Effects of Individual or Combined Inhibition of Interleukin‐1 and Interleukin‐18 on the Resuscitation of Donation after Circulatory Death Murine Heart
Authors: Mezzaroma Eleonora , Kenning Kristine , Toldo Stefano , Quader Mohammed 
Published: April 2020

BackgroundThere is a limited amount of donor hearts to be used for transplantation, which is mainly dependent on Donation after Brain Death (DBD) donors. This pool could be expanded with adoption of Donation after Circulatory Death (DCD) donor hearts. The transplantation of a heart that undergoes the DCD process would expose the organ to ischemia and reperfusion injury (IRI). IRI activates the NLRP3 inflammasome, which promotes the production of interleukin‐1 (IL‐1) and IL‐18.HypothesisCombined pharmacologic inhibition of IL‐1 and IL‐18 confers superior protection compared to individual blockade with IL‐1 or IL‐18 in the DCD heart.MethodsThe DCD mouse model was developed reflecting the clinical DCD protocol. The DCD was induced in C57 mice anesthetized, intubated, ventilated and paralyzed (vecuronium bromide 100 μg/kg) and by removing the ventilatory support to induce anoxia and circulatory failure. Total ischemic time was set at 40 min. Reanimation was achieved on a Langendorff system at 37°C using Krebs Henseleit (KH) buffer alone or supplemented with IL‐1 receptor antagonist (IL‐1Ra, 1 μg/ml), IL‐18 binding protein (IL‐18BP, 1 μg/ml) or their combination (n=8–10/group). Heart rate (HR), perfusion rate, developed pressure (DP) and +/−dP/dt were obtained at 15 min intervals. The rate pressure product (RPP) was calculated. After 90 min, the hearts were collected to measure DNA fragmentation (TUNEL assay). Cardiac troponin‐I (cTnI) levels were measured in the perfusate.ResultsHR and perfusion flow rates were comparable in all groups (table
). IL‐1Ra improved myocardial contractility, although it reached statistical significance only for DP and –dP/dt when compared to the DCD control (table
). IL‐18BP significantly improved all the parameters of myocardial contractility, compared to the control (all p<0.05). Markers of cell injury were significantly better after resuscitation with IL‐18BP, while IL‐1Ra did not reduce the release of cTnI or the DNA fragmentation (Table
). The combination of IL‐1Ra and IL‐18BP did not further improve the contractility nor further reduce damage of the DCD heart compared to the monotherapy treatment.ConclusionPharmacologic blockade of IL‐1 or IL‐18 protects the DCD mouse heart, with better protection with IL‐18BP compared to IL‐1Ra. Combined treatment with IL‐1Ra and IL‐18BP did not give additional benefits compared to the individual inhibitors.Support or Funding InformationThis work was supported by a Merit Review Award and and American Heart Association grant to Dr. Mohammed Quader.

DCD

DCD

KH buffer

KH buffer + inhibitors

N=10

IL‐1Ra
N=8

IL‐18BP
N=9

IL‐1Ra+IL‐18BP
r>N=5

Functional Data

Heart Rate (beats/min)

396±14

401±15

440±10

408±20

Perfusion rate (ml/min)

1.8±0.1

2.2±0.3

2.4±0.1

1.9±0.2

Developed pressure (mmHg)

78.7±5.8

94.1±9.3

108.3±6.5
#

103.0±8.6
#

Rate Pressure Product (mmHgxbeats/min)

29233±3293

34198±3634

43082±3191
#

31715±467

(+)dP/dt (mmHg/ms)

3256±266

3272±695

4744±204
#

5312±120
#

(−)dP/dt (mmHg/ms)

−2278±18

−2992±28
#

−3632±29
#

−3405±24
#

Makers of myocyte damage

cTnI (ng/ml)

7.79±0.98

7.68±0.85

4.21±0.67
#

2.63±0.71
#

TUNEL+ nuclei (%)

2.09±0.53

2.02±0.76

0.99±0.23

1.01±0.23

Values are expressed as mean ± Standard Error of Mean.
#P<0.05 vs DCD K‐H buffer.