Fingolimod (FTY720) Preserves High Energy Phosphates and Improves Cardiac Function in Heterotopic Heart Transplantation Model

Abstract

During heart transplantation, donor heart leads to reduced oxygen supply resulting in low level of high energy phosphate (HEP) reserves in cardiomyocyte. Lower HEP is one of the underlying reasons of cell death due to ischemia. In this study we investigated the role of Fingolimod (FTY720) in heart transplantation ischemia. Eight groups of Sprague-Dawley rats (n = 5 for each subgroup) were made, A1 and C1 were given FTY720 1 mg/kg while B1 and D1 were given normal saline. The hearts were implanted into another set of similar rats after preservation period of 1 h at 4-8 °C. Significantly higher Left ventricular systolic pressure (LVSP), dP/dT maximum (p < 0.05), dP/dT minimum (p < 0.05) were recorded in the FTY720 treated group after 24 h of reperfusion while after 1 h of reperfusion, there were no significant differences in LVSP, maximum and negative dP/dT, and Left ventricular end diastolic pressure (LVEDP) between the control and the FTY720-treated transplant groups. Coronary blood flow (CBF) was enhanced (p < 0.05) in the FTY720 treated group after 1 and 24 h. ATP p < 0.001, p < 0.05 at 1 and 24 h, ADP p < 0.001, p > 0.05 at 1 and 24 h, and phosphocreatine p < 0.05, p > 0.05 at 1 and 24 h were better preserved by FTY720 treatment as compared to control group. The study concluded that pretreatment of grafted hearts with FTY720 improved hemodynamics, CBF, high energy phosphate reserves, reduces the peroxynitrite level and poly (ADP ribose) polymerase (PARP) inhibition that prevents ischemia-reperfusion injury.

Keywords: cardiac function; coronary blood flow; fingolimod (FTY720); high energy phosphates; poly (ADP ribose) polymerase.

Figure 1

Hemodynamic parameters at 1 and 24 h of myocardial reperfusion in FTY720 and Control-saline treated groups. (A) Effect of FTY720 on LVSP, (B) LVEDP, (C) LV dP/dT max, (D) LV dP/dT min. Values are expressed as the means ± SD. dP/dT max: the rate of maximum positive left ventricular pressure development; dP/dT min, the rate of maximum negative left ventricular pressure development LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure.

Figure 2

Coronary blood flow (CBF) measurements. Group A, B: early reperfusion at 1 h, Group C, D: late reperfusion at 24 h of reperfusion. * p < 0.05, ** p < 0.001.

Figure 3

(A) AMP, (B) ADP, (C) ATP, and (D) changes in phosphocreatine levels in left ventricular tissue of the heart in FTY720treated groups and control-saline treated group at 1 and 24 h of reperfusion. AMP: adenosine monophosphate. ADP: adenosine diphosphate. ATP: adenosine triphosphate. Data presented as a mean ± SD. p-value < 0.05 considered as significant. (∗ p < 0.05 and ∗∗ p < 0.001, treated vs. control).

Figure 4

Myocardial nitrotyrosine staining, magnification 20×, scale bar 100 µm, blue color representing nuclei, transplanted heart tissue was collected after 24 h of reperfusion for nitrotyrosine localization by immunohistochemistry. (A) FTY720-treated group, (B) control-saline treated group. Red arrows illustrating decreased expression of peroxynitrite in treated group as compared to control.

Figure 5

Cleaved caspase-3 and PARP protein expressions in myocardial tissue analyzed using Western blot. (A,B) Relative densities of cleaved caspase-3 and PARP protein levels. Data presented as a mean ± SD. p-value < 0.05 considered as significant. (∗ p < 0.05 and ∗∗ p < 0.001, treated vs. control).

Figure 6

Proposed mechanism of action of FTY720 in ischemia reperfusion injury (upward arrows showing increase and downward mean decreasing).

Figure 7

Experimental design.