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Objective: To investigate the impact of circulating histones on cardiac of antihistone antibody (10mg/kg) was studied. Murine blood
injury and dysfunction in a murine model and patients with sepsis. samples were collected serially, and left ventricular function was
Design: Prospective, observational clinical study with in vivo and assessed by intraventricular catheters and electrocardiography.
ex vivo translational laboratory investigations. Measurements and Main Results: Circulating histones and car-
Setting: General ICU and university research laboratory. diac troponins in human and murine plasma were quantified. In 65
Subjects: Sixty-five septic patients and 27 healthy volunteers. patients with sepsis, circulating histones were significantly elevated
Twelve-week-old male C57BL/6N mice. compared with healthy controls (n=27) and linearly correlated with
Interventions: Serial blood samples from 65 patients with sepsis cardiac troponin T levels (rs=0.650; p < 0.001), noradrenaline doses
were analyzed, and left ventricular function was assessed by echo- required to achieve hemodynamic stability (rs=0.608; p < 0.001),
cardiography. Patients sera were incubated with cultured car- Sequential Organ Failure Assessment scores (p=0.028), and mor-
diomyocytes in the presence or absence of antihistone antibody, tality (p=0.008). In a subset of 36 septic patients without prior car-
and cellular viability was assessed. Murine sepsis was initiated diac disease, high histone levels were significantly associated with
by intraperitoneal Escherichia coli injection (108 colony-forming new-onset left ventricular dysfunction (p=0.001) and arrhythmias
unit/mouse) in 12-week-old male C57BL/6N mice, and the effect (p=0.01). Left ventricular dysfunction only predicted adverse out-
comes when combined with elevated histones or cardiac troponin
levels. Furthermore, patients sera directly induced histone-specific
*See also p. 2253.
cardiomyocyte death ex vivo, which was abrogated by antihistone
1
Department of Clinical Infection, Microbiology and Immunology, Institute
antibodies. In vivo studies on septic mice confirmed the cause-effect
of Infection and Global Health, University of Liverpool, Liverpool, United
Kingdom. relationship between circulating histones and the development of
2
The Medical School, Southeast University, Nanjing, China. cardiac injury, arrhythmias, and left ventricular dysfunction.
3
Department of Biostatistics, Institute of Translational Medicine, University Conclusion: Circulating histones are novel and important media-
of Liverpool, Liverpool, United Kingdom. tors of septic cardiomyopathy, which can potentially be utilized
4
Institute of Aging and Chronic Disease, University of Liverpool, Liverpool, for prognostic and therapeutic purposes. (Crit Care Med 2015;
United Kingdom.
43:20942103)
5
Intensive Care Unit, Royal Liverpool University Hospital, Liverpool, United
Key Words: arrhythmias; cardiac troponins; extracellular histones;
Kingdom.
6
Roald Dahl Haemostasis & Thrombosis Centre, Royal Liverpool Univer-
left ventricular function; mortality; sepsis
sity Hospital, Liverpool, United Kingdom.
Supplemental digital content is available for this article. Direct URL citations
appear in the printed text and are provided in the HTML and PDF versions
S
of this article on the journals website (http://journals.lww.com/ccmjournal). epsis is the host response to infection and a major cause of
Supported, in part, by grants from the British Heart Foundation mortality worldwide. Most deaths in sepsis are attributed
(PG/14/19/30751) and National Institute of Health Research (United
Kingdom) i4i (II-FS-0110-14061). to the development of multiple organ failure (1), and car-
Dr. Abrams institution received grant support from the British Heart Founda- diac involvement is very common. This includes cardiomyocyte
tion (BHF). Drs. Toh and Wangs institutions received grant support from the injury, arrhythmia, and echocardiographic abnormalities (24).
BHF and the National Institute of Health Research. The remaining authors In terms of cardiomyocyte injury, cardiac troponin T (cTnT)
have disclosed that they do not have any potential conflicts of interest.
and I (cTnI) are routinely used as specific biomarkers in clinical
For information regarding this article, E-mail: toh@liverpool.ac.uk; wangg@
liverpool.ac.uk practice. As a complex of three regulatory proteins, that is, C, I,
Copyright 2015 by the Society of Critical Care Medicine and Wolters and T that are present only in cardiomyocytes (5), elevated lev-
Kluwer Health, Inc. All Rights Reserved. els of cardiac troponins (cTn) can be detected in approximately
DOI: 10.1097/CCM.0000000000001162 60% of patients with sepsis without coronary artery disease
(6). Indeed, very high levels of more than 10,000 pg/mL can be and the Hospital Governance Committees. Written informed
reached in sepsis as opposed to a threshold of 40 pg/mL cTnI consent was obtained from patients or their next of kin.
(14 pg/mL for cTnT) for diagnosing acute coronary syndrome The diagnosis of sepsis was made according to the definition
(5, 7, 8). More importantly, it is widely recognized that elevated of the American College of Chest Physicians/Society for Critical
cTn levels are strongly associated with cardiac dysfunction and Care Medicine (37, 38). Clinical history was examined in patients
poor prognosis although their use as an independent predictor with sepsis for preexisting cardiac disease and/or arrhythmia.
of mortality remains arguable (6, 914). Those with acute coronary events at admission or in the pre-
New-onset cardiac arrhythmias occur frequently in sepsis (15). ceding 6 months were excluded. Cardiac events were defined
In particular, new-onset atrial fibrillation (AF) may increase mor- as arrhythmias (AF/flutter, recurrent ectopic beats, supraven-
tality by up to three-fold (16). There is, however, less consensus on tricular/ventricular tachycardia [VT]) (39, 40) and/or impaired
the clinical significance of cardiac dysfunction in sepsis, at least if LV function (reduced LV ejection fraction, LV hypokinesia, LV
defined as impaired systolic performance (17). Parker et al (18) dilatation) as judged by echocardiography and detailed clinical
observed that low left ventricular (LV) ejection fraction and LV evaluation by a senior cardiologist and intensivist, using criteria
dilatation were associated with survival in a group of 20 patients published before (20). These were considered new onset if aris-
with sepsis. However, further studies did not support this protec- ing in those without preexisting cardiac disease. Clinical infor-
tive compensatory mechanism (1922) but showed that cardiac mation, including the start date of the septic episode, Sequential
output would increase in early sepsis in compensatory response to Organ Failure Assessment (SOFA) score, Acute Physiology and
reduced systemic vascular resistance (SVR). When SVR changes Chronic Health Evaluation II score, drug history, and the devel-
were accounted for, septic cardiomyopathy (observed in 2040% of opment of any cardiovascular complications were collected.
patients with sepsis) provided prognostic value (23) with its pres-
ence increasing the mortality rate to 7090% (24, 25). The well- Blood Sample Collection
recognized association between cardiac dysfunction and elevated Upon ICU admission, surplus blood samples were collected pro-
cTn in sepsis (10, 11, 13, 14) further suggests that sepsis-induced spectively every 24 hours from all patients up to 7 days of ICU
myocardial injury and dysfunction adversely affect prognosis. stay. Further details are described in the supplemental data (Sup-
The underlying mechanisms of the septic cardiomyopathy plemental Digital Content 1, http://links.lww.com/CCM/B360).
are multifactorial but remain incompletely understood (26).
Impaired coronary perfusion with endothelial and coagulation Measurement of Circulating Histones and
activation, as well as the effect of endotoxins and cytokines, have cTnT in Patients
been proposed as possible mechanisms, but are not universally Circulating histones were measured as described previously
accepted (2730). More recently, toxic effects on endothelial (32). cTnT was quantified by MODULAR ANALYTICS EVO
and hematopoietic cells with increased vascular permeability, analyzer (Roche, Indianapolis, IN) based on electrochemilu-
procoagulant, and proinflammatory consequences (3134) minescence immunoassay, with a cutoff value of 14 pg/mL to
have been attributed to extracellular histones in the circula- suggest cardiac injury and a lower detection limit of 3 pg/mL.
tion. Furthermore, a recent study provided evidence that his- These were performed blind to the clinical information.
tones can induce direct myocardial dysfunction in septic mice
(35). Histones occur normally inside the nucleus as a family of Mouse Septic Model and Animal Experiments
five proteins. During sepsis, histones can be released into the All animal experiments were performed in accordance with state
circulation due to extensive inflammation and cellular death as laws and monitored by local inspectors in compliance with Brit-
a form of damage-associated molecular patterns (31, 35, 36). ish Home Office laws. Twelve-week-old male C57BL/6N mice
In mouse models of sepsis, mortality and cardiac dysfunction were obtained from SLAC Experimental Animal Centre (Shang-
can be ameliorated by antihistone antibodies (31, 35). hai, China) and were housed and used for experiments under
In this study, we examine the hypothesis that circulating sterile conditions at the Research Centre of Genetically Modified
histone levels in patients with sepsis correlate with variables Mice (Southeast University, Nanjing, China). To generate sepsis,
of myocardial injury and LV dysfunction to predict adverse a bacterial peritonitis mouse model was used by injecting mice
cardiac events and outcome. The investigative approach blends intraperitoneally (IP) with fresh, cultured Escherichia coli (41)
clinical studies on septic patients with pathophysiologic in vivo (K-12, 108 colony-forming unit/mouse, n = 6 for septic mice,
studies in septic mice and ex vivo translational studies. n = 8 for septic mice with antihistone antibody, n = 6 for sham
mice injected with normal saline). LV function assessments of
mice using pressure-volume intraventricular catheters and quan-
MATERIALS AND METHODS
tification of histones and cTns in murine plasma are described
Patients in the supplemental data (Supplemental Digital Content 1, http://
Healthy donors and patients with sepsis admitted to the general links.lww.com/CCM/B360).
ICU at the Royal Liverpool University Hospital between Novem-
ber 2010 and October 2011 were recruited prospectively accord- HL-1 Cardiomyocyte Culture and Viability Assay
ing to the protocol approved by the Local Research Ethics (North This is described in supplemental data (Supplemental Digital
West Centre of Research Ethics Committee, United Kingdom) Content 1, http://links.lww.com/CCM/B360).
Copyright 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.
Clinical Investigations
Copyright 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.
Clinical Investigations
b
Some patients have developed more than one type of arrhythmia during the septic episode.
c
One episode of atrial flutter was detected in a patient who also developed new-onset atrial fibrillation.
and cTn in patients with sepsis. Arrhythmias in septic mice Antihistone Intervention Attenuates Cardiac Injury
often occurred after 12 hours and most frequently pre- and Dysfunction in Septic Mice
sented as ectopic beats and Io or IIo atrioventricular (AV) To establish the cause-effect relationship between circulating
block (Supplemental Fig. 2, Supplemental Digital Content histones and cardiac injury and dysfunction, septic mice were
2, http://links.lww.com/CCM/B361). treated with antihistone antibodies (10mg/kg IV 8 and 16
hours after sepsis induction)
to examine for potential pro-
tective effects. The direct infu-
sion of antihistone antibody
significantly reduced cTnI
levels by approximately 70%
(from ~500 pg/mL in septic
mice to 120.8107.3 pg/mL)
(Fig. 6A) (p = 0.002). This find-
ing strongly supports a direct
effect of histones on the devel-
opment of cardiac injury. LV
contractility, measured using
catheters at 20 hours after sep-
sis induction, showed signifi-
cant reduction of LV dp/dtmax
(maximum rate of LV pres-
sure rise) (43) (Fig. 6B) and
LV dp/dtmin (maximum rate
of LV pressure drop) (Fig. 6C)
with significant increases in
Tau (a preload-independent
variable of LV isovolumic
relaxation) (44, 45) (Fig. 6D).
These changes were signifi-
cantly alleviated by ahscFv
Figure 3. High circulating histones and cardiac troponin levels are associated with higher prevalence of new-onset (Fig. 6, BD) to indicate that
left ventricular (LV) dysfunction in patients with sepsis. In 36 septic patients with no history of cardiac disease and/
or arrhythmia, circulating histones (A) and cardiac troponin T (cTnT) levels (B) were compared between those high histone levels are indeed
without (absence, n = 25) and those developing (presence, n = 11) LV dysfunction (*p < 0.01). C, Prevalence of involved in LV dysfunction
LV dysfunction was 75% (9/12) and 8.3% (2/24) in patients with histone levels at least 75 g/mL or less than that is independent of pre-
75 g/mL, respectively (p < 0.001). D, Receiver operating characteristic curve for prediction of new-onset LV
dysfunction in septic patients by circulating histone levels (area under curve [AUC] = 0.865; 95% CI, 0.7031.000; or post-load changes during
p = 0.001). Cutoff value of 75 g/mL circulating histones is arrowed (sensitivity 81.8% and specificity 92%). sepsis.
Copyright 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.
Clinical Investigations
Figure 5. Parallel and concomitant elevation of histones and cardiac troponins in the circulation of septic mice. Three groups of mice (10 per group)
were used to investigate levels of circulating histones and cardiac troponins. A, Representative Western blotting showing typical levels of histone 3 (H3)
at different time points after initiation of sepsis in mice. Recombinant H3 standards were run on the same gel to quantity H3 levels in mice samples.
B, Band quantification histogram of total histone levels in septic mice at different time points after initiation of sepsis (n = 10). C, Western blots of
dynamic changes in cardiac troponin I and T (cTnI, cTnT) and H3 in blood taken from sham group (injection of same volume of saline intraperitoneal) and
sepsis (108 colony-forming units Escherichia coli K12 intraperitoneal) with immunoglobulin-G (IgG) as an endogenous control. D, Linear correlation
(rs = 0.877; p < 0.001) between circulating levels of histones and cTnI in septic mice at different time points (n = 53 events).
from other cells and organs is not known, which requires fur- according to the time or stage of sepsis and is a topic of ongo-
ther investigation. ing debate. However, it is plausible that in the presence of sig-
This study also confirms and extends on previous studies, nificant cardiac injury, when cTn and circulating histone levels
showing that new-onset cardiac events significantly corre- are high, cardiac dysfunction would be directly associated with
late with poor prognosis and high mortality (16, 22, 23, 40, a poor outcome (11, 13, 14). In our group of patients, the non-
48, 49). Specifically, this work provides novel data, suggesting significant association between LV dysfunction and mortality
that the release of histones may represent a new pathologic became significant only when concomitant elevation in cTn or
mechanism for cardiac injury, dysfunction, and arrhythmias circulating histones were considered. This would suggest that
associated with sepsis. Cardiac events were more frequently the accuracy of an echocardiographic diagnosis of myocardial
observed in septic patients with circulating histones at least 75 dysfunction can be further improved by determination of cTn
g/mL (19/65 patients, ~30%), a cutoff value that also pro- concentrations, as has been suggested by others (10, 11, 14).
vided robust sensitivity and specificity to predict the develop- Measurement of circulating histone levels may further add to
ment of new-onset cardiac events and mortality. Therefore, a reliable evaluation of direct cardiac injury and outcome pre-
accurate clinical quantification of circulating histones would diction in patients with sepsis. Future translational and clinical
be required for translational impact. Our study is limited by a studies will have to be tailored to further investigate this.
relatively small sample size of 65 patients with sepsis, of which Further evidence of the direct role of histones in causing
36 patients had no previous cardiac disease. Although we back myocardial dysfunction is provided by our LV catheter find-
up our clinical observations with in vivo studies in septic mice, ings in septic mice, particularly from dP/dt and Tau, which
our results may require further validation with bigger cohort are the volume-independent variables of LV contractile per-
of patients with sepsis and with extended echocardiographic formance. Their significant alterations strongly indicate the
studies and measurements of new markers of cardiac damage existence of primary LV dysfunction in sepsis, which was abro-
to fully assess cardiac injury and dysfunction. gated using antihistone antibodies. Indeed, these volume-inde-
Myocardial dysfunction in sepsis can be caused by direct pendent changes are consistent with a recent report illustrating
cardiomyocyte injury or indirect effects from systemic hemo- the role of circulating histones in inducing LV dysfunction
dynamic changes, which are common during sepsis. The degree using a cecal ligation and puncture (CLP) septic mouse model
to which each contributes to myocardial dysfunction can vary (35). However, the circulating histone levels described in that
Copyright 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.
Clinical Investigations
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