Wistar male rats, 380–500 g, were used for this study. The rats were handed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Pub. No. 8323, Revised 1996). The protocol of the study was approved by the Animal Care and Use Committee of Department of Pharmacology, Medical School, National and Kapodistrian University of Athens (license 842/20-02-2017, ΕL 25BIOexp 10).

In order to assess the effects of cold cardioplegic arrest versus normothermic perfusion on cardiac function and perfusion pressure, the following experiments were performed (Figure 1A).

a. Hearts excised and subjected to cold cardioplegic (CC) arrest for 240 min and then perfused in Langendorff apparatus allowing 30 min recovery (group CC, n = 10),

In order to assess the effects of T3 administration on cardiac function and perfusion pressure during ex vivo normothermic perfusion, the following experiments were performed (Figure 1B).

a. Hearts excised and subjected to normothermic perfusion in Langendorff apparatus for 210 min after an initial period of 30 min perfusion (stabilization period). At the end of stabilization, KH buffer was supplemented with vehicle (group NP, n = 10),

During stabilization both groups were treated the same. At the end of the perfusion, LV was isolated, frozen in liquid nitrogen and kept at −80°C for molecular analysis.

Rats were anaesthetized with ketamine HCl and heparin 1000 IU was given intravenously. Following anesthesia, thoracotomy was performed to expose the heart and major vessels. The heart and large vessels were excised and immersed into ice cold histidine-tryptophan-ketoglutarate (HTK) cardioplegia solution identical to the solution used for cardiopreservation during heart transplantation (composition in mmol/L NaCl 15, KCl 9, MgCl 2 4, Adenosine 5, a-ketoglutarate 1, Tryptophan 2, Histidine 180, Histidine-HCl, 8, Mannitol 30). Changes in cold HTK solution were performed to achieve removal of blood (within 90 s) and then the heart was transferred to 100 mL of new cold HTK cardioplegia solution and stored at 4°C for 240 min. After this period, hearts were cannulated via the ascending aorta and perfused in the Langendorff apparatus with KH buffer allowing 30 min recovery. An intraventricular balloon was inserted into the left ventricle via the left atrium and allowed measurement of left ventricular pressure under isovolumic conditions. In order preload to be similar in both groups and equal to normal preload of the isolated rat heart, balloon volume was set at 250 μL. Constant flow was adjusted at 15 mL/min. The perfusion apparatus was heated to ensure a temperature of 37°C throughout the course of the experiment. Hearts were paced at 320 beats/min with a Harvard pacemaker. Pacing started 5–7 min after perfusion was achieved.

Rats were anaesthetized with ketamine HCl and heparin 1000 IU was given intravenously. The hearts were rapidly excised, placed in ice-cold Krebs-Henseleit (KH) buffer (composition in mmol/L: sodium chloride 118, potassium chloride 4.7, potassium phosphate monobasic 1.2, magnesium sulfate 1.2, calcium chloride 1.4, sodium bicarbonate 25, and glucose 11) and mounted on the aortic cannula of the Langendorff perfusion system. Perfusion with oxygenated (95% O₂/5% CO₂) Krebs-Henseleit buffer was established within 60 s after thoracotomy. The heart perfused to a non-working isolated rat heart preparation at a constant flow according to the Langendorff technique as previously described (12, 14, 19, 20). A water filled balloon, connected to a pressure transducer, was advanced into the left ventricle through an incision in the left atrium. Pressure signal was transferred to a computer using a data analysis software (IOX, Emka Technologies) which allowed continuous monitoring and recording (21). The intraventricular balloon allowed measurement of left ventricular pressure under isovolumic conditions. Left ventricular balloon volume was adjusted to produce an average initial left ventricular end-diastolic pressure of 6–8 mmHg in all groups and was held constant thereafter throughout the experiment. Since the balloon was not compressible, left ventricular contraction was isovolumic. As intraventricular volume was maintained at a constant value, preload, did not change. Thus, the left ventricular peak systolic pressure and the left ventricular developed pressure (LVDP), defined as the difference between left ventricular peak systolic pressure and left ventricular end-diastolic pressure, represented indexes of systolic function obtained under isometric conditions.

The perfusion apparatus was heated to ensure a temperature of 37°C throughout the course of the experiment. Constant flow was adjusted at 15 mL/min. Hearts were paced at 320 beats/min with a Harvard pacemaker. Pacing started 5–7 min after perfusion was achieved.

3,5,3′-triiodothyronine (T3) was purchased from Sigma Chemicals (St Louis MO, USA). T3 was dissolved in ethanol by adding a small volume of 25% NaOH and diluted in 0.9% normal saline to obtain a stock solution. T3 concentration in stock solution was 1 mg/mL. Stock solutions were kept at −20°C and before each experiment a quantity of this solution containing T3 was added in Krebs-Henseleit (KH) buffer to a final concentration of 60 nM. This dose has previously been shown to be cardioprotective against ischemia-reperfusion injury in an isolated rat heart experimental model (14). T3 administration initiated after the first 30 min of perfusion (stabilization period) in the isolated heart apparatus. Vehicle (T3 diluent) was injected in hearts subjected to normothermic perfusion without T3.

Left ventricular function was assessed by recording the left ventricular developed pressure (LVDP, mmHg) and the positive and negative first derivative of LVDP (+dp/dt and –dp/dt). Diastolic function was assessed by monitoring isovolumic left ventricular end-diastolic pressure (LVEDP) as a measure of diastolic chamber distensibility. Perfusion pressure under constant flow conditions was used to assess coronary vessel resistance (PP, mmHg). All parameters were also expressed as percentage of change from the baseline values.

In order to investigate potential molecular mechanisms underlying the effects of T3 in normothermic perfusion, the pattern of stress induced kinase signaling activation was assessed in NP and NP + T3 hearts at the end of perfusion. Molecular analysis was performed as previously described (21,22). A sample from the left ventricular tissue (200–220 mg) was homogenized in ice-cold buffer containing 10 mM Hepes (pH: 7.8), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, 1 mM DTT and 10 μg/mL leupeptin. 200 μL of 10% Igepal was added and samples were left in ice for 30 min. Homogenization was repeated and the homogenate was centrifuged at 1000 g for 5 min, 4°C. The supernatant representing the cytosol-membrane fraction was kept at −80°C for further processing. Protein concentrations were determined by the bicinchoninic acid (BCA) method, using bovine serum albumin as a standard. Samples were prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by boiling for 5 min in Laemmli sample buffer containing 5% 2-mercaptoethanol. Aliquots (40 μg) were loaded onto 9% (w/v) acrylamide gels and subjected to SDS-PAGE in a Bio-Rad Mini Protean gel apparatus. Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane (Hybond ECL) at 100 V and 4°C, for 1.5 h using Towbin buffer for Western blotting analysis. Subsequently, filters were probed with specific antibodies against total p38 MAPK and dual phospho-p38 MAPK, total c-jun NH2-terminal kinases (JNKs) and dual phospho-JNKs, total Akt and dual phospho-Akt (Cell Signaling Technology, dilution 1:1000), total AMPK and phospho (Thr172)-AMPK (Cell Signaling Technology, dilution 1:1000) overnight at 4°C. Filters were incubated with appropriate anti-mouse (Amersham) or anti-rabbit (Cell Signalling) HRP secondary antibodies and immunoreactivity was detected by enhanced chemiluminescence using Lumiglo reagents (New England Biolabs). Chemiluminescence was detected by the image analysis system FluorChem HD2 (AlphaInnotech Corporation, 14743, Catalina Street, San Leandro, CA) equipped with a CCD camera and analysis software. Five samples from each group were loaded on the same gel for comparisons between groups. Data were expressed as the ratio of phosphorylated to total protein expression.

Values are presented as mean (Standard Deviation). Normal distribution of variables was estimated with Shapiro-Wilk test of normality. Normally distributed data were compared using an independent t-test. Skewed data were analysed non-parametrically using Mann-Whitney U test. Serial measurements of LVEDP and PP were compared by mixed, repeated measures analysis of variance (mixed ANOVA) to test for the effect of treatment, time and the interaction (tests for “within-subjects” factor and “between-subjects” factor); the respective non-linear fit-curves with the 95% CI error were produced with non-linear fit analysis. When significant, differences within and between each group were tested by a post hoc analysis using the Bonferroni correction for multiple comparisons if needed. A two-tailed test with a p value less than 0.05 was considered significant. Analysis was performed using the GraphPad 8 software.

Cardiac function and perfusion pressure were assessed at the end of the recovery period in CC group and the end of normothermic perfusion in NP group. After cold cardioplegia, LVEDP was 33.6 (11.7) mmHg in CC group vs.18.5 (13.3) in NP group, p = 0.02. LVDP was similar between the groups; 81 (18.9) mmHg for CC and 87.3 (7.0) for NP, p = n.s. Perfusion Pressure was 64.2 (7.0) mmHg in CC group vs. 132 (51) mmHg in NP group, p = 0.001 (Figure 2).

Cardiac function and perfusion pressure were measured at the end of stabilization period (baseline parameters) and the end of normothermic perfusion. Baseline parameters were similar between NP and NP + T3 groups (Table 1). LVEDP was 19.7 (12) mmHg in NP group and 8.6 (5.8) mmHg in T3 treated group p = 0.026 (Figure 3A). LVDP was 87 (20) mmHg in NP group and 90 (21) mmHg in T3 treated group, p = 0.49. PP was 130 (39.1) mmHg in NP and 92 (10.6) mmHg in T3 treated group, p = 0.019 (Table 1).

Functional parameters and perfusion pressure were also expressed as percentage of change from the baseline values. LVEDP gradually increased by 139% from baseline in NP group, and only by 13% in NP + T3 group, p = 0.048 (Table 1). The decline in LVDP was found to be less after T3 treatment; 18.2% in NP + T3 vs. 25.3% in NP, p = 0.01 (Table 1). PP increased by 91% from baseline in NP group and by 41% in NP + T3 group, p = 0.024 (Table 1).

Measurements of cardiac function and perfusion pressure were also performed every 30 min from the end of stabilization period to the end of normothermic perfusion (Figure 3). Mixed Repeated Measures ANOVA analysis for LVEDP (dependent variable) at different time points between the groups showed that the main effect of treatment (T3, “between-subjects” factor) on LVEDP was statistically significant (F = 4.0, p = 0.04). Thus, T3 treatment induced a sustained improvement of LVEDP over time. Furthermore, there was a statistically significant effect (F = 9.1, p = 0.002) regarding the main effect of time (“within-subjects” factor). There was also a statistically significant interaction effect between time and treatment (p = 0.044).

Mixed Repeated Measures ANOVA analysis for PP (dependent variable) at different time points between the groups showed that the main effect of treatment (T3, “between-subjects” factor) on PP was statistically significant (F = 5.5, p = 0.03). Thus, T3 treatment induced a sustained improvement of PP over time. Furthermore, there was a statistically significant effect (F = 28.8, p = 0.000002) regarding the main effect of time (“within-subjects” factor) on deterioration of PP. Α statistically significant interaction effect between time and treatment was found (p = 0.024).

At the end of normothermic perfusion, the ratio of LV weight to body weight was 2.35 (0.3) for the NP hearts and 2.39 (0.26) for NP + T3 hearts, p = n.s.

Stress induced kinase signaling activation was assessed in NP and NP + T3 hearts at the end of normothermic perfusion. T3 administration during normothermic perfusion resulted in suppression of proapoptotic signalling and upregulation of the pro-survival signalling pathways.

Phosphorylated to total Akt and phosphorylated to total AMPK levels were both significantly increased by 1.85 (p = 0.047) and 2.25-fold (p = 0.01) in T3 treated hearts (NP + T3) as compared to NP hearts, p < 0.05 (Figures 4A,B).

Phosphorylated to total p38 MAPK levels were lower by 3 fold in NP + T3 as compared to NP hearts, p = 0.04. Phosphorylated to total p54 and p46 JNKs levels were lower by 4.0 (p = 0.04) and 3.7-fold (p = 0.03) respectively in NP + T3 as compared to NP hearts (Figures 4C,D).

Ex vivo perfusion is a new strategy for organ preservation and reconditioning prior to transplantation (4, 45). Extensive research in this field is underway to identify optimal perfusate composition and duration. The perfusate commonly used in clinical practice is cell based consisted of leukocyte depleted, packed red blood cells supplemented with anticoagulants and vasodilators. However, the use of red cells may not be suitable for prolonged periods of perfusion. There is now evidence that non cell solutions as Ringer’s lactate or Steen solution supplemented with nutrient and metabolic substrates may optimize normothermic perfusion (24). Along this line, the present study provides evidence that addition of high dose of T3 in K-H can preserve ex vivo rat hearts subjected to normothermic perfusion. Furthermore, for the first time, this therapeutic modality is shown to induce the activation of intracellular repair signaling. Until now, thyroid hormone has been added to solutions at replacement doses and along with other hormones to resuscitate and preserve donor organs (11). Thus, the cardioprotective effect of this hormone has not been evaluated.

There are limitations of this study which have to be taken into consideration. The study was performed in small animals and its findings are needed to be validated in large animals and humans. However, the rat species is shown to be of high translational validity for pharmacological studies of ischemia/reperfusion (17). The potential effects of T3 in extended periods of normothermic perfusion have not been investigated. Cardiac damage was assessed by functional indices and not histological examination. The benefit of T3 in cell-based solutions has not been investigated. However, it has recently been shown that T3 may have favorable effects on erythrocyte aggregation and hemorheology.

In conclusion, T3 appears to limit cardiac and microvascular dysfunction in an experimental model of ex vivo rat heart normothermic perfusion. This effect was associated with a switch of death to survival kinase signaling.