Extracellular histones are a target in myocardial ischaemia–reperfusion injury

Figures

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  Figure 1. Nucleosomes and histone H4 are released from rat hearts subject to ischaemia and reperfusion. (A) Nucleosome concentration in the perfusate of perfused heart measured by ELISA (N = 5 hearts per group). (B) Histone H4 concentration in the perfusate of perfused heart measured by DELFIA. N = 8 hearts per group. (C) Total histone H4 release is correlated to final infarct size (N = 16). Statistical analyses by repeated measures ANOVA followed by Tukey test. **P < 0.01, ***P < 0.001 compared to the measurement taken before reperfusion.

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  Figure 2. Histones exhibit a dose dependent cytotoxic effect on isolated rat cardiomyocytes in vitro, which can be reversed by the addition of mCBS. (A) Primary adult rat cardiomyocytes were incubated for 1 h with pure histones in the presence of 0, 25, or 100 μg/mL mCBS. The percentage of cell death was determined by PI staining. N = 4 independent biological experiments. Analysed by two-way ANOVA with Bonferroni correction for multiple comparison. ****P < 0.0001 vs. control group. ††P < 0.01 ††††P < 0.0001 vs. 40 μg/mL histones alone. (B) LDH release was used to assay cell death in primary cardiomyocytes treated with the drugs at concentrations indicated. N = 5 independent biological experiments. Analysis by one-way ANOVA and Tukey post-test. **P < 0.01, ***P < 0.001, ***P < 0.0001.

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  Figure 3. The histone neutralizing agent mCBS reduces infarct size in an ex vivo rat heart model of ischaemic and reperfusion injury (I/R). (A) Infarct size in hearts exposed to 45 min of global ischaemia followed by 2 h reperfusion, in the presence of vehicle or 100 μg/mL mCBS or pre-treated with ischaemic preconditioning (IPC). N = 6 hearts per treatment group. (B) The concentration of free histone H4 in the perfusate of rat hearts at the point of reperfusion after 45 min of ischaemia, measured by DELFIA. N = 6 (or 4 IPC) hearts per group. Statistical analyses by one-way ANOVA with Tukey post-test. *P < 0.05, ***P < 0.001.

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  Figure 4. Extracellular histones contribute to myocardial infarction during reperfusion, and histone neutralization during reperfusion is cardioprotective. (A–C) Exogenous histones or vehicle were added to the perfusate of Langendorff-perfused rat hearts following 45 min ischaemia and during 2 h reperfusion. Infarct size (B) was measured at the end of the experiment. Left ventricular developed pressure (LVDP) (B) and flow rate of the perfusate (C) were measured 5 min into the reperfusion period. N = 5 hearts per group. (D) Infarct size in hearts subject to 45 min of global ischaemia followed by 2 h reperfusion, in the presence of vehicle or 100 μg/mL mCBS. N = 6 hearts per group. Statistical analyses by one-way ANOVA with Tukey post-test (A–C), or by unpaired T-test (D). *P < 0.05, **P < 0.01, ****P < 0.0001.

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  Figure 5. In an in vivo rat model of ischaemic and reperfusion injury, the histone-H4 specific neutralizing peptide HIPe, but not mCBS, reduced infarct size. (A) Infarct size as a percentage of area at risk (AAR) in an in-vivo rat coronary artery occlusion model of I/R, after administration of vehicle or mCBS. N = 4–7 hearts per group. (B) Infarct size as a percentage of AAR in an in-vivo rat coronary artery occlusion model of I/R, after administration of vehicle or HIPe. N = 6–7 hearts per group. Statistical analyses by one-way ANOVA with Tukey post-test (C), or unpaired T-test (D). **P < 0.01, ***P < 0.001.

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  Figure 6. HEK293 reporter cells expressing a TLR4 reporter (A) or null control construct (B) were exposed to histones, mCBS or LPS overnight, then secreted embryonic alkaline phosphatase was measured as an index of reporter activity. The positive control of LPS caused a huge increase of NFκB activity as expected (N = 6 independent biological experiments) (P < 0.01) analysed by one-way ANOVA and Tukey post-test. A small increase in NFκB activity was seen at the highest concentration of 100 μg/mL histones, but as this occurred similarly in control cells, it was not related to TLR4 stimulation.

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  Figure 7. Histones do not cause activation and nuclear re-localization of NF-κB in HL-1 cardiomyocytes. HL-1 cells were treated with vehicle, 10 ng/mL TNFα or 10 μg/mL histones for 40 min, before fixation and staining of NF-κB. Only TNFα caused significant nuclear relocalization as shown in representative images and quantification (N = 3 experiments). Statistical analysis by one-way ANOVA with Tukey post-test. *=P < 0.0001; ns = non-significant, in comparison to vehicle.