Role of extracellular histones in the cardiomyopathy of sepsis

Abstract

The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca(2+)]i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht-, LRR-, and PYD-domains-containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca(2+)]i, as well as defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction.

Keywords: NLRP3 inflammasome; cardiomyocytes; complement; echocardiogram/Doppler; polymicrobial sepsis.

Figure 1.

Requirements for histone appearance in plasma after CLP and binding of histones to CMs. A) Time course (hours) for extracellular histone appearance in plasma from C57BL/6 (WT) mice after CLP. B) Requirements for both C5a receptors (C5aR1, C5aR2) for histone appearance in plasma 12 hours after CLP. C) PMN dependence for histone appearance in plasma 12 h after CLP, using the mAb that depletes blood PMN via reactivity with the Ly-6G epitope on PMNs. D) Histone appearance in plasma after CLP is dependent on the NLRP3 inflammasome. E) Thirty min after intravenous infusion of FITC-histones (45 mg/kg body weight) into WT mice, heart homogenates contained histones. F) Confocal microscopy of rat CMs exposed in vitro to fluorescein-labeled histones (50 μg/ml) for 30 minutes at 37°C, showing both surface binding of histones (upper), as well as evidence of histone internalization into CM cytosol (lower). Nuclei are defined by DAPI stain (blue). For all frames, n ≥ 6 for each bar. *Differences were significant, P < 0.05 (A) vs. sham and (B) vs. WT, or as indicated.

Figure 2.

Appearance of increased levels of ROS and [Ca²⁺]_i mouse in CMs exposed to a histone mixture. All data were obtained by flow cytometry. A) Cytosolic ROS responses of CMs exposed 30 minutes at 37°C to histones as function of histone concentrations. B) Cytosolic buildup of ROS in CMs exposed to histones (50 μg/ml) at 37°C for the times indicated. C) Increased ROS in CMs from mice infused intravenously with histones 30 minutes earlier (65 mg/kg body weight). D) [Ca²⁺]_i increases in CMs exposed to histones (50 μg/ml) for the indicated periods of time at 37°C. E) [Ca²⁺]_i responses of CMs as a function of concentration of histones. F) [Ca²⁺]_i increases in heart after intravenous infusion of histones (65 mg/kg body weight) into mice 30 minutes earlier. G) [Ca²⁺]_i increases in CMs obtained from sham or CLP mice and exposed in vitro for 30 minutes at 37°C to histones (5 μg/ml). H) [Ca²⁺]_i in CMs obtained from sham mice or from TLR4^−/−, TLR2^−/−, or WT mice infused intravenously 30 minutes earlier with histones or PBS (sham WT mice). For each bar, n ≥ 6. *Differences were significant, P < 0.05 vs. control or as indicated.

Figure 3.

Histones cause increases in [Ca²⁺]_i in CMs and physiologic defects in perfused hearts. Ability of histones to cause defects in paced CMs before or after histone addition (at time 0) (A) or in Langendorff mouse hearts perfused with histones (at arrows) (B–F). LV pressures were measured (B, F) and also monitored by ECG tracings (C–E). A) Isolated cardiomyocytes were loaded with an intracellular calcium indicator (Fluo3 AM) and calcium change was measured during electrical pacing (0.5 Hz). Histones caused calcium overload in cardiomyocytes. B) LV pressures in Langendorff mouse hearts showing falling and narrowing of pressure tracings after perfusion with histones (arrow). C–E) ECG tracings of perfused mouse hearts before (normal sinus rhythm) (C) and after histone exposure, which caused sinus bradycardia (D) and ventricular bigeminy (E), which was confirmed by LV pressure tracings (F). Tracings are representative of 11 independent experiments.

Figure 4.

Expanded tracings of [Ca²⁺]_i in CMs before and after histone addition. Expanded intracellular calcium tracings (from Fig. 3) in electrically paced CMs (0.5 mHz) before (A) and after (B) addition of the histone mixture (5 μg/ml). In the lower panel, there was expanding width of intracellular calcium tracings, indicating defective [Ca²⁺]_i clearance during diastole. Tracings are representative of patterns in which n ≥ 4 each.

Figure 5.

Changes in CMs and CM mitochondria exposed (37°C, 30 minutes) to increasing concentrations of histones. A) Binding of annexin V to CMs after exposure to a range of concentrations of the histone mixture. B) Reductions in mitochondrial membrane potential after exposure to increasing concentrations of the histone mixture. C) Reductions in mitochondrial ATP levels after increasing concentrations of histones (37°C for 60 minutes). D) Increases in mitochondrial mass following exposure to increasing concentrations of the histone mixture (37°C for 60 minutes). For each bar, n ≥ 4. *Differences were significant, P < 0.05 vs. control.

Figure 6.

In vivo induction of cardiac dysfunction by histones after CLP. Heart functional parameters in normal WT mice (WT sham, white bars), CLP mice receiving isotype-matched IgGκ (nsIgG, gray bars), or mAb with neutralizing activity to H2A and H4 (α-histone, black bars) (13); 65 μg of antibody was given i.v. at time 0, and ECHO/Doppler parameters were measured before and 8 h after CLP in each mouse. A) Heart rates. B) Left ventricular stroke volume after CLP. C) Cardiac output. D) LV ejection fraction. E) Isovolumic relaxation. F–H) Doppler tissue imaging parameters of mitral valve function. H) LV volume diastole. For each bar, n ≥ 6 mice. *Differences between groups were significant as indicated, P < 0.05.

Figure 7.

The cardiomyopathy of sepsis requires C5a, its receptors, and PMNs, whereas cardiac dysfunction is linked to appearance of histones. Proposed sequence of events after CLP (polymicrobial sepsis), leading to complement activation, with dependence on C5a receptors and PMNs for histone appearance in plasma. Histones, which interact with TLR2 and TLR4 on a variety of cell types, including CMs, cause cell damage, organ dysfunction, and lethality.