Sequencing of RNAs involved in sepsis-induced myocardial injury: role of Local sympathetic denervation

Sepsis has been a serious public health concern for a long time and is characterized by disrupted development of specific diagnostic criteria and novel therapies for sepsis is critically dependent on an understanding of the basic mechanisms immune homeostasis accompanied by infection and multiple organ dysfunction as determined assessed by the Sequential Organ Failure. Adult (4 to 6 months) male C57BL/6 mice, animal set in three groups, control group, Cecal Ligation and Puncture (CLP) group, CLP+ Local sympathetic denervation group 8 mice in each group. CLP model for created sepsis and local sympathetic denervation procedure for alleviated the effects of sepsis. Assessment of cardiac function by Millar Instruments, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and RNA extraction and sequencing. Our findings showed that the myocardial injury score and TNF-α level in both tissue and serum were significantly increased in the CLP group. CLP with local sympathetic denervation group mice had lower scores of myocardial injuries. Sepsis augments MCP-1 and KC level in myocardium. After 48 hours after CLP, the myocardial injury score. CLP with local sympathetic denervation alleviates sepsis-induced myocardial injury by inhibiting the NF-κB signaling pathway. Moreover, compared with the Ctrl group, the CLP with local sympathetic denervation group obtained fewer ncRNAs and mRNAs. Inc onclusions, the NF-κB signaling pathway is associated with sepsis-induced myocardial injury, and local sympathetic denervation can relieve sepsis-induced myocardial injury by regulating the ncRNAs and mRNAs.


Introduction
As the main cause of death in intensive care units, sepsis has been a serious public health concern for a long time and is characterized by disrupted development of specific diagnostic criteria and novel therapies for sepsis is critically dependent on an understanding of the basic mechanisms immune homeostasis accompanied by infection and multiple organ dysfunction as determined assessed by the Sequential Organ Failure [1].The of the disease.Numerous lines of evidence have concluded that many signaling pathways and pathological processes are involved in sepsis, such as the host immune response, unstable circulation, endothelial barrier dysfunction, and disordered cell function [2,3].Myocardial injury is a major and serious complication of sepsis; sepsis-induced myocardial injury has a high incidence rate and is observed in 40-50% of patients with sepsis [4].Moreover, studies have reported that sepsis-induced cardiac dysfunction may be as high as 70% and is a major predictor of mortality from sepsis [5,6].However, current treatments for myocardial injury following sepsis are not ideal, as mechanisms underpinning sepsis-induced myocardial injury are not fully understood.
Sepsis depresses cardiac function via upregulation of the expression of cardiodepressant cytokines, including TNF-α, IL-1β and IL-6 [7].IL-6 expression is elevated in several tissues of old mice [8].In addition, aging has been shown to exacerbate the cytokine response to proinflammatory insults, including endotoxin, trauma, and ischemia/reperfusion injury [9].Thus, it is likely that aging upregulates the myocardial inflammatory responses to endotoxin and exaggerates sepsis cardiac depression [10] and the mononuclear cells are major sources of tissue pro-inflammatory cytokines [11].The sympathetic nervous system has a wide variety of cardiovascular actions, including heart rate acceleration, increase in cardiac contractility, reduction of venous capacitance, and constriction of resistance vessels.On the contrary, the parasympathetic nervous system affects the cardiovascular system by slowing heart rate through vagal impulses [12].The cardiac sympathetic nerve fibers are located subepicardially and travel along the major coronary arteries representing the predominant autonomic component in the ventricles [13].The parasympathetic fibers run with the vagus nerve subendocardially after it crosses the atrioventricular groove and are mainly present in the atrial myocardium and less abundantly in the ventricular myocardium [14].
The ventricular sympathetic innervation is characterized by a gradient from base to apex (3).The cardiac neuronal system is made up of spatially distributed cell stations comprising afferent, efferent, and interconnecting neurons behaving as a control system [15].The neurons are in constant communication with each other, and each neuronal cell station is involved in cardio-cardiac reflexes that control spatially organized cardiac regions [16].
Cardiac sympathetic neuronal activity or its pharmaceutical inhibition can also be noninvasively assessed by the use of 123 I-metaiodobenzylguanidine (MIBG), an analogue of NE [17], using semiquantitative analyses, namely early heart-to-mediastinum ratio, late heart-to-mediastinum ratio, and myocardial washout.Beta-blockade and renin-angiotensin-aldosterone inhibition are associated with an increase in 123 I-MIBG uptake and a reduced washout.Data from a systematic meta-analysis suggest that patients with decreased late heart-to-mediastinum or increased myocardial 123 I-MIBG washout have a worse prognosis than those patients with normal semiquantitative myocardial MIBG parameters [18].
A prospective study that compared the predictive value of cardiac 123 I-MIBG imaging for sudden cardiac death with that of the signal-averaged electrocardiogram, heart rate variability, and QT dispersion in patients with mild-to-moderate heart failure demonstrated that 123 I-MIBG was the only powerful predictor of sudden cardiac death independently of left ventricular ejection fraction [19].

Animals and treatment
Adult (4 [20], in briefly anesthetize the mouse by injecting intraperitoneally a solution of 1:1 ketamine (75mg/kg) and xylazine (15mg/kg).As a reference, inject 30 μl of the 1:1 solution into a mouse weighing 20 grams [20].The cecum is tightly ligated with a 6.0 silk suture (6-0 PROLENE, 8680G; Ethicon) at its base below the ileo-cecal valve and is perforated once or twice with a 19-gauge needle (4 and 5) on the same side of the cecum.Please note that the length of ligated cecum defined as the distance from the distal end of cecum to ligation point will determine the degree of severity.A distance of >1cm produces high grade sepsis while a distance of ≤1 cm produces mid-to-low grade sepsis.The cecum is then gently squeezed to extrude a small amount of feces from the perforation sites.The cecum is returned to the peritoneal cavity and the peritoneum is closed with 6.0 silk sutures .
The skin is closed with Reflex 7mm clips (RS-9258, Roboz Surgical Instruments) or Michel wound clips (7 mm, RS-9270) .Resuscitate mice by injecting subcutaneously 1 ml of prewarmed 0.9% saline solution using a 25G needle.This fluid resuscitation measure will induce the hyperdynamic phase of sepsis.Mice of sham as control group only received cecum exposure without ligation or puncture.All mice were resuscitated by sterile 0.9% saline at the end of the surgery and had free access to water and food.
Mice were euthanized at 72 hrs after surgery.Tissues of lungs, heart, liver and kidney were harvested and fixed in 4% paraformaldehyde at 4°C or stored at −80°C immediately.Whole blood was centrifuged at 3000 rpm for 15 mins and sera were collected and stored at −80°C.
After analysis of cardiac function, the heart tissue and blood were collected and prepared for analysis.

Local sympathetic denervation
It was performed as described previously for rats [21], briefly after induction of anesthesia, a vertical neck incision (∼10 mm) was made and kept open using placeholders.Next, the salivary glands were carefully dissected from the caudal adhesive tissue and flipped aside.The position was then corrected by adjusting the lower two placeholders (with expanded surface).With blunt forceps, the sternocleidomastoid muscle was lifted, moved aside, and held using the upper two, thinner placeholders.Lateral positioning of the sternocleidomastoid muscle allowed for clear visibility of the carotid artery.The common carotid artery (CCA) was followed in cranial direction to locate the carotid bifurcation (in internal and external carotid arteries).The almond-shaped superior cervical ganglia (SCG) are located behind the carotid bifurcation.This approach exposed the preganglionic and postganglionic branches of the SCG, facilitating complete removal of the ganglion itself.The ganglionic cell body was fully extracted by shifting the external carotid artery to the lateral side using a special 90° angled forceps.Finally, the SCG was detached from the sympathetic chain and the SCG tissue was collected.Complete cardiac sympathetic denervation was achieved by additional removal of the SCG on the contralateral side.

Measurement of cardiac function
We assessed cardiac function at 48 h after CLP as described previously [22].Briefly, mice were anesthetized with pentobarbital sodium (Vortech Pharmaceuticals, Dearborn, MI, USA; 50 mg/kg, intraperitoneal (ip)) and anticoagulated with heparin (Elkins-Sinn, Cherry Hill, NJ, USA; 1,000 units/kg, ip).Animals were laid supine on a heating blanket and core body temperature was maintained at 37°C ± 0.5°C.A microcatheter (Millar Instruments, Houston, TX, USA; 1 F) was inserted into the left ventricle (LV) through the right common carotid artery.Pressurevolume loop was recorded using the MPVS-400 system with the aid of PVAN software (Millar Instruments).Heart rates, LV pressure, LV volume, and related function parameters were analyzed: 10 μL of 30% saline was injected into the inferior vena cava for actual ventricle volume calculation.

Immunofluorescence staining
The optimal cutting temperature (OCT)-embedded tissues were cut into thin sections (5-μm thick).Sections were treated with a mixture of 30% acetone and 70% methanol for 5 minutes, washed with PBS and fixed with 4% paraformaldehyde.Then sections were incubated with a rabbit polyclonal antibody against murine CD68 (a marker of mononuclear cells, including monocytes and macrophages) followed by Cy3-tagged goat anti-rabbit IgG (imaged on the red channel).Nuclei were stained with bis-benzimide (4′,6-diamidino-2-phenylindole (DAPI), imaged on the blue channel), and glycoproteins on cell surfaces were stained with Alexa 488tagged wheat germ agglutinin (imaged on the green channel).Microscopy was performed with a Leica DMRXA digital microscope (Leica Mikroskopie and System GmbH, Wetzlar, Germany).
Images were analyzed in a blinded fashion.Samples and standards were prepared according to manufacturer's instructions.Absorbance of standards and samples were determined spectrophotometrically at 450 nm, using a microplate reader (Bio-Rad Laboratories, Inc, Hercules, CA, USA).Results were plotted against the linear portion of a standard curve.

RNA extraction and sequencing
The mice (n = 8) in each group were anesthetized, and cardiac tissues were quickly transferred to liquid nitrogen and transported to the sequencing laboratory with dry ice.The mirVana™ miRNA Isolation Kit and Ambion-1561 were used to extract total RNA, after which TruSeq Stranded Total RNA with Ribo-Zero Gold was used for library construction.Generally, the RNA was broken into short fragments.The first and second cDNA strands were synthesized in turn.dTTP was replaced with dUTP in the second cDNA strand after end repair, ligation of the sequencing adapters and digestion by the UNG enzyme.PCR amplification was then performed.After quality inspection, an Illumina sequencer was used for sequencing, and clean reads were obtained for subsequent analysis.

Quantification of miRNAs and identification of DE miRNAs
After total RNA was extracted from the samples, a miRNA sequencing library was constructed using the mirVana miRNA Isolation Kit (Ambion) and TruSeq Small RNA Sample Prep Kits.
Total RNA, concentration and integrity were measured by Nanodrop 2000 and Agilent 2100.
After PCR amplification and quality inspection, the library was sequenced using an Illumina HiSeq X Ten platform.High-quality clean reads were obtained after filtering using Fastx-toolkit (0.0.13) software, Bowtie (1.1.1)and NGSQCToolkit.

Sepsis results in attenuated cardiac function
We determined whether sepsis causes attenuated cardiac function in CLP mice than in local sympathetic denervation.Heart rate increased in both CLP mice and local sympathetic denervation in comparison to saline-treated controls (Table 1).Compared to the control group, ejection fraction and developed pressure decreased in CLP mice more than local sympathetic denervation.Similarly, cardiac output was decreased in CLP mice and improved in local sympathetic denervation.

Sepsis augments MCP-1 and KC level in myocardium
Sepsis increased the levels of MCP-1 and KC in plasma and the myocardium (Figure 2A and   B).Interestingly, local sympathetic denervation (LSD) mice had significantly higher levels of MCP-1 and KC in plasma and the myocardium than CLP mice only.The expression levels of miRNA-133b-3p (Fig. 4a) and miR-486b-3p (Fig. 4b) were significantly lower in the CLP group than in the Ctrl group.In comparison, the expression levels of miRNA-133b-3p (Fig. 4a), miR-486b-3p (Fig. 4b) and miRNA-135b-5p (Fig. 4c) were significantly higher in the CLP with local sympathetic denervation group mice than in the CLP group.The expression levels of circRNA-0000246 (Fig. 4d), circRNA-003646 (Fig. 4e), and circRNA-0013022(Fig. 4f) were consistent with the sequencing results.

Discussion
The immune and the nervous systems intensely interact and mutually control each other [24].In peripheral tissues, the sympathetic nervous system has been demonstrated to regulate key processes of immune cell function, such as the release of monocytes from the bone marrow compartment [25] tissue programming of macrophages [27], the cytokine expression profile of various immune cells [28], and the antibody production of B cells [29].
A detailed picture of the signalling pathways and molecules involved that execute these diverse functions is only gradually evolving.Upon their activation, postganglionic sympathetic fibers release norepinephrine together with several cotransmitters (e.g., adenosine triphosphate).
Norepinephrine exerts its function on recipient cells through the binding and activation of adrenoceptors, among which the beta-2-adrenoceptor and the beta-3-adrenoceptor have been assigned critical roles in regulating immune cell function or their mobilization [30].
Sepsis causes whole body dysfunction due to infection and is one of the leading causes of mortality worldwide [31].While it has been studied for ~2,000 years, the incidence of sepsis has not decreased over time [32].Inflammation is the first characteristic feature of sepsis.
Previous studies have revealed that oxidative stress caused by inherent inflammatory responses can lead to the initiation of lipid peroxidation, DNA damage and mitochondrial function deterioration, as well as further the development of organ dysfunction and failure [33].
Therefore, increases in inflammation and oxidative stress both contribute to the damage caused by sepsis [30].Moreover, oxidative stress has been reported to participate in numerous pathological conditions, such as diabetes and myocardial ischemia reperfusion injury [33].
NcRNAs play essential roles in myocardial injury and many other systems such as lung tissue.
To further explore the mechanism of sympathetic nerve regulation in myocardial injury, we investigated whether sympathetic nerves affect circRNA, lncRNA, or miRNA levels during sepsis-induced myocardial injury.We found many differentially expressed circRNAs, lncRNAs, miRNAs, and mRNAs in the CLP group, compared with the Ctrl group that regulated inflammatory signaling pathways indicated by GO and KEGG enrichment.Some previous studies have also used whole-transcriptome sequencing to assess the transcriptome changes in myocardial injury and have proven that multiple circRNAs, lncRNAs, miRNAs, and mRNAs are significantly altered in myocardial injury [34].
The clean reads were aligned to the mouse genome and compared with miRBase (version 22.0).Target genes of DE miRNAs were predicted by Miranda (3.3a).The expression levels of the identified known mature miRNA sequences and the newly predicted miRNAs were quantified as the transcripts per million (TPM).DE miRNAs were analyzed with DESeq2 American Journal of BioMedicine AJBM 2022;10(1):25-34 doi:10.18081/2333-5106/2022.10/2540 Statistical methods Data are presented as mean ± standard error (SE).Statistical analysis was performed using StatView software (Abacus Concepts, Calabasas, CA, USA).Analysis of variance (ANOVA)with Fisher post-hoc test was used to analyze differences between experimental groups, and differences were confirmed using the Mann-Whitney U-test.Statistical significance was defined as P ≤0.05.ResultsIn septic rats, myocardial contraction and diastolic function were weakened, which often manifested as heart failure, impaired myocardial function, increased mortality, and increased inflammatory factors.The representative pressure-volume loops in Figure1, sepsis causes a reduction in LV function, and old mice have worse LV function during sepsis.

Figure 1 .
Figure 1.Sepsis results in greater cardiac depression and worse myocardial injury in old mice.Adult mice were suspected to CLP.Left ventricular pressure-volume loops and plasma levels cardiac troponin-I (cTn-I) were analyzed 48 days after CLP.Sepsis mice displayed greater reduction in left ventricular performance (A) and higher levels of cTn-I in plasma (B).Data are expressed as mean ± standard error; *P <0.05 versus corresponding local sympathetic denervation; #P <0.05 versus adult mice with sepsis only.

Figure 2 .
Figure 2. CLP mice have higher levels of chemokines in the myocardium and plasma.Levels of MCP-1 and KC were analyzed by ELISA 48 h after CLP.CLP mice only had higher levels of MCP-1 and KC in myocardial tissue (A) and plasma (B).Data are expressed as mean ± standard error.*P <0.05 versus corresponding control (Ctrl); # P <0.05 versus CLP only mice.
In the current study, we identified 629 DE circRNAs, 269 DE lncRNAs, 192 DE mRNAs, and 7 DE circRNAs in the CLP with local sympathetic denervation group compared with the CLP group.We performed GO and KEGG enrichment analyses to further understand the roles of these DE ncRNA and DE mRNAs.GO and KEGG enrichment analyses of the DE ncRNAs and DE mRNAs in the CLP group showed that many immune-inflammatory processes and pathways included the NF-κB signaling pathway.Surprisingly, many DE circRNAs, DE lncRNAs, DE miRNAs, and DE mRNAs were found in the CLP with local sympathetic denervation group compared with the CLP group, too.GO and KEGG analyses identified many inflammatory and immune responses such as the TGF-β signaling pathway, TNF signaling, MAPK signaling, Th17 cell differentiation, and B-cell receptor signaling pathways, between the DE ncRNAs and the DE mRNAs.