title: Calpain inhibition decreases myocardial apoptosis in a swine model of chronic myocardial ischemia.
abstract: BACKGROUND
  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 the metabolic syndrome, calpain inhibition (CI) improved collateral-dependent perfusion and increased expression of proteins implicated in angiogenesis and vasodilation. In this study, we hypothesized that CI (by MLD28170) would decrease myocardial apoptosis in the same model.
  
  
  METHODS
  Yorkshire swine, all fed a high-cholesterol diet for 4 weeks underwent placement of an ameroid constrictor on the left circumflex coronary artery. Three weeks later, animals received either no drug, termed the 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 the CI were continued for 5 weeks, after which the pig was humanely killed and the left ventricular myocardium was harvested and analyzed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, oxyblot analysis, and Western blots. Data were analyzed using the Kruskal-Wallis test.
  
  
  RESULTS
  The percentage of apoptotic cells to total cells in ischemic myocardial territory was decreased in the LCI and HCI groups compared with the HCC group as shown by TUNEL staining (P = .018). There was a decrease in proapoptotic proteins, including cleaved caspase 3, caspase 9, cleaved caspase 9, Bax, BAD, p-BAD, and Erk 1/2 (P ≤ .049 each), but no decrease in caspase 3 (P = .737). There was also an increase in antiapoptotic proteins, including BCL-2 and p-BCL2 (P ≤ .025 each). In the ischemic myocardium, several proangiogenic proteins were increased in the LCI and HCI groups compared with the HCC group, including p-AKT, p-eNOS, and eNOS (P ≤ .006 each) but there was no increase in AKT (P = .311). CI decreased tissue oxidative stress in both the LCI and HCI groups compared to the HCC group as shown by oxyblot analysis (P = .021).
  
  
  CONCLUSIONS
  In the setting of hypercholesterolemia, CI decreases apoptosis and the expression of proteins in proapoptotic signaling pathways. CI also increased expression of proteins implicated in anti apoptotic pathways and improves oxidative stress in ischemic myocardial tissue.
authors.affiliation: Division of Cardiothoracic Surgery, Department of Surgery, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI.
authors.forename: Brittany A
authors.initials: BA
authors.lastname: Potz
authors.affiliation: Division of Cardiothoracic Surgery, Department of Surgery, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI.
authors.forename: Ashraf A
authors.initials: AA
authors.lastname: Sabe
authors.affiliation: Division of Cardiothoracic Surgery, Department of Surgery, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI.
authors.forename: Nassrene Y
authors.initials: NY
authors.lastname: Elmadhun
authors.affiliation: Division of Cardiothoracic Surgery, Department of Surgery, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI.
authors.forename: Jun
authors.initials: J
authors.lastname: Feng
authors.affiliation: Division of Cardiothoracic Surgery, Department of Surgery, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI.
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Division of Cardiothoracic Surgery, Department of Surgery, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI.
authors.forename: Hunter
authors.initials: H
authors.lastname: Mitchell
authors.affiliation: Division of Cardiothoracic Surgery, Department of Surgery, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI.
authors.forename: Peter
authors.initials: P
authors.lastname: Quesenberry
authors.affiliation: Division of Cardiothoracic Surgery, Department of Surgery, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI.
authors.forename: M Ruhul
authors.initials: MR
authors.lastname: Abid
authors.affiliation: Division of Cardiothoracic Surgery, Department of Surgery, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI. Electronic address: fsellke@lifespan.org.
authors.forename: Frank W
authors.initials: FW
authors.lastname: Sellke
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002154:Calpain / Q000037:antagonists & inhibitors; D002316:Cardiotonic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D002908:Chronic Disease; D004305:Dose-Response Relationship, Drug; D006023:Glycoproteins / Q000494:pharmacology* / Q000627:therapeutic use; D006321:Heart / Q000187:drug effects*; D006937:Hypercholesterolemia / Q000150:complications; D020287:In Situ Nick-End Labeling; D024821:Metabolic Syndrome / Q000150:complications; D017202:Myocardial Ischemia / Q000150:complications / Q000188:drug therapy* / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D013552:Swine
chemical_list: D015415:Biomarkers; D002316:Cardiotonic Agents; D006023:Glycoproteins; C039304:calpain inhibitors; D002154:Calpain