Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1

doi:10.21203/rs.3.rs-4866738/v2

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Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1

https://doi.org/10.21203/rs.3.rs-4866738/v2

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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, the mechanisms by which MH protects cardiomyocytes remain largely unexplored.

Methods In this study, three- to four-month-old male Wistar rats, weighing between 319.1 and 480.0 grams, were randomly divided into normothermia (NT) groups (acceptable range, 36.0-38.0°C) and MH groups (acceptable range, 32-34°C). Ten minutes after CA, CPR was perfumed with a slow injection of 0.30-0.50 mL epinephrine solution (30.00 μg/mL). Moreover, to investigate the role of TRPV1, capsaicin was administered through the right femoral artery in both NT and MH groups.

Results We found that MH improved the spontaneous breathing recovery, increased survival rate, and inhibited cardiac necroptosis in Wistar rats after CPR. Additionally, MH alleviated primary cardiomyocytes necroptosis after oxygen-glucose deprivation and reperfusion through upregulating TRPV1, downregulating EGFR, and subsequently suppressing MLKL. Furthermore, the combination of capsaicin, a TRPV1 activator, with MH enhanced blood pressure in Wistar rats after CPR compared to MH alone. Capsaicin improved the spontaneous breathing rate and survival rate of Wistar rats after CPR.

Conclusions MH may protect cardiac function by mitigating cardiomyocyte necroptosis through regulating TRPV1.

CPR

Mild hypothermia

Ischemia

TRPV1

Capsaicin

Cardiopulmonary resuscitation (CPR) is a lifesaving technology that has successfully enabled numerous patients experiencing sudden cardiac arrest (CA) to achieve spontaneous circulation. Despite this, survival rates for out-of-hospital cardiac arrest (OHCA) increased only from 5.7% in 1990 to 10.1% in 2011, with little improvement thereafter[1]. Furthermore, many patients with CA succumb to cerebral and cardiac hypoxic ischemia. Additionally, ischemia/reperfusion injury (IRI) poses significant risks of myocardial dysfunction following the return of spontaneous circulation (ROSC). Hence, mitigating ischemia/reperfusion (I/R) induced cardiac injury is crucial for reducing brain damage associated with CPR and improving survival outcomes.

In recent years, mild hypothermia (MH), targeting a body temperature of 32 to 36°C, has demonstrated potential benefits for improving survival and functional outcomes following CA. Some guidelines recommend against actively rewarming comatose patients with MH after ROSC in order to achieve normothermia. Mechanistically, MH has been reported to decrease the production of reactive oxygen species (ROS), ischemia/reperfusion injury (IRI), myocardial infarction area, ventricular remodeling, and cardiomyocyte metabolism, thereby offering myocardial protection. However, the precise mechanisms through which MH enhances survival after CA and protects the heart and brain remain largely unclear.

Cell death occurs through several mechanisms, four of which are relevant to the effects of mild hypothermia: 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 offers myocardial protection by enhancing intra-myocardial anti-inflammatory cytokine balance [2]. Another research team found that mild hypothermia increases the intramyocardial synthesis of heat shock protein 72, which inhibits necrosis but not apoptosis [3]. Based on the knowledge of therapeutic hypothermia, the authors suggest that mild hypothermia may protect the injured central nervous system from tissue damage and inflammatory responses by targeting necroptosis signaling following traumatic brain injury [4]. Additionally, an experiment involving transient spinal cord ischemia in rabbits discovered that mild hypothermia inhibits the expression of receptor-interacting protein kinases 1 and 3 (RIPK1/3), which are potential mediators of necroptosis [5]. However, the precise mechanism by which mild hypothermia inhibits necroptosis remains undefined.

Transient receptor potential (TRP) channels are non-selective cation channels that respond to various chemical and physical stimuli, including temperature, pressure, inflammation, and pain. Among these, the heat-sensitive transient receptor potential cation channel subfamily V member 1 (TRPV1) is predominantly expressed in sensory neurons and afferent nerve terminals within both the somatic and visceral sensory nervous systems. Numerous studies have highlighted the critical role of TRPV1 in pain and neural diseases. Recently, there has been growing interest in TRPV1’s role within the cardiovascular system. Reports indicate that TRPV1 is involved in postischemic recovery in isolated perfused hearts and in protecting rat hearts against IRI through hypoxic preconditioning. For instance, capsaicin (10 mg/kg) pretreatment 40 minutes before heart isolation was shown to reduce infarct size in subsequent IRI [6]. Furthermore, ruthenium red, a TRPV1 inhibitor, eliminated the reduction in infarct size induced by adenosine preconditioning [7]. Additionally, inhibition of TRPV1 by capsazepine has been found to increase apoptosis in colorectal cancer cells [8]. However, whether TRPV1 activation affects other forms of cell death, such as necroptosis, remains underexplored. This study aims to explore the mechanisms through which MH achieves cardioprotective effects by regulating TRPV1.

Rats CA model

The feed provided to the rats complied with the GB 14924.3-2010 Chinese standard for "Laboratory animals -Nutrients for formula feeds". Male Wistar rats, aged three to four months and weighing between 319.1 and 480.0 grams (Supplementary Fig. S1a), were randomly assigned to two groups: normothermia (NT) and mild hypothermia (MH). In the NT group, body temperature was maintained at 37°C (acceptable range: 36.0-38.0°C) using a heating pad after ROSC. In the MH group, body temperature was maintained at 33°C (acceptable range: 32-34°C) using an ice bag after ROSC.

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

CA was induced by administering Rocuronium Bromide (1.25 mg/kg, Merck Sharp & Dohme) and placing a tracheal cannula. Then, CA was confirmed by asystole on the EKG and a drop in MAP to 30 mmHg (Supplementary Fig. S1b). Ten minutes after CA, CPR was performed, involving a slow injection of 0.30-0.50 mL epinephrine solution (30.00 μg/mL) (Supplementary Fig. S1c), chest compressions, and ventilation [9]. ROSC was considered successful when MAP increased above 60 mmHg and remained stable for more than 30 minutes (Supplementary Fig. S1d).

Moreover, to assess the effect of TRPV1, capsaicin (10 mg/kg, MedChemExpress, HY-10448) was administered via the right femoral artery 15 minutes before the Rocuronium Bromide injection [6].

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

Total RNA was isolated from heart tissue or primary cardiomyocytes (PCMs) using the SteadyPure Universal RNA Extraction KitⅡ (Accurate Biology, AG21022). Five hundred nanograms of RNA were utilized for reverse transcription with the Evo M-MLV RT Premix (Accurate Biology, AG11706). Quantitative PCR (qPCR) was performed using a Real-Time PCR System (Roche, LightCycler 480 II) with the SYBR^® Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, AG11702). Melting curves were analyzed to ensure primer specificity. Relative mRNA expression levels were normalized to Actb. Primer sequences, designed using the NCBI database, are listed in Supplementary Table 1. Primers were synthesized by Guangzhou IGE Biotechnology Ltd [9].

Culture of primary cardiomyocytes (PCMs)

Whole hearts were collected from neonatal Sprague-Dawley (SD) rats aged three to ten days. The ventricles were isolated and cut into 1.0 mm³ pieces. These pieces were then digested with 0.125% Trypsin-EDTA (Gibco™, 930004) for eight minutes at 37°C. After removing the supernatant, the pieces were further 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]. Digestion was halted with DMEM supplemented with 10% fetal bovine serum (FBS). The supernatant was then gently collected, filtered through a 200-μm strainer, and centrifuged at 800 revolutions per minute (rpm) for eight minutes at 4°C [12]. Subsequently, the cardiac cells were plated in 10 cm culture dishes with DMEM supplemented with 10% FBS, and incubated at 5% CO₂ and 37°C for 60 minutes to remove the settling down non-cardiomyocytes. The cell supernatant, which primarily contained cardiomyocytes, was then collected and seeded into six-well plates [13].

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

The PCMs were randomly assigned to two groups: normothermia (NT-R) and mild hypothermia (MH-R). The PCMs were maintained in the DMEM without Glucose, glutamine or phenol red (Gibco™, A1443001). The culture plates were first placed in a modular incubator chamber (Billups-Rothenberg, CA92014) filled with 5% CO₂ and 95% N₂. Five minutes after the incoming and outgoing hose tubes were closed, the oxygen detector (SMART SENSOR, AS8801) registered 0.3%. Then, hypoxia was sustained for one to six hours at 37°C to establish the OGD model. Subsequently, the DMEM medium was replaced with complete medium. The PCMs were then cultured at 37°C (NT treatment) or 34°C (MH treatment) for two, six or ten hours to simulate reperfusion [9].

Immunofluorescence

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

Flow cytometry

Annexin V-PI double staining was performed to evaluate apoptosis rates in the PCMs (1 × 10⁵ cells/well, 6-well plate). Briefly, following various treatments, the PCMs were collected by trypsinization and washed twice with ice-cold phosphate-buffered saline (PBS). The cells were then resuspended in 60 μL binding buffer, followed by the addition of 5 μL Annexin V-FITC and 10 μL PI staining solution (Becton Dickinson, USA). The mixture was incubated for 15 minutes at room temperature in the dark. After incubation, 200 μL binding buffer was added to the cells, which were then analyzed using 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 determine whether MH offers protective benefits following CPR. We first established a CPR model in Wistar rats (Fig.1a). There were no significant differences among the groups in terms of rat weight, time to induce CA, volume of epinephrine solution administered, or duration of successful CPR (Supplementary Fig.S1). Thus, capsaicin injection 15 minutes prior to cardiac arrest did not influence CPR outcomes. During ROSC, rat body temperature was controlled with either a heating pad or ice bag. There was no body temperature difference in the NT and Cap-NT groups compared to the Sham group, but the body temperature was significantly lower in the MH and Cap-MH groups (Supplementary Fig.S2). As expected, MH provided a notable advantage in maintaining MAP (Fig.1b). Additionally, rats treated with MH exhibited a higher rate of recovery of spontaneous breathing and an increased survival rate compared to the NT treatment group (Fig.1c, d). Together, these findings support the hypothesis that MH can raise the survival rate after CPR.

MH inhibits necroptosis in rats

To clarify how MH protects the heart from IRI. We first analyzed gene expression associated with myocardial infarction (MI) using 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 found to be upregulated after MI. Subsequently, we assessed the mRNA levels of these cell death-related genes (Fig.2). While the expression of markers for 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) did not show statistically significant differences between the NT group and MH group, receptor interacting serine/threonine kinase 3 (Ripk3), a marker of necroptosis, was downregulated in the MH treatment group (Fig.2c). However, 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), which are also markers of necroptosis (Fig.2b, d). These findings suggest that MH may inhibit necroptosis, thereby attenuating myocardial injury following CPR.

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

To further validate it, we constructed an OGD model using PMCs. The PMCs were isolated from neonatal SD rats (Fig.3a, b). The PMCs were then cultured in DMEM under oxygen-free conditions to simulate CA. Annexin V-FITC/PI staining was performed to assess this model. With the prolongation of OGD duration, both early and late apoptotic cells were observed to rise (Fig.3c, d). Furthermore, after 6 hours of OGD, PMCs exhibited the lowest number of Annexin V-/PI+ cells. Consequently, we determined 6 hours to be the optimal duration for the OGD model.

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

Consistent with the findings from rat experiments, MH increased the survival rate of cardiomyocytes after 6 hours of OGD (Fig.S4). We then investigated how MH inhibited necroptosis after CPR. Given that temperature is the primary difference between NT and MH therapy, we focused on genes sensitive to temperature. As previously mentioned, many transient receptor potential (TRP) channels are polymodal, responding to both physical (including temperature) and chemical stimuli. The TRPV1 channel, originally identified as the ‘capsaicin receptor’ and also known as vanilloid receptor-1 (VR1), is well-known for its role in sensing pain and heat [14, 15]. Additionally, TRPV1 is involved in detecting ischemic stimuli during myocardial ischemia, thereby activating downstream pathways [16]. Moreover, TRPM4 is also reported to be related with the development of arrhythmias [17], and TRPM7 is crucial for executing necroptosis [18]. Therefore, we measured the mRNA level of these TRP channels after 6 hours of OGD (Fig.4). We noticed that MH had no significant effect on Trpv2 (Fig.4a-c) but inhibited the expression of Trpm4 (Fig.4e) and Trpm7 (Fig.4g).

To explore the relationship between TRP channels and cell death, we constructed networks of TRP channels and various cell death marker genes using the MetaCore database, which revealed the Trpv1-Egfr-Mlkl pathway. The TRPV1-EGFR interaction has been documented, with TRPV1 activation by EGFR leading to the activation of protein tyrosine phosphatase 1B (PTP1B), which in turn inhibits EGFR activity [19]. We subsequently verified this pathway (Fig.5). Notably, we found that Trpv1 was significantly upregulated by MH, highlighting 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 mitigating myocardial injury after CPR, we administered capsaicin, a TRPV1 activator, to rats 10 minutes before CA (Fig.6a). Capsaicin not only helped maintain MAP, but also improved the rate of recovery of spontaneous breathing and survival rate (Fig.6b, d, f). Furthermore, Capsaicin combined with MH demonstrated superior efficacy compared to MH alone (Fig.6c, e, g). Together, these results suggest that the efficacy of MH is associated with TRPV1 activation, highlighting its potential as a therapeutic target for CA and other cardiovascular diseases.

MH exerts an inhibitory effect on Trpm4/7. Unfortunately, this effect is inconsistent across the three OGD/R time points. In contrast, the expression pattern of the Trpv1-Egfr-Mlkl pathway remains stable. Our results identified Trpv1 as a crucial enhancer of the cardioprotective effect of MH. MH may alleviate cardiomyocytes necroptosis by upregulating Trpv1 during cardiomyocyte ischemia and hypoxia, thereby improving the survival rate of CPR. Additionally, capsaicin, a TRPV1 activator, in combination with MH, acts synergistically to enhance the MAP and survival rate of rats after CPR.

Our results align with previous research on the cardioprotective effects of capsaicin. Capsaicin is the agonist of TRPV1, which is one of the main integrators of the afferent of pain and temperature stimuli [20]. In the cardiovascular system, TRPV1 is predominantly expressed in the capsaicin-sensitive peptidergic primary sensory neurons that innervate the heart, as well as in ventricular cardiomyocytes, vascular smooth muscle, endothelial cells and epicardial cells [21]. Additionally, TRPV1 channels are expressed in both the cell membrane and mitochondrial membrane [22]. These channels can be activated by various factors during anoxia, tissue injury and inflammation, including heat, ROS, and inflammatory factors [23]. Beyond its role in pain perception, TRPV1 has garnered attention for its involvement in nervous system diseases, cardiovascular protection, and tumor development. C. M Hurt et al. demonstrated that capsaicin dose-dependently reduced the infarct size and decreased mitochondrial membrane potential, which could be counteracted by the TRPV1 antagonist capsazepine [24]. In cellular studies, capsaicin pretreatment protected cardiomyocytes from apoptosis induced by anoxia and reoxygenation [25]. Additionally, topical application of capsaicin has been shown to decrease infarct size via circulating humoral factors [26]. Jiang et al. found that TRPV1 knockout rats, compared with wild type rats, exhibited larger infarct sizes and higher apoptosis levels in response to ex vivo I/R, indicating that TRPV1 served as a protective role against myocardial apoptosis during I/R [27]. Furthermore, Capsaicin also exerts cardioprotective effects through its antioxidant and anti-inflammatory properties. 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]. Despite these findings, there is limited research on the role of capsaicin or TRPV1 in the cardioprotective effects of MH after CA.

In our cellular experiments, we isolated and purified primary cardiomyocytes from neonatal SD rats and constructed an OGD/R model. We observed a time-dependent upregulation of Trpv1 mRNA levels in the MH group, particularly 10 hours after reperfusion, compared with the NT group following 6 hours of OGD. Given previous studies demonstrating the cardioprotective effects of capsaicin and TRPV1, we hypothesize that TRPV1 activation plays a crucial role in the cardioprotective effects of MH, providing evidence for targeted temperature management (TTM) after CA and identifying it as a potential intervention target [29]. However, there is currently insufficient evidence to either support or refute the therapeutic MH for CA patients [30]. Those recommendations for therapeutic MH are primarily based on improvements in neurological outcomes [31, 32]. In contrast, our research indicates that MH directly protects cardiomyocytes by activating TRPV1, thereby mitigating cardiomyocyte necroptosis. Consequently, TRPV1 emerges as a promising target, and the application of capsaicin could be extended to cardiomyocyte protection in emergency settings. Furthermore, clinical trials have shown that therapeutic MH or TTM can lead to QTc prolongation, which may increase the risk of arrhythmias, particularly in early repolarization syndrome [33, 34]. In this respect, capsaicin or TRPV1 targeting may offer a safer alternative. Additionally, combining mild hypothermic perfusion with capsaicin may enhance donor organ quality during preservation and transport.

The IRI of the brain is a primary cause of death and disability in CA patients after ROSC [35]. Clinical research has demonstrated that MH can mitigate cerebral injury after CA [31]. A multicenter cohort study conducted in Japan divided 1,111 OHCA patients from 125 hospitals into two groups: 33-34°C and 35-36°C. The study found that the 33-34°C group was associated with better recovery from moderate nervous system impairment [36]. In a randomized controlled trial involving 574 comatose patients who were resuscitated from CA with a non-shockable rhythm, moderate therapeutic hypothermia at 33°C for 24 hours improved the neurological prognosis [37]. Notably, asphyxia-induced cardiac arrest results in more severe neurological injury and worse outcomes compared to ventricular fibrillation-induced cardiac arrest [38, 39]. In this respect, patients in this group may benefit more from therapeutic MH. Additionally, cerebral injury resulting from CA can exacerbate cardiomyocyte injury, which is recognized as part of the brain-heart axis [40]. Therefore, MH may also protect cardiomyocytes through brain-heart axis, with TRPV1 playing a crucial role in both the cardiovascular and neural systems.

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

Study limitations and future work

Several limitations are noted in our research. Firstly, our study was conducted in rats, and the findings may not be directly applicable to humans. Further experiments using larger animals, such as pigs, would provide more robust evidence for our conclusions. Secondly, the absence of TRPV1 knockout mice and the lack of TRPV1 antagonist injections prevent us from determining whether the cardioprotective effect of MH is mediated by TRPV1. Thirdly, the causes of OHCA are diverse, including anoxia, hypovolemia, electrolyte abnormality, and myocardial infarction, which can be broadly categorized into cardiac and non-cardiac causes [41]. Recently, OHCA has increasingly been recognized as distinct from in-hospital cardiac arrest (IHCA) and studied independently [42]. Therefore, the heterogeneity of CA patients must be considered. In our experiment, the CA model was constructed by respiratory depression induced by muscle relaxants, which limits the generalizability of our conclusions. Furthermore, we have not verified the Trpv1-Egfr-Mlkl pathway at the protein level, and the interactions among TRPV1, EGFR, and MLKL remain 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

Supplementary information

Additional file 1. Figure S1: Weight of rats, time of inducing CA, volume of using epinephrine solution and time of successful CPR in the CPR model of rats. Figure S2: Temperature of rats. Figure S3: FPKM of key genes of apoptosis (Casp3), necroptosis (Ripk1, Ripk3 and Mlkl1), pyroptosis (Casp1 and Gsdmd), ferroptosis (Ptgs2), autophagic death (Becn1). Figure S4: Cell death rate after OGD/R. Table S1: Primer sequences of the target genes.

Acknowledgements

We would like to express our deepest gratitude to Professor Huang for his exceptional guidance and support throughout this research. His expertise and insightful feedback were crucial to the development and success of this study.

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.

Acknowledgments

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

The authors declare no competing interests.

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The authors declare no competing interests.

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Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1

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