Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1

Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1 Meixian Yin, Chuiliang Liu, Yifan Zhu, Haohong Zhan, Yuanzheng Lu, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4866738/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction 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. Materials and methods 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 mm 3 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 × 10 5 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. Results 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. Discussion 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. Conclusions 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. Abbreviations 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 Declarations 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. 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Miyake T, Shirakawa H, Nakagawa T, Kaneko S: Activation of mitochondrial transient receptor potential vanilloid 1 channel contributes to microglial migration . Glia 2015, 63 (10):1870-1882. 23. Randhawa PK, Jaggi AS: TRPV1 and TRPV4 channels: potential therapeutic targets for ischemic conditioning-induced cardioprotection . Eur J Pharmacol 2015, 746 :180-185. 24. Hurt CM, Lu Y, Stary CM, Piplani H, Small BA, Urban TJ, Qvit N, Gross GJ, Mochly-Rosen D, Gross ER: Transient receptor potential vanilloid 1 regulates mitochondrial membrane potential and myocardial reperfusion injury . J Am Heart Assoc 2016, 5 (9). 25. Huang J, Liu Z, Xu P, Zhang Z, Yin D, Liu J, He H, He M: Capsaicin prevents mitochondrial damage, protects cardiomyocytes subjected to anoxia/reoxygenation injury mediated by 14-3-3 η/Bcl-2 . Eur J Pharmacol 2018, 819 :43-50. 26. Redington KL, Disenhouse T, Strantzas SC, Gladstone R, Wei C, Tropak MB, Dai X, Manlhiot C, Li J, Redington AN: Remote cardioprotection by direct peripheral nerve stimulation and topical capsaicin is mediated by circulating humoral factors . Basic Res Cardiol 2012, 107 (2):241. 27. Jiang XX, Liu GY, Lei H, Li ZL, Feng QP, Huang W: Activation of transient receptor potential vanilloid 1 protects the heart against apoptosis in ischemia/reperfusion injury through upregulating the PI3K/Akt signaling pathway . Int J Mol Med 2018, 41 (3):1724-1730. 28. Ahmed RA, Alam MF, Alshahrani S, Jali AM, Qahl AM, Khalid M, Muzafar HMA, Alhamami HN, Anwer T: Capsaicin ameliorates the cyclophosphamide-induced cardiotoxicity by inhibiting free radicals generation, inflammatory cytokines, and apoptotic pathway in rats . Life (Basel) 2023, 13 (3). 29. Taccone FS, Picetti E, Vincent JL: High quality targeted temperature management (TTM) after cardiac arrest . Crit Care 2020, 24 (1):6. 30. Sandroni C, Natalini D, Nolan JP: Temperature control after cardiac arrest . Crit Care 2022, 26 (1):361. 31. Group HaCAS: Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest . N Engl J Med 2002, 346 (8):549-556. 32. Callaway CW, Soar J, Aibiki M, Böttiger BW, Brooks SC, Deakin CD, Donnino MW, Drajer S, Kloeck W, Morley PT et al : Part 4: advanced life support: 2015 international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations . Circulation 2015, 132 (16 Suppl 1):S84-145. 33. Piktel JS, Wilson LD: Targeted temperature management after sudden cardiac arrest: Proarrhythmic or antiarrhythmic? Probably both . J Crit Care 2018, 46 :149-150. 34. Thomsen JH, Hassager C, Erlinge D, Nielsen N, Lindholm MG, Bro-Jeppesen J, Grand J, Pehrson S, Graff C, Køber LV et al : Repolarization and ventricular arrhythmia during targeted temperature management post cardiac arrest . Resuscitation 2021, 166 :74-82. 35. Lemiale V, Dumas F, Mongardon N, Giovanetti O, Charpentier J, Chiche JD, Carli P, Mira JP, Nolan J, Cariou A: Intensive care unit mortality after cardiac arrest: the relative contribution of shock and brain injury in a large cohort . Intensive Care Med 2013, 39 (11):1972-1980. 36. Nishikimi M, Ogura T, Nishida K, Hayashida K, Emoto R, Matsui S, Matsuda N, Iwami T: Outcome related to level of targeted temperature management in postcardiac arrest syndrome of low, moderate, and high severities: a nationwide multicenter prospective registry . Crit Care Med 2021, 49 (8):e741-e750. 37. Lascarrou JB, Merdji H, Le Gouge A, Colin G, Grillet G, Girardie P, Coupez E, Dequin PF, Cariou A, Boulain T et al : Targeted temperature management for cardiac arrest with nonshockable rhythm . N Engl J Med 2019, 381 (24):2327-2337. 38. Zhang Y, Li CS, Wu CJ, Yang J, Hang CC: Comparison of cerebral metabolism between pig ventricular fibrillation and asphyxial cardiac arrest models . Chin Med J (Engl) 2015, 128 (12):1643-1648. 39. Uray T, Lamade A, Elmer J, Drabek T, Stezoski JP, Missé A, Janesko-Feldman K, Garman RH, Chen N, Kochanek PM et al : Phenotyping cardiac arrest: bench and bedside characterization of brain and heart injury based on etiology . Crit Care Med 2018, 46 (6):e508-e515. 40. Chen Z, Venkat P, Seyfried D, Chopp M, Yan T, Chen J: Brain-heart interaction: cardiac complications after stroke . Circ Res 2017, 121 (4):451-468. 41. Myat A, Song KJ, Rea T: Out-of-hospital cardiac arrest: current concepts . Lancet 2018, 391 (10124):970-979. 42. Penketh J, Nolan JP: In-hospital cardiac arrest: the state of the art . Crit Care 2022, 26 (1):376. Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4866738","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":337489927,"identity":"13290a32-e799-43cc-9d97-94e8b25a245d","order_by":0,"name":"Meixian 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University","correspondingAuthor":false,"prefix":"","firstName":"Chunlin","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2024-08-06 08:16:20","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4866738/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-4866738/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63910981,"identity":"50277e8c-66a2-4ca0-82c7-d9019c1e3ef2","added_by":"auto","created_at":"2024-09-03 16:27:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":291861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMild hypothermia increased the survival rate of rats after cardiopulmonary resuscitation (CPR). a\u003c/strong\u003e, Timeline of the experimental workflow. \u003cstrong\u003eb\u003c/strong\u003e, Mean arterial pressure of the Sham, NT (normothermia), and MH (mild hypothermia) groups from pre-operation (pre-op) to 5 hours after the return of spontaneous circulation (ROSC). Mean ± SEM, two-way ANOVA, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ****\u003cem\u003eP\u003c/em\u003e≤0.0001. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e, Rate of recovery of spontaneous breathing (c) and survival rate (d) of the Sham, NT, and MH groups at 5 hours post-ROSC after CPR. \u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4866738/v2/71ef1f68d0c5c6df2f36d583.png"},{"id":63910512,"identity":"10587c91-e8d2-45b5-af48-050d0e06dd52","added_by":"auto","created_at":"2024-09-03 16:19:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":324221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMild hypothermia inhibited necrotic apoptosis in rats after CPR. a-h\u003c/strong\u003e, mRNA level of key genes of apoptosis (\u003cem\u003eCasp3\u003c/em\u003e)\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(a)\u003c/strong\u003e, necroptosis (\u003cem\u003eRipk1\u003c/em\u003e, \u003cem\u003eRipk3\u003c/em\u003e and \u003cem\u003eMlkl1\u003c/em\u003e)\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(b-d)\u003c/strong\u003e, pyroptosis (\u003cem\u003eCasp1\u003c/em\u003e and \u003cem\u003eGsdmd\u003c/em\u003e)\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(e, f)\u003c/strong\u003e, ferroptosis (\u003cem\u003ePtgs2\u003c/em\u003e)\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(g)\u003c/strong\u003e, autophagic death (\u003cem\u003eBecn1\u003c/em\u003e)\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(h)\u003c/strong\u003e; Mean ± SEM, two-tailed ratio unpaired \u003cem\u003et\u003c/em\u003e-test; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05. \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4866738/v2/ad7fb70e624b4e8912c20842.png"},{"id":63910510,"identity":"f70b4700-8f01-49a7-ba7d-81dce69b4acf","added_by":"auto","created_at":"2024-09-03 16:19:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1751281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstructing a primary cardiomyocyte model of oxygen-glucose deprivation. a\u003c/strong\u003e, Phase contrast images of primary cardiomyocytes. \u003cstrong\u003eb\u003c/strong\u003e, Identification of primary cardiomyocytes with an antibody against α-Actin. Green, α-Actin; blue, DAPI.\u003cstrong\u003e c\u003c/strong\u003e, Apoptosis of primary cardiomyocytes was assessed by flow cytometry with Annexin V-FITC/PI double staining. OGD, oxygen-glucose deprivation.\u003cstrong\u003e d\u003c/strong\u003e, Proportions of cells in survival, early apoptosis, and late apoptosis stages. Mean ± SEM, two-tailed ratio unpaired \u003cem\u003et\u003c/em\u003e-test; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4866738/v2/ac2483135b4f3270c20b8c1e.png"},{"id":63910508,"identity":"7a7dd051-7db1-4176-bf33-626df0f39af2","added_by":"auto","created_at":"2024-09-03 16:19:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":139521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMild hypothermia inhibits \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrpm4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrpm7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after primary cardiomyocytes OGD/R. a\u003c/strong\u003e-I, mRNA level of \u003cem\u003eTrpv2\u003c/em\u003e \u003cstrong\u003e(a-c)\u003c/strong\u003e, \u003cem\u003eTrpm4\u003c/em\u003e \u003cstrong\u003e(d-f) \u003c/strong\u003eand \u003cem\u003eTrpm7\u003c/em\u003e \u003cstrong\u003e(g-i)\u003c/strong\u003e.OGD/R, oxygen-glucose deprivation and reperfusion. Mean ± SEM, two-tailed ratio unpaired \u003cem\u003et\u003c/em\u003e-test; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e≤0.0001\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea, d and g\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 8;\u003cstrong\u003e b, e and h\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 6;\u003cstrong\u003ec, f and i\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4866738/v2/b1b6f849a1e4ac387f056f0d.png"},{"id":63910980,"identity":"1ee5c6d0-6567-4569-ae21-b170caf74ad7","added_by":"auto","created_at":"2024-09-03 16:27:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":138885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMild hypothermia inhibits primary cardiomyocytes necroptosis through \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrpv1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEgfr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMlkl\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e pathway after OGD/R. a-i\u003c/strong\u003e, mRNA level of \u003cem\u003eTrpv1 \u003c/em\u003e\u003cstrong\u003e(a-c)\u003c/strong\u003e, \u003cem\u003eEgfr\u003c/em\u003e \u003cstrong\u003e(d-f)\u003c/strong\u003e and \u003cem\u003eMlkl1\u003c/em\u003e \u003cstrong\u003e(g-i)\u003c/strong\u003e. Mean ± SEM, two-tailed ratio unpaired \u003cem\u003et\u003c/em\u003e-test; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e≤0.0001\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea, d and g\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 8;\u003cstrong\u003e b, e and h\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 6;\u003cstrong\u003e c, f and i\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4866738/v2/f13f552ed6f9f6013d03c314.png"},{"id":63910982,"identity":"aaa83886-bf3d-4d12-804a-60696cf282c7","added_by":"auto","created_at":"2024-09-03 16:27:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":378908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCapsaicin improves the survival rate of rats after CPR. a\u003c/strong\u003e, Timeline of the experimental workflow. \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, Mean arterial pressure of rats in the Sham, NT (normothermia), and Cap-NT (Capsaicin + normothermia) groups (\u003cstrong\u003eb\u003c/strong\u003e), and Sham, MH (mild hypothermia), and Cap-MH (Capsaicin + mild hypothermia) groups (\u003cstrong\u003ec\u003c/strong\u003e) from pre-operation (pre-op) to 5 hours after the return of spontaneous circulation (ROSC). \u003cstrong\u003eb, c, \u003c/strong\u003eMean ± SEM, two-way ANOVA, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ****\u003cem\u003eP\u003c/em\u003e≤0.0001. \u003cstrong\u003ed, e, \u003c/strong\u003eRate of recovery of spontaneous breathing of the Sham, NT, and Cap-NT groups (\u003cstrong\u003ed\u003c/strong\u003e), and Sham, MH, and Cap-MH groups (\u003cstrong\u003ee\u003c/strong\u003e) 5 hours after ROSC following 10 minutes of CA. \u003cstrong\u003ef, g,\u003c/strong\u003e Survival rate of the Sham, NT, and Cap-NT groups (\u003cstrong\u003ef\u003c/strong\u003e), and Sham, MH, and Cap-MH groups (\u003cstrong\u003eg\u003c/strong\u003e) 5 hours after ROSC following 10 minutes of CA.Sham,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 3; NT,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 4; MH,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 3; Cap-NT,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 6;Cap-MH,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4866738/v2/d8cf1269962941c2d419d045.png"},{"id":63911492,"identity":"791e2e92-661a-4b18-9760-9a3f5572f359","added_by":"auto","created_at":"2024-09-03 16:35:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4975942,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4866738/v2/e8982284-761a-4b5a-bb7f-55cd95ecf695.pdf"},{"id":63910513,"identity":"02ae4079-4b25-4886-84dc-eb2267b94078","added_by":"auto","created_at":"2024-09-03 16:19:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9955789,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation2024.08.19.docx","url":"https://assets-eu.researchsquare.com/files/rs-4866738/v2/2ded71d72a84713c39105288.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eMild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiopulmonary 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[\u003ca href=\"#_ENREF_1\" title=\"Jerkeman, 2022 #77\"\u003e1\u003c/a\u003e]. 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,\u0026nbsp;mitigating ischemia/reperfusion (I/R) induced cardiac injury is crucial for reducing brain damage associated with CPR and improving survival outcomes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn recent years, mild hypothermia (MH), targeting a body temperature of 32 to 36\u0026deg;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.\u003c/p\u003e\n\u003cp\u003eCell 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\u0026nbsp;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\u0026nbsp;[\u003ca href=\"#_ENREF_2\" title=\"Vazquez-Jimenez, 2001 #72\"\u003e2\u003c/a\u003e]. Another research team found that mild hypothermia increases the intramyocardial synthesis of heat shock protein 72, which inhibits necrosis but not apoptosis\u0026nbsp;[\u003ca href=\"#_ENREF_3\" title=\"Qing, 2002 #62\"\u003e3\u003c/a\u003e]. 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\u0026nbsp;[\u003ca href=\"#_ENREF_4\" title=\"Liu, 2016 #52\"\u003e4\u003c/a\u003e]. 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\u0026nbsp;[\u003ca href=\"#_ENREF_5\" title=\"Motomatsu, 2020 #55\"\u003e5\u003c/a\u003e]. However, the precise mechanism by which mild hypothermia inhibits necroptosis remains undefined.\u003c/p\u003e\n\u003cp\u003eTransient 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\u0026rsquo;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.\u0026nbsp;For instance, capsaicin (10 mg/kg) pretreatment 40 minutes before heart isolation was shown to reduce infarct size in subsequent IRI\u0026nbsp;[\u003ca href=\"#_ENREF_6\" title=\"Randhawa, 2017 #65\"\u003e6\u003c/a\u003e]. Furthermore, ruthenium red, a TRPV1 inhibitor, eliminated the reduction in infarct size induced by adenosine preconditioning\u0026nbsp;[\u003ca href=\"#_ENREF_7\" title=\"Singh, 2017 #79\"\u003e7\u003c/a\u003e]. Additionally, inhibition of TRPV1 by capsazepine has been found to increase apoptosis in colorectal cancer cells\u0026nbsp;[\u003ca href=\"#_ENREF_8\" title=\"Sung, 2012 #80\"\u003e8\u003c/a\u003e]. 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. \u0026nbsp;\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eRats CA model\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe feed provided to the rats complied with the GB 14924.3-2010 Chinese standard for \u0026quot;Laboratory animals -Nutrients for formula feeds\u0026quot;.\u0026nbsp;Male Wistar rats, aged three to four months and weighing between 319.1 and 480.0 grams (\u003cstrong\u003eSupplementary Fig. S1a\u003c/strong\u003e), were randomly assigned to two groups: normothermia (NT) and mild hypothermia (MH). In the NT group, body temperature was maintained at 37\u0026deg;C (acceptable range: 36.0-38.0\u0026deg;C) using a heating pad after ROSC. In the MH group, body temperature was maintained at 33\u0026deg;C (acceptable range: 32-34\u0026deg;C) using an ice bag after ROSC.\u003c/p\u003e\n\u003cp\u003eFollowing 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 \u0026times; diastolic pressure) / 3. Three needle electrodes were positioned subcutaneously in the limbs for continuous electrocardiogram (EKG) monitoring.\u003c/p\u003e\n\u003cp\u003eCA was induced by administering Rocuronium Bromide (1.25 mg/kg, Merck Sharp \u0026amp; Dohme) and placing a tracheal cannula. Then, CA was confirmed by asystole on the EKG and a drop in MAP to 30 mmHg (\u003cstrong\u003eSupplementary Fig. S1b\u003c/strong\u003e). Ten minutes after CA, CPR was performed, involving a slow injection of 0.30-0.50 mL epinephrine solution (30.00 \u0026mu;g/mL) (\u003cstrong\u003eSupplementary Fig. S1c\u003c/strong\u003e), chest compressions, and ventilation\u0026nbsp;[\u003ca href=\"#_ENREF_9\" title=\"Wei, 2019 #74\"\u003e9\u003c/a\u003e]. ROSC was considered successful when MAP increased above 60 mmHg and remained stable for more than 30 minutes (\u003cstrong\u003eSupplementary Fig. S1d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eMoreover, 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\u0026nbsp;[\u003ca href=\"#_ENREF_6\" title=\"Randhawa, 2017 #65\"\u003e6\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse transcription and quantitative polymerase chain reaction (RT-qPCR)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from heart tissue or primary cardiomyocytes (PCMs) using the \u003cem\u003eSteadyPure\u003c/em\u003e Universal RNA Extraction KitⅡ (Accurate Biology, AG21022). Five hundred nanograms of RNA were utilized for reverse transcription with the \u003cem\u003eEvo M-MLV\u003c/em\u003e RT Premix (Accurate Biology, AG11706). Quantitative PCR (qPCR) was performed using a Real-Time PCR System (Roche, LightCycler 480 II) \u0026nbsp;with the SYBR\u003csup\u003e\u0026reg;\u003c/sup\u003e Green Premix \u003cem\u003ePro Taq\u003c/em\u003e HS qPCR Kit II (Accurate Biology, AG11702). Melting curves were analyzed to ensure primer specificity. Relative mRNA expression levels were normalized to \u003cem\u003eActb\u003c/em\u003e. Primer sequences, designed using the NCBI database, are listed in \u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e. Primers were synthesized by Guangzhou IGE Biotechnology Ltd\u0026nbsp;[\u003ca href=\"#_ENREF_9\" title=\"Wei, 2019 #74\"\u003e9\u003c/a\u003e].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eprimary\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;cardiomyocytes\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(PCMs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole hearts were collected from neonatal Sprague-Dawley (SD) rats aged three to ten days. The ventricles were isolated and cut into 1.0 mm\u003csup\u003e3\u003c/sup\u003e pieces. These pieces were then digested with 0.125% Trypsin-EDTA (Gibco\u0026trade;, 930004) for eight minutes at 37\u0026deg;C. After removing the supernatant, the pieces were further digested with Dulbecco\u0026apos;s Modified Eagle\u0026apos;s Medium (DMEM) containing 0.5 mg/mL (62.5 U/mL) collagenase Ⅰ (Gibco\u0026trade;, 17100017) for two hours at 37\u0026deg;C with gentle pendular movement in a water bath\u0026nbsp;[\u003ca href=\"#_ENREF_10\" title=\"Zhou, 2022 #76\"\u003e10\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_11\" title=\"Nicks, 2022 #57\"\u003e11\u003c/a\u003e]. Digestion was halted with DMEM supplemented with 10% fetal bovine serum (FBS). The supernatant was then gently collected, filtered through a 200-\u0026mu;m strainer, and centrifuged at 800 revolutions per minute (rpm)\u0026nbsp;for eight minutes at 4\u0026deg;C\u0026nbsp;[\u003ca href=\"#_ENREF_12\" title=\"Louch, 2011 #53\"\u003e12\u003c/a\u003e]. Subsequently, the cardiac cells were plated in 10 cm culture dishes with DMEM supplemented with 10% FBS, and incubated at 5% CO₂ and 37\u0026deg;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\u0026nbsp;[\u003ca href=\"#_ENREF_13\" title=\"Leone, 2021 #51\"\u003e13\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular oxygen-glucose deprivation and reperfusion (OGD/R) model\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026trade;, 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\u0026deg;C to establish the OGD\u0026nbsp;model. Subsequently, the DMEM medium was replaced with complete medium. The PCMs were then cultured at 37\u0026deg;C (NT treatment) or 34\u0026deg;C (MH treatment) \u0026nbsp;for two, six or ten hours to simulate reperfusion\u0026nbsp;[\u003ca href=\"#_ENREF_9\" title=\"Wei, 2019 #74\"\u003e9\u003c/a\u003e].\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026deg;C for one hour and incubated with anti-\u0026alpha;-Actin antibodies (Abcam, 1:100) at 4\u0026deg;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\u0026deg;C, and then gently washed three times with PBS. After stained with DAPI, the PCMs were observed using fluorescence microscopy\u0026nbsp;[\u003ca href=\"#_ENREF_9\" title=\"Wei, 2019 #74\"\u003e9\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnnexin V-PI double staining was performed to evaluate apoptosis rates in the PCMs (1\u0026nbsp;\u0026times;\u0026nbsp;10\u003csup\u003e5\u003c/sup\u003e 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 \u0026mu;L binding buffer, followed by the addition of 5 \u0026mu;L Annexin V-FITC and 10 \u0026mu;L PI staining solution (Becton Dickinson, USA). The mixture was incubated for 15 minutes at room temperature in the dark. After incubation, 200 \u0026mu;L 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.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMH raises the survival rate of rats after CPR \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether MH offers protective benefits following CPR. We first established a CPR model in Wistar rats (\u003cstrong\u003eFig.1a\u003c/strong\u003e). 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 (\u003cstrong\u003eSupplementary Fig.S1\u003c/strong\u003e). 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 (\u003cstrong\u003eSupplementary Fig.S2\u003c/strong\u003e). As expected, MH provided a notable advantage in maintaining MAP (\u003cstrong\u003eFig.1b\u003c/strong\u003e). 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 (\u003cstrong\u003eFig.1c, d\u003c/strong\u003e). Together, these findings support the hypothesis that MH can raise the survival rate after CPR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMH inhibits necroptosis in rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 (\u003cstrong\u003eSupplementary Fig.S3a\u003c/strong\u003e), necroptosis (\u003cstrong\u003eFig.S3b-d\u003c/strong\u003e), pyroptosis (\u003cstrong\u003eSupplementary Fig.S3e, f\u003c/strong\u003e), ferroptosis (\u003cstrong\u003eSupplementary Fig.S3g\u003c/strong\u003e) and autophagy (\u003cstrong\u003eSupplementary Fig.S3h\u003c/strong\u003e) were found to be upregulated after MI. Subsequently, we assessed the mRNA levels of these cell death-related genes (\u003cstrong\u003eFig.2\u003c/strong\u003e). While the expression of markers for apoptosis (Caspase 3, \u003cem\u003eCasp3\u003c/em\u003e) (\u003cstrong\u003eFig.2a\u003c/strong\u003e), pyroptosis (Caspase 1 and Gasdermin D, \u003cem\u003eCasp1\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Gsdmd\u003c/em\u003e) (\u003cstrong\u003eFig.2e, f\u003c/strong\u003e), ferroptosis (Prostaglandin G/H synthase 2, \u003cem\u003ePtgs2\u003c/em\u003e) (\u003cstrong\u003eFig.2g\u003c/strong\u003e) and autophagy (Beclin 1, \u003cem\u003eBecn1\u003c/em\u003e) (\u003cstrong\u003eFig.2h\u003c/strong\u003e) did not show statistically significant differences between the NT group and MH group, receptor interacting serine/threonine kinase 3 (\u003cem\u003eRipk3\u003c/em\u003e), a marker of necroptosis, was downregulated in the MH treatment group (\u003cstrong\u003eFig.2c\u003c/strong\u003e). However, we didn\u0026rsquo;t observe statistically significant difference of the transcription level of receptor interacting serine/threonine kinase 1(\u003cem\u003eRipk1\u003c/em\u003e) and mixed lineage kinase domain-like protein (\u003cem\u003eMlkl\u003c/em\u003e), which are also markers of necroptosis (\u003cstrong\u003eFig.2b, d\u003c/strong\u003e). These findings suggest that MH may inhibit necroptosis, thereby attenuating myocardial injury following CPR.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA primary cardiomyocyte model of oxygen-glucose deprivation (OGD)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate it, we constructed an OGD model using PMCs. The PMCs were isolated from neonatal SD rats (\u003cstrong\u003eFig.3a, b\u003c/strong\u003e). 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 (\u003cstrong\u003eFig.3c, d\u003c/strong\u003e). 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMH inhibits cardiomyocyte necroptosis through the \u003cem\u003eTrpv1\u003c/em\u003e-\u003cem\u003eEgfr\u003c/em\u003e-\u003cem\u003eMlkl\u003c/em\u003e pathway\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsistent with the findings from rat experiments, MH increased the survival rate of cardiomyocytes after 6 hours of OGD (\u003cstrong\u003eFig.S4\u003c/strong\u003e). 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 \u0026lsquo;capsaicin receptor\u0026rsquo; and also known as vanilloid receptor-1 (VR1), is well-known for its role in sensing pain and heat\u0026nbsp;[\u003ca href=\"#_ENREF_14\" title=\"Koivisto, 2022 #48\"\u003e14\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_15\" title=\"Ramsey, 2006 #63\"\u003e15\u003c/a\u003e]. Additionally, TRPV1 is involved in detecting ischemic stimuli during myocardial ischemia, thereby activating downstream pathways\u0026nbsp;[\u003ca href=\"#_ENREF_16\" title=\"Pan, 2004 #59\"\u003e16\u003c/a\u003e]. Moreover, TRPM4 is also reported to be related with the development of arrhythmias\u0026nbsp;[\u003ca href=\"#_ENREF_17\" title=\"Watanabe, 2008 #73\"\u003e17\u003c/a\u003e], and TRPM7 is crucial for executing necroptosis\u0026nbsp;[\u003ca href=\"#_ENREF_18\" title=\"Cai, 2014 #81\"\u003e18\u003c/a\u003e]. Therefore, we measured the mRNA level of these TRP channels after 6 hours of OGD (\u003cstrong\u003eFig.4\u003c/strong\u003e). We noticed that MH had no significant effect on \u003cem\u003eTrpv2\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFig.4a-c\u003c/strong\u003e) but inhibited the expression of \u003cem\u003eTrpm4\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFig.4e\u003c/strong\u003e) and \u003cem\u003eTrpm7\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFig.4g\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo 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\u0026nbsp;[\u003ca href=\"#_ENREF_19\" title=\"de Jong, 2014 #44\"\u003e19\u003c/a\u003e]. We subsequently verified this pathway (\u003cstrong\u003eFig.5\u003c/strong\u003e). Notably, we found that Trpv1 was significantly upregulated by MH, highlighting its role in the effect of MH (\u003cstrong\u003eFig.5b, c\u003c/strong\u003e). Moreover, both epidermal growth factor receptor (\u003cem\u003eEgfr\u003c/em\u003e) (\u003cstrong\u003eFig.5e, f\u003c/strong\u003e) and \u003cem\u003eMlkl\u003c/em\u003e (\u003cstrong\u003eFig.5h, i\u003c/strong\u003e) were downregulated by MH.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCapsaicin can improve the survival rate of rats after CPR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the role of TRPV1 in mitigating myocardial injury after CPR, we administered capsaicin, a TRPV1 activator, to rats 10 minutes before CA (\u003cstrong\u003eFig.6a\u003c/strong\u003e). Capsaicin not only helped maintain MAP, but also improved the rate of recovery of spontaneous breathing and survival rate (\u003cstrong\u003eFig.6b, d, f\u003c/strong\u003e). Furthermore, Capsaicin combined with MH demonstrated superior efficacy compared to MH alone (\u003cstrong\u003eFig.6c, e, g\u003c/strong\u003e). 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.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMH exerts an inhibitory effect on \u003cem\u003eTrpm4\u003c/em\u003e/\u003cem\u003e7\u003c/em\u003e. Unfortunately, this effect is inconsistent across the three OGD/R time points. In contrast, the expression pattern of the \u003cem\u003eTrpv1\u003c/em\u003e-\u003cem\u003eEgfr\u003c/em\u003e-\u003cem\u003eMlkl\u003c/em\u003e pathway remains stable. Our results identified \u003cem\u003eTrpv1\u003c/em\u003e as a crucial enhancer of the cardioprotective effect of MH. MH may alleviate cardiomyocytes necroptosis by upregulating \u003cem\u003eTrpv1\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003eOur results align with previous research on the cardioprotective effects of capsaicin.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eCapsaicin is the agonist of TRPV1, which is one of the main integrators of the afferent of pain and temperature stimuli\u0026nbsp;[\u003ca href=\"#_ENREF_20\" title=\"Aghazadeh Tabrizi, 2017 #40\"\u003e20\u003c/a\u003e]. In the cardiovascular system, TRPV1 is predominantly expressed in \u0026nbsp;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\u0026nbsp;[\u003ca href=\"#_ENREF_21\" title=\"Randhawa, 2017 #66\"\u003e21\u003c/a\u003e]. Additionally, TRPV1 channels are expressed in both the cell membrane and mitochondrial membrane\u0026nbsp;[\u003ca href=\"#_ENREF_22\" title=\"Miyake, 2015 #54\"\u003e22\u003c/a\u003e]. These channels can be activated by various factors during anoxia, tissue injury and inflammation, including heat, ROS, and inflammatory factors\u0026nbsp;[\u003ca href=\"#_ENREF_23\" title=\"Randhawa, 2015 #64\"\u003e23\u003c/a\u003e]. 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\u0026nbsp;[\u003ca href=\"#_ENREF_24\" title=\"Hurt, 2016 #46\"\u003e24\u003c/a\u003e]. In cellular studies, capsaicin pretreatment protected cardiomyocytes from apoptosis induced by anoxia and reoxygenation\u0026nbsp;[\u003ca href=\"#_ENREF_25\" title=\"Huang, 2018 #45\"\u003e25\u003c/a\u003e]. Additionally, topical application of capsaicin has been shown to decrease infarct size via circulating humoral factors\u0026nbsp;[\u003ca href=\"#_ENREF_26\" title=\"Redington, 2012 #67\"\u003e26\u003c/a\u003e]. 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\u0026nbsp;[\u003ca href=\"#_ENREF_27\" title=\"Jiang, 2018 #47\"\u003e27\u003c/a\u003e]. Furthermore, Capsaicin also exerts cardioprotective effects through its antioxidant and anti-inflammatory properties. \u0026nbsp;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\u0026nbsp;[\u003ca href=\"#_ENREF_28\" title=\"Ahmed, 2023 #41\"\u003e28\u003c/a\u003e]. Despite these findings, there is limited research on the role of capsaicin or TRPV1 in the cardioprotective effects of MH after CA.\u003c/p\u003e\n\u003cp\u003eIn 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 \u003cem\u003eTrpv1\u003c/em\u003e 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\u0026nbsp;targeted temperature management (TTM) after CA and identifying it as a potential intervention target\u0026nbsp;[\u003ca href=\"#_ENREF_29\" title=\"Taccone, 2020 #69\"\u003e29\u003c/a\u003e]. However, there is currently insufficient evidence to either support or refute the therapeutic MH for CA patients\u0026nbsp;[\u003ca href=\"#_ENREF_30\" title=\"Sandroni, 2022 #68\"\u003e30\u003c/a\u003e]. Those recommendations for therapeutic MH are primarily based on improvements in neurological outcomes \u0026nbsp;[\u003ca href=\"#_ENREF_31\" title=\"Group, 2002 #39\"\u003e31\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_32\" title=\"Callaway, 2015 #42\"\u003e32\u003c/a\u003e]. 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\u0026nbsp;[\u003ca href=\"#_ENREF_33\" title=\"Piktel, 2018 #61\"\u003e33\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_34\" title=\"Thomsen, 2021 #70\"\u003e34\u003c/a\u003e]. 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe IRI of the brain is a primary cause of death and disability in CA patients after ROSC\u0026nbsp;[\u003ca href=\"#_ENREF_35\" title=\"Lemiale, 2013 #50\"\u003e35\u003c/a\u003e]. Clinical research has demonstrated that MH can mitigate cerebral injury after CA\u0026nbsp;[\u003ca href=\"#_ENREF_31\" title=\"Group, 2002 #39\"\u003e31\u003c/a\u003e]. A multicenter cohort study conducted in Japan divided 1,111 OHCA patients from 125 hospitals into two groups: 33-34\u0026deg;C and 35-36\u0026deg;C. The study found that the 33-34\u0026deg;C group was associated with better recovery from moderate nervous system impairment\u0026nbsp;[\u003ca href=\"#_ENREF_36\" title=\"Nishikimi, 2021 #58\"\u003e36\u003c/a\u003e]. In a randomized controlled trial involving 574 comatose patients who were resuscitated from CA with a non-shockable rhythm, moderate therapeutic hypothermia at 33\u0026deg;C for 24 hours improved the neurological prognosis\u0026nbsp;[\u003ca href=\"#_ENREF_37\" title=\"Lascarrou, 2019 #49\"\u003e37\u003c/a\u003e]. Notably, asphyxia-induced cardiac arrest results in more severe neurological injury and worse outcomes compared to ventricular fibrillation-induced cardiac arrest\u0026nbsp;[\u003ca href=\"#_ENREF_38\" title=\"Zhang, 2015 #75\"\u003e38\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_39\" title=\"Uray, 2018 #71\"\u003e39\u003c/a\u003e]. \u0026nbsp;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\u0026nbsp;[\u003ca href=\"#_ENREF_40\" title=\"Chen, 2017 #43\"\u003e40\u003c/a\u003e]. Therefore, MH may also protect cardiomyocytes through brain-heart axis, with TRPV1 playing a crucial role in both the cardiovascular and neural systems.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study supports our novel finding that MH may protect cardiac function by inhibiting cardiomyocyte necroptosis through the\u0026nbsp;\u003cem\u003eTrpv1\u003c/em\u003e-\u003cem\u003eEgfr\u003c/em\u003e-\u003cem\u003eMlkl\u003c/em\u003e pathway after CA.\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy limitations and future work\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral 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\u0026nbsp;[\u003ca href=\"#_ENREF_41\" title=\"Myat, 2018 #56\"\u003e41\u003c/a\u003e]. Recently, OHCA has increasingly been recognized as distinct from in-hospital cardiac arrest (IHCA) and studied independently\u0026nbsp;[\u003ca href=\"#_ENREF_42\" title=\"Penketh, 2022 #60\"\u003e42\u003c/a\u003e]. 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.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCPR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Cardio-pulmonary resuscitation\u003c/p\u003e\n\u003cp\u003eCA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Cardiac arrest\u003c/p\u003e\n\u003cp\u003eNT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Normothermia\u003c/p\u003e\n\u003cp\u003eMH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Mild hypothermia\u003c/p\u003e\n\u003cp\u003eTRPV \u0026nbsp; \u0026nbsp; \u0026nbsp; Transient receptor potential cation channel subfamily V\u003c/p\u003e\n\u003cp\u003eEGFR \u0026nbsp; \u0026nbsp; \u0026nbsp; Epidermal growth factor receptor\u003c/p\u003e\n\u003cp\u003eMLKL \u0026nbsp; \u0026nbsp; \u0026nbsp;Mixed lineage kinase domain-like protein\u003c/p\u003e\n\u003cp\u003eOHCA \u0026nbsp; \u0026nbsp; \u0026nbsp;Out-of-hospital cardiac arrest\u003c/p\u003e\n\u003cp\u003eROSC \u0026nbsp; \u0026nbsp; \u0026nbsp; Return of spontaneous circulation\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eTRP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Transient receptor potential\u003c/p\u003e\n\u003cp\u003eMAP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Mean arterial pressure\u003c/p\u003e\n\u003cp\u003eRT-qPCR \u0026nbsp;Reverse transcription and quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003ePCM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Primary cardiomyocyte\u003c/p\u003e\n\u003cp\u003eSD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sprague dawley\u003c/p\u003e\n\u003cp\u003eDMEM \u0026nbsp; \u0026nbsp;Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium\u003c/p\u003e\n\u003cp\u003eFBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eOGD/R \u0026nbsp; \u0026nbsp; Oxygen-glucose deprivation and reperfusion\u003c/p\u003e\n\u003cp\u003eBSA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Bovine serum albumin\u003c/p\u003e\n\u003cp\u003eMI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Myocardial infarction\u003c/p\u003e\n\u003cp\u003eRipk3 \u0026nbsp; \u0026nbsp; \u0026nbsp; Receptor interacting serine/threonine kinase 3\u003c/p\u003e\n\u003cp\u003eRipk1 \u0026nbsp; \u0026nbsp; \u0026nbsp; Receptor interacting serine/threonine kinase 1\u003c/p\u003e\n\u003cp\u003eVR1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Vanilloid receptor-1\u003c/p\u003e\n\u003cp\u003eTRPM \u0026nbsp; \u0026nbsp; \u0026nbsp;Transient receptor potential cation channel subfamily M\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePTP1B \u0026nbsp; \u0026nbsp; Protein tyrosine phosphatase 1B\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIHCA \u0026nbsp; \u0026nbsp; \u0026nbsp; In-hospital cardiac arrest\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 1. Figure S1:\u0026nbsp;\u003c/strong\u003eWeight of rats, time of inducing CA, volume of using epinephrine solution and time of successful CPR in the CPR model of rats. \u003cstrong\u003eFigure S2:\u0026nbsp;\u003c/strong\u003eTemperature of rats. \u003cstrong\u003eFigure S3:\u0026nbsp;\u003c/strong\u003eFPKM of\u0026nbsp;key genes of apoptosis (\u003cem\u003eCasp3\u003c/em\u003e), necroptosis (\u003cem\u003eRipk1\u003c/em\u003e, \u003cem\u003eRipk3\u003c/em\u003e and \u003cem\u003eMlkl1\u003c/em\u003e), pyroptosis (\u003cem\u003eCasp1\u003c/em\u003e and \u003cem\u003eGsdmd\u003c/em\u003e), ferroptosis (\u003cem\u003ePtgs2\u003c/em\u003e), autophagic death (\u003cem\u003eBecn1\u003c/em\u003e). \u003cstrong\u003eFigure S4:\u0026nbsp;\u003c/strong\u003eCell death rate after OGD/R. \u003cstrong\u003eTable S1:\u0026nbsp;\u003c/strong\u003ePrimer sequences of the target genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM.Y.:\u003c/strong\u003e Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Supervision; Validation; Visualization; Writing-original draft; Writing-review and editing. \u003cstrong\u003eC.L.:\u003c/strong\u003e \u003csup\u003e\u0026nbsp;\u003c/sup\u003eWriting-review and editing.\u003cstrong\u003e\u0026nbsp;Y.Z.:\u003c/strong\u003e Data curation; Investigation; Writing-original draft; Writing-review and editing. \u003cstrong\u003eH.Z.:\u003c/strong\u003e Data curation; Investigation; Methodology; Resources.\u003cstrong\u003e\u0026nbsp;Yuanzheng Lu:\u003c/strong\u003e Funding acquisition; Investigation; Writing-review and editing.\u003cstrong\u003e\u0026nbsp;K.L.:\u003c/strong\u003e Visualization; Writing-original draft. \u003cstrong\u003eYawen Lu:\u003c/strong\u003e Data curation; Investigation.\u003cstrong\u003e\u0026nbsp;S.Z.:\u003c/strong\u003e Investigation.\u003cstrong\u003e\u0026nbsp;C.Z.:\u003c/strong\u003e Investigation.\u003cstrong\u003e\u0026nbsp;R.L.:\u003c/strong\u003e \u003csup\u003e\u0026nbsp;\u003c/sup\u003eWriting-review and editing. \u003cstrong\u003eY.L.:\u003c/strong\u003e \u003csup\u003e\u0026nbsp;\u003c/sup\u003eWriting-review and editing.\u003cstrong\u003e\u0026nbsp;Z.G.:\u003c/strong\u003e Funding acquisition.\u003cstrong\u003e\u0026nbsp;X.H.:\u003c/strong\u003e Funding acquisition.\u003cstrong\u003e\u0026nbsp;C.H.:\u003c/strong\u003e Funding acquisition; Supervision; Validation; Visualization; Writing-review and editing.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the following funds: the National Natural Science Foundation of China (81970564, 82170663\u0026nbsp;and\u0026nbsp;82370664,\u0026nbsp;to Dr Guo; 82070670, to Dr He), Medical\u0026ensp;Scientific\u0026ensp;Research\u0026ensp;Foundation\u0026ensp;of\u0026ensp;Guangdong\u0026ensp;Province\u0026ensp;of\u0026ensp;China (A2020275 to Dr\u0026nbsp;Yin), Guangdong Basic and Applied Basic Research Foundation\u0026nbsp;(2024B1515040011\u0026nbsp;to\u0026nbsp;Dr Guo; 2023A1515011792 and 2024A1515010799 to Dr Hu;\u0026nbsp;2020A1515010383\u0026nbsp;to Dr Liu;\u0026nbsp;2020A1515110827\u0026nbsp;to Dr Lu),\u0026nbsp;the Guangdong Provincial Key Laboratory Construction Projection on Organ Donation and Transplant Immunology (2023B1212060020, to Dr He),\u0026nbsp;Shenzhen Fundamental Research Program (jcyj20190809150817414 to Dr Lu).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1.\u0026nbsp;\u0026nbsp;Jerkeman M, Sultanian P, Lundgren P, Nielsen N, Helleryd E, Dworeck C, Omerovic E, Nordberg P, Rosengren A, Hollenberg J\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eTrends in survival after cardiac arrest: a Swedish nationwide study over 30 years\u003c/strong\u003e. \u003cem\u003eEur Heart J\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e43\u003c/strong\u003e(46):4817-4829.\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp;\u0026nbsp;Vazquez-Jimenez JF, Qing M, Hermanns B, Klosterhalfen B, W\u0026ouml;ltje M, Chakupurakal R, Schumacher K, Messmer BJ, von Bernuth G, Seghaye MC: \u003cstrong\u003eModerate hypothermia during cardiopulmonary bypass reduces myocardial cell damage and myocardial cell death related to cardiac surgery\u003c/strong\u003e. \u003cem\u003eJ Am Coll Cardiol\u0026nbsp;\u003c/em\u003e2001, \u003cstrong\u003e38\u003c/strong\u003e(4):1216-1223.\u003c/p\u003e\n\u003cp\u003e3.\u0026nbsp;\u0026nbsp;Qing M, Vazquez-Jimenez JF, Schumacher K, Bhardwaj RS, Klosterhalfen B, Minkenberg R, Messmer BJ, von Bernuth G, Seghaye MC: \u003cstrong\u003eModerate hypothermia during cardiopulmonary bypass increases intramyocardial synthesis of heat shock protein 72\u003c/strong\u003e. \u003cem\u003eJ Thorac Cardiovasc Surg\u0026nbsp;\u003c/em\u003e2002, \u003cstrong\u003e124\u003c/strong\u003e(4):724-731.\u003c/p\u003e\n\u003cp\u003e4.\u0026nbsp;\u0026nbsp;Liu T, Zhao DX, Cui H, Chen L, Bao YH, Wang Y, Jiang JY: \u003cstrong\u003eTherapeutic hypothermia attenuates tissue damage and cytokine expression after traumatic brain injury by inhibiting necroptosis in the rat\u003c/strong\u003e. \u003cem\u003eSci Rep\u0026nbsp;\u003c/em\u003e2016, \u003cstrong\u003e6\u003c/strong\u003e:24547.\u003c/p\u003e\n\u003cp\u003e5.\u0026nbsp;\u0026nbsp;Motomatsu Y, Sakurai M, Onitsuka H, Abe K, Shiose A: \u003cstrong\u003eHypothermia inhibits the expression of receptor interacting protein kinases 1 and 3 after transient spinal cord ischaemia in rabbits\u003c/strong\u003e. \u003cem\u003eEur J Vasc Endovasc Surg\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e59\u003c/strong\u003e(5):824-833.\u003c/p\u003e\n\u003cp\u003e6.\u0026nbsp;\u0026nbsp;Randhawa PK, Jaggi AS: \u003cstrong\u003eInvestigating the involvement of glycogen synthase kinase-3\u003c/strong\u003e\u003cstrong\u003e\u0026beta; 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\u0026nbsp; \u0026nbsp; \u0026nbsp;Penketh J, Nolan JP: \u003cstrong\u003eIn-hospital cardiac arrest: the state of the art\u003c/strong\u003e. \u003cem\u003eCrit Care\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e26\u003c/strong\u003e(1):376.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CPR, Mild hypothermia, Ischemia, TRPV1, Capsaicin","lastPublishedDoi":"10.21203/rs.3.rs-4866738/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4866738/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u0026nbsp;\u003c/strong\u003eTherapeutic 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e \u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e \u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e \u0026nbsp;MH may protect cardiac function by mitigating cardiomyocyte necroptosis through regulating TRPV1.\u003c/p\u003e","manuscriptTitle":"Mild hypothermia alleviates cardiomyocyte necroptosis after cardiopulmonary resuscitation by regulating TRPV1","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2024-09-03 16:19:16","doi":"10.21203/rs.3.rs-4866738/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2024-08-08 05:21:39","doi":"10.21203/rs.3.rs-4866738/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"047ca054-3744-4801-9c20-493d8c74cd81","owner":[],"postedDate":"September 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-03T03:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-03 16:19:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-4866738","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4866738","identity":"rs-4866738","version":["v2"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}