Beneficial Effects of Tacrolimus on Brain-Death-Associated Right Ventricular Dysfunction in Pigs

Abstract

Background: Right ventricular (RV) dysfunction remains a major problem after heart transplantation and may be associated with brain death (BD) in a donor. A calcineurin inhibitor tacrolimus was recently found to have beneficial effects on heart function. Here, we examined whether tacrolimus might prevent BD-induced RV dysfunction and the associated pathobiological changes.

Methods: After randomized tacrolimus (n = 8; 0.05 mg·kg^-1·day^-1) or placebo (n = 9) pretreatment, pigs were assigned to a BD procedure and hemodynamically investigated 1, 3, 5, and 7 h after the Cushing reflex. After euthanasia, myocardial tissue was sampled for pathobiological evaluation. Seven pigs were used as controls.

Results: Calcineurin inhibition prevented increases in pulmonary vascular resistance and RV-arterial decoupling induced by BD. BD was associated with an increased RV pro-apoptotic Bax-to-Bcl2 ratio and RV and LV apoptotic rates, which were prevented by tacrolimus. BD induced increased expression of the pro-inflammatory IL-6-to-IL-10 ratio, their related receptors, and vascular cell adhesion molecule-1 in both the RV and LV. These changes were prevented by tacrolimus. RV and LV neutrophil infiltration induced by BD was partly prevented by tacrolimus. BD was associated with decreased RV expression of the β-1 adrenergic receptor and sarcomere (myosin heavy chain [MYH]7-to-MYH6 ratio) components, while β-3 adrenergic receptor, nitric oxide-synthase 3, and glucose transporter 1 expression increased. These changes were prevented by tacrolimus.

Conclusions: Brain death was associated with isolated RV dysfunction. Tacrolimus prevented RV dysfunction induced by BD through the inhibition of apoptosis and inflammation activation.

Keywords: FK506; apoptosis; brain death; calcineurin inhibitor; heart transplantation; inflammation; right ventricular dysfunction; tacrolimus.

Figure 1

Pulmonary and systemic hemodynamics were evaluated at baseline, 1 (CR + 1 h), 3 (CR + 3 h), 5 (CR + 5 h), and 7 h (CR + 7 h) after Cushing reflex (CR) in the control (n = 7; white bars), placebo (brain death, BD; n = 9; red bars), and tacrolimus pretreated brain death (BD + Tac; n = 8; blue bars) groups. PAP (A) indicates mean pulmonary arterial pressure; PVR (B), pulmonary vascular resistance; SVR (C), systemic vascular resistance; SAP (D), mean systemic arterial pressure; RAP (E), right atrial pressure; LAP (F), left atrial pressure evaluated by the pulmonary arterial wedge pressure; CO (G), cardiac output; HR (H), heart rate; and NA need (I), noradrenaline need. Values are expressed as mean ± standard error of the mean. *** p < 0.05 BD vs. BD + Tac, † p < 0.05 Base vs. CR + 1 h, ‡ p < 0.05 Base vs. CR + 3 h, § p < 0.05 Base vs. CR + 5 h, || p < 0.05 Base vs. CR + 7 h, # p < 0.05 CR + 1 h vs. CR + 3 h, †† p < 0.05 CR + 1 h vs. CR + 5 h, ‡‡ p < 0.05 CR + 1 h vs. CR + 7 h, §§ p < 0.05 CR + 3 h vs. CR + 5 h, |||| p < 0.05 CR + 3 h vs. CR + 7 h. The comparisons between the different recording or sampling times are marked in red for the “BD” group and in blue for the “BD + Tac” group.

Figure 2

Right (A) and left (B) ventriculo-arterial coupling efficiency (Ees/Ea); right (C) and left (D) ventricular contractility (ventricular end-systolic elastance, Ees); and right (E) and left (F) ventricular afterload (effective pulmonary arterial elastance, Ea) were evaluated at baseline, 1 (CR + 1 h), 3 (CR + 3 h), 5 (CR + 5 h), and 7 h (CR + 7 h) after Cushing reflex (CR) in the control (n = 7; white bars), placebo (brain death, BD; n = 9; red bars), and tacrolimus pretreated brain death (BD + Tac; n = 8; blue bars) groups. Values are expressed as mean ± standard error of the mean. *** p < 0.05 BD vs. BD + Tac, † p < 0.05 Base vs. CR + 1 h, ‡ p < 0.05 Base vs. CR + 3 h, § p < 0.05 Base vs. CR + 5 h, || p < 0.05 Base vs. CR + 7 h, # p < 0.05 CR + 1 h vs. CR + 3 h, †† p < 0.05 CR + 1 h vs. CR + 5 h, ‡‡ p < 0.05 CR + 1 h vs. CR + 7 h, §§ p < 0.05 CR + 3 h vs. CR + 5 h, |||| p < 0.05 CR + 3 h vs. CR + 7 h. The comparisons between the different recording or sampling times are marked in red for the “BD” group and in blue for the “BD + Tac” group.

Figure 3

Serum C-reactive protein (A), troponin I (B), urea (C), and creatinine (D) concentrations in the control (n = 7; white bars), placebo (brain death, BD; n = 9; red bars), and tacrolimus pretreated brain death (BD + Tac; n = 8; blue bars) groups 7 h after the Cushing reflex (CR + 7 h). Values are expressed as mean ± standard error of the mean. * p < 0.05 control vs. BD.

Figure 4

Right and left myocardial apoptosis in brain-death–induced right ventricular dysfunction with/without tacrolimus pretreatment. Pro-apoptotic Bax−to−Bcl−2 (A) and Bax−to−Bcl−XL (B) gene ratios in the right (RV) and left ventricles (LV) in the control (n = 7; white bars), placebo (brain death, BD; n = 9; red bars), and tacrolimus pretreated brain death (BD + Tac; n = 8; blue bars) groups. The cardiac apoptotic rate (C) (percentage) was evaluated as the ratio between the numbers of terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL)-positive cardiomyocytes (brown nuclei) and the total number of cardiomyocytes (brown + blue nuclei) in the RV and LV from the control (n = 7; white bars), placebo (brain death, BD; n = 9; red bars), and tacrolimus pretreated brain death (BD + Tac; n = 8; blue bars) groups 7 h after the Cushing reflex (CR + 7 h). Scale bars: 200 µm. Values are expressed as mean ± standard error of the mean. * p < 0.05 Control vs. BD, ** p < 0.05 Control vs. BD + Tac, *** p < 0.05 BD vs. BD + Tac. Correlations between the Ees/Ea ratio and the pro-apoptotic Bax−to−Bcl−2 (D) and Bax−to−Bcl−XL (E) ratios and apoptotic rate (F) in the RV. The arrows mark the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL)-positive cardiomyocytes (brown nuclei).

Figure 5

Myocardial expression of cytokines, adhesion molecules, heme oxygenase (HO) −1, and inducible nitric oxide synthase (iNOS) in brain-death–induced right ventricular dysfunction with/without tacrolimus pretreatment. The interleukin (IL) −1b (A), receptor for IL−1 (RN) (B), IL−6−to−IL−10 ratio (C), the ratio of the IL−6 receptor (R) to IL−10 R (D), receptor Gp130 for IL−6 (E), intercellular adhesion molecule (ICAM−1 (F), ICAM−2 (G)), vascular cell adhesion molecule (VCAM−1) (H), heme oxygenase (HO) −1 (I), and iNOS (J) relative mRNA content in the right (RV) and left ventricles (LV) from the control (n = 7; white bars), placebo (brain death, BD; n = 9; red bars), and tacrolimus pretreated brain death (BD + Tac; n = 8; blue bars) groups 7 h after the Cushing reflex (CR + 7 h). Values are expressed as mean ± standard error of the mean. * p < 0.05 Control vs. BD, ** p < 0.05 Control vs. BD + Tac, *** p < 0.05 BD vs. BD + Tac. Correlations between the Ees/Ea ratio and expression of IL−1β (K), IL−6/IL−10 (L), VCAM−1 (M), and iNOS (N) in the RV.

Figure 6

Characterization of inflammatory cells infiltrating the myocardium in brain-death-induced right ventricular dysfunction. The number of extravascular neutrophils (myeloperoxidase, MPO−positive cells) was determined in the right (RV) (A) and left ventricles (LV) (B) from the control (n = 7; white bars), placebo (brain death, BD; n = 9; red bars), and tacrolimus pretreated brain death (BD + Tac; n = 8; blue bars) groups 7 h after the Cushing reflex (CR + 7 h). Values are expressed as mean ± standard error of the mean. * p < 0.05 Control vs. BD, ** p < 0.05 Control vs. BD + Tac, *** p < 0.05 BD vs. BD + Tac. Correlations between the Ees/Ea ratio and the number of extravascular neutrophils in the right (C) and left (D) ventricles.

Figure 7

Myocardial expression of β-adrenergic receptors, regulators controlling calcium homeostasis, molecules regulating contractile function, natriuretic hormones, and signaling components involved in contraction-inducible substrate in brain-death–induced right ventricular dysfunction with/without tacrolimus pretreatment. b1 (A), 2 (B), and 3 (C) adrenergic receptors, sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase 2a (SERCA2A)-to-phospholamban (PLB) ratio (F), ryanodine receptor type 2 (RyR2) (G), α1c subunit of the L-type calcium channel (CACNA1C) (H), natriuretic peptide A (NPPA) (L), brain natriuretic peptide (NPPB) (M), α-myosin heavy chain (MYH6) to β-myosin heavy chain (MYH7) ratio (N), endothelial (e)-NOS (P), glucose transporters (GLUT-) 1 (Q) and 4 (R), and thrombospondin receptor CD36 molecule (CD36) (S) relative mRNA content in right (RV) and left ventricles (LV) from the control (n = 7; white bars), placebo (brain death, BD; n = 9; red bars), and tacrolimus pretreated brain death (BD + Tac; n = 8; blue bars) groups 7 h after the Cushing reflex (CR + 7 h). Values are expressed as mean ± standard error of the mean. * p < 0.05 Control vs. BD, ** p < 0.05 Control vs. BD + Tac, *** p < 0.05 BD vs. BD + Tac. Correlations between the Ees/Ea ratio and gene expression for the β 1 (D) and β 3 (E) adrenergic receptors, SERCA2/PLB (I), Ryr2 (J), CACNA1C (K), MYH7/MYH6 (O), eNOS (T), GLUT1 (U), and GLUT4 (V) in the RV and GLUT1 in the LV (W).

Figure 8

Myocardial expression of Toll-like receptor signaling pathways in brain-death–induced right ventricular dysfunction with/without tacrolimus pretreatment. Toll-like receptor (TLR-) 2 (A), 4 (B), and 9 (C), and NLR family pyrin domain containing 3 (NLRP3) (D) relative mRNA content in the right (RV) and left ventricles (LV) from the control (n = 7; white bars), placebo (brain death, BD; n = 9; red bars), and tacrolimus pretreated brain death (BD + Tac; n = 8; blue bars) groups 7 h after the Cushing reflex (CR + 7 h). Values are expressed as mean ± standard error of the mean. * p < 0.05 Control vs. BD, ** p < 0.05 Control vs. BD + Tac, *** p < 0.05 BD vs. BD + Tac.