B. A. Potz1, A. A. Sabe1, N. Y. Elmadhun1, J. Feng1, Y. Lui1, H. Williams1, F. W. Sellke1 1Brown University School Of Medicine,Division Of Cardiothoracic Surgery, Department Of Surgery,Providence, RI, USA
Introduction: Calpain is a family of cysteine proteases that has an important role in the initiation, regulation and execution of cell death. Our recent studies using a hypercholesterolemic swine model demonstrated that in the setting of metabolic syndrome calpain inhibition improved collateral dependent perfusion, and increased expression of proteins implicated in angiogenesis and vasodilation. In this study, we hypothesized that calpain inhibition (CI) would decrease myocardial apoptosis in the same model.
Methods: Yorkshire swine, fed a high cholesterol diet for four weeks, then underwent placement of an ameroid constrictor on the left circumflex artery. Three weeks later animals received either: no drug, high cholesterol control group (HCC; n= 8); low dose CI (0.12 mg/kg; LCI, n= 9); or high dose CI (0.25 mg/kg; HCI, n= 8). The high cholesterol diet and CI were continued for five weeks, after which the pig was euthanized and the left ventricular myocardium was harvested and analyzed via TUNEL staining, oxiblot analysis and western blots. Data was analyzed via the Kruskal-Wallis test.
Results: The percentage of apoptotic cells to total cells in ischemic myocardial territory was significantly decreased in the LCI and HCI groups compared to the HCC group as shown by TUNEL Staining (p= 0.018). There was a significant decrease in pro-apoptotic proteins including cleaved caspase 3 (p =0.001), caspase 9 (p = 0.003), cleaved caspase 9 (p=0.004), Bax (p=0.0262) and an insignificant decrease in caspase 3 (LCI 0.973 +/- 0.261 fold, HCI 0.981 +/- 0.290, HCC 1.0 fold, p= 0.737). There was a significant increase in anti-apoptotic proteins including BCL-2 (p= 0.025) and p-BCL2 (p= 0.004). In the ischemic myocardium there was a significant increase in several pro-angiogenic proteins in the LCI and HCI groups compared to the HCC group including p-AKT (p=0.0001), p-ENOS (p= 0.003) and ENOS (LCI 1.26 +/- 0.665 fold, HCI 1.87+/- 0.761 fold, HCC 1.0 fold, p=0.006) with an insignificant increase seen in AKT (LCI 1.263+/- 0.613 fold, HCI 1.180+/-0.636 fold, HCC 1.0 fold, p=0.311). CI significantly decreased tissue oxidative stress in both the LCI and HCI groups as compared to the HCC group as shown by Oxiblot analysis (p= 0.021).
Conclusion: In the setting of hypercholesterolemia, CI decreases apoptosis and the expression of proteins in the pro apopotic signaling pathway. CI also increased expression of proteins implicated in angiogenesis and anti apoptotic pathways.
