1.
Baseline and CPR characteristics. A, Body weight. B, Heart rate. C, Mean arterial pressure. D, pH. E, Lactate. F, Coronary perfusion pressure. G, Duration of CPR. H, Dosage of epinephrine. I, Number of defibrillations.
Six pigs were included in each group.
The data were shown as mean ± SD, and group comparisons were performed by 1-way ANOVA with Bonferroni test or Student t test.
CA, cardiac arrest; CPR, cardiopulmonary resuscitation; TubA, tubastatin A.
Myocardial function and cardiac injury biomarkers
After resuscitation, stroke volume and global ejection fraction were significantly decreased at all time points in the two groups experiencing the CA/CPR procedure when compared with the Sham group. However, both of these two indicators of myocardial function were significantly increased at all time points after resuscitation in the CA/CPR + TubA group when compared to the CA/CPR group (Fig. 2A, B).
Fig. 2.
The changes of myocardial function indicators and cardiac injury biomarkers. A, Stroke volume. B, Global ejection fraction. C, Cardiac troponin I. D, creatine kinase-MB.
Six pigs were included in each group.
The data were shown as mean ± SD, and group comparisons were performed by 1-way ANOVA with Bonferroni test.
*P < 0.05 vs. Sham group; #P < 0.05 vs. CA/CPR group.
BL, baseline; CA, cardiac arrest; CPR, cardiopulmonary resuscitation; TubA, tubastatin A.
After resuscitation, the serum levels of cardiac troponin I and creatine kinase-MB were significantly increased at all time points in those pigs experiencing the CA/CPR procedure when compared with the Sham group. However, both of these two biomarkers of cardiac injury were lower after resuscitation in the CA/CPR + TubA group than in the CA/CPR group, in which significant differences in them were observed at 4 h and 24 h after resuscitation between the two groups (Fig. 2C, D).
Myocardial apoptosis
At 24 h after resuscitation, myocardial apoptosis indicated by those TUNEL-positive cells was observed in the CA/CPR and CA/CPR + TubA groups, in which the differences were significant in these two groups when compared with the Sham group. However, in those pigs receiving TubA treatment, the apoptosis ratio was significantly decreased in post-resuscitation myocardium when compared to the CA/CPR + TubA group (Fig. 3).
Fig. 3.
The evaluation of myocardial apoptosis at 24 h post-resuscitation in each group. A, Representative photomicrographs of TdT-mediated dUTP nick end labeling (TUNEL) staining in myocardial tissue (×200 magnification). B, Apoptosis ratio.
Six pigs were included in each group.
The data were shown as mean ± SD, and group comparisons were performed by 1-way ANOVA with Bonferroni test.
*P < 0.05 vs. Sham group; #P < 0.05 vs. CA/CPR group.
CA, cardiac arrest; CPR, cardiopulmonary resuscitation; TubA, tubastatin A.
Myocardial pyroptosis-related proteins
At 24 h after resuscitation, the expression levels of those proteins related to cell pyroptosis including Caspase 3 and GSDME were both significantly increased in myocardium in the CA/CPR and CA/CPR + TubA groups when compared with the Sham group. However, both of them were significantly decreased in those pigs receiving TubA treatment when compared to the CA/CPR group (Fig. 4A). Additionally, at 24 h post-resuscitation, the IOD value of GSDME-N positive staining in myocardium was significantly higher in the CA/CPR and CA/CPR + TubA groups than in the Sham group. Nevertheless, TubA treatment significantly decreased myocardial GSDME-N expression when compared to the CA/CPR group (Fig. 4B).
Fig. 4.
The evaluation of pyroptosis-related proteins in myocardium at 24 h post-resuscitation in each group. A, The bands of Caspase 3 and gasdermin E (GSDME) proteins, and their relative expression levels. B, Representative photomicrographs of immunostaining of GSDME N-terminal (GSDME-N) protein (×200 magnification), and it’s integrated optical density (IOD) value of positive staining.
Six pigs were included in each group.
The data were shown as mean ± SD, and group comparisons were performed by 1-way ANOVA with Bonferroni test.
*P < 0.05 vs. Sham group; #P < 0.05 vs. CA/CPR group.
CA, cardiac arrest; CPR, cardiopulmonary resuscitation; TubA, tubastatin A.
Myocardial necroptosis-related proteins
At 24 h after resuscitation, significantly higher expression of key proteins related to cell necroptosis including RIP1, RIP3, and MLKL was observed in myocardium in the CA/CPR and CA/CPR + TubA groups when compared with the Sham group. However, the expression levels of these proteins were significantly lower in those pigs receiving TubA treatment when compared to the CA/CPR group (Fig. 5A). Likewise, the IOD value of p-MLKL positive staining in myocardium was significantly higher at 24 h post-resuscitation in the CA/CPR and CA/CPR + TubA groups than in the Sham group. Nevertheless, TubA treatment significantly decreased myocardial p-MLKL expression when compared to the CA/CPR group (Fig. 5B).
Fig. 5.
The evaluation of necroptosis-related proteins in myocardium at 24 h post-resuscitation in each group. A, The bands of receptor interacting protein-1 (RIP1), RIP3, and mixed lineage kinase domain-like (MLKL) proteins, and their relative expression levels. B, Representative photomicrographs of immunostaining of phosphorylated-MLKL (p-MLKL) protein (×200 magnification), and it’s integrated optical density (IOD) value of positive staining.
Six pigs were included in each group.
The data were shown as mean ± SD, and group comparisons were performed by 1-way ANOVA with Bonferroni test.
*P < 0.05 vs. Sham group; #P < 0.05 vs. CA/CPR group.
CA, cardiac arrest; CPR, cardiopulmonary resuscitation; TubA, tubastatin A.
Myocardial proinflammatory cytokines
At 24 h after resuscitation, the contents of proinflammatory cytokines related to cell pyroptosis and necroptosis including HMGB1, IL-1β, and IL-18 were all significantly increased in the myocardium in the CA/CPR and CA/CPR + TubA groups when compared with the Sham group. However, all of them were significantly less in those pigs treated with the TubA when compared to the CA/CPR group (Fig. 6).
Fig. 6.
The evaluation of proinflammatory cytokines in myocardium at 24 h post-resuscitation in each group. A, high mobility group box 1 (HMGB1). B, interleukin-1β (IL-1β). C, interleukin-18 (IL-18).
Six pigs were included in each group.
The data were shown as mean ± SD, and group comparisons were performed by 1-way ANOVA with Bonferroni test.
*P < 0.05 vs. Sham group; #P < 0.05 vs. CA/CPR group.
CA, cardiac arrest; CPR, cardiopulmonary resuscitation; TubA, tubastatin A.
Discussion
In the present study, we demonstrated that a specific HDAC6 inhibitor, TubA produced effective post-resuscitation myocardial protection by improving myocardial dysfunction, alleviating cardiac injury, and reducing cardiomyocyte apoptosis in a porcine model of CA and resuscitation. Furthermore, the phenomenon of cell pyroptosis mediated by GSDME activation and cell necroptosis mediated by MLKL activation were observed in myocardium after resuscitation; however, both of them were effectively inhibited in those pigs receiving TubA treatment. Thus, the potential mechanism of myocardial protection provided by TubA treatment was possibly related to the inhibition of GSDME-mediated pyroptosis and MLKL-mediated necroptosis.
It is reported that 44 % CA victims after resuscitation develop myocardial dysfunction, and the latter can result in circulatory failure and even death.5, 6 Some studies have confirmed that multiple forms of programmed cell death participate in the pathophysiologic process of myocardial damage induced by CA and resuscitation, and further promoted the occurrence of myocardial dysfunction after resuscitation.8, 9, 10 Thus, the exploration of those common therapeutic targets for regulating programmed cell death would facilitate to find the effective strategies for improving post-resuscitation myocardial dysfunction. Recently, HDACs, known as the key epigenetic modifying enzymes for regulating gene expression, have been shown to trigger multiple forms of programmed cell death in disease states.18 In addition, the HDAC6, considered as one representative member of HDACs due to its unique structure, cellular localization, and wider range of biological functions, has been proven to be an effective target for protecting the heart against global and regional I/R stimulation.13, 19, 20 Based on the evidence, the effects of HDAC6 inhibition on programmed cell death including apoptosis, pyroptosis, and necroptosis in post-resuscitation myocardium were investigated in this study. Our results showed that TubA, a specific HDAC6 inhibitor, significantly inhibited myocardial apoptosis, pyroptosis, and necroptosis, and further improved myocardial dysfunction and cardiac injury after resuscitation in pigs. Hence, HDAC6 could become a common therapeutic target for alleviating myocardial programmed cell death after resuscitation; however, its regulatory mechanism requires further investigation.
Nowadays, inflammation is proven to be a key factor for promoting myocardial dysfunction after resuscitation.21, 22 Additionally, some lytic types of programmed cell death including pyroptosis and necroptosis are shown to play an important role in aggravating inflammatory response under the condition of various stimuli.23 In theory, cell pyroptosis can be triggered by the following several pathways including the caspase-1-mediated canonical pathway, the caspase-4/5/11-mediated noncanonical pathway, the caspase-8-gasdermin D (GSDMD) pathway, and the caspase-3-GSDME pathway, and the latter activates the gasdermin family proteins to form holes in the cell membrane and release massive proinflammatory cytokines.24 In the setting of CA and resuscitation, some studies have demonstrated that cell proptosis mediated by the caspase-1-GSDMD pathway was involved in the pathophysiologic process of myocardial damage after resuscitation, and therefore could become a therapeutic target for improving post-resuscitation myocardial dysfunction.9, 13, 25 Recently, some studies have demonstrated that the caspase-3-GSDME pathway also played an important role in promoting cell pyroptosis in the setting of I/R injury of vital organs, such as the brain and kidney.26, 27 However, no investigation has confirmed the change of the caspase-3-GSDME pathway and its potential regulation by TubA treatment in the myocardium after resuscitation. Based on the evidence, the effects of TubA treatment on the caspase-3-GSDME pathway in post-resuscitation myocardium were investigated in this study. Our results showed that the caspase-3-GSDME pathway was significantly activated in the myocardium after resuscitation; however, this pathway was markedly inhibited by TubA treatment. Hence, GSDME-mediated pyroptosis also participated in post-resuscitation myocardial damage, and further became a therapeutic target by which the HDAC6 inhibitor TubA produced effective post-resuscitation myocardial protection.
The necroptosis, known as another type of lytic cell death, has also been shown to trigger an inflammatory response and further promote myocardial dysfunction after resuscitation.10, 25 In theory, cell necroptosis is mainly triggered by the RIP1/RIP3/MLKL pathway, in which RIP1 interacts with RIP3 to form a necrosome, then RIP3 phosphorylates its substrate MLKL to promote its oligomerization, subsequently oligomerized MLKL forms pore structures in cell membrane or interacts with cation channels to increase cellular osmotic pressure, and eventually results cell death and the release of inflammatory cytokines.28 However, the regulatory mechanism of myocardial necroptosis mediated by the RIP1/RIP3/MLKL pathway remains to be investigated in the setting of CA and resuscitation. Recently, one study demonstrated that the inhibition of HDAC6 activity by tubacin treatment significantly alleviated neuronal necroptosis after oxygen-glucose deprivation in the cultured rat cortical neurons.29 Currently, another study demonstrated that the inhibition of HDAC6 by TubA treatment could inhibit the activation of the RIP1/RIP3/MLKL pathway and further decrease inflammation and tubular cell death in acute oxalate nephropathy.30 Based on the evidence, the effects of HDAC6 inhibition on cell necroptosis mediated by the RIP1/RIP3/MLKL pathway in post-resuscitation myocardium were investigated in this study. Our results showed that TubA treatment significantly inhibited the activation of the RIP1/RIP3/MLKL pathway and further decreased cardiomyocytes necroptosis in the myocardium after resuscitation. Hence, the inhibition of HDAC6 by TubA treatment could improve post-resuscitation myocardial dysfunction by inhibiting cardiomyocyte necroptosis via inactivating the RIP1/RIP3/MLKL pathway.
This study has several limitations. First, more doses of epinephrine and a greater number of defibrillations were observed during CPR in the CA/CPR group than in the CA/CPR + TubA group although statistically insignificantly; however, both of which might result in more severe myocardial injury after resuscitation. Thus, the same administration of epinephrine doses and defibrillation shocks should be achieved between the two groups in the future study. Second, one single dose of TubA was used at one time point in this study, and thus its feasible dose range and therapeutic window require further investigation. Third, although post-resuscitation myocardial damage was evaluated by its function, injury biomarker, and tissue apoptosis; however, a shorter duration of 24 h of observation was difficult to fully confirm the effectiveness of TubA treatment in alleviating post-resuscitation myocardial damage. Fourth, although the inactivation of HDAC6 by TubA treatment inhibited GSDME-mediated pyroptosis and MLKL-mediated necroptosis in myocardium after resuscitation; however, the key molecular mechanism by which HDAC6 governs myocardial pyroptosis and necroptosis remains to be investigated. Fifth, cell pyroptosis and necroptosis were only evaluated by their protein markers and proinflammatory productions in this study; however, their special ultrastructure should be further confirmed using an electron microscope. Sixth, the proportion of pyroptotic and necroptotic effects contributing to post-resuscitation myocardial damage is important but unclear in this study. Thus, the relative contributions of pyroptosis and apoptosis inhibition should be further explored in the future studies.
Conclusions
TubA treatment could effectively alleviate post-resuscitation myocardial damage possibly via inhibiting GSDME-mediated pyroptosis and MLKL-mediated necroptosis in a porcine model of CA and resuscitation.
Funding
Pin Lan was funded by the Lishui Municipal Science and Technology Project (2022GYX36) and the Zhejiang Provincial Chinese Medical Science Foundation (2024ZL207). Xue Zhao was funded by the Zhejiang Provincial Medical Science Foundation (2023KY924).
CRediT authorship contribution statement
Linjie Lai: Writing – original draft, Methodology, Conceptualization. Yuanhua Fang: Investigation, Formal analysis. Lutao Xie: Investigation, Formal analysis. Xue Zhao: Project administration, Funding acquisition, Data curation. Jiefeng Xu: Visualization, Supervision, Project administration. Pin Lan: Writing – review & editing, Validation, Supervision, Resources, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
None.
Consent for publication
All authors have consented to publication.
Footnotes
Supplementary material to this article can be found online at https://doi.org/10.1016/j.resplu.2025.101158.
Contributor Information
Jiefeng Xu, Email: z2jeffxu@zju.edu.cn.
Pin Lan, Email: lszxlp@wmu.edu.cn.
Appendix A. Supplementary material
The following are the Supplementary material to this article:
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