Key role of Extracellular RNA in hypoxic stress induced myocardial injury

Figures

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  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).

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  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).

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  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).

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  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).

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  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).

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  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).