Brittany A. Potz

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

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 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). CONCLUSION 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.

Calpain inhibition improves collateral-dependent perfusion in a hypercholesterolemic swine model of chronic myocardial ischemia

PURPOSE Calpain overexpression is implicated in aberrant angiogenesis. We hypothesized that calpain inhibition (MDL28170) would improve collateral perfusion in a swine model with hypercholesterolemia and chronic myocardial ischemia. METHODS 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 no drug, high cholesterol control group (n = 8); low-dose calpain inhibition (0.12 mg/kg; n = 9); or high-dose calpain inhibition (0.25 mg/kg; n = 8). The heart was harvested after 5 weeks. RESULTS Myocardial perfusion in ischemic myocardium significantly improved with high-dose calpain inhibition at rest and with demand pacing (P = .016 and .011). Endothelium-dependent microvessel relaxation was significantly improved with low-dose calpain inhibition (P = .001). There was a significant increase in capillary density, with low-dose calpain inhibition and high-dose calpain inhibition (P = .01 and .01), and arteriolar density with low-dose calpain inhibition (P = .001). Calpain inhibition significantly increased several proangiogenic proteins, including vascular endothelial growth factor (P = .02), vascular endothelial growth factor receptor 1 (P = .003), vascular endothelial growth factor receptor 2 (P = .003), and talin, a microvascular structural protein (P = .0002). There was a slight increase in proteins implicated in endothelial-dependent (nitric oxide mediated) relaxation, including extracellular signal-regulated kinase, phosphorylated extracellular signal-regulated kinase, and inducible nitric oxide synthase with calpain inhibition. CONCLUSIONS In the setting of hypercholesterolemia, calpain inhibition improved perfusion, with a trend toward increased collateralization on angiography and increased capillary and arteriolar densities in ischemic myocardium. Calpain inhibition also improved endothelium-dependent microvessel relaxation and increased expression of proteins implicated in angiogenesis and vasodilatation.

Calpains and Coronary Vascular Disease.

Despite many advances in percutaneous and surgical interventions in the treatment of coronary artery disease (CAD), up to one-third of patients are still either not candidates or receive suboptimal revascularization. Calpains are a class of calcium-activated non-lysosomal cysteine proteases that serve as a proteolytic unit for cellular homeostasis. Uncontrolled activation of calpain has been found to be involved in the pathogenesis of myocardial reperfusion injury, cardiac hypertrophy, myocardial stunning and cardiac ischemia. Inhibition of calpains has been shown to significantly attenuate myocardial stunning and reduced infarct size after ischemia-reperfusion. Calpain inhibition therefore serves as a potential medical therapy for patients suffering from a number of diseases, including CAD.

Endothelial ROS and Impaired Myocardial Oxygen Consumption in Sepsis-induced Cardiac Dysfunction.

Sepsis is known as the presence of a Systemic Inflammatory Response Syndrome (SIRS) in response to an infection. In the USA alone, 750,000 cases of severe sepsis are diagnosed annually. More than 70% of sepsis-related deaths occur due to organ failure and more than 50% of septic patients demonstrate cardiac dysfunction. Patients with sepsis who develop cardiac dysfunction have significantly higher mortality, and thus cardiac dysfunction serves as a predictor of survival in sepsis. We have very little understanding about the mechanisms that result in cardiac dysfunction in the setting of sepsis. At present, the factors involved in sepsis-related cardiac dysfunction are believed to include the following: persistent inflammatory changes in the vascular endothelium and endocardium leading to circulatory and micro vascular changes, increase in endothelial reactive oxygen species (ROS), abnormal endothelium-leukocyte interaction resulting in a feed-forward loop for inflammatory cytokines and ROS, contractile dysfunction of the heart due to autonomic dysregulation, metabolic changes in myocardium leading to impaired oxygen delivery and increased oxygen consumption, mitochondrial dysfunction, and persistent inflammatory signaling. In this review article, we will briefly discuss the clinical challenges and our current understanding of cardiac dysfunction in sepsis. Major focus will be on the pathological changes that occur in vascular endothelium, with an emphasis on endocardium, and how endothelial ROS, impaired endothelium-leukocyte interaction, and microcirculatory changes lead to cardiac dysfunction in sepsis. The importance of the ongoing quest for the clinical biomarkers for cardiac dysfunction will also be discussed.

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

Background Inhibition of glycogen synthase kinase 3β (GSK‐3β) has been reported to be cardioprotective during stressful conditions. Methods and Results Pigs were fed a high‐fat diet for 4 weeks to develop metabolic syndrome, then underwent placement of an ameroid constrictor to their left circumflex artery to induce chronic myocardial ischemia. Two weeks later, animals received either: no drug (high cholesterol control group [HCC]) or a GSK‐3β inhibitor (GSK‐3β inhibited group [GSK‐3βI]), which were continued for 5 weeks, followed by myocardial tissue harvest. Coronary blood flow and vessel density were significantly increased in the GSK‐3βI group compared to the HCC group. Expression levels of the following proteins were greater in the GSK‐3βI group compared to the HCC group: vascular endothelial growth factor receptor 1 , vascular endothelial cadherin, γ‐catenin, β‐catenin, protein kinase B, phosphorylated forkhead box O1, and superoxide dismutase 2. Conclusions In the setting of metabolic syndrome, inhibition of GSK‐3β increases blood flow and vessel density in chronically ischemic myocardium. We identified several angiogenic, cell survival, and differentiation pathways that include β‐catenin signaling and AKT/FOXO1, through which GSK‐3β appears to improve vessel density and blood flow. These results may provide a potential mechanism for medical therapy of patients suffering from coronary artery disease and metabolic syndrome.

Calpain inhibition modulates glycogen synthase kinase 3β pathways in ischemic myocardium: A proteomic and mechanistic analysis

Background: Calpain inhibition has an enhancing effect on myocardial perfusion and improves myocardial density by inhibiting glycogen synthase kinase 3&bgr; (GSK‐3&bgr;) and up‐regulating downstream signaling pathways, including the insulin/PI3K and WNT/&bgr;‐catenin pathways, in a pig model of chronic myocardial ischemia in the setting of metabolic syndrome. Methods: Pigs were fed a high‐fat diet for 4 weeks, then underwent placement of an ameroid constrictor to the left circumflex artery. Three weeks later, the animals received no drug (high‐cholesterol controls [HCC]), a high‐dose calpain inhibitor (HCI), a low‐dose calpain inhibitor (LCI), or a GSK‐3&bgr; inhibitor (GSK‐3&bgr;I). The diets and drug regimens were continued for 5 weeks and the myocardial tissue was harvested. Results: Calpain and GSK‐3&bgr; inhibition caused an increase in myocardial perfusion ratios at rest and during pacing compared with controls. Pigs in the LCI and HCI groups had increased vessel density in the ischemic myocardium, and pigs in the GSK‐3&bgr;I group had increased vessel density in the ischemic and nonischemic myocardium compared with the HCC group. Calpain inhibition modulates proteins involved in the insulin/PI3K and WNT/&bgr;‐catenin pathways. Quantitative proteomics revealed that calpain and GSK‐3&bgr; inhibition significantly modulated the expression of proteins enriched in cytoskeletal regulation, metabolism, respiration, and calcium‐binding pathways. Conclusions: In the setting of metabolic syndrome, calpain or GSK‐3&bgr; inhibition increases vessel density in both ischemic and nonischemic myocardial tissue. Calpain inhibition may exert these effects through the inhibition of GSK‐3&bgr; and up‐regulation of downstream signaling pathways, including the insulin/PI3K and WNT/&bgr;‐catenin pathways.

Glycogen synthase kinase 3β inhibition reduces mitochondrial oxidative stress in chronic myocardial ischemia

Objectives: Glycogen synthase kinase 3&bgr; (GSK‐3&bgr;) inhibition has been reported to increase microvascular density and improve myocardial blood flow in a porcine model of chronic myocardial ischemia and metabolic syndrome. Inhibition of GSK‐3&bgr; can also be cardioprotective by modulating fibrosis signaling and mitochondrial‐induced apoptosis. We hypothesized GSK‐3&bgr; inhibition would have a beneficial effect on myocardial fibrosis and oxidative stress in a porcine model of chronic myocardial ischemia and metabolic syndrome. Methods: Pigs were fed a high fat diet for 4 weeks followed by placement of an ameroid constrictor to the left circumflex coronary artery. Three weeks later animals received either no drug or a GSK‐3&bgr; inhibitor. The diets and placebo/GSK‐3&bgr; inhibition were continued for an additional 5 weeks, the pigs were then euthanized, and the myocardial tissue was harvested. Collagen expression was analyzed via Picrosirius staining. Oxidative stress was analyzed via Oxyblot analysis. Protein expression was analyzed via Western blot. Results: GSK‐3&bgr; inhibition was associated with decreased collagen expression and oxidative stress in the ischemic and nonischemic myocardial tissue compared with control. There was a decrease in profibrotic proteins transforming growth factor‐&bgr;, p‐SMAD2/3, and matrix metalloproteinase‐9, and in proapoptotic and oxidative stress proteins, apoptosis inducing factor, the cleaved caspase 3/caspase 3 protein ratio and phosphorylated myeloid cell leukemia sequence‐1 in the GSK‐3&bgr; inhibited group compared with the control. Conclusions: In the setting of metabolic syndrome and chronic myocardial ischemia, inhibition of GSK‐3&bgr; decreases collagen formation and oxidative stress in myocardial tissue. GSK‐3&bgr; inhibition might be having this beneficial effect by downregulating transforming growth factor‐&bgr;/SMAD2/3 signaling and decreasing mitochondrial induced cellular stress.

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

Background Mesenchymal stem cell–derived extracellular vesicles (EVs) are believed to be cardioprotective in myocardial infarct. The objective of this study was to examine the effects of human mesenchymal cell–derived EV injection on cardiac function, myocardial blood flow, and vessel density in the setting of chronic myocardial ischemia. Methods and Results Twenty‐three Yorkshire swine underwent placement of an ameroid constrictor on their left circumflex artery. Two weeks later, the animals were split into 2 groups: the control group (CON; n=7) and the EV myocardial injection group (MVM; n=10). The MVM group underwent myocardial injection of 50 μg of EVs in 2 mL 0.9% saline into the ischemic myocardium. Five weeks later, the pigs underwent a harvest procedure, and the left ventricular myocardium was analyzed. Absolute blood flow and the ischemic/nonischemic myocardial perfusion ratio were increased in the ischemic myocardium in the MVM group compared with the CON group. Pigs in the MVM group had increased capillary and arteriolar density in the ischemic myocardial tissue compared with CON pigs. There was an increase in expression of the phospho–mitogen‐activated protein kinase/mitogen‐activated protein kinase ratio, the phospho–endothelial nitric oxide synthase/endothelial nitric oxide synthase ratio, and total protein kinase B in the MVM group compared with CON. There was an increase in cardiac output and stroke volume in the MVM group compared with CON. Conclusions In the setting of chronic myocardial ischemia, myocardial injection of human mesenchymal cell–derived EVs increases blood flow to ischemic myocardial tissue by induction of capillary and arteriolar growth via activation of the protein kinase B/endothelial nitric oxide synthase and mitogen‐activated protein kinase signaling pathways resulting in increased cardiac output and stroke volume.

Calpain inhibition decreases inflammatory protein expression in vessel walls in a model of chronic myocardial ischemia

Background. Emerging data suggest a link between calpain activation and the enhanced inflammatory response of the cardiovascular system. We hypothesize that calpain activation associates with altered inflammatory protein expression in correlation with the proinflammatory profile of the myocardium. Our pig hypercholesterolemic model with chronic myocardial ischemia was treated with calpain inhibitors to establish their potential to improve cardiac function. Methods. Yorkshire swine, fed a high cholesterol diet for 4 weeks then underwent placement of an ameroid constrictor on the left circumflex artery. Two weeks later, animals received either no drug (high‐cholesterol control group, n = 8), a low dose of calpain inhibitors (0.12 mg/kg, n = 9), or a high dose of calpain inhibitors (0.25 mg/kg; n = 8). The high‐cholesterol diet and calpain inhibitors were continued for 5 weeks, after which the pig was euthanized. The left ventricular myocardial tissue (ischemic and nonischemic) was harvested and analyzed for inflammatory protein expression. Data were statistically analyzed via the Kruskal‐Wallis and Dunn post hoc test. Results. Calpain inhibitor treatment coincides with increased expression of IKB‐&agr; and decreased expression of macrophages, NFkB, IL‐1, and tumor necrosis factor (TNF)‐&agr; in the ischemic myocardial tissue as compared with the control group. An NFkB array revealed decreased expression of IRF5, JNK1/2, JNK2, CD18, NFkB p65, c‐Rel, Sharpin, TNF R1, TNF R2, and DR5 in the ischemic myocardium of the group treated with a high dose of calpain inhibitors compared with the control. Conclusion. Calpain activation in metabolic syndrome is a potential contributor to cardiac dysfunction in metabolic disorders with ischemic background. We suggest that calpain inhibition downregulates NFkB signaling in the vessel walls, which might be useful for improving myocardial blood flow in ischemic conditions.

Alcohol attenuates myocardial ischemic injury

Background: Moderate alcohol consumption is cardioprotective but the mechanism of action remains unclear. Nuclear factor &kgr;‐B regulates the expression of genes involved in inflammation, stress, and apoptosis. We used a swine model of diet‐induced metabolic syndrome to investigate the effects of red wine and vodka on nuclear factor &kgr;‐B signaling and cytokine activity in chronically ischemic myocardium. Methods: Yorkshire swine were given a high‐fat diet for 4 weeks; an ameroid constrictor was then placed on the left circumflex artery. The high‐fat diet was continued and the swine were divided into 3 groups for 7 weeks: hypercholesterolemic diet alone (control, n = 8), hypercholesterolemic diet with vodka (vodka, n = 8), and hypercholesterolemic diet with wine (wine, n = 8). Ischemic myocardium was analyzed by Western blot and cytokine array. Results: Administration of alcohol was associated with decreased expression of inhibitor of &kgr;‐B kinase complex &agr;, inhibitor of &kgr;‐B kinase complex &bgr;, and phosphorylated inhibitor of &kgr;‐B &bgr; in the ischemic myocardium compared with the control group. Alcohol administration demonstrated an increase in nuclear factor &kgr;‐B in the ischemic myocardium. Both wine and vodka demonstrated a significant decrease in leptin, interleukin‐1&agr;, IL‐13, IL‐15, and interferon‐&ggr;. Vodka demonstrated a significant decrease in phosphorylated BCL‐2 and caspase‐9. Conclusion: In ischemic myocardium, alcohol modulates the nuclear factor &kgr;‐B pathway, which may contribute to the adaptive response of tissues to the stress of ischemia. Furthermore, both wine and vodka decreased multiple proinflammatory cytokines. This study provides a mechanism by which alcohol may be cardioprotective in ischemic myocardium.