| PART ONEElevated expression of urotensin II and its receptor indiabetic cardiomyopathyBackgroundDiabetes is a major risk factor for cardiovascular disease. Increasing amount of evidence has accumulated for the presence of myocardial dysfunction in diabetic patients in the absence of discernible coronary artery, valvular or hypertensive heart disease. In particular, it has been recently shown that diabetes is independently associated with nonischemic cardiomyopathies. Several mechanisms for the pathogenesis of this diabetic cardiomyopathy (DCM) have been proposed. These include the metabolic derangement, potentially adverse effects of hyperglycemia on endothelial function, autonomic dysfunction, myocardial fibrosis and myocyte hypertrophy. Myocardial fibrosis and myocyte hypertrophy are the most frequently proposed mechanisms to explain cardiac changes in diabetic cardiomyopathy. Several vasoconstrictors have been involved in these processes, such as angiotensin II (Ang II) and endothelin-1 (ET-1).Recently, the novel vasoconstrictor peptide urotensin II (U II) has emerged as a likely contributor to cardiovascular physiology and pathology. U II is a somatostatin-like cyclic peptide synthesized by proteolytic cleavage from a precursor molecule, prepro-U II, and has been identified as the most potent vasoconstrictor in mammals. In humans, U II binds to a 389 amino acid G-protein coupled receptor termed UT. The G-protein associated with the UT receptor is of the Gq class, which is the same class of G-proteins that bind to angiotensin, endothelin, and α-adrenoceptors. UII induced both endothelium-independent vasoconstriction and endothelium-dependent vasorelaxation, the order and magnitude of which were dependent on the species tested and anatomical location. UII also exerted inotropic effects on the isolated human atrial trabeculea and mitogenic effects on smooth muscle cells. Bolus injection of UII into cynomolgus monkeys resulted in the development of cardiovascular collapse. More recently, a close relationship has been found between U II and congestive heart failure (CHF). Plasma levels of U II are elevated in patients with CHF compared with control subjects. Expression of U II and its receptor is increased in the myocardium of patients with end-stage CHF, in the myocardium of rats with myocardial infarction and in the myocardium of rats with chronic hypoxia induced-right ventricular hypertrophy. Blockage of the UT receptor reduced mortality and improved cardiac function in the rat model of myocardial infarction and CHF. In vitro, U II increases collagen synthesis of cardiac fibroblasts independently and stimulates cellular hypertrophy of cardiac myocytes in conditions of UT upregulation, and the UT receptor antagonist BIM-23127 can inhibit U II-induced hypertrophy in H9c2 cardiomyocytes. All of these suggest an important role for U II/ UT system in the pathogenesis of CHF and in the progression of cardiac remodeling.Besides its important role in cardiovascular system, U II is also a peptide that has been implicated in metabolic regulation and plays significant roles in diabetes and its complications. The U II gene is localized to 1p36-p32, one of the regions showing potential linkage with type 2 diabetes in Japanese affected sib-pairs. Wenyi showed that S89N polymorphism in the U II gene was associated with development of Type 2 diabetes via insulin sensitivity in the Japanese population. Ong found that haplotypes in the urotensin II gene and urotensin II receptor gene are associated with insulin resistance and impaired glucose tolerance. Following ICV infusion of U II in conscious sheep, plasma glucose increases by 7.0±1.4 mmol/L, compared with vehicle. In the rat pancreas, infusion of U II inhibits insulin's response to glucose through a direct influence of U II on the B-cells. Plasma U II levels are increased in Type 2 diabetic patients and further increased by renal failure. An increased expression of U II and UT was found in diabetic nephropathy. Long-term treatment of STZ-induced diabetic rats with U II receptor antagonist palosuran not only improves survival, increases insulin and slows the increase in glycemia, glycosylated hemoglobin and serum lipids, but also increases renal blood flow and delays the development of proteinuria and renal damage. All of these data strongly suggest an important role of U II in the pathophysiology of diabetes and diabetic nephropathy.Important role of U II/UT has been found in the pathophysiology of both CHF and diabetes, but whether this system plays a role in diabetic cardiomyopathy still remains unknown. Our aim is to investigate myocardial expression of U II and UT in the hearts of normal and diabetic rats. The data show for the first time that U II and UT are upregulated within diabetic myocardium.Objective1. To establish a DCM animal model.2. To observe the changes of cardiac structure and cardiac function in DCM model.3. To observe the expression of U II and UT in the myocardium of diabetic rats and controls.Methods1. Establishment of DCM animal modelTwenty-seven male Wistar rats were randomly divided into 2 groups: control group (n=12) and DCM group (n=15). Diabetes was induced by a single intraperitoneal injection of STZ (65 mg/kg body wt and dissolved in 0.1 mol/1 citrate buffer, pH 4.2) in rats of DCM group. Control rats received citrate buffer alone. One week after injection of STZ, fasting plasma glucose levels were measured, and rats with plasma glucose at least two times higher than 16.7mmol/L were used. All rats were fed for 5 months after STZ or citrate buffer injections and had free access to standard rat diet and water.2. Echocardiogram examinationAt the beginning and at the end of the study, transthoracic echocardiogram was performed in diabetic and control animals. Rats were placed supine and the anterior chest wall was shaved. Echocardiograms were performed with a Hewlett-Packard Sonos 7500 sector scanner equipped with a 7.5-MHz phased-array transducer. Conventional images included 2-dimensional, M-mode, and continuous wave and pulsed Doppler images.3. HE staining and Masson stainingHE staining was used to study the pathological changes and Masson staining was used to quantify the collagen content in this study.4. Real-time RT-PCRThe total RNA was extracted from left ventricles, right ventricles or atriums of control and diabetic rats. The mRNA expression of U II and UT were determined by real-time RT-PCR.5. ImmunohistochemistryImmunohistochemistry was used to detect the protein expression of U II and UT.6. Western-blot analysisWestern-blot analysis was used to determine the protein expression of UT.Results1. General features of the experimental ratsAt the end of the experiment, 11 diabetic rats induced by STZ and 12 control rats survived. Glucose levels were significantly elevated in diabetic rats compared with control rats after STZ injection. Other symptoms, such as lower body weights, polyuria and polyphagia, which are normally associated with diabetic state were also observed in the diabetic rats.2. Echocardiographic examinationAt the beginning of the study, no significant differences of echocardiogram were found between two groups. After 5 months period of diabetes, outstanding echocardiographic changes were detected in diabetic rats, including increased LVIDs and LVIDd; elevated E peak velocity and ration of E/A; decreased A peak velocity, FS and APV; prolongation of IVRT' and increased incidence of valvular regurgition.3. HE stainingThe myocytes from the control group arranged regularly. The size of the nuclear was uniform. The staining cytoplasm was homogeneous. The myocytes from the DCM group arranged irregularly. The nuclear was irregular and the interrupted myofibril arranged irregularly.4. Masson stainingAfter five months of diabetes, the cardiac collagen deposition was obviously enhanced in the diabetic groups compared with control groups.5. Myocardial expression of U II in DCMReal-time PT-PCR demonstrated a significant increase in U II mRNA transcripts in left ventricles, right ventricles and atriums of diabetic rats compared with controls. There is no difference of mRNA expression among different heart chambers of the same group.Immunohistochemistry using anti-U II antibody showed little or no U II protein expression in the normal rat heart. Occasionally, certain cell types, such as cardiomyocytes and endothelial cells expressed weak U II immunoreactivity. The myocardium of rats with DCM showed strong U II protein expression, which was mainly concentrated in cardiomyocytes, and to a lesser extent expressed in endothelial cells and cardiac fibroblasts. 6. Myocardial expression of UT in DCMSimilar to U II, real-time RT-PCR also demonstrated a significant increase in UT mRNA transcripts in left ventricles, right ventricles and atriums of diabetic rats compared with controls. There was also no difference of mRNA expression among different heart chambers of the same group.Immunohistochemistry using anti-UT antibody demonstrated weak UT protein expression in the normal rat heart, which was mainly located in cardiomyocytes. The myocardium of rats with DCM showed strong UT protein expression, which was concentrated in cardiomyocytes, cardiac fibroblasts, endothelial cells and smooth muscle cells.Western blot analysis demonstrated a significant increase in UT protein expression in left ventricles, right ventricles and atriums of diabetic rats compared with controls.Conclusions1. A DCM animal model was established by 5 months period of STZ-induced diabetes in male Wistar rats.2. The main histopathologic changes of DCM were collagen deposition and myocardial fibrosis.3. Diastolic dysfunction is characteristic in this DCM model.4. U II and UT expression were significantly enhanced in the myocardium of DCM group compared with healthy controls.Part TwoSignal transduction pathways in urotensin II-inducedcollagen synthesis in cardiac fibroblastsBackground Urotensin II (U II) is a somatostatin-like cyclic peptide synthesized by proteolytic cleavage from a precursor molecule, prepro-U II, and has been identified as the most potent vasoconstrictor in mammals. In humans, U II binds to a 389 amino acid G-protein coupled receptor termed GPR14, also known as UT. The G-protein associated with the UT receptor belongs to the Gq class, which is the same class of G-proteins that bind to angiotensin, endothelin, and α-adrenoceptors. Recently, U II has emerged as a likely contributor to cardiovascular physiology and pathology. U II induced endothelium-independent vasoconstriction and endothelium-dependent vasorelaxation, the orders and magnitudes of which were dependent on the species tested and anatomical location. U II also exerted inotropic effects on the isolated human atrial trabecular muscles. Bolus injection of U II into cynomolgus monkeys resulted in the development of cardiovascular collapse.In addition to its short-term roles which include vasoconstriction and chronotropic and inotropic effects on cardiac muscle, recently U II has been implicated in the long-term regulation of growth in the cardiovascular system. A number of studies have suggested an important role of U II in the development of cardiac remodeling. Plasma levels of U II were elevated in patients with congestive heart failure (CHF) compared with control subjects, and this increased plasma level of U II correlates with left ventricular end-diastolic pressure significantly. In vivo studies found that expression of U II and its receptor increased in the myocardium of patients with end-stage CHF, in the myocardium of rats with myocardial infarction and in the myocardium of rats with chronic hypoxia induced-right ventricular hypertrophy. In our previous study, it has been found that the expression of U II/UT system was significantly elevated in diabetic cardiomyopathy on both mRNA and protein levels. Blockade of the UT receptor can result in mortality reduction and cardiac function improvement in the rat model of myocardial infarction and CHF. Moreover, Tzanidis demonstrated that U II promoted collagen synthesis of cardiac fibroblasts independently and stimulated cellular hypertrophy of cardiac myocytes in conditions of UT upregulation. It is also found that the UT receptor antagonist BIM-23127 inhibited U II-induced hypertrophy in H9c2 cardiomyocytes. This U II -induced hypertrophy of cardiac myocytes is promoted by extracelluar signal-regulated kinase1/2 (ERK1/2) and p38 signaling pathways in an epidermal growth factor receptor-dependent manner. Nevertheless, the mechanisms of action of U II in cardiac remodeling, especially in cardiac fibrosis, are still incompletely understood. Further insights into the mechanisms of this effect will have important therapeutic implications.Objective1. To identify the function of U II in collagen synthesis in CFBs.2. To determine whether ERK1/2 pathway and TGF-β1 can be activated by U II in CFBs.3. To investigate the possible role of UT, ERK1/2 and TGF-β1 in U II-induced collagen synthesis of CFBs.Methods1. Cell cultureNeonatal rat cardiac fibroblasts were prepared by the following procedures: three to four hearts from 1- to 3-day-old Wistar rats were finely minced and placed together in 0.25% trypsin. Pooled cell suspensions were centrifuged and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The resuspension was plated onto culture flasks for 90 min, which allowed for preferential attachment of fibroblasts to the bottom of the culture flask. Non-adherent and weakly attached cells were removed and the medium was changed. Cells were grown to confluence and subsequently passaged 1:2 by trypsin. Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2/95% air. Studies were conducted on cardiac fibroblasts (passages two through four) that were grown to subconfluence in serum-containing media and then growth arrested for 24 h in serum-free medium before treatment.2. Study design1) To examine the function of U II on collagen synthesis, cells were stimulated with U II (10-9mol/L, 10-8mol/L and 10-7mol/L) for 24 h (for real-time RT-PCR) or for 48 h (for 3H-proline incorporation) before harvest, part of the cells were pretreated with urantide. Then real-time RT-PCR for type I ,III collagen and 3H-proline incorporation were performed and the concentration point for maximal effect of U II was used in subsequent experiments.2) To examine the function of U II on ERK1/2 activation, cells were treated with 10-7mol/L U II for 5 min- 60 min before harvest. Part of the cells were pretreated with urantide or PD98059. Then western blot was performed to evaluate U II -induced phosphorylation of ERK1/2.3) To examine the function of U II on TGF-β1 production, cells were incubated with 10-7mol/L U II for 4 h to 48 h, and then real-time RT-PCR and sandwish ELISAwere performed to determine the expression of TGF-β1. Pre-treatment of PD98059 was also used to examine the role of ERK1/2 in U II -induced TGF- β1 production.4) To examine the mechanisms involved in U II-induced collagen synthesis, cells were pretreated with PD98059 or TGF-β1 antibody or both of them, then stimulated with 10-7mol/L U II for 48 h and 3H-proline incorporation was performed.3. Real-time RT-PCRThe total RNA was extracted from collected cells. The mRNA expression of collagen I , collagen III and TGF-β1 were determined by real-time RT-PCR.4. Western-blot analysisThe total protein was extracted from collected cells.Western blot analysis was used to determine the protein expression of p-ERK1/2. 4. ELISA analysis ELISA analysis was used to determine the concentration of TGF-β1 in the supernatants collected from cell cultures. 5. 3H-proline incorporation3H-proline incorporation examination was used to determine the collagen synthesis in CFBs.Results1. Effects of U II on collagen synthesis by neonatal cardiac fibroblastsThe mRNA expression of both type I and type III collagen mRNA expression was significantly increased by U II in a concentration-dependent manner, with the maximal effect at 107mol/L. 3H-proline incorporation was also increased by U II in a concentration dependent manner with the same maximal effect at 10-7 mol/L. Pretreatment of urantide can significantly inhibited these U II-induced collagen synthesis in CFBs.2. Effects of U II on ERK1/2 activationU II stimulation resulted in a robust activation of ERK1/2 at 5-10 min and remained above basal up to 60 min after stimulation in CFBs. Pretreatment of urantide or PD98059 can significantly inhibited this U II-induced ERK1/2 activation.3. Induction of TGF-β1 by U IIIncubation of cardiac fibroblasts with U II (10-7mol/L) induced the expression of both TGF-β1 mRNA and protein in a time-dependent manner. This effect of U II on TGF-β1 mRNA expression began to increase at 4 h, reached a peak at 8 h, and then gradually decreased to the control level at 48 h. Meanwhile, protein expression started to increase at 12 h and reached a plateau at 24 h. Pretreatment of urantide or PD98059 can significantly inhibite this U II-induced TGF-β1 expression.4. Involvement of ERK1/2 activation and TGF-β1 production in UII-induced collagen synthesisTo determine whether or not ERK1/2 activation and TGF-β1 production are involved in U II-induced collagen synthesis, cardiac fibroblasts were pretreated with TGF-β1 antibody or PD98059 or both of them. Significant inhibition of UII -induced collagen synthesis was observed by using TGF-β1 antibody alone. U II-induced collagen synthesis was reduced about 70%, from 141% to 112% (P<0.01). Similar effect was also observed for ERK1/2 inhibitor PD98059. U II-induced collagen synthesis was reduced by about 50%, from 141% to 120% (P<0.05). Furthermore, the combined inhibition of TGF-β1 and ERK1/2 further reduced U II -induced collagen synthesis by about 90%, from 141% to 105% (P<0.01).Conclusions1. U II promoted collagen synthesis in CFBs in a dose-dependent manner.2. U II promoted ERK1/2 activation and TGF-β1 secrection in CFBs in a time-dependent manner.3. UT/ERK1/2/TGF-β1 pathway was probably involved in U II -induced collagen synthesis in CFBs.
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