The dynamic changes in autophagy activity and its role in lung injury after deep hypothermic circulatory arrest

Abstract Deep hypothermic circulatory arrest (DHCA) can cause acute lung injury (ALI), and its pathogenesis mimics ischaemia/reperfusion (I/R) injury. Autophagy is also involved in lung I/R injury. The present study aimed to elucidate whether DHCA induces natural autophagy activation and its role in DHCA‐mediated lung injury. Here, rats were randomly assigned to the Sham or DHCA group. The sham group (n = 5) only received anaesthesia and air intubation. DHCA group rats underwent cardiopulmonary bypass (CPB) followed by the DHCA procedure. The rats were then sacrificed at 3, 6 and 24 h after the DHCA procedure (n = 5) to measure lung injury and autophagy activity. Chloroquine (CQ) was delivered to evaluate autophagic flux. DHCA caused lung injury, which was prominent 3–6 h after DHCA, as confirmed by histological examination and inflammatory cytokine quantification. Lung injury subsided at 24 h. Autophagy was suppressed 3 h but was exaggerated at 6 h. At both time points, autophagic flux appeared uninterrupted. To further assess the role of autophagy in DHCA‐mediated lung injury, the autophagy inducer rapamycin and its inhibitor 3‐methyladenine (3‐MA) were applied, and lung injury was reassessed. When rapamycin was administered at an early time point, lung injury worsened, whereas administration of 3‐MA at a late time point ameliorated lung injury, indicating that autophagy contributed to lung injury after DHCA. Our study presents a time course of lung injury following DHCA. Autophagy showed adaptive yet protective suppression 3 h after DHCA, as induction of autophagy caused worsening of lung tissue. In contrast, autophagy was exaggerated 6 h after DHCA, and autophagy inhibition attenuated DHCA‐mediated lung injury.


| INTRODUC TI ON
The deep hypothermic circulatory arrest (DHCA, a type of cardiopulmonary bypass (CPB)) procedure is required in certain cases undergoing aortic arch surgery and complex congenital heart surgery, as it not only increases the body's tolerance to the harsh environment of ischaemia and hypoxia due to a decrease in tissue metabolism and oxygen consumption but also provides a relatively bloodless surgical field, thus increasing the likelihood of a successful surgery. 1 Although continuous improvements in DHCA technology and CPB circuit materials have significantly reduced postoperative complications and mortality, approximately 20%-30% of patients undergoing CPB with DHCA still experience varying degrees of postoperative pulmonary dysfunction, ranging from transient lung injury in mild cases to acute respiratory distress syndrome in severe cases. 2,3 The incidence of acute respiratory distress syndrome after cardiac surgery related to CPB is 0.4%-1.3%, and mortality rates of 15%-68.4% are reported among these patients. 4,5 The underlying mechanisms for acute respiratory distress have been suggested to be multifactorial; in fact, its related pathological process primarily recapitulates that of ischaemia/reperfusion (I/R)induced lung injury 6,7 and systemic inflammatory response syndrome. 8 As an essential biological process for maintaining normal cell homeostasis, autophagy plays key roles not only in physiological states of the lung tissue 9 but also in various pathophysiological processes of lung injury, [10][11][12] including I/R-induced lung injury. 6,7 Different signalling pathways are involved in and contribute to lung injury, such as the mTOR/TLR4/NF-κB, 13 ERK 1/2, 14 and MAPK and NF-κB 15 signalling pathways. Intriguingly, autophagy can exert either beneficial (or protective) 14,16 or detrimental effects 7 on lung tissue during the pathological process of reperfusion injury, and these effects depend on the stage and severity of a specific disease, which are closely related to dynamic microenvironmental conditions. 17,18 On the other hand, studies have been performed to elucidate the underlying mechanisms of DHCA-mediated lung injury [19][20][21] ; however, few studies have focused on the role of autophagy in DHCA-mediated lung injury. Therefore, the aims of the present research were (a) to investigate whether DHCA induces time course damage in lung tissue and whether this damage was associated with dynamic changes in autophagy activity, (b) to quantify phase-dependent changes in autophagic flux and finally (c) to evaluate whether manipulation of autophagy activity affects DHCA-mediated lung injury.

| ME THODS
We declare that all data that support the findings of this study are available within the article and its Appendix S1.
A more detailed description of the experimental methods

| Animals
Twelve-week-old male Sprague-Dawley (SD) special pathogen-free rats weighing between 400 g and 450 g (the Slaccas Animal Center, Shanghai, China) were studied. The

| Surgical preparation
The detailed methods for the rat model of DHCA have been described in our previous report with minor modifications. 22 Briefly, 12-week-old rats were anaesthetized with sodium pentobarbital (50 mg/kg) by intraperitoneal injection (i.p.) and endotracheally intubated with a 16-gauge cannula, which was then connected to an animal respirator (Model alc-v8s, ALCOTT, Shanghai). Anaesthesia was maintained with 1.5%-2.0% sevoflurane (Hengrui Pharmaceutical, Shanghai, China) ventilation and mechanical ventilation was performed with a tidal volume of 8 ml/kg and at a respiratory rate of 60 cycles per minute, 23 both of which were adjusted according to blood gas analyses obtained at five time points. The left superficial femoral artery, right external jugular vein and tail artery were dissected. Using a surgical approach, the left superficial femoral artery was cannulated with a preheparinized 24-gauge catheter for both the delivery of heparin sodium (500 IU/kg, H32022088, Qianhong Biopharma, Changzhou, China) to perform systemic anticoagulation and facilitate connection to a multidirectional physiological monitor (Powerlab, Harvard Apparatus, America) to continuously monitor both mean artery pressure (MAP) and heart rate (HR). The right external jugular vein was also cannulated with a homemade 24-side venous drainage tube to serve as a venous outflow. The median tail artery was catheterized using a 20-gauge catheter, which served as the arterial inflow for the CPB circuit. Rectal temperature was continuously monitored (Powerlab, Harvard Apparatus, America).

| Execution of CPB and DHCA
The CPB device (for details, Figure 1)  China). Blood from the jugular vein was drained into the venous reservoir from which the blood was immediately pumped into the membrane oxygenator with the aid of a roller for gas exchange. A heat exchange process then followed before the blood finally entered the body through the caudal artery. During CPB, the flow rate started at 135 ml/kg/min and gradually decreased during the cooling phases.
Once the CPB was established, systemic cooling was initiated via a heat exchanger assisted by ice bags. Controlled cooling was maintained for 30 min, and ventilation was maintained at a lower tidal volume and frequency, which was adjusted based on the blood gas analysis at the dedicated time points described above. When the rectal temperature reached 18°C, CPB and ventilation were completely interrupted to generate a 50-min circulatory arrest, that is, all the organs of the rats were exposed to ischaemia. Once the circulation arrest period was completed, the CPB was restarted. The rats were rewarmed with a heated blanket to ensure that a rectal temperature of 35°C was reached within 30 min. Once the rectal temperature was stable at 35°C, CPB and anaesthesia were terminated, whereas ventilation was maintained for an additional 60 min, allowing for full recovery from anaesthesia. The remaining priming solution was infused if needed to maintain a MAP greater than 80 mmHg.
To maintain stable homeostasis, arterial blood gas analysis was regularly sampled and analysed at the following five time points, which were selected based on previous reports both from other 23 and our own laboratory 22 : 10 min pre-CPB (T1), precardiac arrest (T2), cardiac rebeating (T3), weaning of CPB (T4) and before sacrifice (T5).

| Protocol I
To assess whether DHCA can induce lung injury, 20 SD rats were randomly assigned and divided into 4 groups (n = 5). In the sham group (S group), all rats underwent anaesthesia, air intubation, ventilation and vascular insertion of the cannulas without standard CPB and DHCA procedures. The other three groups included DHCA 3 h group (D3 h group), DHCA 6 h group (D6 h group) and DHCA 24 h group (D24 h group), each of these group rats experienced standard CPB and DHCA intervention as described above and were sacrificed at 3 h, 6 h and 24 h after weaning of CPB respectively. These time points were determined based on previous reports from other groups 24,25 and our own results. 22 The extent of lung injury, including lung histology examination, wet/dry lung weight ratio and inflammatory cytokine quantification, was assessed for each group of rats.

| Protocol II
To describe the dynamic changes in autophagy activities in the lung tissue obtained from the rats subject to Protocol I (including the S, D3 h, D6 h and D24 h groups), the anterior lobe and middle lobe of the right lung were used for Western blotting to quantify the protein F I G U R E 1 The Schematic Diagram of Our Model Establishment. The right external jugular vein, left superficial femoral artery and tail artery were catheterized. The cardiopulmonary bypass device mainly includes a venous reservoir, roller pump, membrane oxygenator and heat exchanger. Blood from the jugular vein was drained into the venous reservoir from which the blood was then, with the aid of a roller, pumped into the membrane oxygenator for gas exchange. A heat exchanger followed before finally entering the body through the caudal artery. Mean arterial pressure, heart rate anal temperature, and other parameters were continuously monitored expression levels of LC3-II, Beclin 1, ATG5 and p62, and immunofluorescence staining for LC3-II was also performed.

| Protocol III
To further assess the dynamic details of the autophagy process, autophagic flux was also evaluated using chloroquine (CQ, C6628, Sigma Aldrich, America, 20 mg/kg in PBS, i.p.). The D3 h and S group rats were given CQ 2 h before euthanization to assess the autophagic flux at the early phase after the DHCA procedure, and similarly, CQ was also delivered to the other two groups (including the D6 h and S groups) rats hours before the end of the experiments to assess the late phase changes in autophagic flux after DHCA intervention.

| Protocol IV
To assess whether activation (rapamycin, V900930, Sigma Aldrich, America, 4 mg/kg in 1.8% DMSO + PBS, i.p.) or suppression (3-methyladenine, 3-MA, M9281, Sigma Aldrich, America, 30 mg/kg in PBS, jugular vein injection) of autophagic activity can modulate the lung injury induced by the DHCA procedure. Rapamycin or 3-MA was delivered to either the D3 h group or the D6 h group (with their corresponding solvents serving as the placebo for a control group respectively) to further delineate whether autophagy plays protective or detrimental roles in lung injury induced by the DHCA procedure.

| Animal euthanization and tissue handling
Once each experimental protocol was completed, the rats from each group were sacrificed with an overdose of anaesthesia (sodium pentobarbital, 100 mg/kg (i.p.)), and the lung tissue was obtained. The right lung tissue was processed for the following studies:   In contrast, type II alveolar epithelial from the DHCA 6 h group display relatively electron-lucent nuclei and cytoplasm, reduced microvilli, fine vacuolar-like changes in the cytoplasm and increased lysosomes. In addition, mitochondrial swelling and hydropic change were observed (Scale bar = 2 μm). *p < 0.05; **p < 0.01; ***p < 0.001 of the left lung was used to assess the wet/dry weight ratio. Blood samples were collected for cytokine quantification.

| Histological assessment of lung injury
As

| Quantification of cytokines in blood serum and bronchoalveolar lavage fluid
The lung tissues obtained from each group of rats as described above were individually removed and placed in disposable Petri dishes.
The bronchi were fixed with forceps, and a 1-ml syringe with a 20- Blood samples were collected immediately after the rats were euthanized and kept on ice. The blood samples were centrifuged at 1000 g for 20 min at 4°C, and the serum was collected and stored for further analysis.

| Lung wet/dry weight ratio
As described above, a portion of the left lung was weighed after removing superficial blood (wet weight, W). The lung samples were then dried in an oven (at 58°C) for 48 h, and weighed to obtain the dry weight (D). The ratio of wet lung weight to dry lung weight was calculated (W/D).

| Western blot
The anterior lobe and middle lobe of the right lung were obtained as described above and stored in liquid nitrogen for protein quan-

| Transmission electron microscopy
The lung tissues from the indicated group rats as described above were prepared for transmission electron microscope examination following the standard methods as described previously. 27 Briefly, samples approximately 1 mm 3 in size were fixed with 2.5%

| Immunofluorescence staining
The lung tissues obtained from group rats that were designated for haematoxylin and eosin staining were fixed in 10% neutral forma-

| Statistical analysis
Data with normal distribution confirmed by Kolmogorov-Smirnov method were presented as mean ± standard deviation. One  was not altered based on the DHCA procedure, and lactic acid accumulation occurred with the completion of a 50-min circulation arrest for which sodium hydrogen carbonate was deliberately administered to correct acidosis. These data indicate that our modified CPB-aided DHCA procedure was successfully delivered to most of the rats with reasonable results obtained in Protocol I (Figure 2A). DHCA-exposed rats exhibited a dynamic phase-distinct phe-

| Dynamic changes in autophagic activities following DHCA
A set of autophagy machinery proteins, including LC3-II, Beclin 1, ATG5, and p62, 28 was carefully measured using lung tissue obtained from rats in different designated groups to assess autophagy activity. The dynamic changes in autophagy activity were quantified according to Protocol II ( Figure 3A). Compared with the S group, the expression levels of LC3-II, Beclin 1, and ATG5 were slightly lower in the D3 h group rats ( Figure 3B, D, full blots are shown in Figure   S1A,  Figure 3B, D), which was eventually even higher than that in the Consistent with Western blot findings, the immunofluorescence staining showed a decrease in LC3-II expression level at the early stage (3 h) after DHCA compared with S group rats; however, the level was even higher than that of S group rats at the late stage (6 h) ( Figure 3C, E) after DHCA. Transmission electron microscopy examination further confirmed this pattern of changes in autophagy activity at the two specific time points after the DHCA procedure ( Figure 3F).
To further evaluate the dynamic changes in autophagy activity in the lung tissue following the DHCA procedure, CQ was delivered to rats in both the D3 h and D6 h groups to measure autophagic flux (see Protocol III for details, Figure 4A). Interestingly, we observed that

| Autophagy activation at the early phase of DHCA aggravated lung injury
Rapamycin, a typical autophagy inducer, was delivered at 24 h and 30 min pre-operation (i.p.) to re-activate the suppressed autophagy activity at the early phase (3 h) after DHCA (see Protocol IV for details, Figure 5A). Rapamycin increased autophagic protein expression levels in D3 h group rats, which reached similar levels as S group rats ( Figure 5B, C, whole blots on PVDF membrane shown in Figure Figure 5F). Thus, our data indicated that suppressed autophagy is probably an adaptive response to changes in microenvironments at the early phase after DHCA.

| Inhibition of autophagy at the late stage of DHCA alleviated pulmonary injury
Given that increased autophagic activity was observed at a late phase after (6 h post) DHCA, 3-MA, an autophagy inhibitor, was then administered 2 h before euthanization (jugular vein injection) to rats to further investigate the role of autophagy in lung injury late after the DHCA procedure (Protocol V, Figure 6A). We noted that 3-MA exposure resulted in decreased levels of LC3-II, Beclin 1 and ATG5 in lung tissue at 6 h post DHCA ( Figure 6B, C, full blots are shown in Figure   S1D,  Figure 6D, E). Moreover, the improved lung injury was associated with a trend of, albeit not statistically significant, reductions in the levels of inflammatory factors in lung tissue ( Figure 6F).
Thus, these data indicated that overactivation of autophagy leads to pathological lung damage in the relatively late phase after DHCA.

| DISCUSS ION
In our study, we showed that a natural inflammatory response occurred in the lung tissue following the DHCA procedure, which caused a unique phase-dependent phenotype of lung injury.
Coincidently, the activity of autophagy after DHCA was decreased at the early phase (3 h following DHCA procedure) but was increased at the late phase (6 h after). Interestingly, autophagic flux was not interrupted at either the early or late phase following DHCA, as CQ delivery caused further increases in the expression levels of autophagy machinery proteins. Importantly, when rapamycin was administered at the early phase after DHCA to augment autophagic activity, it caused a worsening of lung injury. These results indicate that suppressed autophagy at the early phase following DHCA is probably an adaptive response that plays a protective role. In contrast, 3-MA delivered at the late stage following DHCA to inhibit excessive activated autophagy attenuated lung injury, indicating that augmented autophagy is responsible for the pathological injury A large body of data indicate that autophagy plays key roles not only in maintaining cellular homeostasis but also in the pathological process of many diseases, 29  accordingly, inhibition of autophagy aggravates lung injury. 16 On the other hand, overactivation of autophagy can cause further damage to the lung tissue. A study has shown that upregulated autophagy exacerbates lung I/R injury. 7 The different data obtained from these studies could be due to the different extents of ischaemia (ie the infarct area) or different experimental setups that exist in different research laboratories. Indeed, the different data actually indicate that a moderately functioning autophagy process is essential for maintaining normal homeostasis in lung tissue. In the present study, our data suggest that autophagy in general contributes to DHCA-induced lung injury. In the early phase after DHCA, a reversal of autophagy activity by rapamycin resulted in augmented lung injury. However, in the late phase following DHCA, suppression of autophagy activity attenuated lung injury. Thus, our data strongly suggest that in our specific rat model of DHCA-induced lung injury, autophagy has a detrimental effect on lung injury rather than a protective effect.

| Study limitations and future perspectives
Several limitations in our study should be emphasized.  39 Nevertheless, deep insights into this differential regulatory mechanism would reveal unique molecular markers that will guide our therapeutic treatment for better clinical outcomes.
In conclusion, DHCA can induce a time-dependent pathological process in lung tissue, for which a dynamic change in autophagy activity characterized by suppressed autophagy at the early phase followed by considerably augmented autophagy at the late phase after DHCA plays an important role. Importantly, induction of autophagy activation by rapamycin at the early phase actually worsened lung injury, suggesting an adaptive decrease in autophagy. In contrast, 3-MA-mediated suppression of autophagy attenuates lung injury at the late phase after DHCA. These data suggest that DHCAmediated autophagic activation contributes to the lung injury observed in our present study. Further studies are needed to elucidate the underlying mechanisms of how autophagy is regulated following the DHCA procedure.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science Foundation of China.

F I G U R E 6
Inhibition of Autophagy at the Late Stage of DHCA Alleviated Pulmonary Injury. (A) Experimental Protocol V. Autophagy inhibitor 3-methyladenine (3-MA) was given by intraperitoneal injection at 2 h before euthanization in Sham group and DHCA 6 h group (n = 3 independent experiments). (B and C) The expression levels of autophagy-related proteins were significantly decreased after 3-MA was given. Quantitative analysis is shown in (C; n = 5). (D) Representative haematoxylin and eosin staining images in Sham group (as Control), DHCA 6 h group (as Control), Sham + 3-MA group and DHCA 6 h + 3-MA group. Compared with the DHCA 6 h group, the degree of lung injury in DHCA 6 h + 3-MA group decreased (Scale bar = 100 μm). (E) Inhibition of autophagy significantly decreased lung injury score at the late stage of DHCA. (F) The levels of some inflammatory factors in blood serum and BALF showed a trend of decreases, albeit not statistically significant, after the inhibition of autophagy. *p < 0.05; **p < 0.01; ***p < 0.001

CO N FLI C T S O F I NTE R E S T
No conflicts of interest, financial or otherwise, are declared by the authors.