M. Ruhul Abid

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.

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.

Antioxidant Therapy: Is it your Gateway to Improved Cardiovascular Health?

General use and popularity of over-the-counter supplemental antioxidants have rapidly spread all over the world and are believed to promote cardiovascular health and wellbeing. However, there is a paucity of information and lack of proof that physiological and above-physiological levels of oxidants do harm at the cellular and organismal levels. Instead, several reports demonstrated that reduction in Reactive Oxygen Species (ROS) did not improve vascular function. Interestingly, recent studies show that increased ROS levels play protective role in vascular endothelium and may improve coronary endothelial function. In the current review, we introduce the concept that increased ROS levels, often seen in association with cardiovascular disease, probably is an endothelial-way or ‘oxidative response’ to cope with vascular pathology.

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.

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

A disease burden analysis of garment factory workers in Bangladesh: proposal for annual health screening

Background To characterize the health status of selected garment and textile factory workers in Bangladesh using a workplace mobile health clinic. Methods A retrospective review of de-identified worker health information was performed using 1906 medical records. Results The mean age of the workers was 27.9±7.3 y, with 60.3% (1150/1906) female and 39.7% (756/1906) male workers. The most common medical conditions identified were anemia (19.0%; 362/1906), elevated BP (EBP) (11.9%; 226/1906) and elevated fasting blood glucose (FBG) (8.0%; 151/1906). Among the workers who had EBP, 86.3% (195/226) were previously undiagnosed, while out of the workers with elevated FBG, 72.8% (110/151) were newly diagnosed. Men were more likely than women to have EBP (OR 3.5, 95% CI [2.12-5.56], p<0.001), a family history of diabetes (OR 3.6, 95% CI [2.1-5.9], p<0.001) and no formal education (OR 4.9, 95% CI [3.6-6.7], p<0.001). Conclusions Despite the relatively young mean age, significant percentages of workers were identified as having undiagnosed chronic health conditions, including EBP and elevated FBG, that require urgent medical attention and health education. The findings suggest that provision for annual health screening, either by mobile on-site clinics or by training the existing in-house paramedics, will help improve health of the factory workers.

Role of Calpains (Calcium-Dependent Proteases) on Coronary Artery Disease and Metabolic Syndrome

Coronary artery disease and metabolic syndrome together are two major causes of death in the United States and the numbers of people afflicted by these diseases are increasing around the world. Overactivation of calpain has been shown to contribute to cardiovascular disease and its associated comorbidities. Therefore, calpain inhibition may offer a novel potential medical therapy for treating not only coronary artery disease but also the individual aspects of the associated comorbidities.