Calpain inhibition decreases myocardial apoptosis in a swine model of chronic myocardial ischemia

Abstract

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.

Glycogen Synthase Kinase 3β Inhibition Improves Myocardial Angiogenesis and Perfusion in a Swine Model of Metabolic Syndrome

“…These risk factors include (1) greater weight gain, (2) higher total cholesterol and low‐density lipoprotein/HDL ratios, (3) increased mean arterial pressure, and (4) elevated blood glucose levels in response to a glucose tolerance test 10. Using this model, we found that moderate inhibition of calpain decreases myocardial apoptosis, improves collateral dependent perfusion, and increases expression of signaling proteins that promote angiogenesis 11, 12. Interestingly, GSK‐3β is a substrate for calpain.…”

Extracellular Vesicle Injection Improves Myocardial Function and Increases Angiogenesis in a Swine Model of Chronic Ischemia

“…Twenty‐three Yorkshire swine (Tufts, Boston, MA) underwent placement of an ameroid constrictor to their left circumflex artery at 11 weeks of age, in a manner similar to that previously reported13, 14, 15 (Figure 1). Pigs received aspirin (10 mg/kg) 1 day before surgery and 5 days postoperatively.…”

“…The data, analytic methods, and study materials have been and will continue to be made available to other researchers for purposes of reproducing the results or replicating the procedure 13, 14, 15, 16, 17, 18, 19, 20…”

Chronic heart failure: Ca 2+ , catabolism, and catastrophic cell death

“…Furthermore, Ca 2+ can stimulate calpain, a cysteine protease which can cleave Bax, creating pro-apoptotic fragments [90]. …”