Calpain inhibition decreases myocardial fibrosis in chronically ischemic hypercholesterolemic swine.

OBJECTIVES: Calpain activation during ischemia is known to play critical roles in myocardial remodeling. We hypothesize that calpain inhibition (CI) may serve to reverse and/or prevent fibrosis in chronically ischemic myocardium. METHODS: Yorkshire swine were fed a high-cholesterol diet for 4 weeks followed by placement of an ameroid constrictor on the left circumflex artery to induce myocardial ischemia. 3 weeks later, animals received either: no drug; high-cholesterol control group (CON; 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 5 weeks, after which myocardial tissue was harvested. Tissue samples were analyzed by western blot for changes in protein content. RESULTS: In the setting of hypercholesterolemia and chronic myocardial ischemia, CI decreased the expression of collagen in ischemic and nonischemic myocardial tissue. This reduced collagen content was associated with a corresponding decrease in Jak/STAT/MCP-1 signaling pathway, suggesting a role for Jak 2 signaling in calpain activity. CI also decreases the expression of focal adhesion proteins (vinculin) and stabilizes the expression of cytoskeletal and structural proteins (N-cadherin, α-fodrin, desmin, vimentin, filamin, troponin-I). CI had no significant effect on metabolic and hemodynamic parameters. CONCLUSIONS: Calpain inhibition may be a beneficial medical therapy to decrease collagen formation in patients with coronary artery disease and associated comorbidities.

Effects of High Fat Versus Normal Diet on Extracellular Vesicle-Induced Angiogenesis in a Swine Model of Chronic Myocardial Ischemia.

Background Mesenchymal stem cell-derived extracellular vesicles (EVs) promote angiogenesis in the ischemic myocardium. This study examines the difference in vascular density, myocardial perfusion, molecular signaling, and gene expression between normal diet (ND) and high fat diet (HFD) groups at baseline and following intramyocardial injection of EVs. Methods and Results Intact male Yorkshire swine fed either an ND (n=17) or HFD (n=14) underwent placement of an ameroid constrictor on the left circumflex coronary artery. Subsequently, animals received either intramyocardial injection of vehicle-saline as controls; (ND-controls n=7, HFD-controls, n=6) or EVs; (ND-EVs n=10, HFD-EVs n=8) into the ischemic territory. Five weeks later, myocardial function, perfusion, vascular density, cell signaling, and gene expression were examined. EVs improved indices of myocardial contractile function, myocardial perfusion, and arteriogenesis in both dietary cohorts. Interestingly, quantification of alpha smooth muscle actin demonstrated higher basal arteriolar density in HFD swine compared with their ND counterparts; whereas EVs were associated with increased CD31-labeled endothelial cell density only in the ND tissue, which approached significance. Levels of total endothelial nitric oxide synthase, FOXO1 (forkhead box protein O1) , transforming growth factor-ß, phosphorylated VEGFR2 (vascular endothelial growth factor receptor 2), and phosphorylated MAPK ERK1/ERK2 (mitogen-activated protein kinase) were higher in ischemic myocardial lysates from ND-controls compared with HFD-controls. Conversely, HFD-control tissue showed increased expression of phosphorylated endothelial nitric oxide synthase, phosphorylated FOXO1, VEGFR2, and MAPK ERK1/ERK2 with respect to ND-controls. Preliminary gene expression studies indicate differential modulation of transcriptional activity by EVs between the 2 dietary cohorts. Conclusions HFD produces a profound metabolic disorder that dysregulates the molecular mechanisms of collateral vessel formation in the ischemic myocardium, which may hinder the therapeutic angiogenic effects of EVs.

Intravenous injection of extracellular vesicles to treat chronic myocardial ischemia.

BACKGROUND: Mesenchymal stem cell-derived extracellular vesicles (EVs) appear to be a very exciting treatment option for heart disease. Here, we used a swine model of chronic myocardial ischemia to evaluate the efficacy of a less-invasive method of injection of EVs via a peripheral intravenous route. METHODS: Sixteen Yorkshire swine underwent placement of an ameroid constrictor on the left circumflex (LCx) artery at age 11 weeks to induce chronic myocardial ischemia. Two weeks later, they were divided into two groups: control (CON; n = 8), and intravenous injection of EVs (EVIV; n = 8). At 18 weeks of age, animals underwent final analysis and euthanasia. The chronically ischemic myocardium (LCx territory) was harvested for analysis. RESULTS: Intravenous injection (IV) of EVs induced several pro-angiogenic markers such as MAPK, JNK but not Akt. Whereas IV injections of EVs decreased VEGFR2 expression and inhibited apoptotic signaling (caspase 3), they increased expression of VEGFR1 that is believed to be anti-angiogenic. Injection of EVs did not result in an increase in vessel density and blood flow when compared to the control group. CONCLUSIONS: Although IV injection of EVs upregulated several pro-angiogenic signaling pathways, it failed to induce changes in vascular density in the chronically ischemic myocardium. Thus, a lack of increase in vascular density at the doses tested failed to elicit a functional response in ischemic myocardium.

Lower preoperative hematocrit, longer hospital stay, and neurocognitive decline after cardiac surgery.

BACKGROUND: Cardiopulmonary bypass may be associated with postoperative neurocognitive dysfunction; however, risk factors have not been clearly identified. We hypothesize that lower hematocrit levels are correlated with postoperative neurocognitive dysfunction. METHODS: A total of 30 patients underwent cardiac operations utilizing cardiopulmonary bypass and screening for neurocognitive dysfunction preoperatively and on postoperative day 4. Patients were analyzed according to hematocrit preoperatively, 6 hours postoperatively, and on postoperative day 4, and whether they received intra or postoperative transfusions of packed red blood cells. Neurocognitive data is presented as a difference in Repeatable Battery for the Assessment of Neuropsychological Status standardized score from baseline to postoperative day 4 and analyzed by unpaired two-tailed Spearman test and unpaired Mann-Whitney U test. RESULTS: There was a significant correlation between patients with lower hematocrit before surgery and a decline in neurocognitive function at postoperative day 4 (P < .05). All patients experienced a decrease in hematocrit during their hospital stay, but the hematocrit 6 hours postoperatively and postoperative day 4 did not impact cognition. Receiving a transfusion was also not associated with neurocognitive dysfunction. Patients with low hematocrit preoperatively had a consistently lower hematocrit throughout their stay. Prolonged total length of stay was also significantly associated with neurocognitive decline. CONCLUSION: A lower preoperative hematocrit and prolonged length of hospital stay are correlated with neurocognitive decline after cardiac surgery utilizing cardiopulmonary bypass.

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.

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

OBJECTIVES: Glycogen synthase kinase 3ß (GSK-3ß) 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-3ß can also be cardioprotective by modulating fibrosis signaling and mitochondrial-induced apoptosis. We hypothesized GSK-3ß 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-3ß inhibitor. The diets and placebo/GSK-3ß 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-3ß 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-ß, 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-3ß inhibited group compared with the control. CONCLUSIONS: In the setting of metabolic syndrome and chronic myocardial ischemia, inhibition of GSK-3ß decreases collagen formation and oxidative stress in myocardial tissue. GSK-3ß inhibition might be having this beneficial effect by downregulating transforming growth factor-ß/SMAD2/3 signaling and decreasing mitochondrial induced cellular stress.

Diabetes and Cardioplegia.

Cardiac surgery with cardiopulmonary bypass and cardioplegic arrest is associated with injury to the vasculature and microcirculation leading to coronary microvascular dysfunction, permeability changes and cardiac dysfunction. In the setting of cardiopulmonary bypass with cardioplegia, poorly-controlled diabetes is associated with significant changes in endothelium-dependent and independent vascular dysfunction, vascular reactivity, vascular permeability, protein expression, cell death, coronary/peripheral microcirculation and reduced vasomotor tone leading to hypotension and impaired endothelial function. The gene expression profiles after cardiopulmonary bypass with cardioplegic arrest is quantitatively and qualitatively different in patients with diabetes. Gene expression profiling capitalizing on the differences between patients with and without diabetes is a good place to identify potential medical targets.

Novel molecular targets for coronary angiogenesis and ischemic heart disease.

Coronary artery disease (CAD) is the number one cause of death among men and women in the USA. Genetic predisposition and environmental factors lead to the development of atherosclerotic plaques in the vessel walls of the coronary arteries, resulting in decreased myocardial perfusion. Treatment includes a combination of revascularization procedures and medical therapy. Because of the high surgical risk of many of the patients undergoing revascularization procedures, medical therapies to reduce ischemic disease are an area of active research. Small molecule, cytokine, endothelial progenitor cell, stem cell, gene, and mechanical therapies show promise in increasing the collateral growth of blood vessels, thereby reducing myocardial ischemia.

Alcohol attenuates myocardial ischemic injury.

BACKGROUND: Moderate alcohol consumption is cardioprotective but the mechanism of action remains unclear. Nuclear factor κ-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 κ-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 κ-B kinase complex α, inhibitor of κ-B kinase complex ß, and phosphorylated inhibitor of κ-B ß in the ischemic myocardium compared with the control group. Alcohol administration demonstrated an increase in nuclear factor κ-B in the ischemic myocardium. Both wine and vodka demonstrated a significant decrease in leptin, interleukin-1α, IL-13, IL-15, and interferon-γ. Vodka demonstrated a significant decrease in phosphorylated BCL-2 and caspase-9. CONCLUSION: In ischemic myocardium, alcohol modulates the nuclear factor κ-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.

Clinical Significance of Spontaneous Pneumomediastinum.

BACKGROUND: Spontaneous pneumomediastinum (SPM) is classified as free air in the mediastinum in the absence of any precipitating cause. It is relatively uncommon, and the clinical significance and risk associated with SPM is not well understood and has not been widely documented in the literature. Our goals were to determine the outcomes of patients who presented with SPM and to determine predictors of severe pathology associated with SPM. METHODS: From 2004 through 2013, a retrospective review was conducted of patients who presented with SPM to our institution. Patient demographics, comorbidities, laboratory tests, and esophageal perforation were recorded. RESULTS: In all, 249 patients were discovered to have SPM on chest radiograph or computed tomography scan. Mean age was 38.7 years (range, 17 to 81). Sixty-one percent of patients (151 of 249) were male. Ten percent of all patients (24 of 249) were ultimately discovered to have esophageal perforation, determined by upper endoscopy, upper gastrointestinal series, or intraoperatively during emergent surgery. Age (p < 0.01), pleural effusion (p < 0.01), and elevated white blood cell count (p < 0.01) were the only significant risk factors for esophageal perforation on multivariate analysis. CONCLUSIONS: Spontaneous pneumomediastinum is usually associated with a benign clinical course. Risk factors for esophageal perforation in these patients include age, elevated white blood cell count, and a pleural effusion. In the absence of abnormal laboratory values or associated radiologic findings, the majority of patients with SPM may be safely observed without the need for further diagnostic testing.

Continuous Glucose Monitoring in the Cardiac ICU: Current Use and Future Directions.

Perioperative glucose control is highly important, particularly for patients undergoing cardiac surgery. Variable glucose levels before, during and after cardiac surgery lead to increased post-operative complications and patient mortality. [1] Current methods for intensive monitoring and treating hyperglycemia in the Intensive Care Unit (ICU) usually involve hourly glucose monitoring and continuous intravenous insulin infusions. With the advent of more accurate subcutaneous glucose monitoring systems, the role of improved glucose control with newer systems deserves consideration for widespread adoption.

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-α and decreased expression of macrophages, NFkB, IL-1, and tumor necrosis factor (TNF)-α 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.

Calpain inhibition modulates glycogen synthase kinase 3ß 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 3ß (GSK-3ß) and up-regulating downstream signaling pathways, including the insulin/PI3K and WNT/ß-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-3ß inhibitor (GSK-3ßI). The diets and drug regimens were continued for 5 weeks and the myocardial tissue was harvested. RESULTS: Calpain and GSK-3ß 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-3ßI 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/ß-catenin pathways. Quantitative proteomics revealed that calpain and GSK-3ß 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-3ß inhibition increases vessel density in both ischemic and nonischemic myocardial tissue. Calpain inhibition may exert these effects through the inhibition of GSK-3ß and up-regulation of downstream signaling pathways, including the insulin/PI3K and WNT/ß-catenin pathways.

Surgical palliation of gastric outlet obstruction in advanced malignancy.

Gastric outlet obstruction (GOO) is a common problem associated with advanced malignancies of the upper gastrointestinal tract. Palliative treatment of patients' symptoms who present with GOO is an important aspect of their care. Surgical palliation of malignancy is defined as a procedure performed with the intention of relieving symptoms caused by an advanced malignancy or improving quality of life. Palliative treatment for GOO includes operative (open and laparoscopic gastrojejunostomy) and non-operative (endoscopic stenting) options. The performance status and medical condition of the patient, the extent of the cancer, the patients prognosis, the availability of a curative procedure, the natural history of symptoms of the disease (primary and secondary), the durability of the procedure, and the quality of life and life expectancy of the patient should always be considered when choosing treatment for any patient with advanced malignancy. Gastrojejunostomy appears to be associated with better long term symptom relief while stenting appears to be associated with lower immediate procedure related morbidity.

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

BACKGROUND: Inhibition of glycogen synthase kinase 3ß (GSK-3ß) 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-3ß inhibitor (GSK-3ß inhibited group [GSK-3ßI]), which were continued for 5 weeks, followed by myocardial tissue harvest. Coronary blood flow and vessel density were significantly increased in the GSK-3ßI group compared to the HCC group. Expression levels of the following proteins were greater in the GSK-3ßI group compared to the HCC group: vascular endothelial growth factor receptor 1 , vascular endothelial cadherin, γ-catenin, ß-catenin, protein kinase B, phosphorylated forkhead box O1, and superoxide dismutase 2. CONCLUSIONS: In the setting of metabolic syndrome, inhibition of GSK-3ß increases blood flow and vessel density in chronically ischemic myocardium. We identified several angiogenic, cell survival, and differentiation pathways that include ß-catenin signaling and AKT/FOXO1, through which GSK-3ß 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.

Alcohol modulates autophagy and apoptosis in pig liver tissue.

BACKGROUND: Autophagy serves as a cellular protective mechanism against alcohol-induced tissue injury but excessive autophagy can also be detrimental leading to apoptosis. Our laboratory has previously shown that moderate alcohol consumption alters expression of proteins in the insulin signaling pathway and worsens glucose metabolism in the liver in a swine model of metabolic syndrome. We examined the effect of alcohol consumption on apoptosis and autophagy signaling in the liver in our clinically relevant animal model of chronic hypercholesterolemia. MATERIAL AND METHODS: Twenty-six Yorkshire swine were fed a high-fat diet for 4 wks and were then split into three groups: hypercholesterolemic diet alone (HCC, n = 9), hypercholesterolemic diet with vodka (hypercholesterolemic vodka [HCV], n = 9), and hypercholesterolemic diet with wine (hypercholesterolemic wine [HCW], n = 8) for 7 wks. Animals underwent euthanasia, and liver tissue samples were harvested for analysis. Liver tissue was analyzed via Western blot analysis. Protein density data were normalized to GAPDH and is reported as fold-change values ± standard error of the mean compared to the high-cholesterol diet control group. A Kruskal-Wallis test with a Dunn's multiple comparison test was used to compare the means among groups. RESULTS: The HCV group showed significant increases in several proapoptotic proteins (including caspase 3, caspase 8, caspase 9, and cleaved caspase 9) compared with the HCC group. There was a decrease in the proapoptotic protein (BAD) and an increase in anti-apoptotic signal (B-cell lymphoma-2) in the HCW group compared with HCC control. There were increases in pro-survival proteins (AKT, p-AKT, mTOR, p-mTOR) in the HCW and the HCV group compared with control (HCC). There were decreases in autophagy protein LCB-3 in the HCW and HCV compared with the control. CONCLUSIONS: We found that moderate alcohol consumption altered protein expression related to apoptosis and autophagy signaling in pig liver in the setting of hypercholesterolemia. Interestingly, vodka may induce proapoptotic pathways in liver tissue, whereas wine may induce anti-apoptotic signaling. These results provide a mechanism by which vodka may contribute to alcoholic liver disease and supports the notion that wine, containing resveratrol, may prevent cellular apoptosis in liver tissue in the setting of hypercholesterolemia.

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.

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.