Dexmedetomidine ameliorates myocardial ischemia-reperfusion injury through regulating FASN-associated cholesterol homeostasis

doi:10.21203/rs.3.rs-5310056/v1

Download PDF

Research Article

Dexmedetomidine ameliorates myocardial ischemia-reperfusion injury through regulating FASN-associated cholesterol homeostasis

https://doi.org/10.21203/rs.3.rs-5310056/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Dexmedetomidine (Dex) has been used in sedation in intensive care units and as an anesthetic adjunct. However, the mechanism of the protective function of Dex against myocardial ischemia/reperfusion (I/R) injury remains unclear. We applied in vivo rat model as well as in vitro cardiomyocyte models (H9c2 cells and neonatal rat cardiomyocytes, NRCMs) to evaluate the effects of Dex against myocardial I/R injury. In the results, protective effects of Dex were observed in rat heart tissues after I/R injury. Next, transcriptomic sequencing was performed to determine the global change of gene expression, and identified genes related to cholesterol metabolism were significantly upregulated by Dex, where the change of fatty acid synthase (FASN) was the most significant. Furthermore, shRNAs targeting FASN were transfected into H9c2 cells and NRCMs to knock down FASN. By comparing the effects of Dex on both wild type and FASN-knockdown cells under the OGD/R challenge, the protection of Dex was absent in knockdown cells supported by the dataset including the cell viability and apoptosis as well as key gene expressions. Overall, this study systematically evaluates the protective effects of Dex on myocardial I/R injury and provides a better understanding of the role of cholesterol metabolism in the function of Dex.

Dexmedetomidine

myocardial I/R injury

cholesterol metabolism

FASN

As previously reported, myocardial infarction (MI) is one of the leading global concerns in public health that results in considerable deaths worldwide, which is the irreversible death of heart muscle secondary to prolonged lack of oxygen supply [1]. Compatibly, myocardial reperfusion has long been recognized as the main strategy for MI treatment. Nevertheless, due to the direct restoration of blood supply to the impacted area, evident ischemia/reperfusion (I/R) injury such as arrhythmia, functional impairment, and aggravation of apoptosis was observed [2, 3]. Consequently, there is a pressing need to develop therapies that can rescue myocardial I/R injury.

In the I/R injury, the main mechanism has been referred to overproduction of reactive oxygen species (ROS) and the presence of pro-inflammatory chemokines and cytokines [4, 5], which further outnumber the antioxidant and anti-inflammatory defenses, and eventually lead to I/R injury [6]. Specifically, the crosstalk between fatty acid and myocardial I/R injury has been investigated and the role of fatty acid metabolism in myocardial I/R injury has been well recognized. Studies have demonstrated that during myocardial reperfusion, overshoot occurred in the rate of fatty acid oxidation, impaired pyruvate oxidation, and accelerated anaerobic glycolysis [7–9]. In addition, the correlation between fatty acid synthesis and the homeostasis of cholesterol has been well established, which could potentially play role in myocardial I/R injury. On the other hand, the specific gene has been identified in the cardiac system that is essential for fatty acid metabolism and myocardial inflammation during I/R injury of the heart [10].

Dexmedetomidine (Dex) is not only widely used in sedation in intensive care units and as an anesthetic adjunct but also been reported to have protective effects against I/R injury in several organ systems including heart [11], brain [12], and kidney [13], where the main contribution was reported as the antioxidant and anti-inflammatory properties of this compound [14]. It has also been reported that Dex can affect the homeostasis of glucose and fatty acid in animal models [15, 16] and cardiac surgery patients [17]. However, whether Dex affects the myocardial I/R injury via the homeostasis of cholesterol remains unclear and the key gene/protein that plays the mediation role is yet to be explored.

In this study, the protective effect of Dex against myocardial I/R injury was investigated using both in vitro cardiomyocyte models and in vivo rats. I/R injury rat model and OGD/R-challenged cardiomyocytes models were established and applied to determine the potential effects of Dex. In the animal model, pathological detection analysis was performed to provide direct evidence and transcriptomic sequencing was conducted to identify the global change of gene expressions that were involved in Dex functional effects and further validated by qRT-PCR. In the H9c2 cells and neonatal rat cardiomyocytes (NRCMs), cytotoxicity endpoints including the cell viability and apoptosis as well as key gene expressions were evaluated. Furthermore, to confirm the mediation role of a key gene that is related to cholesterol metabolism, gene knockdown was performed in both cell and rat models by transfecting shRNA using a lentivirus-based system and followed by determining the response change caused by Dex in OGD/R challenge and myocardial I/R injury, respectively. Overall, the present study will expansively assess the effects of Dex on the myocardial I/R injury and provide a better understanding of the role of cholesterol metabolism in the function of Dex, which further offers more comprehensive evidence for assessment and wide application of Dex.

Animals, cell models, and experimental design. Male BALB/c rats were purchased from the Center of Experimental Animals at Xi’an Jiaotong University (Shaanxi, China). All the animals were maintained in a temperature and humidity-controlled environment with a 12-hour light/dark cycle and had access to food and water. All animal procedures were performed by the approvement provided by the Animal Use and Care Committee of Xi’an Jiaotong University. In the treatment group, rats were intraperitoneally injected with 20 µg/kg body weight of dexmedetomidine (Dex, obtained from Nhwa Pharmaceutical Corporation Ltd., Jiangsu, China) prior the 30-min ischemia. Cell models used in this study include H9c2 cardiomyocytes and neonatal rat cardiac myocytes (NRCMs). Cells were exposed to 1 µM of Dex or 100 µM of palmitate (Sigma Aldrich) for 24 h before endpoint measurement.

Myocardial ischemia/reperfusion (I/R) model. The I/R model establishment procedures were conducted according to previous study [18]. In detail, the rats were anesthetized with 4% chloral hydrate (1 mL/100 g body weight, i.p.) and anesthesia was maintained by periodically intraperitoneal injection of 4% chloral hydrate (0.5 mL/10 g body weight). Following the tracheal intubation, the respiratory rates of rats were adjusted and maintained at 70–80 times/min. Medlab biological signal collecting and processing system was applied to continuously measure the hemodynamic parameters including heart rate (HR), left ventricular filling pressures (LVFP), left ventricular end-diastolic pressure (LVEDP), and maximum rise/fall rate of left ventricular internal pressure (LV ± dp/dtmax). A left parasternal incision was made through the third and fourth ribs, and the pericardium was gently opened to expose the heart. The circumflex branch and the suture ends were threaded through a polyethylene tube to form snares for reversible coronary artery occlusion. Myocardial ischemia was induced by tightening the silk suture around the polyethylene tube following 20 min stabilization. An elevated ST-segment in the ECG indicated the success of ischemia. After 30 min of ischemia, the polyethylene tubes were loosened for 120 min to mimic reperfusion.

Infarct area assessment and histological analysis. Infarct size was determined using 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining as previously reported [19]. In brief, heart tissues were collected after the reperfusion and the excess blood was cleaned with prechilled PBS, followed by storage under − 20°C for 10 min. The tissues were then sliced transversely into 2 mm sections and subsequently incubated in 20 mg/mL TTC solution for 10 min at 37°C under dark. Slices were rinsed with PBS three times and then fixed using 4% PFA for 24 hours before imaging. Infarct (pale) and risk (red) areas were calculated by planimetry using Image-Pro Plus software (v6.0, Media Cybernetics, Inc., Rockville, MD, USA). Hematoxylin and eosin staining (H&E staining) was performed for hearts as reported elsewhere [20]. Hearts after reperfusion were excised and fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned at 5µm thickness. The samples were stained with hematoxylin and eosin and examined by light microscopy.

High throughput transcriptomic sequencing. Total RNA was extracted from the heart tissue samples TRIzol (Magen, China) according to the manufacture’s instruction and Illumina sequencing was performed following the manufacturer’s protocols (Illumina, San Diego, CA). Briefly, the poly (A) mRNA was purified from the total RNA using Sera-mag Magnetic Oligo (dT) beads from Illumina, and the mRNA-enriched RNAs were chemically fragmented into short sequences using the fragmentation solution (Ambion, USA). The double-stranded cDNA was synthesized using the HiFiScript cDNA synthesis kit (CoWin Biosciences, China). Finally, the pair-end RNA-seq libraries were constructed using the Illumina Paired End Sample Prep kit and were subsequently sequenced on the Illumina HiSeqTM 2000 platform. Following this, the differentially expressed genes were screened and gene set enrichment analysis (GSEA) was performed to explore the underlying bio-function of these differentially expressed genes.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted as described above and followed by reverse-transcription to cDNA with the HiFiScript cDNA synthesis kit (CoWin Biosciences, China). Real-time quantitative PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, China). β-actin was used the housekeeping gene to normalize the expression changes. The genes analyzed and the primer information are listed as follow, (FASN, F: 5ʹ-CAGCGGTTGGAGGTTGT-3ʹ and R: 5ʹ-TTTGAGGGTGGAGGTGG-3ʹ; HMGCR, F: 5ʹ- AGCTTGCCCGAATTGTATGTG-3ʹ and R: 5ʹ TCTGTTGTGAACCATGTGACTTC-3ʹ; ApoE, F: 5ʹ-GGCCCAGGAGAATCAATGAG-3ʹ and R 5ʹ-CCTGGCTGGATATGGATGTTG-3ʹ; CYP46A1, F: 5ʹ-TCCTCTCCTGTTCAGCACC-3ʹ and R, 5ʹ-CAGCTTGGCCATGACAACT-3ʹ; and β-actin, F: 5ʹ-GCTCTTTTCCAGCCTTCCTT-3ʹ and R: 5ʹ-CTTCTGCATCCTGTCAGCAA-3ʹ. The relative gene expression among the treatment groups was quantified using the 2 − ΔΔCt method.

Myocardial cell models of Oxygen-glucose deprivation/reoxygenation (OGD/R). Myocardial cells used in this study including H9c2 cardiomyocytes and neonatal rat cardiac myocytes (NRCMs) were treated with oxygen-glucose deprivation/reoxygenation (OGD/R) as previously reported [21]. In brief, three million cells were seeded in 6-cm plate and with 90% confluency, culture medium was changed into DMEM without serum and glucose for 2 h and followed by hypoxic condition (94% N₂, 5% CO₂, and 37°C) for 6 hours as reported. Then the medium was changed into normal culture medium under regular condition. 1 µM of Dex was added into cells prior the OGD/R treatment.

Transfection of shRNA in Myocardial cells and rats. To knockdown FASN in cells and animal model, shRNA expression cassettes were constructed (GenePharm) for the transfection. For the cells, when H9c2 cardiomyocytes and neonatal rat cardiac myocytes (NRCMs) reached 70% confluency, shRNA1/2 and corresponding negative control (NC) was transfected using Lipofectamine®3000 (Invitrogen. L3000-015) according to the manufacturer’s instruction and the cells were incubated for 2–4 days before Dex treatment.

In the animal model, rats were anesthetized with sodium pentobarbital at a dose of 100 mg/kg. The heart was exposed upon opening the left pleural cavity by cutting the left third and fourth ribs and intercostal muscles with a cautery pen. The pericardium was removed, and a syringe fitted with a 30-G needle was inserted near the apex of the heart and tunneled intramuscularly to the anterior LV at the mid-ventricular level. Then, 30 µl of adenovirus-expressing either negative control or shRNA1/2 were injected into from the apex of the left ventricle into the aortic root. The chest was closed in layers with disinfection by ethanol.

Palmitate treatments.

Western blot analysis. Western blot for the target proteins was performed using the proteins extracted from the heart tissue and cells. Total protein was extracted using RIPA buffer. Protein concentration was measured using BCA according to the manufacture’s protocol. Equal amounts of proteins from each sample were subjected to 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA, blots were incubated with the following antibodies at 4°C overnight: anti-FASN (1:10000 dilution, #ab128870; Abcam, USA), anti-HMGCR (1:2000 dilution, #ab242315; Abcam), anti-ApoE (1:2000 dilution, #ab183597; Abcam), anti-CYP46A1 (1:1000 dilution, #ab244241; Abcam), anti-cleaved PARP (1:2000 dilution, #ab32064; Abcam) anti-PARP (1:1000 dilution, #ab191217; Abcam), anti-cleaved caspase-3/caspase-3 (1:2000 dilution, #ab184787; Abcam), anti-Bax (1:2000 dilution, #ab32503; Abcam), anti-Bcl-2 (1:2000 dilution, #ab182858; Abcam), and β-actin (1:5000 dilution, #ab8226; Abcam). After three washes with TBST, these membranes were incubated with HRP-conjugated antibody (Biosharp, BL003A). Immune complexes were visualized with enhanced chemiluminescence. β-actin (Proteintech, 20536-1-AP) was used as the endogenous control. The optical density of bands was analyzed by ImageJ software.

Cytotoxicity assays and apoptosis analysis. The cytotoxicity was after Dex treatment was evaluated using CCK 8 analysis. In brief, the cells were seeded on the 96-well plate at a density of 5 x 10³ cells/well. After the Dex treatment, the cell viability was measured by adding 10 µL of CCK-8 solution (Beyotime, C0038) to each well and incubate for 1 h according to the manufacturer’s instruction. Then the absorbance of the medium was read at a wavelength of 450 nm. Apoptosis analysis was performed using Annexin V-FITC Apoptosis Staining/Detection Kit (Beyotime, C1062S). In detail, cells were rinsed with PBS three times and resuspended in 195 µL of Annexin V-FITC binding buffer, followed by adding 5 µL of Annexin V-FITC and incubation at 4°C under dark for 15 min. Then 5 µL of propidium iodide was added and the mix was incubated at 4°C under dark for 5 min before proceeding the quantification by flow cytometry.

Cholesterol measurements. To determine the concentration of cholesterol in plasma samples collected from in vivo experiments, Mice blood was collected from the eyes afer successful anesthesia with ether. Te blood was immediately placed in centrifuge tubes pre-treated with sodium heparin and centrifuged at 4°C, 4000 rpm for 15 min to collect the plasma. Then, cholesterol concentration was quantitatively estimated using the EnzyChrom™ AF Cholesterol Assay kit (#E2CH-100, Bioassay Systems, Hayward, CA, USA) according to the manufacturer’s protocol.

Statistical Analysis. The experimental results are collected from three independent experiments and presented as means ± SD. For the in vivo mouse study, each treatment group includes at least 5 biological replicates. For the in vitro cell experiments, each treatment group contains at least 5 biological replicates. Statistical significance between treatment and control groups was determined by Student’s t test and p < 0.05 indicates the statistically significant difference. Histograms were prepared with GraphPad Prism 8.0 (La Jolla, CA, USA).

Dexmedetomidine alleviated the myocardial ischemia-reperfusion (I/R) injury in rats. To evaluate the potential role of dexmedetomidine (Dex) in myocardial I/R injury, rats were treated with Dex prior to I/R injury and the overall impacts on myocardia morphological changes as well as the expression of involved genes were comprehensively determined. As shown in Fig. 1A, the representative TTC-stained slides of the myocardium sections from different treatment groups indicated that Dex effectively reduced the myocardial infarction size compared to the vehicle groups. A similar result was obtained from the hematoxylin & eosin (H&E) staining of heart sections as depicted in Fig. 1B, where the I/R injury resulted in disordered myocardial fibers with obvious infarction and inflammatory infiltration (left panel); however, these effects were rescued to be less severe by the treatment of Dex prior to I/R injury as shown in the right panel. In addition, Dex treatment significantly increased the concentration of cholesterol (Fig. 1C).

Next, myocardium samples were collected for transcriptomic sequencing to identify the global gene expression changes. As shown in Fig. 1D, GSEA demonstrated that cholesterol homeostasis-related genes were significantly enriched in the Dex-treated group compared to the vehicle, indicating the status of cholesterol is involved in the protective effects of Dex on the myocardial I/R injury. The volcanic plot (Fig. 1E) further identified the expression of genes with significant changes, among which the gene FASN, encoding for fatty acid synthase, was profoundly upregulated.

Furthermore, expressions of genes related to the biosynthesis (HMGCR), recycling (ApoE), and degradation (CYP46A1) of cholesterol were determined. As depicted in Fig. 1F-H, all these genes were significantly upregulated in the Dex-treated groups compared to the vehicle control, indicating that the whole biological process of cholesterol is involved in the protective effects of Dex on myocardial I/R injury.

Dexmedetomidine protected rat H9c2 cardiomyocytes and neonatal rat cardiomyocytes (NRCMs) from oxygen deprivation/reoxygenation challenges. H9c2 cells were divided into groups including non-treatment (control) and pretreated with vehicle or Dex before oxygen-glucose deprivation/reoxygenation (OGD/R) challenge. Cytotoxicity endpoints including cell viability and apoptosis were evaluated to assess the effects of Dex as well as the expressions of genes/proteins related to cholesterol metabolism. As shown in Fig. 2A, cell viability was significantly decreased in the vehicle group after the OGD/R challenge compared to the control (P < 0.01), which was profoundly rescued in the Dex-treated group (P < 0.05). Similarly, the apoptosis results provided by flow cytometry were consistent with the cell viability results, where the OGD/R treatment induced more than a two-time apoptosis rate compared to the control but reversed by prior treatment with Dex (Fig. 2B-C).

The expression of FASN at both mRNA and protein levels under different treatments was further investigated. As shown in Figs. 2D and 2F, expression of FASN was significantly inhibited in OGD/R-treated cells at mRNA and protein levels, respectively. However, prior treatment with Dex did increase the expression of FASN, indicating that FASN is involved in the function of Dex rescuing the adverse effects caused by the OGD/R challenge. Furthermore, expressions of genes related to the metabolism of cholesterol in both H9c2 cells and NRCMs were determined. As shown in Fig. 2E and 2G, in both cell types, the OGD/R challenge significantly decreased the expression of genes related to biosynthesis (HMGCR), recycling (ApoE), and degradation (CYP46A1) of cholesterol (P < 0.005), respectively, and these reductions were rescued by the treatment of Dex, where all the gene expressions were enhanced comparing to the vehicle groups.

Inhibition of FASN in H9c2 cells and NRCMs abrogated the protective effects of Dex against OGD/R-induced injury. To further investigate the role of FASN in the function of Dex, FASN in both H9c2 cells and NRCMs were knocked down using a lentivirus-based system expressing shRNAs that target FASN. After 48-h virus infection, both shRNAs showed successful knockdown of FASN expression, where shFASN-1 exhibited higher knockdown efficiency compared to shFASN-2 as the qPCR and Western blot evidence provided in Fig. 3A (H9c2 cells) and Fig. 4A (NRCMs), respectively. Next, both cell models were subjected to pretreatment with vehicle (as control) or Dex and OGD/R exposure, and different endpoints were evaluated and compared among different groups (Figs. 3B and 4B). First, cell viability results showed that in the lentivirus negative control groups, OGD/R exposure induced significant cytotoxicity without Dex-pretreatment, and Dex reversed the adverse effects with statistical significance. However, in the FASN-knockdown cells, the control groups without any treatments showed a significant decrease in cell viability compared to wild-type cells. Furthermore, ODG/R exposure resulted in significantly lower cell viability with or without pretreatment of Dex, and no significant difference was observed in these two groups. The apoptosis results were consistent with the cell viability results in both cell types (Fig. 3C-D and 4C-D), where the pretreatment of Dex significantly reduced the apoptosis rates in both cell types under OGD/R exposures, but the knockdown of FASN eliminated the protective function of Dex and the apoptosis rate remained significantly high compared to the control groups. In addition, expression of genes related to cholesterol metabolism was further determined at protein levels, where the Western blot results showed that all the selected genes were down-regulated under OGD/R exposures and rescued by the pretreatment of Dex, whereas in FASN-knockdown groups, expression of these genes was also significantly reduced but the protective effects of Dex were also eliminated (Fig. 3E-G and 4E-G).

FASN-driven cholesterol synthesis is essential to prevent OGD/R-induced injury in H9c2 cells and NRCMs. The effects of knocking down FASN on the protective function of Dex were unveiled in the last section. Next, the question was whether FASN-associated cholesterol synthesis was essential to the function of Dex. As shown in Fig. 5A, knocking down FASN significantly reduced the cholesterol levels in both cell types. Furthermore, cholesterol levels in both cell types were increased significantly, backing to similar levels as the controls, under the acetoacetyl-CoA or HMG-CoA treatment, which can reverse the phenotype of FASN suppression caused by reduction in cholesterol biosynthesis (Fig. 5B). In the groups under OGD/R exposure, pretreatment with Dex can rescue the cholesterol levels caused by the OGD/R challenging. However, once FASN was suppressed, Dex was not able to restore cholesterol levels in both H9c2 cells and NRCMs (Fig. 5C). All these results indicate that cholesterol synthesis driven by FASN is needed for Dex to exhibit the protective effects against OGD/R challenging.

Role of FASN in protection function of Dex against myocardial I/R injury in rats. Previous sections provided in vitro evidence that supports FASN is essential and responsible for the protective effect of Dex towards OGD/R-induced injury. Furthermore, the role of FASN for Dex was evaluated in vivo using an AAV-based system. As shown in Fig. 6A, FASN was successfully knocked down in rats, where the protein level of FASN was significantly lower as depicted in Western blot results. Furthermore, H&E staining for myocardial tissues collected from different groups showed that in the AAV-negative control, Dex rescued the myocardial damages induced by I/R injury. However, in the FASN-knockdown group, severe myocardia damage still can be observed in both vehicle and Dex-pretreatment (Fig. 6B).

Next, expressions of apoptosis-related proteins including PARP, caspase 3, Bax, and Bcl2 were determined in rats after myocardial I/R injury by Western blot, and the levels were quantified and compared (Fig. 6C). Overall, in the AAV-negative control groups, pretreatment with Dex resulted in lower expressions of cleaved-PARP, cleaved-caspas 3, and Bax, but increased the level of Bcl-2 compared to vehicle control. In the FASN-knockdown groups, no significant differences were observed in these above-mentioned protein levels between the vehicle control and rats pretreated with Dex after myocardial I/R injury. It should be noted that Dex exhibited its protective function against I/R injury in both AAV-negative control and knockdown rats, where pretreatment with Dex could significantly enhance the expression of FASN in both groups.

Furthermore, mRNA expression of genes related to the metabolism of cholesterol was evaluated. As shown in Fig. 6D, all the genes (HMGCR, ApoE, and CYP46A1) were significantly upregulated in Dex-treated rats under myocardial I/R injury. However, no effects of Dex-pretreatment were observed in the FASN-knockdown rats under myocardial I/R injury, where all these genes were expressed at similar levels.

In this study, the protective functions of dexmedetomidine (Dex) against myocardial ischemia-reperfusion (I/R) injury were comprehensively evaluated using in vitro cell and in vivo rat models. As a highly selective α2-adrenergic receptor agonist, Dex has been widely used for sedation and analgesia in the intensive care unit or as an anesthetic adjuvant [22, 23]. Additional to the medical applications, Dex has also been extensively reported to attenuate myocardial I/R injury through different mechanisms and pathways, including endoplasmic reticulum stress [24, 25], pyroptosis [26, 27], and inflammatory pathways [28, 29]. Overall, we demonstrated in the present study that the protective effects of Dex against myocardial I/R injury were essentially mediated by cholesterol homeostasis via actively regulating the expression of FASN, the gene that encodes the synthesis of fatty acid.

First, protective effects of pretreatment with Dex were observed in both rat and cell models, which were under myocardial I/R injury and oxygen-glucose deprivation/reoxygenation (OGD/R) exposures, respectively. In the animal model, the myocardial damage after I/R injury and the effect of Dex was clearly demonstrated by TTC and H&E staining of heart tissues, which was significantly reversed by the pretreatment with Dex (Fig. 1A-B), and results were consistent with previous literature [25, 26]. In rat H9c2 cardiomyocytes culture, the cytotoxicity dataset including cell viability and apoptosis also showed the protective effects against OGD/R exposures, where the addition of Dex significantly reversed the adverse effects caused by OGD/R challenges (Fig. 2A-C). Next, to identify the involved genes and pathways for Dex functions, transcriptomic sequencing was performed, and genes related to cholesterol metabolism were found to be significantly upregulated by Dex, including FASN that responsible for fatty acid synthesis, HMGCR for cholesterol biosynthesis, ApoE for recycling, and CYP46A1 for degradation (Fig. 1D-G). Similar results were obtained in H9c2 cells (Fig. 2D-G). These results further indicate that the homeostasis of cholesterol is involved in the action of Dex and specifically, FASN can potentially play an essential role in this process.

Fatty acids and cholesterol in intracellular contents contribute to the fuel storage as well as a source for necessary components to increase the membrane production [30]. FASN is a gene encoding fatty acid synthase, and it has been reported that FASN/cholesterol axis can facilitate a resistant response for cancer cells under stress [31]. However, whether FASN is involved in the function of Dex towards myocardial I/R injury remains unknown. To solve this puzzle, we knocked down FASN in both H9c2 cells and neonatal rat cardiomyocytes (NRCMs) using the lentivirus-based system with shRNA targeting FASN (Fig. 3–4). In the in vitro models, both cell types exhibited similar results that, with the presence of FASN (negative control groups without knockdown), cytotoxicity occurred after OGD/R exposure as well as downregulation of genes related to cholesterol metabolism, and these adverse effects were rescued by Dex. However, in FASN-knockdown groups, cytotoxicity in Dex-pretreated groups remained as high as that in the vehicle group, and no significant differences were observed between Dex and vehicle groups in terms of the regulation of genes that are involved in cholesterol metabolism. Similarly, FASN has been reported to be related to oxidative stress caused by other factors in the cells [32, 33]. Here we demonstrated that the inhibition of FASN resulted in abrogating the protective effects of Dex against OGD/R-induced injury in cardiomyocytes.

Besides, to determine whether FASN-driven cholesterol synthesis is needed for Dex to prevent OGD/R-induced stress in cardiomyocytes, we measured and compared the levels of cholesterol in wild type and knockdown cells, with and without treatment of Dex. First cholesterol level significantly decreased in FASN-knockdown groups (Fig. 5A), confirming the correlation between FASN and cholesterol synthesis as previously reported [34]. The level of cholesterol can be restored back by acetoacetyl-CoA and HMG-CoA, which are commonly used to reverse the phenotype of FASN suppression-caused reduction in the cholesterol biosynthesis [34, 35]. However, the decrease of cholesterol levels also occurred in OGD/R-treated cells but can be rescued by Dex in the wild-type cells, but not in the FASN-knockdown cells (Fig. 5C). These results denote that Dex cannot exhibit its protective function against OGD/R injury in cardiomyocytes with FASN suppressed, further indicating that FASN-driven cholesterol synthesis is not only essential but needed for Dex’s protective function.

Furthermore, the role of FASN in the protective function of Dex was determined in the in a vivo rat model (Fig. 6). Similar to the in vitro results, Dex was only able to reverse the adverse effects caused by myocardial I/R injury with the presence of FASN, supported by the H&E evidence as well as the data of key proteins involved in apoptosis. And no significant differences were identified in FASN-knockdown groups whether the rats were pretreated with Dex. Indeed, FASN was reported to mediate apoptosis in several cell types [36, 37], which was consistent with the findings in this study that suppression of FASN did result in more severe apoptosis, and further confirmed that dependence of FASN for Dex to exhibit the protective functions.

In summary, this study investigated the protective role of dexmedetomidine (Dex) against myocardial ischemia/reperfusion injury, and the mechanism involved in cholesterol homeostasis was determined by evaluating the role of gene FASN. We demonstrated that in both in vitro cardiomyocyte models and in vivo rat models FASN is an essential and responsible gene for Dex to exhibit the function against myocardial I/R injury in rats and OGD/R-induced stress in cardiomyocytes, supported by the evidence that with FASN knockdown, pretreatment with Dex failed to reverse the adverse effects caused by myocardial I/R injury. Overall, this study provided the mechanism of action of Dex from a novel insight and better understanding of the function of Dex, which further offers comprehensive evidence for the future application of Dex.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Availability of data and material

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Ethics approval and consent to participate

All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiao Tong University.

Acknowledgments

Thanks for the support from the First Affiliated Hospital of Xi'an Jiao Tong University.

Author Contribution

Hong Gao: Conceptualization, Methodology, Software.Xinyan Zeng and Jiansheng Wang: Data curation. Shenghui Zhang: Visualization, Investigation. Jia Zhang: Supervision.: Kun Zhao: Software, Validation.: Yapeng Guo: Writing- Reviewing and EditingAll the authors have read and approved the final version of the manuscript.

White HD and Chew DP. Acute myocardial infarction. The Lancet. 2008; 372(9638):570-584.
Fan Z, Cai L, Wang S, Wang J and Chen B. Baicalin prevents myocardial ischemia/reperfusion injury through inhibiting ACSL4 mediated ferroptosis. Frontiers in Pharmacology. 2021; 12.
Weinreuter M, Kreusser MM, Beckendorf J, Schreiter FC, Leuschner F, Lehmann LH, Hofmann KP, Rostosky JS, Diemert N and Xu C. Ca M Kinase II mediates maladaptive post‐infarct remodeling and pro‐inflammatory chemoattractant signaling but not acute myocardial ischemia/reperfusion injury. EMBO molecular medicine. 2014; 6(10):1231-1245.
Land W, Schneeberger H, Schleibner S, Illner W-D, Abendroth D, Rutili G, Arfors KE and Messmer K. The beneficial effect of human recombinant superoxide dismutase on acute and chronic rejection events in recipients of cadaveric renal transplants. Transplantation. 1994; 57(2):211-217.
Land WG. The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation. Transplantation. 2005; 79(5):505-514.
Cai Y, Xu H, Yan J, Zhang L and Lu Y. Molecular targets and mechanism of action of dexmedetomidine in treatment of ischemia/reperfusion injury. Molecular medicine reports. 2014; 9(5):1542-1550.
Jaswal JS, Keung W, Wang W, Ussher JR and Lopaschuk GD. Targeting fatty acid and carbohydrate oxidation—a novel therapeutic intervention in the ischemic and failing heart. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2011; 1813(7):1333-1350.
Lopaschuk G. AMP-activated protein kinase control of energy metabolism in the ischemic heart. International journal of obesity. 2008; 32(4):S29-S35.
Lopaschuk GD and Stanley WC. Malonyl-CoA decarboxylase inhibition as a novel approach to treat ischemic heart disease. Cardiovascular drugs and therapy. 2006; 20(6):433-439.
Bonney S, Kominsky D, Brodsky K, Eltzschig H, Walker L and Eckle T. Cardiac Per2 functions as novel link between fatty acid metabolism and myocardial inflammation during ischemia and reperfusion injury of the heart. PloS one. 2013; 8(8):e71493.
Yoshitomi O, Cho S, Hara T, Shibata I, Maekawa T, Ureshino H and Sumikawa K. Direct protective effects of dexmedetomidine against myocardial ischemia-reperfusion injury in anesthetized pigs. Shock. 2012; 38(1):92-97.
Engelhard K, Werner C, Eberspächer E, Bachl M, Blobner M, Hildt E, Hutzler P and Kochs E. The effect of the α2-agonist dexmedetomidine and the N-methyl-D-aspartate antagonist S (+)-ketamine on the expression of apoptosis-regulating proteins after incomplete cerebral ischemia and reperfusion in rats. Anesthesia & Analgesia. 2003; 96(2):524-531.
Gu J, Sun P, Zhao H, Watts HR, Sanders RD, Terrando N, Xia P, Maze M and Ma D. Dexmedetomidine provides renoprotection against ischemia-reperfusion injury in mice. Critical care. 2011; 15(3):1-11.
Lai Y-C, Tsai P-S and Huang C-J. Effects of dexmedetomidine on regulating endotoxin-induced up-regulation of inflammatory molecules in murine macrophages. Journal of Surgical Research. 2009; 154(2):212-219.
Tao L, Guo X, Xu M, Wang Y, Xie W, Chen H, Ma M and Li X. Dexmedetomidine ameliorates high‐fat diet‐induced nonalcoholic fatty liver disease by targeting SCD1 in obesity mice. Pharmacology Research & Perspectives. 2021; 9(1):e00700.
Bouillon J, Duke T, Focken AP, Snead EC and Cosford KL. Effects of dexmedetomidine on glucose homeostasis in healthy cats. Journal of Feline Medicine and Surgery. 2020; 22(4):344-349.
Xue FS, Li RP, Liu GP and Sun C. Assessing renoprotective effect of perioperative dexmedetomidine in cardiac surgery patients. Kidney International. 2016; 89(5):1163-1164.
Zhou H, Hou SZ, Luo P, Zeng B, Wang JR, Wong YF, Jiang ZH and Liu L. Ginseng protects rodent hearts from acute myocardial ischemia–reperfusion injury through GR/ER-activated RISK pathway in an endothelial NOS-dependent mechanism. Journal of ethnopharmacology. 2011; 135(2):287-298.
Cheng XY, Gu XY, Gao Q, Zong QF, Li XH and Zhang Y. Effects of dexmedetomidine postconditioning on myocardial ischemia and the role of the PI3K/Akt-dependent signaling pathway in reperfusion injury. Molecular Medicine Reports. 2016; 14(1):797-803.
Fang X, Wang H, Han D, Xie E, Yang X, Wei J, Gu S, Gao F, Zhu N and Yin X. Ferroptosis as a target for protection against cardiomyopathy. Proceedings of the National Academy of Sciences. 2019; 116(7):2672-2680.
Xing M, Jiang Y, Bi W, Gao L, Zhou Y-L, Rao S-L, Ma L-L, Zhang Z-W, Yang H-T and Chang J. Strontium ions protect hearts against myocardial ischemia/reperfusion injury. Science advances. 2021; 7(3):eabe0726.
Chrysostomou C and Schmitt CG. Dexmedetomidine: sedation, analgesia and beyond. Expert opinion on drug metabolism & toxicology. 2008; 4(5):619-627.
Bajwa S and Kulshrestha A. Dexmedetomidine: an adjuvant making large inroads into clinical practice. Annals of medical and health sciences research. 2013; 3(4):475-483.
Li J, Zhao Y, Zhou N, Li L and Li K. Dexmedetomidine attenuates myocardial ischemia-reperfusion injury in diabetes mellitus by inhibiting endoplasmic reticulum stress. Journal of diabetes research. 2019; 2019.
Tang C, Hu Y, Gao J, Jiang J, Shi S, Wang J, Geng Q, Liang X and Chai X. Dexmedetomidine pretreatment attenuates myocardial ischemia reperfusion induced acute kidney injury and endoplasmic reticulum stress in human and rat. Life Sciences. 2020; 257:118004.
Zhong Y, Li Y-P, Yin Y-Q, Hu B-L and Gao H. Dexmedetomidine inhibits pyroptosis by down-regulating miR-29b in myocardial ischemia reperfusion injury in rats. International Immunopharmacology. 2020; 86:106768.
Wu Y, Qiu G, Zhang H, Zhu L, Cheng G, Wang Y, Li Y and Wu W. Dexmedetomidine alleviates hepatic ischaemia‐reperfusion injury via the PI3K/AKT/Nrf2‐NLRP3 pathway. Journal of Cellular and Molecular Medicine. 2021; 25(21):9983-9994.
Zhang J, Xia F, Zhao H, Peng K, Liu H, Meng X, Chen C and Ji F. Dexmedetomidine-induced cardioprotection is mediated by inhibition of high mobility group box-1 and the cholinergic anti-inflammatory pathway in myocardial ischemia-reperfusion injury. PLoS One. 2019; 14(7):e0218726.
Cheng X, Hu J, Wang Y, Ye H, Li X, Gao Q and Li Z. Effects of dexmedetomidine postconditioning on myocardial ischemia/reperfusion injury in diabetic rats: role of the PI3K/Akt-dependent signaling pathway. Journal of Diabetes Research. 2018; 2018.
Pombo JP and Sanyal S. Perturbation of intracellular cholesterol and fatty acid homeostasis during flavivirus infections. Frontiers in immunology. 2018; 9:1276.
Jin Y, Chen Z, Dong J, Wang B, Fan S, Yang X and Cui M. SREBP1/FASN/cholesterol axis facilitates radioresistance in colorectal cancer. FEBS Open bio. 2021; 11(5):1343-1352.
Tao Z, Zhang L, Wu T, Fang X and Zhao L. Echinacoside ameliorates alcohol-induced oxidative stress and hepatic steatosis by affecting SREBP1c/FASN pathway via PPARα. Food and Chemical Toxicology. 2021; 148:111956.
Huang W-C, Li X, Liu J, Lin J and Chung LW. Activation of androgen receptor, lipogenesis, and oxidative stress converged by SREBP-1 is responsible for regulating growth and progression of prostate cancer cells. Molecular Cancer Research. 2012; 10(1):133-142.
Carroll RG, Zasłona Z, Galván-Peña S, Koppe EL, Sévin DC, Angiari S, Triantafilou M, Triantafilou K, Modis LK and O’Neill LA. An unexpected link between fatty acid synthase and cholesterol synthesis in proinflammatory macrophage activation. Journal of Biological Chemistry. 2018; 293(15):5509-5521.
Yoshii Y, Furukawa T, Saga T and Fujibayashi Y. Acetate/acetyl-CoA metabolism associated with cancer fatty acid synthesis: overview and application. Cancer letters. 2015; 356(2):211-216.
Wu X, Qin L, Fako V and Zhang J-T. Molecular mechanisms of fatty acid synthase (FASN)-mediated resistance to anti-cancer treatments. Advances in biological regulation. 2014; 54:214-221.
Zhang W, Huang J, Tang Y, Yang Y and Hu H. Inhibition of fatty acid synthase (FASN) affects the proliferation and apoptosis of HepG2 hepatoma carcinoma cells via the β-catenin/C-myc signaling pathway. Annals of Hepatology. 2020; 19(4):411-416.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Dexmedetomidine ameliorates myocardial ischemia-reperfusion injury through regulating FASN-associated cholesterol homeostasis

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Results

Discussion

Declarations

Author Contribution

References

Additional Declarations

Status:

Version 1