Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1

doi:10.21203/rs.3.rs-4866738/v1

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Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1

https://doi.org/10.21203/rs.3.rs-4866738/v1

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Background Therapeutic mild hypothermia (MH) is expected to improve the neurological outcome and the survival rate of CPR after cardiac arrest (CA). However, whether and how mild hypothermia protects cardiomyocytes largely remains to be explored.

Methods Three to four-month-old (age), 319.1-480.0 g (weight), male (sex) Wistar rats were randomly divided into normothermia (NT) groups (acceptable range, 36.0-38.0°C) and MH groups (acceptable range, 32-34°C). Then ten minutes after CA, CPR was perfumed by a slow injection of 0.30-0.50 mL epinephrine solution (30.00 μg/mL). Moreover, to test the effect of TRPV1, capsaicin was injected through the right femoral artery both in NT groups and MH groups.

Results We found that MH improved the recovery of spontaneous breathing, increased survival rate, and inhibited cardiac necroptosis of Wistar rats after CPR. Moreover, MH alleviated primary cardiomyocytes necroptosis after oxygen-glucose deprivation and reperfusion through up-regulating TRPV1, down-regulating EGFR, and subsequently suppressing MLKL. Furthermore, capsaicin, the TRPV1-activator, combined with MH, improved the blood pressure of Wistar rats after CPR when compared with MH alone. Capsaicin improved the spontaneous breathing rate and survival rate of Wistar rats after CPR.

Conclusions MH may protect cardiac function by TRPV1-inhibited cardiomyocyte necroptosis.

CPR

Mild hypothermia

Ischemia

TRPV1

Capsaicin

As a lifesaving technology, cardiopulmonary resuscitation (CPR) has successfully aided numerous patients with sudden cardiac arrest (CA) to recover spontaneous circulation. However, survival in out-of-hospital cardiac arrest (OHCA) just increased from 5.7% in 1990 to 10.1% in 2011 and basically unchanged thereafter [1]. Meanwhile, most patients with CA lost their lives due to cerebral and cardiac hypoxic ischemia. Moreover, ischemia/reperfusion injury (IRI) exposes patients with CA to risks of myocardial dysfunction after return of spontaneous circulation (ROSC). Hence, suppressing the ischemia/reperfusion (I/R) induced cardiac injury is critical for reducing CPR-linked brain damage and subsequent death.

In recent years, mild hypothermia (MH) with a target of 32 to 36°C body temperature has shown a potential benefit for improved survival and function outcome after CA. And some guidelines recommend not actively rewarming comatose patients with MH after ROSC to achieve normothermia. Mechanistically, MH has been reported to reduce the production of reactive oxygen species (ROS), IRI, myocardial infarction area, ventricular remodeling, and cardiomyocyte metabolism so as to protect myocardium. However, to a large extent, how MH improves survival after CA and helps to protect heart and brain remains poorly understood.

Cell death works in many ways, four related to the effects of mild hypothermia, namely apoptosis, necroptosis, pyroptosis, and ferroptosis. In cardiac surgery, the study from Vazquez-Jimenez JF, et al tested the hypothesis that mild hypothermia during cardiopulmonary bypass provides myocardial protection by enhancing intra-myocardial anti-inflammatory cytokine balance [2]. Another team discovered mild hypothermia's role of increasing the intramyocardial synthesis of heat shock protein 72 to inhibit necrosis but not apoptosis [3]. Based on the knowledge of therapeutic hypothermia, the authors reveal that mild hypothermia may protect the injured central nervous system from tissue damage and inflammatory responses by targeting the necroptosis signaling after traumatic brain injury [4]. The experiment of transient spinal cord ischemia in rabbits found that MH inhibits the expression of receptor receptor-interacting protein kinases 1 and 3 (RIPK1/3) as potential suppressors of necroptosis [5]. However, the mechanism by which MH inhibits necroptosis are still undefined.

Transient receptor potential (TRP) channels are non-selective cation channels which are sensitive to various chemical and physical stimuli such as temperature, press, inflammation, pain and so on. Among them, heat-sensitive transient receptor potential cation channel subfamily V member 1 (TRPV1) is predominantly expressed in sensory neurons and afferent nerve terminals in both the somatic and visceral sensory nervous systems. And numerous studies have revealed the vital role of TRPV1 in pain and neural diseases. Moreover, the protection of TRPV1 in cardiovascular system earns more and more attention recently. It has been reported that TRPV1 is involved in the postischemic recovery in isolated perfused heart and protecting rat hearts against IRI by hypoxic preconditioning. Capsaicin (10 mg/kg) pretreatment 40 minutes before the heart isolation reduced infarct size in subsequent IRI [6]. Furthermore, ruthenium red, a TRPV inhibitor, abolished adenosine preconditioning-induced decreased infarct size [7]. Besides, inhibition of TRPV1 by capsazepine sensitized the apoptosis of colorectal cancer cells [8]. Whether activation of TRPV1 antagonizes other forms of cell death, such as necroptosis, is rarely reported. The aim of this study was to explore the mechanism by which MH achieves cardioprotective efficacy by regulating TRPV1.

Rats CA model

The feed given to rats complied with the GB 14924.3–2010 Chinese standard for "Laboratory animals-Nutrients for formula feeds". Three to four-mouth-old (age), 319.1–480.0 g (weight) (Supplementary Fig. S1a), male (sex) Wistar rats were randomly divided into groups, normothermia (NT) groups, in which the animal body temperature was maintained at 37°C (acceptable range, 36.0–38.0°C) with heating pad after ROSC, and mild hypothermia (MH) groups, in which the animal body temperature was maintained at 33°C (acceptable range, 32–34°C) with ice bag after ROSC.

After fasted overnight but had access to water, rats were anesthetized by intraperitoneal injection of pentobarbital (30.00 mg/kg, Sigma, Natick, MA, USA). An electronic underarm thermometer was inserted into the rectum to monitor body temperature. A PE-10 catheter was inserted into the right femoral artery for drug administration and blood pressure monitoring. Mean arterial pressure (MAP) evaluated using the MAP = (systolic pressure + 2 × diastolic pressure) / 3 formula. Three needle electrodes were placed subcutaneously in limbs for continuous electrocardiogram (EKG) monitoring.

Then, CA was induced with an injection of Rocuronium Bromide Injection (1.25 mg/kg, Merk Sharp & Dohme) and blocked tracheal cannula, confirmed by asystole on EKG and MAP dropping to 30 mmHg (Supplementary Fig. S1b). Ten minutes after CA, CPR was perfumed by a slow injection of 0.30–0.50 mL epinephrine solution (30.00 µg/mL) (Supplementary Fig. S1c), chest compressions, and ventilation [9]. ROSC succeed when MAP rose above 60 mmHg and remained stable for more than 30 minutes (Supplementary Fig. S1d).

Moreover, to test the effect of TRPV1, capsaicin (MedChemExpress, HY-10448, 10 mg/kg) was injected through the right femoral artery, 15 minutes prior to the injection of Rocuronium Bromide [6].

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from the heart tissue or PCMs by the SteadyPure Universal RNA Extraction KitⅡ (Accurate Biology, AG21022). 500 nanograms of the RNA was used in reverse transcriptional responses with the Evo M-MLV RT Premix (Accurate Biology, AG11706). A Real-Time PCR System (Roche, LightCycle480 II) was used to performed qPCR with the SYBR® Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, AG11702). Melting curves were detected to check for primer specificity. Relative mRNA expression levels were normalized to Actb. Primer sequences designed with the NCBI database, were listed in Supplementary Table 1. Primers were synthesized from the Guangzhou IGE Biotechnology Ltd [9].

Culture of primary cardiomyocytes (PCMs)

Whole hearts were collected from three to ten-day-old neonatal sprague dawley (SD) rats. The ventricles were isolated and cut into 1.0 mm³ pieces. Next, the pieces were digested with 0.125% Trypsin-EDTA (Gibco™, 930004) for eight minutes at 37°C. After supernatant removal, the pieces continued to be digested with Dulbecco's modified Eagle's medium (DMEM) containing 0.5 mg/mL (62.5 U/mL) collagenase Ⅰ (Gibco™, 17100017) for two hours at 37°C with gentle pendular movement in a water bath [10, 11]. DMEM supplemented with 10% fetal bovine serum (FBS) was used to stop digestion. The supernatant was then gently collected, filtered with a 200-µm strainer, and centrifuged at 800 revolutions per minute (rpm) for eight minutes at 4°C [12]. Afterward, the cardiac cells were plated on 10 cm culture dishes with DMEM supplemented with 10% FBS at 5% CO₂ and 37°C for 60 minutes to remove the settling down non-cardiomyocytes. The cell supernatant containing mainly cardiomyocytes was then collected and seeded into six-well plates [13].

Cellular oxygen-glucose deprivation and reperfusion (OGD/R) model

PCMs were assigned randomly to the following groups, NT-R and MH-R. PCMs were bathed in the DMEM without Glucose, glutamine and phenol red (Gibco™, A1443001). First the culture plates were placed in a modular incubator chamber (Billups-Rothenberg, CA92014), which was filled with 5% CO₂ and 95% N₂. Incoming and outgoing hose tubes were closed, five minutes after the oxygen detector (SMART SENSOR, AS8801) showed 0.3%. Then, hypoxia lasted one to six hours at 37°C to establish the OGD model. Next, the DMEM medium was replaced with the complete medium. Finally, PCMs were cultured at the 37°C (NT treatment) or 34°C (MH treatment) to reperfusion for two, six or ten hours [9].

Immunofluorescence

The PCMs seeded on chamber slides were fixed with 4.0% paraformaldehyde for 15 minutes, rinsed with 0.01M phosphate-buffered saline (PBS) three times, permeabilized in 0.3% Triton X-100 for 20 minutes and washed with PBS three times. PCMs were blocked in 5% bovine serum albumin (BSA) at 37°C for one hour, then incubated with anti-α-Actin antibodies (Abcam, 1:100) at 4°C more than 16 hours. Next, after incubated with secondary antibody (1:200) in a wet box sheltered from light for one hour at 37°C, PCMs were washed gently with PBS three times. After stained with DAPI, PCMs were viewing with a fluorescence microscopy [9].

Flow cytometry

Annexin V-PI double staining was performed for evaluating apoptosis rates in the PCMs (1 × 10⁵ cells/well, 6-well plate). Briefly, after following various treatments, the PCMs were collected by trypsinization and washed 2 times with ice-cold phosphate buffered saline (PBS). The PCMs were resuspended by 60 µL binding buffer and then adding 5 µL Annexin V-FITC and 10 µL PI staining solution (Becton Dickinson, USA). The mixture was incubated for 15 minutes at room temperature avoiding light. Then the PCMs were added 200 µL binding buffer and detection by flow cytometry equipped with a laser for FITC (488 nm) and PI (535 nm) excitation.

MH raises the survival rate of rats after CPR

To confirm whether MH has a protective effect after CPR. First, we established a Wistar rat's CPR model (Fig. 1a). There were no significant differences in weight of rats, time of inducing CA, volume of using epinephrine solution, and time of successful CPR among all groups (Supplementary Fig.S1). Therefore, capsaicin injection 15 min prior to induction CA did not affect CPR. During ROSC, the temperature of rats was maintained by heating pad or ice bag. There was no difference in the NT and Cap-NT groups compared to the Sham group, but the temperature was significantly lower in the MH and Cap-MH groups (Supplementary Fig.S2). As anticipated, MH gave it an edge in maintaining MAP (Fig. 1b). Moreover, rats treated by MH demonstrated higher rate of recovery of spontaneous breathing and survival rate relative to NT treatment group (Fig. 1c, d). Together, these data support the hypothesis that MH can raise the survival rate after CPR.

MH inhibits necroptosis in rats

To clarify that MH protects the heart in IRI. First, we analyzed the gene expression after Myocardial infarction (MI) from public datasets (GSE114695). Genes associated with apoptosis (Supplementary Fig.S3a), necroptosis (Fig.S3b-d), pyroptosis (Supplementary Fig.S3e, f), ferroptosis (Supplementary Fig.S3g) and autophagy (Supplementary Fig.S3h) were upregulated after MI. Then, we tested the mRNA level of these cell death associated genes (Fig. 2). The expression of markers of apoptosis (Caspase 3, Casp3) (Fig. 2a), pyroptosis (Caspase 1 and Gasdermin D, Casp1 and Gsdmd) (Fig. 2e, f), ferroptosis (Prostaglandin G/H synthase 2, Ptgs2) (Fig. 2g) and autophagy (Beclin 1, Becn1) (Fig. 2h) had no statistically significant difference between group NT and MH. However, receptor interacting serine/threonine kinase 3(Ripk3, the marker of necroptosis) was downregulated in MH treatment group, although we didn’t observe statistically significant difference of the transcription level of receptor interacting serine/threonine kinase 1(Ripk1) and (mixed lineage kinase domain-like protein Mlkl), another two markers of necroptosis (Fig. 2b-d). Together, we hypothesized that MH could inhibit necroptosis to attenuate myocardial injury after CPR.

A primary cardiomyocyte model of oxygen-glucose deprivation (OGD)

To further validate it, we constructed OGD model with PMCs. PMCs were obtained from neonatal SD rats (Fig. 3a, b). And then PMCs were cultured with DMEM in oxygen-free environment to construct OGD model in order to mimic CA. Annexin V-FITC/PI staining was performed to test this model. With the prolongation of OGD, both early apoptosis cells and late apoptosis cells were increased (Fig. 3c, d). Moreover, after OGD 6h, PMCs presented least Annexin V-/PI + cells, Therefore, we set 6h as the optimal time of ODG model.

MH inhibits cardiomyocyte necroptosis through the Trpv1-Egfr-Mlkl pathway

Consistent with the conclusion drawn from rat experiments, MH increased the survival rate of cardiomyocytes after 6 hours of OGD (Fig.S4). Then we sought to explore how MH inhibited necroptosis after CPR. Since temperature is the most fundamental change between NT and MH therapy, we pay attention to the genes sensitive to temperature. As mentioned before, most TRP channels are polymodal channels which can be activated by both physical (including temperature) and chemical factors. TRPV1(originally cloned as the ‘capsaicin receptor’), also named as vanilloid receptor-1 (VR1), is famous for its role in the afference of pain and heat [14, 15]. Apart from this, TRPV1 also participates in perceiving ischemic stimulation during myocardial ischemia, further activating downstream pathways [16]. Moreover, transient receptor potential cation channel subfamily M member 4 (TRPM4) is also reported to be related with the development of arrhythmias [17], and TRPM7 is reported to be essential for the execution of necroptosis [18]. Therefore, we detected the mRNA level of target members of TRP channels after 6 hours of OGD (Fig. 4). We noticed that MH seemingly had no effect on Trpv2 (Fig. 4a-c) but inhibited Trpm4 (Fig. 4e) and Trpm7 (Fig. 4g).

To investigate the relationship between TRP channels and cell death, we tried to construct networks of TRP channels and various cell death marker genes in the MetaCore database and obtained the Trpv1-Egfr-Mlkl pathway. TRPV1-EGFR loop has been reported. In De Jong PR’s research, TRPV1 was activated by EGFR, and then activated protein tyrosine phosphatase 1B (PTP1B), which can in turn inhibit the activity of EGFR [19]. Subsequently, we verified this pathway (Fig. 5). We were pleasantly surprised to find that Trpv1 was significantly upregulated by MH, indicating its role in the effect of MH (Fig. 5b, c). Moreover, both epidermal growth factor receptor (Egfr) (Fig. 5e, f) and Mlkl (Fig. 5h, i) were downregulated by MH.

Capsaicin can improve the survival rate of rats after CPR

To validate the role of TRPV1 in attenuating myocardial injury after CPR, we injected the rats with capsaicin, the activator of TRPV1, 10 minutes before CA (Fig. 6a). Capsaicin not only contributed to the maintenance of MAP, but also improved the rate of recovery of spontaneous breathing and survival rate (Fig. 6b, d, f). Additionally, Capsaicin combined with MH demonstrated better efficacy relative to MH alone (Fig. 6c, e, g). Together, all these indicate that the efficacy of MH is linked to the activation of TRPV1, which harbors potential of being a target for CA and other cardiovascular diseases.

MH has an inhibitory effect on TRPM4/7. Unfortunately, this effect is not stable at the three OGD/R time points. In contrast, the expression trend of the Trpv1-Egfr-Mlkl pathway is stable. In summary, our results identified Trpv1 as a key amplifier of the cardioprotective effect of MH. MH may alleviate cardiomyocytes necroptosis by upregulating Trpv1 during cardiomyocyte ischemia and hypoxia, thereby increasing the survival rate of CPR. And capsaicin, the activator of TRPV1, combined with MH, can work synergistically to improve the MAP and survival rate of rats after CPR.

Our results are consistent with work by other authors concerning the cardioprotective effect of capsaicin. Capsaicin is the agonist of TRPV1, which is one of the main integrators of the afferent of pain and temperature stimuli [20]. As for cardiovascular system, TRPV1 is located mainly on the capsaicin-sensitive peptidergic primary sensory neurons that innervate the heart, but also in ventricular cardiomyocytes, vascular smooth muscle, endothelial cells and epicardial cells [21]. TRPV1 channels are also located in the cell membrane and mitochondrial membrane [22]. They can be activated by many factors during anoxia, tissue injury and inflammation, such as heat, ROS, inflammatory factors [23]. Apart from the pain, its role in nervous system diseases, cardiovascular protective effect and the development of tumor have earned more and more attention recently. C. M Hurt et al. demonstrated that capsaicin dose-dependently reduced the infarct size and decreased mitochondrial membrane potential. Further, this effect can be counteracted by the TRPV1 antagonist capsazepine [24]. Moreover, in cellular experiments, pretreatment with capsaicin protected cardiomyocytes by inhibiting anoxia and reoxygenation induced apoptosis [25]. Interestingly, topical capsaicin application was also shown to reduce the infarct size mediated by the circulating humoral factors [26]. Jiang et al. found that TRPV1 knockout rats, compared with wild type rats, were subjected to larger infarct size and higher level of apoptosis in response to ex vivo I/R, indicating that TRPV1 served as a protective role against myocardial apoptosis during I/R [27]. Capsaicin also protects cardiomyocytes through antioxidant and anti-inflammatory effects. Rayan A. et al. demonstrated that capsaicin significantly reduced the level of inflammatory factor, caspase 3, cardiomyocyte damage markers (lactate dehydrogenase, troponin-I) and increases the level of glutathione and catalase [28]. However, few research concerning the role of capsaicin or TRPV1 in the cardioprotective effects of MH after CA has been reported.

In our cellular experiments, we isolated and purified primary cardiomyocytes from neonatal SD rats and then constructed OGD/R model. We found that mRNA level of Trpv1 was up-regulated time-dependently (especially 10 hours after reperfusion) in MH group compared with NT group after OGD 6 hours. In consideration of the previous studies demonstrating the cardioprotective effects of capsaicin and TRPV1, we speculated that the activation of TRPV1 played a vital role in the cardioprotective effects of MH, providing evidence for targeted temperature management (TTM) after CA and an intervenable target [29]. Actually, there has been no sufficient evidence to support or oppose therapeutic MH for CA patients [30]. And those recommending therapeutic MH are mainly based on the improvement of the neurological outcome [31, 32]. However, our research found that MH could directly protect cardiomyocytes by activating TRPV1, thus alleviating cardiomyocyte necroptosis. Therefore, TRPV1 is a promising target and the application of capsaicin can be extended to the protection of cardiomyocyte in first aid. Moreover, clinical trials demonstrated that therapeutic MH or TTM could cause the QTc prolongation, which may be associated with higher risk of arrhythmia, especially in early repolarization syndrome [33, 34]. In this respect, capsaicin or targeting TRPV1 may be a safer choice. Additionally, mild hypothermic perfusion combined with capsaicin may also give it an edge on improving donor organ quality during preservation and transport.

IRI of brain is the main death and disability cause for CA patients after ROSC [35]. Clinical researches have demonstrated that MH can alleviate cerebral injury after CA [31]. A multicenter cohort study conducted in Japan divided 1111 OHCA patients from 125 hospitals into two groups: 33–34°C and 35–36°C, in which 33–34°C was associated with better recovery of moderate nervous system impairment [36]. In a randomized controlled trial, conducted on 574 patients with coma who had been resuscitated from CA with nonshockable rhythm, moderate therapeutic hypothermia at 33°C for 24 hours improved the neurological prognosis [37]. Especially, asphyxia-induced cardiac arrest can cause heavier neurologic injury and worser neurological outcome than ventricular fibrillation-induced cardiac arrest [38, 39]. In this respect, this group of patients may be more suitable for therapeutic MH. Additionally, cerebral injury caused by CA can aggravate cardiomyocyte injury, which is recognized as brain-heart axis or intervention [40]. Therefore, MH may also protect cardiomyocytes through brain-heart axis, in which TRPV1 may play a vital role in both cardiovascular and neural system.

This study supports our novel finding that MH may protect cardiac function by inhibiting cardiomyocyte necroptosis through the Trpv1-Egfr-Mlkl pathway after OGD/R. To our knowledge, we first report that capsaicin, the TRPV1-activator, combined with MH, can improve the blood pressure and survival rate of Wistar rats after CPR. This may provide a new treatment strategy for cardiac arrest patients.

Study limitations and future work

Several limitations are noted for our research. Firstly, our subjects were rats, and the findings are not directly applicable to humans. Further experiments on large animals, such as pigs, would provide stronger evidence for our conclusion. Secondly, it's a pity that we had no TRPV1 knockout mice and we didn't inject an antagonist of TRPV1 to explore whether the cardioprotective effect of MH was TRPV1-mediated. Thirdly, the causes of OHCA are diverse, such as anoxia, hypovolemia, electrolyte abnormality, myocardial infarction and so on, which can be broadly categorized into cardiac and non-cardiac causes [41]. Recently, OHCA has increasingly been recognized as different from in-hospital cardiac arrest (IHCA) and studied independently from each other [42]. Thus, the heterogeneity of CA patients cannot be neglected. In our experiment, the CA model is constructed by respiratory depression induced by muscle relaxant, which limits the extrapolation of our conclusion. What's more, we have not verified the Trpv1-Egfr-Mlkl pathway at the level of protein. And the interaction among TRPV1, EGFR and MLKL remains to be clarified.

CPR Cardio-pulmonary resuscitation

CA Cardiac arrest

NT Normothermia

MH Mild hypothermia

TRPV Transient receptor potential cation channel subfamily V

EGFR Epidermal growth factor receptor

MLKL Mixed lineage kinase domain-like protein

OHCA Out-of-hospital cardiac arrest

ROSC Return of spontaneous circulation

ROS Reactive oxygen species

TRP Transient receptor potential

MAP Mean arterial pressure

RT-qPCR Reverse transcription and quantitative polymerase chain reaction

PCM Primary cardiomyocyte

SD Sprague dawley

DMEM Dulbecco's modified Eagle's medium

FBS Fetal bovine serum

OGD/R Oxygen-glucose deprivation and reperfusion

BSA Bovine serum albumin

MI Myocardial infarction

Ripk3 Receptor interacting serine/threonine kinase 3

Ripk1 Receptor interacting serine/threonine kinase 1

VR1 Vanilloid receptor-1

TRPM Transient receptor potential cation channel subfamily M

PTP1B Protein tyrosine phosphatase 1B

IHCA In-hospital cardiac arrest

Acknowledgements

Not applicable

Author contributions

M.Y.: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Supervision; Validation; Visualization; Writing-original draft; Writing-review and editing. C.L.: Writing-review and editing. Y.Z.: Data curation; Investigation; Writing-original draft; Writing-review and editing. H.Z.: Data curation; Investigation; Methodology; Resources. Yuanzheng Lu: Funding acquisition; Investigation; Writing-review and editing. K.L.: Visualization; Writing-original draft. Yawen Lu: Data curation; Investigation. S.Z.: Investigation. C.Z.: Investigation. R.L.: Writing-review and editing. Y.L.: Writing-review and editing. Z.G.: Funding acquisition. X.H.: Funding acquisition. C.H.: Funding acquisition; Supervision; Validation; Visualization; Writing-review and editing.

Funding

This work was supported by the following funds: the National Natural Science Foundation of China (81970564, 82170663 and 82370664, to Dr Guo; 82070670, to Dr He), Medical Scientific Research Foundation of Guangdong Province of China (A2020275 to Dr Yin), Guangdong Basic and Applied Basic Research Foundation (2024B1515040011 to Dr Guo; 2023A1515011792 and 2024A1515010799 to Dr Hu; 2020A1515010383 to Dr Liu; 2020A1515110827 to Dr Lu), the Guangdong Provincial Key Laboratory Construction Projection on Organ Donation and Transplant Immunology (2023B1212060020, to Dr He), Shenzhen Fundamental Research Program (jcyj20190809150817414 to Dr Lu).

Availability of data and materials

All data needed to evaluate the conclusions in this study are present in the article or the Supplementary Material. Reasonable requests for materials should be addressed to Meixian Yin.

Ethical approval

This study was approved by the Animal Investigation Committee of Sun Yat-sen University. The animal experiment was in line with the animal ethical research of the National Institutes of Health Guidelines.

Consent for publication

Not applicable.

Competing interests

All authors declare no competing interests.

Author details

¹Department of Emergency Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. ²Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. ³Organ Transplant Centre, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. ⁴Department of Anesthesiology, Foshan Fosun Chancheng Hospital, Foshan, China. ⁵Guangdong Provincial Key Laboratory of Organ Medicine, Guangzhou, China. ⁶NHC Key Laboratory of Assisted Circulation (Sun Yat-sen University), Guangzhou, China. ⁷Department of Emergency Medicine, The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, China. ⁸Department of Emergency Medicine, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, China. ⁹Panyu Experimental Middle School, Guangzhou, China. ¹⁰Department of Critical Care Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. ¹¹Department of Emergency, The First Afﬁliated Hospital of Guangzhou Medical University, Guangzhou, China. ¹²Department of Emergency, Outpatient of Cardiovascular, State Key Laboratory of

Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China. ¹³Guangdong Provincial International Cooperation Base of Science and Technology, Guangzhou, China.

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No competing interests reported.

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Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1

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Abstract

Figures

Introduction

Materials and methods

Rats CA model

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)

Culture of primary cardiomyocytes (PCMs)

Cellular oxygen-glucose deprivation and reperfusion (OGD/R) model

Immunofluorescence

Flow cytometry

Results

MH inhibits necroptosis in rats

A primary cardiomyocyte model of oxygen-glucose deprivation (OGD)

Capsaicin can improve the survival rate of rats after CPR

Discussion

Conclusions

Study limitations and future work

Abbreviations

Declarations

Funding

References

Additional Declarations

Supplementary Files

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