Background Calpain over-expression is implicated in aberrant angiogenesis. We hypothesized that calpain inhibition (CI, MDL28170) would improve collateral perfusion in a swine model with hypercholesterolemia and chronic myocardial ischemia. Methods and Results Yorkshire swine fed a high cholesterol diet for 4 weeks underwent surgical placement of an ameroid constrictor to their left circumflex coronary 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 heart was harvested after 5 weeks. There was a trend toward increased right to left collateral vessels on angiography with HCI. Myocardial perfusion in ischemic myocardium significantly improved with HCI at rest and with demand pacing (p = 0.016 and 0.011). Endothelium-dependent microvessel relaxation was significantly improved with LCI (p = 0.001). There was a significant increase in capillary density, with LCI and HCI (p= 0.01 and 0.01), and arteriolar density with LCI (p= 0.001). CI significantly increased several proangiogenic proteins including VEGF (p= 0.02), VEGFR1 (p= 0.003), VEGFR2 (p= 0.003) and talin, a microvascular structural protein (p= 0.0002). There was a slight increase in proteins implicated in endothelial-dependent (NO Mediated) relaxation including ERK, p-ERK and iNOS with CI. Conclusions In the setting of hypercholesterolemia, CI improved perfusion, with a trend toward increased collateralization on angiography and increased capillary and arteriolar densities in ischemic myocardium. CI also improved endothelium-dependent microvessel relaxation and increased expression of proteins implicated in angiogenesis and vasodilatation.

Ossabaw miniswine have been naturally selected to efficiently store large amounts of lipids offering them a survival advantage. Our goal was to evaluate the myocardial response to chronic ischemia of the Ossabaw consuming a hypercaloric, high-fat/cholesterol diet with and without metformin supplementation. At 6 weeks of age animals were fed either a regular diet (OC, n = 9), a hypercaloric high-fat/cholesterol diet (OHC, n = 9), or a hypercaloric high-fat/cholesterol diet supplemented with metformin (OHCM, n = 8). At 9 weeks, all animals underwent ameroid constrictor placement to the left circumflex coronary artery to simulate chronic ischemia. Seven weeks after ameroid placement, all animals underwent hemodynamic and functional measurements followed by cardiac harvest. Both OHC and OHCM animals developed significantly greater weight gain, total cholesterol, and LDL:HDL ratio compared to OC controls. Metformin administration reversed diet-induced hypertension and glucose intolerance. There were no differences in global and regional contractility, myocardial perfusion, capillary and arteriolar density, or total protein oxidation between groups. Myocardial protein expression of VEGF, PPAR-α, γ, and δ was significantly increased in the OHC and OHCM groups. Microvessel reactivity was improved in the OHC and OHCM groups compared to controls, and correlated with increased p-eNOS expression. Overfed Ossabaw miniswine develop several components of metabolic syndrome. However, impairments of myocardial function, neovascularization and perfusion were not present, and microvessel reactivity was paradoxically improved in hypercholesterolemic animals. The observed cardioprotection despite metabolic derangements may be due to lipid-dependant upregulation of the PPAR pathway which is anti-inflammatory and governs myocardial fatty acid metabolism.

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, oxyblot 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), BAD (p= 0.049), p-BAD (p= 0.007) , Erk 1/2 (p= 0.016) and a statistically insignificant decrease in caspase 3 (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 (p=0.006) with a statistically insignificant increase seen in AKT (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 Oxyblot analysis (p= 0.021). Conclusions In the setting of hypercholesterolemia, CI decreases apoptosis and the expression of proteins in pro apoptotic signaling pathways. CI also increased expression of proteins implicated anti apoptotic pathways and improves oxidative stress in ischemic myocardial tissue.