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SEVENTH FRAMEWORK PROGRAMME

The Project - WP 12 Molecular pathways, cell cultures

Objectives

This part is focused on study of sarcoplasmic reticulum dysfunction and platelet regulatory systems at heart failure associated with diabetes mellitus.  At the end of the project we will demonstrate how interaction between sarcoplasmic reticulum function and contractile dysfunction of myocardium is altered during the development of heart failure associated with diabetes mellitus.  Data obtained during this project will show the role of insulin resistance, proinflammatory cytokines, prooxidant-antioxidant disbalance and cAMP-, NO/cGMP-dependent intracellular signaling systems in platelet hemostasis activation and platelet aggregation in the association of type 2 diabetes and heart failure. We will also show how metabolic alterations, such as hyperglycemia, hyperinsulinemia, hypoxia, altered production of adipokines and inflammatory conditions affect functional activity of progenitor cells, isolated from blood  and adipose tissue using in vitro models.

Objectives:

  • Evaluation the correlation between expression of phospholamban, Ca2+-ATPase SERCA, calseqvestrin, ryanodine receptors and contractile function post-infarct remodeled rat
    hearts and biopsy of human heart failure (ischemic and diabetic cardiomyopathies). 
  • Estimation the state of extracellular and intracellular signaling systems of platelets' aggregation regulation in vitro at failure of platelet functional activity in patients with the type 2 diabetes and heart failure.
  • Demonstrate how metabolic alterations, such as hyperglycemia, hyperinsulinemia, hyperlipidemia,  hypoxia and inflammatory conditions affect functional activity of progenitor cells using in vitro models of metabolic alterations.

Task 1. Investigate the force-frequency dependence diabetes remodeled rat hearts and human failing hearts associated with diabetes after alteration the sarcoplasmic reticulum Ca2+-handling proteins activity (Ca2+-ATPase and ryanodine receptors). 
Determination of contractile properties of myocardial preparations from human and rat failing hearts will be carried out. Experiments will be performed in right atrial trabeculae obtained from patients undergoing aortocoronary bypass operation and papillary muscle from myocardium of streptozotocin (STZ)-induced diabetic rats. Contractile activity of myocardium preparation will be recorded. The role of the sarcoplasmic reticulum (SR) in the inotropic responses will be investigated by post-extrasystole and post-rest tests. Isometric contraction will be evaluated by calculation of contraction amplitude (T), maximum contraction rate (+dT/dt), and maximum relaxation rate (-dT/dt).

Task 2. Evaluate the alterations in expression of sarcoplasmic reticulum Ca2+ handling proteins. The amount of SR Ca2+-ATPase, phospholamban and ryanodine receptor will be assessed using the Western blot technique.

Task 3. Investigate a prevailing type of energy substrate and intensity of processes of oxidative phosphorilation at diabetic cardiomyopathy. The oxidative phosphorylation speed in various conditions will be determined using polarografy and membrane potential measurements in diabetic remodeled rat hearts and biopsies of human heart failure associated with diabetes. Mitochondrial ATPase activity on inorganic phosphate will be measured on inorganic phosphate by spectrophotometric method then amount and degree of mitochondria swelling will be estimated by electronic microscopy.

Task 4. Estimate participation of platelet (NADPH-dehydrogenase platelet system) and extraplatelet enzyme systems (xanthine- xanthine-oxidase, glucose-glucoseoxidase) generating reactive oxygen species in regulation of platelets' aggregation activity in patients with combination of the type 2 diabetes and heart failure.

Task 5. Estimate the role of NO-dependent signaling pathway in the realization of cytokines' effect on the platelet aggregation activity in patients with combination of the type 2 diabetes and heart failure and in healthy volunteers. Isolated platelets will be incubated with tumor necrosis factor and interleukin 1 beta and inhibitor and activator of NO-synthase (NG-mono-methyl-L-arginine L-NMMA and L-arginine respectively).

Task 6. Study the state of cAMP-, cGMP-dependent intracellular signaling systems in the insulin-mediated regulation of platelet aggregation activity in patients with combination of the type 2 diabetes and heart failure and in healthy volunteers. Influence of insulin and NO-synthase blockers (NG-mono-methyl-L-arginine L-NMMA), cAMP- and cGMP-depending signaling pathway modifiers (forscolin, zaprinast, 3-isobutil-1-methyl xanthine, genistein, methylene blue, NG-mono-methyl-L-arginin) on the platelet aggregation activity is supposed to be evaluated.

Task 7. Assessment of progenitor cell functioning in  in vitro models of metabolic alterations. Circulating progenitor cells will be isolated from peripheral blood of healthy donors using immunomagnetic celection or preparative cell sorting. Adipose stromal cells (ASCs) will be obtained from liposuction patients. Cells will be cultured in conditions resembling DM/HF metabolic alterations, such as hyperglycemia, hyperinsulinemia, hypoxia, altered production of adipokines and inflammatory conditions. We will evaluate cell viability, proliferation and apoptosis using TUNEL assay, flow cytometry analysis of Annexin V binding and 7AAD incorporation. Also, we will study cell migration in Boyden chamber as well as in a "wound closure" model. Analysis of these models will be performed together with the evaluation of the expression and activity of the matrix degrading proteases (urokinase, MMPs, cathepsins). Furthermore, we will evaluate the cells' abilities to induce angiogenesis in the Matrigel implants, to incorporate into growing vessels as well as enhance capillary-like formation in vitro will be evaluated together with the analysis of growth factors expression and secretion profiling.

Workpackage Leader: Tomsk
Involved Partners: IRCCS, Hannover, St. Petersburg

Workpackage description

In human heart failure Ca2+ homeostasis is disturbed, as indicated by the smaller and more slowly decaying Ca2+ transients recorded from failing versus nonfailing myocytes. These Ca2+ transient derangements are thought to be largely responsible for the depressed contractility of the failing heart. Reduced SR function is described in various forms of hypertrophy and heart failure. Although it is agreed, that SR Ca2+ content is reduced in HF, the nature of this phenomenon remains controversial.

The project supposes study of calcium handling in heart failure developed during the ischemic and diabetes cardiomyopathy. Project will help to find opportunities to correct contractile dysfunction by modulation intracellular calcium homeostasis. Performance project will study the influence of antiarrhythmic drugs on SR function of remodeled and intact myocardium. The project will study contractile function post-infarct remodeled rat hearts and biopsy of human heart failure and alteration the sarcoplasmic reticulum function.  This project supposes study the alteration protein expression of the sarcoplasmic Ca2+-handling proteins, including sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban, ryanodine receptor in ischemic, idiopathic dilated and diabetic cardiomyopathies. Then the correlation between expression of phospholamban, Ca2+-ATPase SERCA, calseqvestrin, ryanodine receptors and cardiac contractile dysfunction will be evaluated. Moreover, influence of antiarrhythmic drugs on sarcoplasmic reticulum function of heart failure will be studied It is supposed to use the electrophysiology and biochemistry study myocardium preparation of intact rats, post-infarct remodeled rat hearts following left coronary occlusion and bioptates of patient with heart failure following ischemic and diabetic cardiomyopathies in this project.
Myocardium damage in the case of chronic heart failure and type 2 diabetes association is accompanied by the changes of vessels in the form of micro- and macroangiopathy and microcirculation failure. Platelet activation may play significant role in the heart failure progression because of microthrombus formation in the microvessels of myocardium. Several works demonstrated increased platelet sensitivity to different aggregation inducers, including ADP, thrombin and collagen, in patients with type 2 diabetes. According to many authors insulin resistance, nonenzymatic glycosylation, oxidative stress and inflammation may play the central role in disturbances of platelet hemostasis in patients with diabetes. Intracellular and extracellular systems of platelet functional activity regulation by which reactive oxygen species, proinflammatory cytokines and insulin may influence on the platelet aggregation activity in patients with association of type 2 diabetes and heart failure remain poorly studied. Reactive oxygen species (ROS) and nitric oxide, along with already well described factors, regulating platelet functions, are known to take part in the regulation of platelet aggregation activity.  According to experimental data platelets can generate both superoxide anion and nitric oxide. Type 2 diabetes and heart failure are considered to be free radical pathologies, which accompanied by oxidative stress characterized by disbalance in the prooxidant-antioxidant system. At the same time mechanisms by which ROS influence on platelet functional activity at diabetes and heart failure remain undiscovered.

Task 1. Investigate the force-frequency dependence diabetes remodeled rat hearts and human failing hearts associated with diabetes after alteration the sarcoplasmic reticulum Ca2+-handling proteins activity (Ca2+-ATPase and ryanodine receptors). Determination of contractile properties of myocardial preparations from human and rat failing hearts will be carried out. Experiments will be performed in right atrial trabeculae obtained from patients undergoing aortocoronary bypass operation and papillary muscle from myocardium of streptozotocin (STZ)-induced diabetic rats. Contractile activity of myocardium preparation (basic stimulation rate = 0.5 Hz) will be recorded. The role of the sarcoplasmic reticulum (SR) in the inotropic responses will be investigated by post-extrasystole and post-rest tests. Isometric contraction will be evaluated by calculation of contraction amplitude (T), maximum contraction rate (+dT/dt), and maximum relaxation rate (–dT/dt). Muscle strips will be superfused with normal Krebs-Henzelaite solution and after addition of ryanodine (1 microM), cyclopiazonic acid (10 microM) and thapsigargin (1 microM). Patients including in the trial will be chosen according to the standards elaborated by the WP03 to standardize the whole project. During the work on this task there will be used biopsy data and cellular research data, obtained by Doehner and Rosano (WP07 and WP08).

Task 2. Evaluate the alterations in expression of sarcoplasmic reticulum Ca2+ handling proteins. The amount of SR Ca2+-ATPase, phospholamban and ryanodine receptor will be assessed using the Western blot technique. Data obtained during this part of the trial will be essential for the progress of WP08, in particular to estimate the alterations of contractile ability.

Task 3. Investigate a prevailing type of energy substrate and intensity of processes of oxidative phosphorilation at diabetic cardiomyopathy. The oxidative phosphorylation speed in various conditions will be determined using polarografy and membrane potential measurements in diabetic remodeled rat hearts and biopsies of human heart failure associated with diabetes. Mitochondrial ATPase activity on inorganic phosphate will be measured on inorganic phosphate by spectrophotometric method then amount and degree of mitochondria swelling will be estimated by electronic microscopy. Data obtained during the work on the task 3 will be important for the progress of WP07 and WP08, specifically in order to estimate the energetic metabolism of the heart during heart failure.

Task 4. Estimate participation of platelet (NADPH-dehydrogenase platelet system) and extraplatelet enzyme systems (xanthine- xanthine-oxidase, glucose-glucoseoxidase) generating reactive oxygen species in regulation of 'platelets' aggregation activity in patients with combination of the type 2 diabetes and heart failure. Platelets will be isolated from platelet reach plasma, obtained from the venous blood. Platelet suspension is washed twice in buffer pH 7.4, containing 136.8 mM NaCl, 2.8 mM KCl, 11.9 mM NaHCO3, 1.1 mM MgCl2, 0.33 mM NaH2PO4, 1 mM CaCl2, 11.2 mM glucose and 3.5 mg/ml BSA. The final concentration of platelets in suspension for further aggregation studies is 440106 cells in 1 ml of buffer. Aggregation activity of isolated platelets and platelet reach plasma will be studied by turbidimetric method using dual-channel laser aggregometer (220 LA "NPF Biola", Russia) with simultaneous registration of light transmission and mean radius of aggregates. The following aggregation inducers will be used: collagen, thrombin, ADP, epinephrine, sodium arachidonate (Sigma). The following parameters of aggregation will be estimated: degree and rate of aggregation according to the light transmission and mean radius curves. Platelets will be incubated with NADPH-oxidase inhibitor – diphenyleneiodonium, and with such systems as xanthine – xanthine-oxidase, glucose-glucose-oxidase. It was discovered recently that proinflammatory cytokines may influence on the platelet aggregation activity. Thus in patients with heart failure, characterized by the high level of tumor necrosis factor in plasma, intensification of collagen-induced platelet aggregation was revealed. There are experimental data, that tumor necrosis factor in high concentrations (lowers platelet aggregation activity. It is supposed that proinflammatory cytokines realize their antithrombotic action through modulation of nitric oxide synthesis by inducible NOS in platelets. At the same time the role of NO-dependent signaling pathway in realization of cytokine effects on the platelet aggregation activity during diabetes mellitus and heart failure, pathologies characterized by the increased secretion of proinflammatory cytokines, is still studied insufficiently. Data obtained during the work on the task 4 will contribute to the work of WP07 and WP12, and will help to create the integrated vision of processes taking part in the circulation during heart failure.

Task 5. Estimate the role of NO-dependent signaling pathway in the realization of cytokines' effect on the platelet aggregation activity in patients with combination of the type 2 diabetes and heart failure and in healthy volunteers. Isolated platelets will be incubated with tumor necrosis factor and interleukin 1 beta and inhibitor and activator of NO-synthase (NG-mono-methyl-L-arginine L-NMMA and L-arginine respectively). Platelets are known to contain receptors for insulin which decreases 'platelets' sensitivity to aggregation inducers in healthy people. It is ascertained that realization of antiaggregatory insulin effects may be mediated by different intracellular mechanisms both by increased level of cAMP and cGMP. Decreasing of aggregation in patients with type 2 diabetes is observed only at very high insulin concentrations. Nowadays it is known that the cause of lowering of insulin antiaggregation effect on platelets of patients with diabetes is the decreasing of platelet receptors' number or decreasing of platelet sensitivity to insulin, caused by the failure of intracellular insulin signaling pathway. Data obtained on this stage of the trial will contribute to the progress of WP07 and display the degree of immunologic activation during the heart failure.

Task 6. Study the state of cAMP-, cGMP-dependent intracellular signaling systems in the insulin-mediated regulation of platelet aggregation activity in patients with combination of the type 2 diabetes and heart failure and in healthy volunteers. Influence of insulin and NO-synthase blockers (NG-mono-methyl-L-arginine L-NMMA), cAMP- and cGMP-depending signaling pathway modifiers (forscolin, zaprinast, 3-isobutil-1-methyl xanthine, genistein, methylene blue, NG-mono-methyl-L-arginin) on the platelet aggregation activity is supposed to be evaluated. Data obtained during the work on this task will be essential for the progress of WP07. In particular we will reveal one of the possible mechanisms of insulin resistance during heart failure.

Task 7. Assessment of progenitor cell functioning in  in vitro models of metabolic alterations. Circulating progenitor cells will be isolated from peripheral blood of healthy donors using immunomagnetic celection or preparative cell sorting. Adipose stromal cells (ASCs) will be obtained from liposuction patients. Cells will be cultured in conditions resembling DM/HF metabolic alterations, such as hyperglycemia, hyperinsulinemia, hypoxia, altered production of adipokines and inflammatory conditions. We will evaluate cell viability, proliferation and apoptosis using TUNEL assay, flow cytometry analysis of Annexin V binding and 7AAD incorporation. Also, we will study cell migration in Boyden chamber as well as in a "wound closure" model. Analysis of these models will be performed together with the evaluation of the expression and activity of the matrix degrading proteases (urokinase, MMPs, cathepsins). Furthermore, we will evaluate the cells' abilities to induce angiogenesis in the Matrigel implants, to incorporate into growing vessels as well as enhance capillary-like formation in vitro will be evaluated together with the analysis of growth factors expression and secretion profiling. Basing on these data we will develop a scheme for the evaluation of the functional activity of progenitor cells in patients with CHF complicated by diabetes mellitus or the metabolic syndrome. CT will interact closely with Moscow State University and Almazov Heart, Blood and Endocrinology Centre working on this task to achieve a rapid progress through the study. Specifically, we will combine our data on proliferative capacity and differentiation abilities of progenitor cells to work out a system of criteria for the assessment of functional activity of those cells in patients with HF complicated by type 2 diabetes mellitus or other metabolic disorders. We will also exchange our data/experience with JJ working on this task. These data will also be utilized in WP14 for understanding the possibilities to use autologous cells for cell therapy in patients with heart failure.

WP 12 Molecular pathways, cell cultures