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ERK1/2 are known key players in the pathophysiology of heart failure, but the members of the ERK cascade, in particular Raf1, can also protect the heart from cell death and ischemic injury. An additional autophosphorylation (ERK1 at Thr208, ERK2 at Thr188) empowers ERK1/2 translocation to the nucleus and phosphorylation of nuclear targets which take part in the development of cardiac hypertrophy. Thereby, targeting this additional phosphorylation is a promising pharmacological approach.
In this thesis, an in silico model of ERK cascade in the cardiomyocyte is introduced. The model is a semi-quantitive model and its behavior was tested with different softwares (SQUAD and CellNetAnalyzer). Different phosphorylation states of ERK1/2 as well as different stimuli can be reproduced. The different types of stimuli include hypertrophic as well as non-hypertrophic stimuli. With the introduced in-silico model time courses and synergistic as well as antagonistic receptor stimuli combinations can be predicted. The simulated time courses were experimentally validated. SQUAD was mainly used to make predictions about time courses and thresholds, whereas CNA was used to analyze steady states and feedback loops.
Furthermore, new targets of ERK1/2 which partially contribute, also in the formation of cardiac hypertrophy, were identified and the most promising of them were illuminated. Important further targets are Caspase 8, GAB2, Mxi-2, SMAD2, FHL2 and SPIN90.
Cardiomyocyte gene expression data sets were analyzed to verify involved components and to find further significantly altered genes after induced hypertrophy with TAC (transverse aortic constriction). Changes in the ultrastructure of the cardiomyocyte are the final result of induced hypertrophy.
b-adrenergic receptors (b-ARs) participate strongly in the development of cardiac hypertrophy and human heart failure. Stimulation of b-adrenergic receptors with catecholamines as well as cardiac overexpression of b1-ARs or of Gas-proteins in transgenic mice induces cardiac hypertrophy. However, direct activation of their downstream targets, such as adenylyl cyclase (AC) or protein kinase A do not promote a significant degree of cardiac hypertrophy. These findings suggest that additional events may occur and that these events require Gas-protein activation. A hypertrophic pathway involving Gaq-protein coupled receptors has recently been described. Upon activation of Gaq-coupled receptors Gbg-subunits are released from Gaq and bind directly to the activated Raf/Mek/Erk cascade. Direct interaction between bg-subunits and activated Erk1/2 leads to an additional autophosphorylation of Erk2 at threonine 188, which mediates cardiac hypertrophy. Murine hearts, as well as isolated cardiomyocytes present an increase in Erk2Thr188-phosphorylation upon b-AR activation. Similarly overexpression of phosphorylation deficient Erk2 mutants (Erk2T188S and Erk2T188A) reduces b-AR mediated cardiomyocyte hypertrophy. Increase in left ventricular wall thickness, fibrosis and up-regulation of natriuretic peptide synthesis, which are physiological features for cardiac hypertrophy, are strongly inhibited in transgenic mice with a cardiac expression of Erk2T188S after two weeks of sustained isoproterenol treatment. It could further be shown in this work that b-AR mediated cardiac hypertrophy requires two distinct pathways initiated by Gs-protein activation: the canonical phosphorylation of Erk1/2 via adenylyl cyclase and the direct interaction of released bg-subunits with activated Erk1/2. Coincidence of both events leads to Erk2Thr188-phosphorylation, which activates then different transcription factors responsible for cardiac hypertrophy. Sequestration of bg-subunits by overexpression of the C-terminus of GRK2 bark-ct and inhibition of adenylyl cyclase efficiently reduced the hypertrophic response to isoproterenol, whereas direct activation of AC by forskolin failed to induce Erk2Thr188-phosphorylation and cardiomyocyte hypertrophy. These findings may help to develop new therapeutic strategies for the prevention of cardiac hypertrophy and maladaptive remodeling of the heart.
The incidence of cardiovascular diseases including cardiac hypertrophy and failure in pre-menopausal women is lower compared to age-matched men but the risk of heart disease increases substantially after the onset of menopause. It has been postulated that female sex hormones play an important role in cardiovascular health in pre-menopausal women. In animal studies including spontaneously hypertensive (SHR) rats, the development of cardiac hypertrophy is attenuated by 17β-estradiol treatment. Cardiac energy metabolism is crucial for normal function of the heart. In cardiac hypertrophy and heart failure, the myocardium undergoes a metabolic shift from fatty acid as primary cardiac energy source to glucose, which re-introduces the fetal type of metabolism that representing the glucose as a major source of energy. Many studies have reported that the disruption of the balance between glucose and fatty acid metabolism plays an important role in cardiac pathologies including hypertrophy, heart failure, diabetes, dilative cardiomyopathy and myocardial infarction. Glucose enters cardiomyocytes via GLUT1 and GLUT4 glucose transporters and GLUT4 is the major glucose transporter which is insulin-dependent. Cardiac-selective GLUT4 deficiency leads to cardiac hypertrophy. This shows that the decrease in cardiac glucose uptake may play a direct role in the pathogenesis of cardiac hypertrophy. Estrogens modulate glucose homeostasis in the liver and the skeletal muscle. But it is not known whether estrogens affect also cardiac glucose uptake which could provide another mechanism to explain the prevention of cardiac hypertrophy by female sex hormones. In the present study, SHR Rats were ovariectomized (OVX), not ovariectomized (sham) or ovariectomized and treated with subcutaneous 17β-estradiol. After 6 weeks of treatment, body weight, the serum levels of estrogen, insulin, intra-peritoneal glucose tolerance test (IP-GTT), myocardial glucose uptake by FDG-PET (2-(18F)-fluoro-deoxyglucose (18FDG) and Positron Emission Tomography), cardiac glucose transporter expression and localization and cardiac hexokinase activity were analyzed. As results of this study, PET analysis of female SHR revealed decreased cardiac glucose uptake in OVX animals compared to intact that was normalized by estrogen supplementation. Interestingly, there was no change in global glucose tolerance among the treatment groups. Serum insulin levels and cardiac hexokinase activity were elevated by E2 substitution. The protein content of cardiac glucose transporters GLUT-4 and GLUT-1, and their translocation as determined by fractionation studies and immuno-staining did not show any significant change by ovariectomy and estrogen replacement. Also levels of insulin receptor substrate-1 (IRS-1) and its tyrosine phosphorylation, which is required for activation and translocation of GLUT4, was un-affected in all groups of SHR. Cardiac gene expression analysis in SHR heart showed that ei4Ebp1 and Frap1 genes which are involved in the mTOR signaling pathway, were differentially expressed upon estrogen treatment. These genes are known to be activated in presence of glucose in the heart. As a conclusion of this study, reduced myocardial FDG uptake in ovariectomized spontaneously hypertensive rat is normalized by 17β-estradiol treatment. Increased myocardial hexokinase appears as a potential mechanism to explain increased myocardial glucose uptake by 17β-estradiol. Increased cardiac glucose uptake in response to 17β-estradiol in ovariectomized SHR may provide a novel mechanism to explain the reduction of cardiac hypertrophy in E2 treated SHR. Therefore, 17β-estradiol improves cardiac glucose utilization in ovariectomized SHR which may give rise to possible mechanism for its protective effects against cardiac hypertrophy.
Integrins are transmembrane receptors transmitting mechanical signals from the extracellular matrix (ECM) to the cytoskeleton (outside-in-signaling). Many molecular defects in the link between cytoskeleton and ECM are known to induce cardiomyopathies. alpha v integrin appears to play a major role in several processes relevant to remodeling, such as binding and activation of matrix metalloproteinases as well as regulation of cell proliferation, migration, and differentiation. We hypothesized that alpha v integrin-mediated signaling is required for the compensatory hypertrophy after aortic banding (AB) and associated with the modulation of ECM protein expression. Mice were treated in vivo with a specific integrin alpha v inhibitor or vehicle via osmotic minipumps starting 1 day prior to aortic banding (AB). At day 2 and day 7 following AB or sham-operation, the mice were examined by echocardiography and hemodynamic analyses were performed. Treatment of alpha v Integrin inhibitor led to a dilated cardiomyopathy and congestive heart failure in AB mice (dilated left ventricle, depressed LV function, and pulmonary congestion), but not to hypertrophy as observed in mice without inhibitor treatment. Investigation of downstream signaling revealed significant activation of the p38 Mitogen-Activated Protein Kinase (MAPK), the Extracellular signal-Regulated Kinases 1 and 2 (Erk 1/2), Focal Adhesion Kinase (FAK) and tyrosine-phosphorylation of c-Src in mice 7 days after AB. This response was blunted in mice treated with integrin alpha v inhibitor. Microarrays probing for a total of 96 cell adhesion and ECM genes identified various genomic targets of integrin alpha v mediated signalling. 7 days after AB 18 ECM genes were up-regulated more than 2-fold (n=6), e.g. collagen (8.11 ± 2.2), fibronectin (2.32 ± 0.94), secreted protein, acidic and rich in cysteine (SPARC, 3.78 ± 0.12), A disintegrin-like and metalloprotease (reprolysin type) with trombospondin type 1 (Adamts-1, 3.51 ± 0.81) and Tissue inhibitor of metalloproteinase 2 (TIMP2, 2.23 ± 0.98), whereas this up-regulation was abolished in mice that were treatd by integrin alpha v inhibitor via mini pumps. We conclude that signaling downstream of integrin alpha v is mediated by the MAPK, FAK and c-Src pathways leading to an up-regulation of extracelluar matrix components necessary for the compensatory response of the heart under a condition of pressure overload.