Key role of Extracellular RNA in hypoxic stress induced myocardial injury.

Myocardial infarction (MI), atherosclerosis and other inflammatory and ischemic cardiovascular diseases (CVDs) have a very high mortality rate and limited therapeutic options. Although the diagnosis is based on markers such as cardiac Troponin-T (cTrop-T), the mechanism of cTrop-T upregulation and release is relatively obscure. In the present study, we have investigated the mechanism of cTrop-T release during acute hypoxia (AH) in a mice model by ELISA & immunohistochemistry. Our study showed that AH exposure significantly induces the expression and release of sterile inflammatory as well as MI markers in a time-dependent manner. We further demonstrated that activation of TLR3 (mediated by eRNA) by AH exposure in mice induced cTrop-T release and Poly I:C (TLR3 agonist) also induced cTrop-T release, but the pre-treatment of TLR3 immuno-neutralizing antibody or silencing of Tlr3 gene or RNaseA treatment two hrs before AH exposure, significantly abrogated AH-induced Caspase 3 activity as well as cTrop-T release. Our immunohistochemistry and Masson Trichrome (MT) staining studies further established the progression of myocardial injury by collagen accumulation, endothelial cell and leukocyte activation and adhesion in myocardial tissue which was abrogated significantly by pre-treatment of RNaseA 2 hrs before AH exposure. These data indicate that AH induced cTrop-T release is mediated via the eRNA-TLR3-Caspase 3 pathway.

Introduction

Ascent to high altitudes generates a hypoxic environment in the body, causing hypoxemia in the circulation that leads to inflammation and hypercoagulation [1]. Several disorders such as CVDs, pulmonary and cerebral edema are concomitant with hypoxia-induced inflammation [2]. The fundamental cause of morbidity and mortality in MI [3] and acute lung injury [4] has been observed to be vascular inflammation due to hypoxia.

Clinically, acute myocardial infarction (AMI) is a condition wherein cTrop-T concentration is elevated. Traditional diagnostic checks for AMI include an onset of chest pain and abnormalities in electrocardiographic (ECG) tests. These are either often absent and/or nonspecific. Therefore, the diagnosis is mainly dependent upon the elevated levels of cTrop-T which is a reliable AMI marker. It is well known that more than 90% of intracellular Troponin are localised in the sarcomere and the rest circulate in the cytoplasm. Therefore, the release of cTrop-T into the circulation followed by the events of myocyte necrosis, apoptosis, formation and release of membranous blebs, increases membrane permeability and release of proteolytic troponin degradation products [5].

Hypoxia induces stress which stimulates “sterile (non-pathogenic) inflammation”. In response to such a stress, a synchronised inflammatory response repairs and/or eliminates the damaged cells, further aggravating the injury. This response is facilitated by the cellular components of the innate immune system (neutrophils, monocytes, macrophages, etc.) and also endothelial cells (ECs), fibroblasts and pericytes, which recognizes various Damage Associated Molecular Patterns (DAMPs) molecules that are inappropriately discharged from dying cells [6,7]. SI induced activation of the innate immune system plays a significant role in the genesis of several chronic diseases viz. atherosclerosis, heart failure, etc [8].

Studies have revealed that post-hypoxic exposure cardiac injury may be reversible (except for papillary muscle necrosis and infarction) [9]; although, cardiomyocyte injury occurs, even if reperfusion therapy is given as soon as possible following the hypoxic insult [10]. Tissue injury due to microbial pathogens or sterile stress such as oxygen free radicals, hypoxia, or hyperthermia upregulates various DAMPs, which may be released into the circulation or can remain at the site of injury and trigger the inflammatory response [11]. Furthermore, DAMPs also trigger and engage the cells of the innate immune system in the heart [12].

Extracellular RNA (eRNA), a DAMP, released during hypoxia causes inflammation and vascular diseases [13]. However, its role in hypoxia induced AMI is not fully understood. eRNA is predominantly derived from ischemic myocardium and plays an adverse role in myocardial ischemia [11]. Apoptosis and necrosis of the tissues release endogenous nucleic acids which are recognized by TLR3,7,8 & 9 and are vital response to viral infection [14]. Previous study in TLR3 deficient mice that were subjected to cardiac I/R have shown reduction in infarct size [15]. The vaso-protective role of RNaseA administration has been studied in cardioprotection for myocardial I/R (ischemia-reperfusion) injury in mice and in the isolated I/R Langendorff-perfused rat heart [16].

Given these links between hypoxia and AMI, we hypothesized that the diminished oxygen level stimulates eRNA release and subsequently triggers AMI through TLR3-caspase-3 pathway. Herein, we presented a possible mechanism of cTrop-T release and the progression of AMI in a murine model of AH. So, this paper assesses (i) the influence of hypoxia on the generation of SI molecules and TLR3 activation and (ii) the implication of TLR3 activation due to the release of cTrop-T and Caspase-3.

Materials and methods

Ethical clearance

Experimental protocols were approved by the Internal Review Board of Defence Institute of Physiology and Allied Sciences (DIPAS) (IAEC/DIPAS/2015-03; authorization no:27/GO/RBi/SL/99/CPCSEA) in accordance with the guidelines of the committee for the purpose of control and supervision of experiments on animal, Ministry of environment and forest, Government of India. This study was done on adult Swiss Albino mice weighing 25-30g.The mice were housed in the animal facility of the institute and provided with chow diet and water ad libitum. All experimental procedures have been carried out in DIPAS and animal procedures were performed under urethane (1.2g/kg) induced general anaesthesia to minimize suffering [1].

Chemicals

The commercial details of the materials used for all experiments are as follows: Mouse monoclonal Troponin-T antibody (ab8295), Rabbit polyclonal Neutrophil Elastase antibody (ab21595), Mouse monoclonal PECAM-1 antibody (ab24590), Mouse monoclonal CD11b+CD18 antibody (ab13219), Mouse monoclonal CD41 antibody (ab11024), TLR3 neutralizing antibody (ab12085) and Rabbit polyclonal alpha smooth muscle Actin antibody (ab5694) were procured from Abcam (MA, USA). Mouse monoclonal Myoglobin Antibody (sc74525) was procured from Santa Cruz Biotechnology (TX, USA). RNaseA, DNase1, TLR3 siRNA sense (5´-CGUUAUCACACACCAUUUA-3´) and antisense (5´-UAAAUGGUGUGUGAUAACG-3´), negative-control siRNA (scrambled sequence) and Trichrome stain (Masson) kit (HT15) were procured from Sigma-Aldrich (MO, USA); MaxSuppressor In Vivo RNA-LANCEr II, a formulation that enables highly efficient siRNA delivery into animals was purchased from Bio Scientific Corporation (TX, USA). HRP conjugated secondary antibodies [anti-mouse (62–6520), anti-rabbit (65–6120), anti-goat (81–1620)], Alexa Fluor 488-conjugated anti-mouse (A-10680) secondary antibody and Rabbit polyclonal s100b antibody (PA5-78161) were procured from Invitrogen (CA, USA). Trizol (15596026), DAPI (D1306) and poly I:C (20148E) were procured from Thermo Fischer (MA, USA). Caspase-3 assay kit (K105-100) was purchased from BioVision Inc (CA, USA). Quantitative ELISA kits were procured as follows: Mouse HMGB1 protein ELISA kit (MBS722248) and Mouse vWF ELISA kit (MBS260427) from MyBiosource Inc (CA, USA); HSP70 high sensitivity ELISA kit (ADI-EKS-715) from Enzo Life Sciences (NY, USA); Mouse HSP90 ELISA kit (NBP2-76449) from Novus Biologicals (CO, USA); Mouse S100B ELISA kit (E-EL-M1033) from Elabscience (TX, USA).

AH and other treatments

The mice were exposed to AH in an animal decompression chamber with conditions equivalent to atmospheric conditions at an altitude of 7628 m (282 mm Hg), with an O2 content of ~8.5%, and standard temperature and humidity, as previously described [13,17]. This setting for hypoxia exposure has been previously standardized in our lab for research purposes [1,13,21,22]. As per experimental requirement, the durations of exposure to mice to hypobaric hypoxia are 0 hr, 6 hrs, 12 hrs, 24 hrs, 3 days and 7 days. Intravenous injection of RNaseA (1 mg/kg BW (body weight)) and DNase1 (5 mg/kg BW) [1,18,19] were done prior to AH exposure and blood was extracted post-exposure. The intravenous injection was performed with a 30-gauge needle insulin syringe (BD Biosciences). In vivo siRNA delivery was carried out as per protocols described elsewhere [13,20]. Poly I:C (TLR3 agonist) (1 mg/kg BW), TLR3 neutralizing (nt)-antibody (40 mg/kg BW) [21], Lipoteichoic Acid (TLR2 agonist) (100 μg/kg BW) [22] and HMGB1 nt-antibody (200 μg/kg BW) [22] were also administered intravenously [13,23]. 80 μg/kg BW of eRNA and eDNA (isolated from plasma of exposed animals, and purified by DNase1 and RNaseA treatment respectively) were injected into the animal intravenously through the tail vein six hrs prior to the sacrifice [1]. Plasma samples from hypoxia exposed mice were pooled to get better yield of eRNA and eDNA for further experiments. Sacrifice of control and treated animals was done by cervical dislocation by trained professional prior to harvest of organs and tissues for further studies [1].

Blood collection and tissue harvest

Retro-orbital blood collection was done in 2 mL citrate vials from non-anesthetized mice [24]. The plasma was separated and stored for further experiments. The whole heart was excised from the sacrificed mice and stored separately for molecular work as well as tissue sectioning for further experiments and studies.

eRNA and eDNA isolation eRNA and eDNA were isolated from the plasma samples of hypoxia exposed mice by the Trizol method and a commercially available kit (Quick-cfDNA, Zymo Research) respectively, and stored after purification by DNase1/RNaseA treatment.

Enzyme linked immunosorbent assay

ELISA was performed as previously described [25]. Briefly, 50μg total protein from plasma or tissue lysate samples was incubated overnight with an equal volume of coating buffer (0.5 M carbonate buffer [pH 9.6]) in an assay plate at 4°C. Non-specific sites were blocked by BSA followed by incubation with primary antibodies for 2hrs (HMGB1, vWF, HSP70, HSP90, s100b, c-Trop T and Myoglobin at 1:3000 dilution in PBS buffer) and then specific HRP-conjugated secondary antibodies for 2hrs (anti-mouse, anti-rabbit and anti-goat secondary antibodies at 1:5000 dilution in PBS buffer). Protein detection was done using OPD substrate and color intensity absorbance was measured at 450 nm.

In vivo gene silencing by TLR3 siRNA

In vivo TLR3 inhibition was done by intravenous injection of TLR3 siRNA using the MaxSuppressor kit as per manufacturer protocol as well as our previous studies [1,13]. Based on the delivery route (intravenous), the injection volume was 100 μl per dose. The injection was done intravenously into the mouse tail vein using an insulin syringe at 20 l/sec approximately. After the injection of the RNAi agent, the recommended incubation time in the animal is 3–4 days. Retro-orbital blood was drawn at the indicated times and the tissues were harvested for further processing.

Histological assessment

Myocardial tissue was fixed in a formaldehyde solution and immersed in neutral buffered saline overnight. The tissues were processed for paraffin embedding and sectioning done using a standard microtome. Deparaffinization and rehydration of the section was done by xylene and alcohol gradient and the antigen retrieval was done by sodium citrate buffer. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 and then permeabilized with 0.25% Triton X in PBS. After incubation of the sections with specific primary (α-SMA, PECAM-1 and NE at 1: 200 dilution) and secondary antibodies (anti-rabbit and anti-mouse at 1:500 dilution), the antibody binding sites were stained with DAB reagent and the images analyzed using ImageJ software (open-source imaging software), NIH.

For immunofluorescence microscopy, the sections were incubated overnight with primary antibodies (CD11/CD18 and CD41 at 1:250 dilution) at 4°C. The sections were then washed thoroughly and incubated in the dark with Alexa 488 conjugated secondary antibodies (anti-mouse at 1:500 dilution) for 60 minutes at room temperature. The samples were treated with 0.01% DAPI for 15 minutes at room temperature for nuclear counterstaining. The sections were then viewed under a fluorescence microscope (Ti2-E Motorized Inverted Microscope; Nikon, Tokyo, Japan).

MT staining was done using a commercially available kit (HT15, Sigma Aldrich).

Activated Caspase assay

The estimation of activated Caspase-3 was done using a commercial kit (Caspase-3 Fluorometric Assay Kit, BioVision Inc.), following the manufacturer’s protocol.

Statistical analysis

All experiments were performed in triplicate and the data expressed as means ± SEM. The statistical significance between experimental groups was determined by one-way ANOVA followed by Bonferroni’s multiple comparison tests. A p-value of <0.05 was considered statistically significant.

Results

1. Time dependent induction of SI molecules by AH

Time-dependent effect of AH exposure on the levels of circulating SI molecules was determined. Blood was collected from the mice exposed to AH for different time points (0–24 hrs) and the plasma was isolated. The mice that were subjected to AH exposure showed significantly higher levels of SI markers i.e., HMGB1, vWF, HSPs, s100b, eRNA and eDNA (Fig 1A–1G; p < 0.05). Thus, the six hrs exposure time remained constant for subsequent experiments unless otherwise stated.

Fig 1

AH induces release of circulating nucleic acids and expression of sterile inflammatory protein molecules.

Level of SI markers in plasma was analysed after exposure to hypoxia for different time durations (0–24 hrs); (a) eDNA (μg/ml), (b) eRNA (μg/ml), (c) HMGB1 (ng/ml), (d) vWF (ng/ml), (e) HSP70 (ng/ml), (f) HSP90 (ng/ml), and (g) s100b (pg/ml). Data are shown as mean ± SEM (n = 5/group/each time point) from one experiment representative of three independent experiments, all performed in triplicate. One-way ANOVA revealed statistical significance in the results (*p< 0.05).

2. AH induces myocardial injury in time dependant manner

To evaluate the time dependant effects of AH exposure on the myocardial injury, we analysed cardiac myocardial injury marker i.e., cTrop-T & myoglobin in plasma by ELISA (0–24 hrs and 3–7 days). It was observed that the mice that were subjected to AH exhibited a time dependent increase of cTrop-T and myoglobin when compared to the control until 7 days (Fig 2A and 2B; p < 0.05). This suggests that hypoxia exposure induced myocardial injury in a time dependent manner.

Fig 2

AH exposure induces myocardial injury in time dependant manner.

Expression of cardiac injury markers in plasma samples after exposure of animals to hypoxia for different time durations (0–24 hrs, 3-7days); (a) cTrop-T, (b) Myoglobin. Data are shown as mean ± SEM (n = 5/group/treatment) from one experiment representative of three independent experiments, all performed in triplicate. One-way ANOVA revealed statistical significance in the results (*p< 0.05).

3. Contribution of eRNA in the AH induced cTrop-T liberation

The above results showed that AH exposure notably increased the circulating concentration of SI as well as cardiac injury markers. However, the involvement of particular sterile inflammatory molecules in the up-regulation of cTrop-T is obscure. Hence, we carried out treatment experiments on mice using RNaseA (RNA degrader) or DNase1 (DNA degrader) or HMGB1 neutralizing antibody or purified endogenous eRNA or eDNA [1], and the plasma level of cTrop-T was measured. Our analysis confirmed that the pre-treatment of RNaseA significantly decreased the plasma level of cTrop-T when compared to the control (Fig 3A; p<0.05). However, the pre-treatment of DNase1 /or HMGB1 neutralizing antibodies failed to do so (Fig 3B and 3C; p<0.05). Endogenous eRNA or eDNA is the positive control. These data suggest that eRNA is only involved in AH induced cTrop-T releases.

Fig 3

eRNA is involved in hypoxia induced expression of cTrop-T.

ELISA estimation of circulating cTrop-T in plasma was done after (a) eRNA injection (80 μg/kg BW; 6 hrs prior to AH exposure) and RNaseA pre-treatment (1mg/kg BW; 2 hrs prior to AH exposure), (b) eDNA injection (80 μg/kg BW; 6 hrs prior to AH exposure) and DNase1 pre-treatment (5 mg/kg BW; 2 hrs prior to AH exposure), (c) HMGB1 IgG pre-treatment (200 μg/kg BW; 2 hrs prior to AH exposure), (d) pre-treatment with LTA (TLR2 agonist, 100 μg/kg BW; 2 hrs prior to AH exposure) and Poly I:C (TLR3 agonist, 1 mg/kg BW; 2 hrs prior to AH exposure). Control treatment with eRNA, eDNA, RNaseA, DNase 1, HMGB1 IgG, LTA and Poly I:C were done 6 hrs prior to sacrifice and the duration of AH exposure after different treatments was also 6 hrs. Data are shown as mean ± SEM (n = 5/group/treatment) from one experiment representative of three independent experiments, all performed in triplicate. One-way ANOVA revealed statistical significance in the results (*p< 0.05 groups w.r.t control; #p<0.05 groups w.r.t RNaseA).

4. AH induced eRNA facilitates cTrop-T release via TLR3 signalling

Our earlier study showed that AH stimulated expression of TLR2 and TLR3 [22]. However, the involvement of TLRs in the hypoxia induced upregulation of cTrop-T is not clear. The above results revealed that the pre-treatment of HMGB1 neutralizing antibody failed to inhibit hypoxia induced cTrop-T release, also mice that were treated with LTA, a TLR2 agonist, failed to induce cTrop–T release. However, Poly I:C, a TLR3 agonist showed positive effect (induce cTrop–T release) (Fig 3D; p<0.05). This suggests the non-involvement of TLR2 in hypoxia induced upregulation of cTrop-T, which was similar to earlier results, showing that HMGB1 worked though TLR2 [22].

To further validate the association of TLR3 in release of cTrop-T under hypoxic condition, mice were treated with TLR3-immune neutralizing antibody, two hrs before exposure to AH, and the plasma cTrop-T was quantified by ELISA. Our result showed that pre-treatment of TLR3-immune neutralizing antibody significantly inhibited cTrop-T release (Fig 4A; p<0.05). To affirm the involvement of TLR3, we performed Tlr3 gene silencing (70%) [1,13] in vivo by using Tlr3 siRNA and the plasma level of cTrop-T was measured. Our results showed that AH-induced release of cTrop-T was substantially reduced in Tlr3-silenced mice as compared to the control (Fig 4B; p<0.05). This clearly demonstrates that hypoxia-induced release of cTrop-T is mediated through TLR3.

Fig 4

AH induced eRNA facilitates release of cTrop-T via TLR3 signalling.

ELISA estimation of circulating cTrop-T in plasma was done after (a) TLR3 IgG treatment (40 mg/kg BW; 2 hrs prior to AH exposure). Poly I:C (1 mg/kg BW; 2 hrs prior to AH exposure) and eRNA (80 μg/kg BW; 6 hrs prior to AH exposure) treatments were used as positive controls and (b) TLR3 siRNA treatment. Non-specific (NS) siRNA control and non-immune IgG control were used similarly in another group of mice. Data are shown as mean ± SEM (n = 5/group/treatment) from one experiment representative of three independent experiments, all performed in triplicate. One-way ANOVA revealed statistical significance in the results (*p< 0.05 groups w.r.t control; #p<0.05 groups w.r.t Hypoxia and eRNA).

5. AH-induced eRNA enriched collagen accumulation, leukocyte infiltration and activation in cardiomyocytes

Vascular remodelling is one of the predominant factors to reduce the blood flow which consequently enhances hypoxic effect. To evaluate whether AH exposure has any role in vascular remodelling and subsequently myocardial injury, experiments were carried out to analyse expression of collagen and different markers for leukocyte activation in the cardiomyocytes. Fig 5A and 5B shows MT staining (for collagen) and immunohistochemistry staining for α-SMA (myofibroblast marker) in the myocardial tissue after various treatments, depicting collagen deposition in the peripheral vascular regions. Hypoxia-induced necrosis in myocardia initiates conversion of fibroblasts to myofibroblasts which requires increased synthesis of collagen, and this leads to its accumulation in myocardia. Collagen deposition and expression of α-SMA increases upon hypoxia exposure or eRNA treatment but is reduced upon pre-treatment with RNaseA, 2 hrs before hypoxia exposure. Furthermore, immunohistochemistry/or immunofluorescence studies were also performed to identify the presence of CD31 (PECAM-1), Neutrophil Elastase (NE) (Fig 5D and 5E), CD11/CD18 (MAC-1) and CD41 (megakaryocyte and platelet) markers on myocardial tissues (Fig 5E and 5F). The relative quantitative densitometry analysis of the respective images (Fig 5A–5F) is presented in S1A–S1F Fig.

Fig 5

Hypoxia induced eRNA stimulates collagen accumulation, leucocyte infiltration and activation in cardiomyocytes.

(a) MT staining (Collagen stained purple whereas tissue was stained pink); immunohistochemistry/immunofluorescence analysis of (b) α-SMA, (c) PECAM-1, (d) NE, (e) CD11/CD18 and (f) CD41 in myocardial tissue sections have been presented with higher magnification images in the subset. Magnification of the images in subset is 40X. Images were acquired at 20X and 40X resolution. DAPI (blue) was used as a nuclear stain. Data are representative of three independent times with three mice per experiment. (Scale bar: 50 μm).

It was observed that the increased leukocyte infiltration (depicted by PECAM-1, NE and CD11/CD18) and platelet accumulation (CD41) due to hypoxic stress was ameliorated by RNaseA pre-treatment, whereas eRNA was used as the positive control.

6. AH induced cTrop-T release is mediated by activation of caspase-3

Traditionally, MI was shown as manifestation of cardiomyocyte necrosis [26,27]. However, the concept of caspase-dependent regulated necrosis (CDRN) involving liberation of nucleosomes and attached DAMPs with no fragmentation of nuclei, etc [28] correlates with myocardial injury observed upon AH exposure. Hence, the activation and release of caspase-3 was studied in this context. Activated Caspase-3 was analysed in plasma and heart tissue lysate of mice after AH exposure and other pre-treatments such as eRNA, RNaseA, TLR3 siRNA and TLR3 IgG. eRNA dissolved in DDW (vehicle 1) and siRNA dissolved in RNALancer II solution (vehicle 2). Non-specific siRNA and non-immune IgG were used as negative controls and Poly I:C as positive control in the experiment. Our results elucidate that AH exposure and/or eRNA induction significantly activated Caspase-3 (Fig 6A and 6D; p<0.05). However, to validate the connection of TLR3 in caspase-3 release, mice were treated with TLR3-immunoneutralizing antibody two hrs before AH exposure and the caspase-3 was quantified in plasma and tissue (heart) lysate by ELISA. Our result illustrated that the pre-treatment with TLR3-immunoneutralizing antibody significantly inhibited caspase-3 release (Fig 6B and 6E; p<0.05). To affirm the connection of TLR3 we performed Tlr3 gene silencing in vivo by using Tlr3 siRNA, and the plasma and tissue lysate level of caspase-3 was estimated. Our results showed that AH-induced caspase-3 level was substantially reduced in Tlr3-silenced mice as compared to the control (Fig 6C and 6F; p<0.05). This indicates that AH induced release of activated caspase-3 is mediated through TLR3.

Fig 6

AH induced cTrop-T release is mediated by activation of caspase-3.

Estimation of activated Caspase-3 was done after (a) eRNA injection (80 μg/kg BW; 6 hrs prior to AH exposure) and RNaseA pre-treatment (1mg/kg BW; 2 hrs prior to AH exposure) in plasma, (b) TLR3 IgG treatment in plasma (c) TLR3 siRNA treatment (40 mg/kg BW; 2 hrs prior to AH exposure) in plasma, (d) eRNA injection (80 μg/kg BW; 6 hrs prior to AH exposure) and RNaseA pre-treatment (1mg/kg BW; 2 hrs prior to AH exposure) in heart lysate, (e) TLR3 IgG treatment (40 mg/kg BW; 2 hrs prior to AH exposure) in heart lysate and (f) TLR3 siRNA treatment in heart lysate. Data are shown as mean ± SEM (n = 5/group/treatment) from one experiment representative of three independent experiments, all performed in triplicate. One-way ANOVA revealed statistical significance in the results (*p< 0.05 groups w.r.t control; #p<0.05 groups w.r.t AH).

Discussion

This study explored the mechanism of myocardial injury due to exposure of AH. The precise role of eRNA and its receptor TLR3 has been evaluated in the induction of cardiomyocyte necrosis as well as infiltration of leukocytes into the cardiomyocytes that lead to tissue damage and release of cTrop-T. The functional implications of TLR3 signaling was studied using genetic as well as pharmacological approaches. To investigate the importance of eRNA, we injected RNaseA in vivo, which significantly diminished not only AH-induced release of SI and cardiac injury markers but also collagen accumulation, leukocyte infiltration and activation in cardiomyocytes. Our genetic study, in vivo silencing of TLR3 also abrogated the AH-induced release of cTrop-T due to myocardial injury. Thus, eRNA-TLR3 signalling pathway could be the potential target for the amelioration of AH-induced AMI.

The inflammatory response triggered by myocardial injury, primarily for healing, may initiate cardiac dysfunction [29] by releasing DAMPs from injured myocardia [30] which triggers innate immune response involving amplified cytokine expression, neutrophil infiltration [31] and cardiomyocyte apoptosis/necrosis [32]. Here, we have observed that AH exposure facilitates release of sterile inflammatory molecules such as eRNA, eDNA, HMGB1, HSPs, vWF, and s100b into the circulation. TLR3, a known receptor for ds-viral RNA, which recognizes endogenous RNA was released from necrotic cells in vitro [33]. This cellular RNA, released from injured cardiomyocytes or ischemic myocardium induces cytokine production [34]. Here, we have shown that eRNA released from the injured murine myocardial tissue significantly escalates cTrop-T release via TLR3 signalling. Whereas, RNaseA pre-treatment or TLR3 silencing significantly abrogated cTrop-T release in the circulation.

cTrop-T and myoglobin are specific cardiac markers used for diagnosis of myocardial injury. In contrast to other cell types, cardiomyocytes are more prone to necrosis triggered by calcium or oxygen insult that result in the release of cTrop-T along with other cellular components [35]. Here, we observed cTrop-T elevation upon AH exposure as early as six hrs suggesting myocardial injury is an early event of AMI.

Stress mediated cardiomyopathy involves agranulocytes infiltration, microvascular dysfunction, and cytokine storm which consequently trigger a strong immune response. The process of leukocyte activation due to inflammation following the recruitment of neutrophil is mediated by selectins, a member of β2-intergin family (MAC-1), which regulates neutrophil and leukocyte adhesion via ICAM-1 on endothelial cells [36]. Neutrophil elastase (NE) is expressed not only by neutrophils but also by monocytes, macrophages and endothelial cells [37] and this modulates cytokine activity during inflammation. Its elevated expression has also been reported in CVDs [38], atherosclerotic plaques 37 and other cardiac complications. Neutrophils serve as host defenses against viral pathogens and are activated by poly I:C to mediate immune response [39], promotes megakaryocyte fragmentation into platelets, leading to platelets activation and subsequent thrombosis in coronary arteries [40]. A disarray in the coagulation and the fibrinolytic system due to myocardial injury has been observed as increased D-dimer (thrombogenesis indicator), fibrinogen (coagulation factor), vWF (endothelial dysfunction marker) and CD41 level (platelet and megakaryocyte surface marker) [41]. PECAM-1, expressed on leukocytes, macrophages and some T-cells is implicated in their infiltration and implicated in the rat model of myocardial [42] and intestinal I/R injury [43]. Here, we observed marked leukocytes infiltration in cardiomyocytes due to AH exposure. However, the pre-treatment of RNaseA two hrs before of AH exposure mitigates it. This shows the role of eRNA in leukocytes infiltration in cardiomyocytes during AH exposure.

Cytokine induced conversion of fibroblasts to myofibroblasts initiates the process of collagen production [44] as well as its accumulation which results in myocardial stiffness and arteriosclerosis. This also dampens the Windkessel effect, which causes a rise in the pulse as well as systolic pressure and subsequently upsurge the risk of MI, stroke and other CVDs. Our results showed marked collagen accumulation in coronary vasculature due to AH exposure or/eRNA induction. However, RNaseA pre-treatment (two hrs before exposure to AH) significantly inhibits it. This demonstrates the role of eRNA in collagen accumulation as well as stiffness of coronary vasculature due to AH exposure.

Caspase-3, a predominant apoptotic marker, is involved in CDRN which induces the release of nucleosomes and DAMPs. These remains in the inactive form in cytosol and becomes active by self-sustained proteolytic autocatalysis [45]. Here, we observed the presence of active caspase-3 in heart tissue due to AH exposure as well as eRNA induction which was significantly ameliorated by either RNaseA treatment and or/ TLR3 silencing.

Briefly, we delineated that AH-induced SI molecules elevations specially eRNA leads to neutrophil activation and leucocyte infiltration in myocardial tissue, which causes cell injury and death via the caspase dependent pathways. The resulting myocardial necrosis releases cTrop-T from the sarcomeres into the circulation where by the tropomyosin remains attached to actin filaments and prevents the binding of myosin; hence developing contractile dysfunction. The subsequent collagen accumulation culminates into myocardial stiffness and dysfunction which ultimately may lead to heart failure. Here, we have validated that the release of eRNA under AH condition triggers TLR3 which facilitate myocardial injury as well as necrosis through leucocyte infiltration. We further showed that this process could mitigate though RNaseA pre-treatment and/ or TLR3 silencing. Thus, this raises a note-worthy possibility of RNaseA or TLR3-silencing therapy as a potential therapeutic option in the prevention of AH-induced AMI or CVDs.

Densitometry analysis of (a) MT staining depicting collagen, (b) immunohistochemistry for α-SMA, (c) immunohistochemistry for PECAM, (d) immunohistochemistry for NE, (e) immunofluorescence for CD11/CD18 and (f) immunofluorescence for CD41. Data are shown as representative of three independent experiments, all performed in triplicate. One-way ANOVA revealed statistical significance in the results (*p< 0.05 groups w.r.t control; #p<0.05 groups w.r.t AH).

(PDF)

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Agarose gel image depicting TLR3 expression upon TLR3 siRNA knockdown.

Lane 1- Control (no siRNA treatment), Lane 2- TLR3 siRNA treatment and Lane 3- non-specific siRNA treatment.

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18 Jun 2021

PONE-D-21-14652

Key role of Extracellular RNA in hypoxic stress induced myocardial injury

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Review Comments to the Author

Authors have studied the key role for eRNA and TLR3 in hypoxia-induced myocardial ischemic injury. The authors presented the mechanism of cTrop-T release and progression of acute myocardial infarction through TLR3-caspase -3 pathway in a murine model of acute hypoxia. The proposed aim and choice of testing the eNA is novel and has current interest in the field of cardiovascular biology. Although there is data provided in support of the aim and objectives, I have some concerns with the manuscript, primarily with methodology and some aspects where statements appear to confuse cause and effect. The paper will be of significant interest, provided the issues below are addressed and modifications are made.

Major comments:

1. The background should clearly state the purpose of the study.

2. In the abstract section, (page 2, line 25) the sentence, “Although the diagnosis is based on……” is incomplete.

3. Details of methodology are missing in several places.

a. How long were these animals exposed to acute hypoxia conditions?

b. In the method section, authors mentioned that 80ug of eRNA and eDNA were injected into the animal by intravenously through the tail vein. What is the starting material for isolating eRNA and eDNA from plasma? What is the yield eRNA and eDNA isolated from plasma of exposed animal?

c. Is eDNA and eRNA from each animal or is it from pooled samples of exposed animals? Please give addition details in methods section.

d. The methodology for isolating eDNA reported in the manuscript is missing. Please add this to methodology.

e. In addition, the time point for the proposed experiments are not described. Please provide specific time point for histological and immunochemical study. Is the time point the same for all parameters?

f. Under the methodology section, line 142, ELISA, the authors should provide detailed information about specific antibodies (primary and secondary antibody) and the dilution used for the assay. Methods do not have proper references and are not explained in detail.

g. For Immunofluorescence, please detail antibody information for both primary and secondary antibodies for CD31, CD11/CD18, neutrophil elastase etc.

h. Please provide higher magnification image showing leukocyte infiltration along with low magnification images.

Minor comments

4. In page 7, line 143 50ug plasma, it should be in uL

5. The quality of the Figure 1-4 is poor. Please maintain the same consistency in numbering all figure panels.

6. The manuscript needs critical editing for language and syntax errors.

Results from the present study agree with previously published reports. The above issues do not abrogate the potential importance of the paper; however, I do think it would be very interesting to see the modified manuscript including the answers for these queries.

Reviewer #2: This manuscript by Bhagat S et al., titled “key role of extracellular RNA... myocardial injury” investigates the role of extracellular RNA (eRNA) in causing myocardial injury caused by exposure of mice to acute hypoxia (AH). The authors show that AH results in increase in eRNA leading to increased cardiac troponin-T (cTrop-T) in plasma through a TLR3-dependent pathway and that blocking the signal through either RNase treatment, or TLR-3 neutralizing antibodies or siRNA against TLR3 reduces levels of cTrop-T. They also show that poly(I:C) a surrogate of eRNA causes a similar increase in cTrop-T. While the studies are interesting the manuscript is difficult to follow and is poorly written. Experimental details are lacking in several places.

Comments:

1. Animals were exposed to acute hypoxia (~8.5% O2) simulating an altitude of 7628 m. The relevance of this level of hypoxia is not clear. Please add details of hypoxic exposure in the Methods section.

2. In Figure 1, fold change in eRNA and eDNA have been shown. Please give the actual amount/unit of the plasma. Assuming that standards were run in ELISA as per reference, the amount of HMGB1, vWF, HSP70, HSP90 and s100b should be given. Details of the methodology are sketchy. For instance, it is stated that 50 ug of plasma or tissue lysate was used in ELISA. What is the volume of plasma used and whether it was diluted to achieve the desired protein concentration? Also, ‘expression analysis’ of SI markers are mentioned. These are eRNA/eDNA, which are not necessarily expressed genes. Most measured molecules are protein, levels of which change following AH. The title of the legend and the legend needs to be changed accordingly.

3. Figure 3 shows changes in troponin. Please correct the y-axis to specify the type of troponin and provide quantitation. The time point of evaluation should also be given in the figure legend. Additionally, it is not clear how much eRNA, eDNA or Poly(I:C) was used as a positive control. Please add these details to the figure legend as well.

4. Figure 4: while the figure shows expression of troponin-T, apparently protein levels were determined by ELISA. Please label accordingly with quantitation rather than fold change. Please also provide details in the figure legends on when the TLR3 neutralizing antibodies were added, and the duration of hypoxic exposures used.

5. Figure 5 (I) shows PECAM -1 and neutrophil elastase for staining of endothelial cells and neutrophils respectively. Apparently PECAM-1 should be in the blood vessels. The images are not of sufficient resolution to appreciate the staining differences in the different groups. Please also provide high resolution images to demonstrate specificity in staining. Similarly, also provide high resolution images of the NE stained sections so the reader can appreciate specificity.

6. Figure 5 (II) shows staining of CD11/CD18 and CD41. The structure within these images is not discernable. Please provide high resolution images to show structures.

7. It is stated that TLR3 levels were knocked out in vivo using siRNA. While siRNA approaches are known to knockdown genes of interest, they are less likely to knockout genes. Please provide more details and demonstrate efficient knockdown or knockout of TLR3.

8. Figure 6 shows fold changes in activated caspase-3 in plasma and heart tissue lysates. Please plot the plasma and tissue lysate findings separately. Please provide details of the commercial kit used. If an ELISA based kit was used, provide quantitation.

9. In writing figure legends, avoid describing the results.

Minor comments:

Sentences are incomplete in several places and the language has syntax errors.

In the ‘Abstract’ line 25 appears incomplete.

Throughout the manuscript PECAM-1 is written as PECAM. Please correct it.

Page 5, lines 107-119. Please write complete sentences.

Page 9, lines 181, please correct the word ‘compression’

Page 9, lines 191, please correct the word ‘dependant’.

Page 10, lines 214, inappropriate use of the word ‘whereas’.

Page 11, lines 241, consider using an alternate word of ‘abridged’. The sentence, as written, lacks clarity.

Page 12, lines 251, please clarify the word ‘caNerspase-3’ or change the title to make it understandable.

Page 12, lines 258-259, please complete the sentence or correct the syntax.

Reviewer #3: This study aims to evaluate the effects of acute hypoxia on cardiomyocytes and role of eRNA in this process through potential activation of TLR3-Caspase 3. The authors try to link this in vivo hypoxia model to clinical acute myocardial pathology in human. This by itself dampen the enthusiasm for the initial question and the readouts collected by the authors. It would have been more logical if authors would have initiated the question using this animal model as to determine the effect of hypoxia on cardiac microvascular inflammation and the role of eRNA-TLR3 axis in this process without the necessity to discuss troponin and cardiomyocytes necrosis etc which was never confirmed based on the data shown.

Other major criticism that might help the impact of this study are as below:

1- All experimental readouts are based on ELIZA and Calorimetric assays. It is necessary for authors to include and add more supporting data even as supplemental data including but not limited to mRNA/protein analysis using PCR and western analysis.

2- There is no data included to confirm some of the proposed methodological approaches have been effective. This will help to better judge some of the data presented in Figures 2, 3 and 4.

3- In Figure 3a, why troponin is lower in RNAse treated control animals when compared to sham and with no hypoxia.

4- In Figure 3b, why the reduction in troponin in animals treated with RNAse and DNAse seems to be similar?

5- Figures 3 legend mentions that eRNA is involved in hypoxia induced release of cTrop-T while on figure it mentions “expression”.

6- Not clear what does “Cardiomyocytes activation” means in figure 5?

**********

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Reviewer #1: Yes: NITHYA MARIAPPAN

Reviewer #2: No

Reviewer #3: No

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Submitted filename: Review Comments to the Author.docx

Click here for additional data file.

4 Aug 2021

First Revision – author’s’ response 2rd August, 2021

All editing in the manuscript is presented as track change.

I am indebted to you for the valuable comments for overall improvement of the manuscript. Accordingly, in this revised paper, I give due weightage to comments and the resultant responses are:

The answer to the Reviewer # I

Major Comments:

Point: 1

The background should clearly state the purpose of the study.

Author Response: I wish to thank reviewer for the comments. The background of the study is mentioned in abstract (page 1 line 25-28) and in Introduction (page 4 line 69-93).

Point: 2

In the abstract section, (page 2, line 25) the sentence, “Although the diagnosis is based on……” is incomplete.

Author Response: I thank the reviewer for the pointing out the mistake. We have addressed the concern and necessary correction was made in the abstract (page 2, line 27-28).

Point: 3

Details of methodology are missing in several places.

a. How long were these animals exposed to acute hypoxia conditions?

b. In the method section, authors mentioned that 80ug of eRNA and eDNA were injected into the animal by intravenously through the tail vein. What is the starting material for isolating eRNA and eDNA from plasma? What is the yield eRNA and eDNA isolated from plasma of exposed animal?

c. Is eDNA and eRNA from each animal or is it from pooled samples of exposed animals? Please give addition details in methods section.

d. The methodology for isolating eDNA reported in the manuscript is missing. Please add this to methodology.

e. In addition, the time point for the proposed experiments are not described. Please provide specific time point for histological and immunochemical study. Is the time point the same for all parameters?

f. Under the methodology section, line 142, ELISA, the authors should provide detailed information about specific antibodies (primary and secondary antibody) and the dilution used for the assay. Methods do not have proper references and are not explained in detail.

g. For Immunofluorescence, please detail antibody information for both primary and secondary antibodies for CD31, CD11/CD18, neutrophil elastase etc.

h. Please provide higher magnification image showing leukocyte infiltration along with low magnification images.

Author Response: I thank reviewer for raising these concerns. The details as per the comments of the reviewer have been incorporated in the methodology section of the manuscript as follows:

a. In time dependent study, these animals were exposed to AH for 0, 6, 12, 24 hrs., 3 and 7 days which are mentioned in “Acute hypoxia and other treatments” (page 6, line 127-133). However, we found that as early 6 hrs. of AH exposure significantly up regulated all the protein molecules. For our further experiments, we chose to use the 6 hrs. time point of hypoxia exposure for mice model in all the experiments unless otherwise mentioned as on Page 10, line 207-208 which is in line of our previous publication reference [Bhagat et al, BCMD,84 (2020),102459].

b. In brief, approximately 1mL of blood we had collected from each AH exposed mice where we were able to separate approximately 0.55 mL of plasma. Therefore, 0.55mL/mice plasma was our starting materials for isolation of eRNA and eDNA. The yield of eRNA and eDNA in AH treated each mouse was approximately 100 µg/mL and 60 µg/mL respectively. Therefore, yield of eRNA & eDNA was 55 µg/mice & 33 µg/mice respectively. The injected doses of eRNA or eDNA were 80 µg/kg BW in our experiments. Each mice weighted approximately 20g. Therefore, the required amount of eRNA or eRNA were 1.6 µg/mice which we obtained form once mice. We had performed similar experiments in our previous manuscript [Bhagat et al, BCMD, 84 (2020),102459]. We hope that we have clearly explain to the reviewer.

c. The eRNA and eDNA used in our experiments were isolated form each AH exposed animal then pooled and used. The same has been mentioned in the methods section (page 7, line 143-146). Similar method, we used in our previous experimentation [Bhagat et al, BCMD, 84 (2020), 102459].

d. In our experiments, the eDNA was isolated though a commercially available kit (Quick-cfDNA, Zymo Research) as per the manufacture instruction and it is also mentioned in the manuscript (page 7, line 153-156). Same method we used in our previous experimentation also [Bhagat et al, BCMD, 84 (2020), 102459].

e. Animals were exposed to hypoxia for 0, 6, 12 and 24 hrs., 3 days and 7 days. However, we used 6 hrs. time points in all the experiments as well as immune histochemistry and IF study because as early in this time point all the parameters are significantly high.

f. Details of specific primary and secondary antibodies used along with their dilutions have been mentioned in the methodology section (page 8, line 161-164).

g. Details of specific primary and secondary antibodies used for IHC and IF along with their dilutions have been mentioned in the methodology section (page 8, line 182-183).

h. Higher resolution image (40X) is added as sublet in respective picture.

Point: 4

In page 7, line 143 50ug plasma, it should in uL

Author response: I wish to thank the reviewer for pointing out the mistake. It is now corrected in page 7 line 158 (50 µg total protein per well in equal volume).

Point: 5

The quality of the Figure 1-4 is poor. Please maintain the same consistency in numbering all figure panels.

Author response: I wish to thank the reviewer for pointing out the mistake. The figures 1-4 have been modified as per the reviewers’ suggestions, same numbering and formatting has been maintained in all the figures.

Point: 6

The manuscript needs critical editing for language and syntax errors.

Author response: I thank the reviewer for the concern. The manuscript has been proofread and syntax errors taken care.

The answer to the Reviewer # 2

Comments

Point: 1

Animals were exposed to acute hypoxia (~8.5% O2) simulating an altitude of 7628 m. The relevance of this level of hypoxia is not clear. Please add details of hypoxic exposure in the Methods section.

Author Response: I thank the reviewer for raising the concern. In brief, animals were subjected to AH in a specially fabricated animal decompression chamber in which conditions were equivalent to atmospheric conditions at an altitude of 7628 m (282 mm Hg), with an O2 content of ~8.5%, as previously described [Biswas et al, Eur. J. Immunol. 2015. 45: 3158–3173]. The temperature and humidity were maintained at 25 ± 3°C and 55% ± 5%, respectively. In our study, we explored the effect of AH in MI. Therefore, we used same AH conditions which have been standardized in our lab and previously published in our papers [Bhagat et al, BCMD, 84 (2020), 102459 & Biswas et al, Eur. J. Immunol. 2015. 45: 3158–3173]. It is also mentioned in the manuscript page 6 line 127- 130).

Point: 2

In Figure 1, fold change in eRNA and eDNA have been shown. Please give the actual amount/unit of the plasma. Assuming that standards were run in ELISA as per reference, the amount of HMGB1, vWF, HSP70, HSP90 and s100b should be given. Details of the methodology are sketchy. For instance, it is stated that 50 ug of plasma or tissue lysate was used in ELISA. What is the volume of plasma used and whether it was diluted to achieve the desired protein concentration? Also, ‘expression analyses of SI markers are mentioned. These are eRNA/eDNA, which are not necessarily expressed genes. Most measured molecules are protein, levels of which change following AH. The title of the legend and the legend needs to be changed accordingly.

Author Response: I thank the reviewer for raising the concern. The absolute values of eRNA and eDNA at different time-points of hypoxia exposure are mentioned below:

Time-point (hr.) eRNA (µg/ml) Time-point (hr.) eDNA (µg/ml)

0 0.87 ± 0.09 0 0.28 ± 0.01

6 2.07 ± 0.37 6 0.83 ± 0.03

12 1.73 ± 0.12 12 1.22 ± 0.05

24 3.14 ± 0.51 24 1.24 ± 0.05

It is further mentioned that we assayed the different protein in plasma or tissue lysate by ELISA with standard established procedures we used in our several previous publications papers [Bhagat et al, BCMD, 84 (2020), 102459 & Biswas et al, Eur. J. Immunol. 2015. 45: 3158–3173]. We did quantitative ELISA with standard, but the data were presented in fold of expression. This is not a clinical study we would rather a basic science research where fold change is signifies the results. We had done similar study and results also presented previously. [Bhagat et al, BCMD, 84 (2020), 102459; Biswas et al, Eur. J. Immunol. 2015. 45: 3158–3173; Biswas et al, BCMD, 49 (2012) 92–10; Singh et al, Biochemistry 2014, 53, 115−126]. As per the reviewer suggestion all the corrections are incorporated. It is now corrected in page 7 line 158 (50 µg total protein per well in equal volume). Figure-1 shows the relative expression of different SI markers, nucleic acids and proteins, upon exposure to different durations of hypoxia after statistical analysis to obtain significance. The term “expression analysis” has been corrected in the figure legend no.1 (page 18, line 390-391). I hope the reviewer will consider it.

Point: 3

Figure 3 shows changes in troponin. Please correct the y-axis to specify the type of troponin and provide quantitation. The time point of evaluation should also be given in the figure legend. Additionally, it is not clear how much eRNA, eDNA or Poly(I:C) was used as a positive control. Please add these details to the figure legend as well.

Author response: I wish to thank the reviewer for the suggestions. As per the suggestions, the figure-3 have been edited. We have shown the expression of cardiac Troponin-T (cTrop-T) in the plasma of mice after different treatments. The dosage concentration for each treatment has been mentioned in the figure legend (page 18, line 409-412). AH exposure in every experiment is 6 hrs.

Point: 4

Figure 4: while the figure shows expression of troponin-T, apparently protein levels were determined by ELISA. Please label accordingly with quantitation rather than fold change. Please also provide details in the figure legends on when the TLR3 neutralizing antibodies were added, and the duration of hypoxic exposures used.

Author response: I thank the reviewer for the suggestion. The graphs of figure-4 have been edited as per reviewer suggestion. We have shown the expression of cardiac Troponin-T (cTrop-T) in the plasma of mice after TLR3 siRNA and TLR3 IgG treatments.

It is further mentioned that we assayed the Troponin-T by ELISA with standard established procedures we used in our several previous publications papers [Bhagat et al, BCMD, 84 (2020), 102459 & Biswas et al, Eur. J. Immunol. 2015. 45: 3158–3173]. We did quantitative ELISA, but the data were presented in fold of expression. This is not a clinical study we would rather a basic science research where fold change is signifies the results. We had done similar study and results also presented previously. [Bhagat et al, BCMD, 84 (2020), 102459; Biswas et al, Eur. J. Immunol. 2015. 45: 3158–3173; Biswas et al, BCMD, 49 (2012) 92–10; Singh et al, Biochemistry 2014, 53, 115−126]. As per the reviewer suggestion all the corrections are incorporated Details of TLR3 neutralizing antibody treatment has been included in the figure legend (page 18, line 423-424).

Point: 5

Figure 5 (I) shows PECAM -1 and neutrophil elastase for staining of endothelial cells and neutrophils respectively. Apparently PECAM-1 should be in the blood vessels. The images are not of sufficient resolution to appreciate the staining differences in the different groups. Please also provide high resolution images to demonstrate specificity in staining. Similarly, also provide high resolution images of the NE-stained sections so the reader can appreciate specificity.

Author response: I thank the reviewer for the concern. As per suggestion high resolution image also included in the figure as subset (Supplementary Figure 1 a-c).

Point: 6

Figure 5 (II) shows staining of CD11/CD18 and CD41. The structure within these images is not discernable. Please provide high resolution images to show structures.

Author response: I thank the reviewer for the concern. As per suggestion high resolution image also included in the figure as subset (Supplementary Figure 1 d, e).

Point: 7

It is stated that TLR3 levels were knocked out in vivo using siRNA. While siRNA approaches are known to knockdown genes of interest, they are less likely to knockout genes. Please provide more details and demonstrate efficient knockdown or knockout of TLR3.

Author response: I thank the reviewer for raising the concern. The details results are below. We used the similar methodology in our previously polished papers and details are also there [Bhagat et al, BCMD, 84 (2020), 102459; Biswas et al, Eur. J. Immunol. 2015. 45: 3158–3173]. For this reason, we did not include TLR3 siRNA mediated knockout data. Appropriate changes in the manuscript have been made. Page 8 line 168-174 & Page 11 line 240-249. Upon TLR3 siRNA treatment we observed significant silencing of the TLR3 gene, and proceeded with further experiments.

This agarose gel image shows the expression of TLR3 gene under different conditions; Lane 1- Control (no siRNA treatment), Lane 2- TLR3 siRNA treatment and Lane 3- non-specific siRNA treatment.

Point: 8

Figure 6 shows fold changes in activated caspase-3 in plasma and heart tissue lysates. Please plot the plasma and tissue lysate findings separately. Please provide details of the commercial kit used. If an ELISA based kit was used, provide quantitation.

Author response: I thank the reviewer for raising the concern.

Graphs of Figure-6 have been modified as per the reviewer suggestions and changes made to the manuscript accordingly. Estimation was done using the Caspase-3 Fluorometric Assay Kit, BioVision Inc. (Page 9 line 196-198). Since the samples have been collected after various treatments and hypoxia exposure to mice, control mice with no treatment or hypoxia exposure were taken as assay control where caspase-3 activity was un induced. Fold change analysis of all sample sets was done in comparison to control.

Point: 9

In writing figure legends, avoid describing the results.

Author response: I thank the reviewer for raising the concern. Figure legends have been modified as per the suggestions of the reviewer (Page 18-20).

Point: 10

Sentences are incomplete in several places and the language has syntax errors.

In the ‘Abstract’ line 25 appears incomplete.

Throughout the manuscript PECAM-1 is written as PECAM. Please correct it.

Page 5, lines 107-119. Please write complete sentences.

Page 9, lines 181, please correct the word ‘compression’

Page 9, lines 191, please correct the word ‘dependant’.

Page 10, lines 214, inappropriate use of the word ‘whereas’.

Page 11, lines 241, consider using an alternate word of ‘abridged’. The sentence, as written, lacks clarity.

Page 12, lines 251, please clarify the word ‘caNerspase-3’ or change the title to make it understandable.

Page 12, lines 258-259, please complete the sentence or correct the syntax.

Author response: I thank the reviewer for raising the concern. All minor comments raised by the reviewer have hereby been addressed in the manuscript.

Abstract line 27 has been completed correctly.

Page 5, lines 105-125, appropriate changes as per reviewer comments have been incorporated.

Page 9, line 202, the word “compression” has been corrected to “comparison”.

Page 10, line 213, the word “dependant” has been corrected.

Page 11, line 235, the sentence has been rectified.

Page 12, line 262, the word abridged has been replaced and the sentence modified accordingly.

Page 12, line 273, the word has been corrected to Caspase-3.

Page 13, lines 279-282, sentence has been corrected for errors.

The answer to the Reviewer # 3

Comments

Point: 1

All experimental readouts are based on ELIZA and Calorimetric assays. It is necessary for authors to include and add more supporting data even as supplemental data including but not limited to mRNA/protein analysis using PCR and western analysis.

Author response: I thank the reviewer for the comments. ELISA is a state of art technique that is why used it. In spite of it in support of the ELISA we used Immunohistochemistry as well as immune florescent studies. These are the direct proof of the expression of the different molecules. However, the protein detection/analysis by Western Blotting are not most reliable. Even if we done the western blotting the we need to reproof by direct evidences. I belive ELISA, Immunohistochemistry as well as IF are more accurate and specific in this context. I hope reviewer will understand.

Point: 2

There is no data included to confirm some of the proposed methodological approaches have been effective. This will help to better judge some of the data presented in Figures 2, 3 and 4.

Author response: I thank reviewer for the concern. In this manuscript we used Pharmacological approach (inhibitors) as well as genetic approach (siRNA mediated gene silencing) and the respective protein estimated by state-of-the-art method ELISA. We also sued immunohistochemistry and IF to revalidate the data. We have some limitation at this moment. I believe the methods were used are scientifically well established to proof the concept. I believe reviewer may please understand and consider.

Point: 3

In Figure 3a, why troponin is lower in RNAse treated control animals when compared to sham and with no hypoxia.

Author response: I thank reviewer for the concern. We repeated these experiments several times and presented the data. Explanation: Basically, RNase A is an endoribonuclease that specifically degrades single-stranded RNA i.e., eRNA. In normal situation (control animals), a basal RNase A is present which maintain/regulate basal eRNA level which in turn control/maintain basal Troponin-T level in plasma. However, when we treated animals with exogenous RNase A (extra/excess than basal level), this might degrade basal eRNA. Therefore, basal eRNA level would decrease which in turn decrease the basal Troponin-T level than the normal or control animals.

Point: 4

In Figure 3b, why the reduction in troponin in animals treated with RNAse and DNAse seems to be similar?

Author response: I thank the reviewer for the comments. We would like to explain our observation as follows, in figure 3a, there is reduction in circulating cTrop-T after RNaseA treatment as compared to control and after RNaseA pre-treatment prior to AH as compared to AH exposed animals. However, in figure 3b, we observe that DNase 1 treatment does not reduce circulating cTrop-T levels as compared to control nor after DNase 1 pre-treatment prior to AH as compared to AH exposed animals. Hence, the effect of RNaseA and DNase1 treatments are not similar.

Point: 5

Figures 3 legend mentions that eRNA is involved in hypoxia induced release of cTrop-T while on figure it mentions “expression”.

Author response: I thank the reviewer for the comment. The discrepancy between the figures and figure legends have been rectified as per the suggestions of the reviewer.

Point: 6

Not clear what does “Cardiomyocytes activation” means in figure 5?

Author response: I thank the reviewer for the comment Addressing the comment of the reviewer, we would like to mention that in figure 5 we intend to show the infiltration and activation of leukocytes in the cardiomyocytes and not the activation of cardiomyocytes themselves.

Submitted filename: Response to Reviewer.pdf

Click here for additional data file.

20 Sep 2021

PONE-D-21-14652R1Key role of Extracellular RNA in hypoxic stress induced myocardial injuryPLOS ONE

Dear Dr. Khan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As you can see from the comments, Reviewer 2 still has significant concerns, which have not been addressed. The reviewer’s request seems reasonable, and I encourage you to resubmit the manuscript after addressing each of the comments.

Please submit your revised manuscript by Nov 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewer #3: Yes

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Reviewer #1: (No Response)

Reviewer #2: This revised manuscript by Bhagat S et al., titled “key role of extracellular RNA... myocardial injury” investigates the role of extracellular RNA (eRNA) in causing myocardial injury caused by exposure of mice to acute hypoxia (AH). Changes have been made to the manuscript. However, there are still significant concerns that have not been addressed satisfactorily.

Comments:

1. In Figure 1, quantitation of the eDNA, eRNA as well as SI markers were requested. While the quantitation of eNA and eRNA were provided in the response letter, the plots were not changed to reflect quantitation. The authors fail to realize the significance of adding quantitation to the graphs in Figure 1. The intent is not to convince the reviewer but the reader. These numbers will serve as a guide to investigators in the field who may be doing similar experiments. The justification for not providing these numbers since it is a basic science study and not a clinical study is baseless and without any merit. In similar basic science studies, the authors have themselves published concentrations of eRNA and eDNA (Refs 1 and 13 of the manuscript). They have also published quantitative values for ELISA in these publications that they have authored. Interestingly, the y-axis values in Figure 1a and 1b correspond to the values provided in the response letter and are not ‘fold changes’ as indicated in the plots.

2. The supplementary figures 1a, 1b and 1c should be shown alongside Figure 5(I) to bring more clarity to the changes. The arrows in the lower and higher magnification images do not match and sometimes they show different regions (Supp Fig 1a: Arrows in Poly I:C and RNase A+AH pairs; Supp Fig 1b: Arrows in Poly I:C and RNase A+AH pairs; Supp Fig 1c: Arrows in Poly I:C and eRNA pairs). The figures in the main body of the manuscript should convey the reported findings without having to go to the supplementary data.

3. Similarly, the structures within Figure 5 (II) shows staining of CD11/CD18 and CD41 are still not discernible. Supplemental Figure 1d and 1e should be added to the main figure to bring additional clarity.

4. Thank you for providing the agarose gel images of the TLR3 siRNA knockdowns. Please add these to the supplemental data.

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Reviewer #1: Yes: Nithya Mariappan

Reviewer #2: No

Reviewer #3: No

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8 Nov 2021

Response to Reviewer-2:

Second Revision – author’s’ response 7th Nov., 2021

All editing in the manuscript is presented as track change yellow.

I am indebted to you for the valuable comments for overall improvement of the manuscript. Accordingly, in this revised paper, I give due weightage to comments and the resultant responses are:

The answer to the Reviewer # 2

Major Comments:

Point: 1

In Figure 1, quantitation of the eDNA, eRNA as well as SI markers were requested. While the quantitation of eDNA and eRNA were provided in the response letter, the plots were not changed to reflect quantitation. The authors fail to realize the significance of adding quantitation to the graphs in Figure 1. The intent is not to convince the reviewer but the reader. These numbers will serve as a guide to investigators in the field who may be doing similar experiments. The justification for not providing these numbers since it is a basic science study and not a clinical study is baseless and without any merit. In similar basic science studies, the authors have themselves published concentrations of eRNA and eDNA (Refs 1and 13 of the manuscript). They have also published quantitative values for ELISA in these publications that they have authored. Interestingly, the y-axis values in Figure 1a and 1b correspond to the values provided in the response letter and are not ‘fold changes’ as indicated in the plots.

Author Response: I wish to thank reviewer for the comments. We have addressed the comment of the reviewer and provided the bar graphs for quantitative analysis of all the SI markers (Figure 1 (c-g). Specific quantitative ELISA kits were used for this purpose. The plots for eRNA and eDNA have been corrected to represent the quantitation details.

Point: 2

The supplementary figures 1a, 1b and 1c should be shown alongside Figure 5(I) to bring more clarity to the changes. The arrows in the lower and higher magnification images do not match and sometimes they show different regions (Supp Fig 1a: Arrows in Poly I:C and RNase A+AH pairs; Supp Fig 1b: Arrows in Poly I:C and RNase A+AH pairs; Supp Fig 1c: Arrows in Poly I:C and eRNA pairs). The figures in the main body of the manuscript should convey the reported findings without having to go to the supplementary data.

Author Response: I thank the reviewer for the suggestions. We have addressed the concern and necessary correction were made. We have merged the higher magnification images for figure 5 with the existing image data set Figure 5 (b-d). The arrow pointers of all the images have been checked and rectified of errors.

Point: 3

Similarly, the structures within Figure 5 (II) shows staining of CD11/CD18 and CD41 are still not discernible. Supplemental Figure 1d and 1e should be added to the main figure to bring additional clarity.

Author Response: I appreciate the reviewer for the comments. Accordingly, we have addressed the suggestion of the reviewer and merged the higher magnification images for figure 5 with the existing image data set Figure 5 (e-f).

Point: 4:

Thank you for providing the agarose gel images of the TLR3 siRNA knockdowns. Please add these to the supplemental data. The background should clearly state the purpose of the study.

Author Response: Acknowledging the comment by the reviewer, we have included the agarose gel image as Supplementary figure 2.

Submitted filename: response to reviewer.docx

Click here for additional data file.

18 Nov 2021

Key role of Extracellular RNA in hypoxic stress induced myocardial injury

PONE-D-21-14652R2

Dear Dr. Khan,

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Aftab Ahmad, Ph.D.

Academic Editor

PLOS ONE

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1 Dec 2021

PONE-D-21-14652R2

Key role of Extracellular RNA in hypoxic stress induced myocardial injury

Dear Dr. Khan:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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