Supplemental Digital Content
DETAILED MATERIALS AND METHODS
Patients’ blood samples collection
Upon ICU admission, surplus blood samples were collected prospectively every 24h
from all patients up to 7 days of ICU stay.
Blood samples were drawn into citrated
vacutainers (4.5ml 0.109M + buffered sodium citrate 3.2%, Becton Dickinson, Plymouth,
UK) and blood was centrifuged at 2600xg for 20 min and the generated plasma was stored as
aliquots in -80oC. In some patients matched sera were also produced and stored at -80 oC.
HL-1 cardiomyocyte culture and maintenance
Murine HL-1 cells that have typical phenotypic features of adult cardiomyocytes (1,
2) were a kind gift from Professor William Claycomb (Louisiana State University, USA) and
cultured as described previously in Claycomb medium supplemented with 10 % fetal bovine
serum (Sigma Aldrich), 100 µM Norepinephrin, 2 mM L-Glutamine and 100 U/ml
Penicillin/Streptomycin (2). When fully confluent, cells showed spontaneous contractions at a
rate of 4-5 Hz (4-5 beats/second) at 37°C. Medium was changed every 24 hours and cells
were passaged only when they have reached full confluency (twice a week) as evident by the
presence of several clusters of spontaneously contracting cells (approximately 70% of cells).
All flasks, tissue culture dishes and well- plates were pre-coated with 5 µg/ml fibronectin and
0.02% gelatine for at least one hour before being seeded with HL-1 cardiomyocytes. Cells
were grown in 5 %CO2 and at 37°C.
HL-1 cardiomyocyte viability assay
Viability of HL-1 cardiomyocytes following incubation with septic patients’ sera
(citrated plasma was not used in these experiments) was assessed using the WST-8 cell
proliferation assay kit (Enzo Life Sciences). 5x104 cells were seeded into each well of a 96well plate (pre-coated with fibronectin/gelatine) and grown until fully confluent. Test agents
were then added to the cells in a total volume of 100 µl/ well (50 µl fully supplemented
Claycomb media and 50 µl Septic patient’s Sera). After 1 hour of incubation at 37°C, cells
were washed with PBS twice. 100 µl of fresh medium was added to each well with 10 µl of
the WST-8 dye, followed by further incubation for one hour. Finally, after gentle shaking, the
absorbance of each well was measured using a microplate reader (Multiskan Spectrum,
Thermo electron Corporation) at a wavelength of 450 nm and a reference wavelength of 650
nm. Absorbance correlates with the amount of viable cells in each well. For septic sera + antihistone antibody treatment (ahscFv), 50μg/ml of ahscFv was added to HL-1 cardiomyocytes
for 5 minutes followed by the addition of septic sera, and viability was assessed as described
here after 1 hour of incubation.
Animal experiments
Septic peritonitis was induced by intra-peritoneal injection of fresh, cultured E.coli (K12, 108 CFU/ mouse) (3). Blood samples were withdrawn from mouse tail veins and sera were
isolated by centrifugation before initiation of sepsis and at 6, 10, 14, 16 and 24 hours after
induction of sepsis.
In vivo hemodynamic analyses were performed using a protocol described previously
(4) with a 1.4F pressure-volume catheter (SPR-839) and pressure volume system (Millar
Instruments). Mice were anaesthetised with Avertin (200 mg/kg i.p.) at 20h after bacterial
infection and a catheter was inserted into the left ventricle (LV) via the right common carotid.
Data were calibrated and analysed with Miller PVAN 2.9 software (Millar Instruments). No
blood samples were taken from mice that were used for haemodynamic assessment.
Haemodynamic changes were monitored for an hour. In this model, blood bacteria were
cultured positive after a few hours and circulating histones reached a peak value around 1618h. The measurement at 20h was designated to detect the effect of histones on
cardiomyocyte injury and contractility with minimum effect from both systemic
haemodynamic changes and heart rates based on our preliminary testing.
Western blotting
Western blotting was used to quantify circulating histones (5) and cardiac troponins in septic
patients and murine plasma samples. 3 µl from each sample was mixed with SDS lysis buffer
(1% SDS, 120 mM Tris, 25 mM EDTA, pH 6.8) and run into 15% SDS-PAGE gels. The
proteins were then transferred at 400mAMP over 1 hour into Immobilon-P PVDF transfer
membrane (Immobilon, UK). The membrane was then blocked with 5% milk in TBS-T for 1
hour at room temperature. Primary antibodies against cardiac troponin I (abcam) (1:500),
cardiac troponin T (Santa Cruz) (1:500) and histone 3 (abcam) (1:2000) were diluted in (5 %
milk in TBS-T) or (5 % BSA in TBS-T) and incubated with the membrane for 12 hours (over
night at 4ºC). The membrane was then washed for 15 minutes with TBS-T (3 times) and then
incubated with a secondary anti-rabbit or anti-mouse antibody-tagged with horseradish
peroxidise (HRP) in a concentration of 1:10,000 in (5% milk in TBS-T) or (5% BSA in TBST) for one hour. The membrane was then washed with TBS-T for 15 minutes, and proteins
signals were assessed by chemiluminiscent detection using G box gel imaging system
(Syngene) and software GeneSnap (Syngene).
REFERENCES
1.
Claycomb WC, Lanson NA, Jr., Stallworth BS, et al. HL-1 cells: a cardiac muscle cell
line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc
Natl Acad Sci U S A 1998;95:2979-2984.
2.
White SM, Constantin PE, Claycomb WC. Cardiac physiology at the cellular level:
use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and
function. Am J Physiol Heart Circ Physiol 2004;286:H823-829.
3.
Frimodt-Moller N, J. D. Knudsen, and F. Espersen. The mouse peritonitis/sepsis
model. In: Sande OZaMA, editor. Handbook of animal models of infection. London, United
Kingdom: Academic Press; 1999. p. 127-136.
4.
Oceandy D, Cartwright EJ, Emerson M, et al. Neuronal nitric oxide synthase
signaling in the heart is regulated by the sarcolemmal calcium pump 4b. Circulation
2007;115:483-492.
5.
Abrams ST, Zhang N, Manson J, et al. Circulating Histones Are Mediators of
Trauma-associated Lung Injury. Am J Respir Crit Care Med 2013;187:160-169.