Heart protection by combination therapy with esmolol and milrinone at late-ischemia and early reperfusion

Abstract

Introduction: The present study determined whether late-ischemia/early reperfusion therapy with the β(1)-adrenergic receptor (AR) blocker esmolol and phosphodiesterase III inhibitor milrinone reduced left ventricular (LV) myocardial infarct size (IS).

Methods and results: In an ischemia/reperfusion rat model (30-min ischemia/4-hr reperfusion), esmolol, milrinone or esmolol + milrinone were intravenous (IV) infused over 10 min (from the last 5 min of ischemia to the first 5 min of reperfusion). LV-IS were 48.9 ± 8.9%, 41.5 ± 5.4%, 25.8 ± 7.7% and 16.8 ± 7.3% for saline, esmolol, milrinone, and esmolol + milrinone, respectively (n = 12/group). Esmolol + milrinone further reduced LV-IS compared with esmolol or milrinone alone (p < 0.05). LV-IS-reduction induced by esmolol + milrinone was eliminated in the presence of protein kinase A-(PKA)-inhibitor (Rp-cAMPS) or Akt-inhibitor (AKT 1/2 kinase inhibitor). In mixed rat ventricular cardiomyocyte cultures, intra-ischemic application of esmolol, milrinone or esmolol + milrinone reduced myocyte death rates by 5.5%, 13.3%, and 16.8%, respectively, compared with saline (p < 0.01). This cell protective effect by esmolol + milrinone was abrogated in the presence of PKA-inhibitor or Akt-inhibitor. Esmolol, milrinone or esmolol + milrinone increased myocardial PKA activity by 22%, 28% and 59%, respectively, compared with saline (n = 6, p < 0.01). No non-specific adverse effect of Rp-cAMPS on myocytes was identified in a purified myocyte preparation during hypoxia/re-oxygenation. Antiapoptotic pathways were assessed by measuring myocardial phosphorylated Akt (pAkt) levels combined with terminal dUTP nick-end labelling staining analysis. Ten minutes following infusion of esmolol, milrinone or esmolol + milrinone, there were 1.7-, 2.7-, and 6-fold increase in tissue pAkt levels, respectively. This esmolol + milrinone induced pAkt activation was abolished in the presence of PKA inhibitor. Esmolol, milrinone and esmolol + milrinone reduced myocyte apoptosis rates by 22%, 37% and 60%, respectively, compared with saline (p < 0.01).

Conclusions: Late-ischemia/early reperfusion therapy with esmolol + milrinone additively reduces LV-IS associated with robust activation of myocardial PKA and subsequent Akt-antiapoptotic pathway.

Conflict of interest statement

No potential interest conflicts.

Figures

Fig. 1

Panels A, B and C show representative cases of IS in the animals treated with saline (a), esmolol + milrinone (E + M) (b) and E + M in the presence of Rp-cAMPS (c). Infarcted tissue is indicated with yellow color. Panel d shows the IS affected by the therapy with esmolol, milrinone or E + M in the absence and presence of Rp-cAMPS or AKT ½ kinase inhibitor (n = 12/group). IS in E + M-treated group is significantly smaller compared with all other groups (*: p < 0.05, **: p < 0.01). Panel E: the effects of intra-ischemic application of esmolol, milrinone or E + M in the absence and presence of Rp-cAMPS or AKT 1/2 kinase inhibitor on myocyte death rates during hypoxia/re-O2 in myocyte culture preparation (n = 8/group). Panel f: effects of β2-AR antagonist ICI-118,551 (ICI), Rp-cAMPS, or AKT-inhibitor alone on IS, comparing with saline (n = 8/group). Effects of esmolol, milrinone and esmolol + esmolol in the presence of their respective antagonists were plotted for comparison. Myocyte death rate in E + M treated group is significantly lower compared with all other groups (*: p < 0.05, **: p < 0.01)

Fig. 2

Effect of Rp-cAMPS on myocyte survival during hypoxia/re-oxygenation in ICA cell-free ventricular myocyte culture. There is no difference in myocyte survival rates between saline and Rp-cAMPS groups (p = ns, n = 6/group). Rp-cAMPS abolishes myocyte protective effect conferred by PKA agonists (Terbutaline + CGRP). Hypoxia/re-oxygenation markedly increases myocyte death rates compared with non-hypoxic condition in ICA cell-free myocyte culture. **: p < 0.01

Fig. 3

Effects of reperfusion therapy with esmolol, milrinone or E + M on LV myocardial tissue PKA during ischemia/reperfusion (n = 6/group). PKA activity in saline treated group is significantly lower than all other groups. *: p < 0.05, **: p < 0.01

Fig. 4

Panel A: Effects of reperfusion therapy with esmolol, milrinone or E + M on myocardial tissue Akt activity during ischemia/reperfusion (n = 5/group). The pAkt activity in saline treated group is significantly lower compared with all other group (**: p < 0.01). The phosphorylated Akt levels were normalized to total Akt. Insert: an individual case of myocardial tissue pAkt levels in response to different treatments. Panel B: A typical case demonstrating that in the presence of PKA inhibitor Rp-cAMPS, E + M exerts no effect on myocardial pAkt activity. Overall, application of E + M increases myocardial pAkt activity by 7 ± 1-fold, compared with saline (p < 0.01, n = 4/group). In the presence of Rp-cAMPS, E + M has no effect on myocardial tissue pAkt activity during ischemia/reperfusion, compared with saline (n = 4/group)

Fig. 5

(a) Effects of reperfusion therapy with esmolol, milrinone or E + M on LV myocyte apoptosis during ischemia/reperfusion. Panels A-C show a case of total cardiocyte apoptosis in a saline-treated heart. A: Propidium iodide-positive nuclei (red) of cardiocytes. B: TUNEL-positive nuclei (green, arrows) in the same field. C: Superimposed panels A and B. Panels D-F show a case of cardiocyte apoptosis in an E + M-treated heart. D: Popidium iodide-positive nuclei of cardiocytes. E: TUNEL-positive nuclei in the same field of D. F: Superimposed panels D and F. Panels G-H: Immunohistochemical double staining confirms myocyte (red)-specific apoptosis in a field of saline-treated heart (G) and field esmolol + milrinone-treated heart (H). TUNEL positive nuclei are stained with green color. Scale bar = 10 μm. (b). A: Percentage changes in cardiocyte apoptosis in the hearts treated with saline, esmolol, milrinone and E + M, respectively (n = 6/group). The cardiocyte apoptosis rate in hearts treated with E + M is smaller than all other groups (p < 0.01). B: Comparison of myocyte-specific apoptosis among 4 different treatment groups. The extent of ventricular myocyte apoptosis in esmolol + milrinone group is significantly smaller than all other groups (p < 0.01). *: p < 0.05, **: p < 0.01