Dissociation of natriuresis and diuresis by oxytocin molecular forms in rats.

In the rat, oxytocin (OT) produces dose-dependent diuretic and natriuretic responses. Post-translational enzymatic conversion of the OT biosynthetic precursor forms both mature and C-terminally extended peptides. The plasma concentrations of these C-terminally extended peptides (OT-G; OT-GK and OT-GKR) are elevated in newborns and pregnant rats. Intravenous injection of OT-GKR to rats inhibits diuresis, whereas injection of amidated OT stimulates diuresis. Since OT and OT-GKR show different effects on the urine flow, we investigated whether OT-GKR modulates renal action by inhibition of the arginine-vasopressin (AVP) receptor V2 (V2R), the receptor involved in renal water reabsorption. Experiments were carried out in the 8-week-old Wistar rats receiving intravenous (iv) injections of vehicle, OT, OT-GKR or OT+OT-GKR combination. OT (10 µmol/kg) increased urine outflow by 40% (P<0.01) and sodium excretion by 47% (P<0.01). Treatment with OT-GKR (10 µmol/kg) decreased diuresis by 50% (P<0.001), decreased sodium excretion by 50% (P<0.05) and lowered potassium by 42% (P<0.05). OT antagonist (OTA) reduced diuresis and natriuresis exerted by OT, whereas the anti-diuretic effect of OT-GKR was unaffected by OTA. The treatment with V2R antagonist (V2A) in the presence and absence of OT induced diuresis, sodium and potassium outflow. V2A in the presence of OT-GKR only partially increased diuresis and natriuresis. Autoradiography and molecular docking analysis showed potent binding of OT-GKR to V2R. Finally, the release of cAMP from CHO cells overexpressing V2 receptor was induced by low concentration of AVP (EC50:4.2e-011), at higher concentrations of OT (EC50:3.2e-010) and by the highest concentrations of OT-GKR (EC50:1.1e-006). OT-GKR potentiated cAMP release when combined with AVP, but blocked cAMP release when combined with OT. These results suggest that OT-GKR by competing for the OT renal receptor (OTR) and binding to V2R in the kidney, induces anti-diuretic, anti-natriuretic, and anti-kaliuretic effects.

Oxytocin treatment prevents the cardiomyopathy observed in obese diabetic male db/db mice.

Oxytocin (OT) is involved in the regulation of energy metabolism and in the activation of cardioprotective mechanisms. We evaluated whether chronic treatment with OT could prevent the metabolic and cardiac abnormalities associated with diabetes and obesity using the db/db mice model. Four-week-old male db/db mice and their lean nondiabetic littermates (db/+) serving as controls were treated with OT (125 ng/kg · h) or saline vehicle for a period of 12 weeks. Compared with db/+ mice, the saline-treated db/db mice developed obesity, hyperglycemia, and hyperinsulinemia. These mice also exhibited a deficient cardiac OT/natriuretic system and developed systolic and diastolic dysfunction resulting from cardiomyocyte hypertrophy, fibrosis, and apoptosis. These abnormalities were associated with increased reactive oxygen species (ROS) production, inflammation, and suppressed 5'-adenosine monophosphate kinase signaling pathway. The db/db mice displayed reduced serum levels of adiponectin and adipsin and elevated resistin. OT treatment increased circulating OT levels, significantly reduced serum resistin, body fat accumulation (19%; P<.001), fasting blood glucose levels by (23%; P<.001), and improved glucose tolerance and insulin sensitivity. OT also normalized cardiac OT receptors, atrial natriuretic peptide, and brain natriuretic peptide, expressions and prevented systolic and diastolic dysfunction as well as cardiomyocyte hypertrophy, fibrosis, and apoptosis. Furthermore, OT reduced cardiac oxidative stress and inflammation and normalized the 5'-adenosine monophosphate-activated protein kinase signaling pathway. The complete normalization of cardiac structure and function by OT treatment in db/db mice contrasted with only partial improvement of hyperglycemia and hyperinsulinemia. These results indicate that chronic treatment with OT partially improves glucose and fat metabolism and reverses abnormal cardiac structural remodeling, preventing cardiac dysfunction in db/db mice.

All-trans retinoic acid stimulates gene expression of the cardioprotective natriuretic peptide system and prevents fibrosis and apoptosis in cardiomyocytes of obese ob/ob mice.

In hypertensive rodents, retinoic acid (RA) prevents adverse cardiac remodelling and improves myocardial infarction outcome, but its role in obesity-related changes of cardiac tissue are unclear. We hypothesized that all-trans RA (ATRA) treatment will improve the cardioprotective oxytocin-natriuretic peptides (OT-NP) system, preventing apoptosis and collagen accumulation in hearts of ob/ob mice, a mouse model of obesity and insulin resistance. Female 9-week-old B6.V-Lep/J ob/ob mice (n = 16) were divided into 2 groups: 1 group (n = 8) treated with 100 µg of ATRA dissolved in 100 µL of corn oil (vehicle) delivered daily (∼2 µg·g body weight(-1)·day(-1)) by stomach intubation for 16 days, and 1 group (n = 8) that received the vehicle alone. A group of nonobese littermate mice (n = 9) served as controls. Ob/ob mice exhibited obesity, hyperglycaemia, and downregulation of the cardiac OT-NP system, including the mRNA for the transcription factor GATA4, OT receptor and brain NP, and the protein expression for endothelial nitric oxide synthase. Hearts from ob/ob mice also demonstrated increased apoptosis and collagen accumulation. ATRA treatment induced weight loss and decreased adipocytes diameter in the visceral fat, thus reducing visceral obesity, which is associated with a high risk for cardiovascular disease. RA treatment was associated with a reduction in hyperglycemia and a normalization of the OT-NP system's expression in the hearts of ob/ob mice. Furthermore, ATRA treatment prevented apoptosis and collagen accumulation in hearts of ob/ob mice. The present study indicates that ATRA treatment was effective in restoring the cardioprotective OT-NP system and in preventing abnormal cardiac remodelling in the ob/ob mice.

Treatment with brain natriuretic peptide prevents the development of cardiac dysfunction in obese diabetic db/db mice.

AIMS/HYPOTHESIS: Obesity and diabetes increase the risk of developing cardiovascular diseases and heart failure. These metabolic disorders are generally reflected by natriuretic peptide system deficiency. Since brain natriuretic peptide (BNP) is known to influence metabolism and cardioprotection, we investigated the effect of chronic exogenous BNP treatment on adverse myocardial consequences related to obesity and diabetes. METHODS: Ten-week-old C57BL/KsJ-db/db obese diabetic mice (db/db) and their lean control littermates (db/+) were treated with BNP (0.6 µg kg(-1) h(-1)) or saline for 12 weeks (n = 10/group). Serial blood and tomography analysis were performed. Cardiac function was determined by echocardiography, and biochemical and histological heart and fat analyses were also performed. RESULTS: BNP treatment resulted in an average increase in plasma BNP levels of 70 pg/ml. An improvement in the metabolic profile of db/db mice was observed, including a reduction in fat content, increased insulin sensitivity, improved glucose tolerance and lower blood glucose, despite increased food intake. db/db mice receiving saline displayed both early systolic and diastolic dysfunction, whereas these functional changes were prevented by BNP treatment. The cardioprotective effects of BNP were attributed to the inhibition of cardiomyocyte apoptosis, myocardial fibrosis, cardiac hypertrophy and the AGE-receptor for AGE (RAGE) system as well as normalisation of cardiac AMP-activated protein kinase and endothelial nitric oxide synthase activities. CONCLUSIONS/INTERPRETATION: Our results indicate that chronic BNP treatment at low dose improves the metabolic profile and prevents the development of myocardial dysfunction in db/db mice.

Oxytocin-Gly-Lys-Arg stimulates cardiomyogenesis by targeting cardiac side population cells.

The functional oxytocin (OT) system is expressed in the human and rodent hearts. OT stimulates differentiation of cardiac stem cells into contracting cardiomyocytes (CM). In this study, we investigated OT receptors (OTR) expressed in the cells of cardiac side population (SP) and the abilities of these cells to differentiate into CM in response to the treatment with OT-Gly-Lys-Arg (OT-GKR), a dominant and biologically active form of OT, in the fetal rodent heart. Immunocytochemistry of whole rat embryo at mid gestation (E11) revealed parallel staining in the heart of OTR and the ATP-binding cassette sub-family G member 2 (brcp1) antigen the marker of the SP phenotype. Using flow cytometry, the SP cells were selected from the newborn CM stained with Höechst 33342: 5.32%±0.06% of SP and 15.2%±1.10 of main population expressed OTR on the cell surface. The OTR was detected in CD29 (6.6%) and then in CD31 (4.7%) but less frequently in CD45 (0.7%) positive SP cell subpopulations. Specifically, the phenotype of SP CD31- cell, but not SP CD31+ cells, proliferates in the presence of OT-GKR and develops large cell aggregates. Then, OT-GKR treatment induced the apparition of beating cell colonies after 11 days (10±2.78%), which increased until day 16 (52±1.21%). The cells in contractile colonies expressed the markers of a CM phenotype, such as troponin, cardiac myosin light chain-2, and actinin. Finally, SP cells stimulated by OT-GKR induced endothelial phenotype. These results suggest that the C-terminally extended OT molecule stimulates cardiac differentiation of SP CD31- cells and is involved in heart growth.

Preconditioning of stem cells by oxytocin to improve their therapeutic potential.

Principal limitation of cell therapy is cell loss after transplantation because of the interplay between ischemia, inflammation, and apoptosis. We investigated the mechanism of preconditioning of mesenchymal stem cells (MSCs) with oxytocin (OT), which has been proposed as a novel strategy for enhancing therapeutic potential of these cells in ischemic heart. In this study, we demonstrate that rat MSCs express binding sites for OT receptor and OT receptor transcript and protein as detected by RT-PCR and immunofluorescence, respectively. In response to OT (10(-10) to 10(-6) M) treatment, MSCs respond with rapid calcium mobilization and up-regulation of the protective protein kinase B (PKB or Akt) and phospho-ERK1/2 proteins. In OT-stimulated cells, phospho-Akt accumulates intracellularly close to the mitochondrial marker cytochrome c oxidase subunit 4. Functional analyses reveal the involvement of Akt/ERK1/2 pathways in cell proliferation, migration, and protection against the cytotoxic and apoptotic effects of hypoxia and serum deprivation. In addition, OT preconditioning increases MSC glucose uptake. Genes with angiogenic, antiapoptotic, and cardiac antiremodeling properties, such as heat shock proteins (hsps) HSP27, HSP32, HSP70, vascular endothelial growth factor, thrombospondin, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, and matrix metalloproteinase-2, were also up-regulated upon OT exposure. Moreover, coculture with OT-preconditioned MSC reduces apoptosis, as measured using terminal transferase dUTP nick end labeling assay in newborn rat cardiomyocytes exposed to hypoxia and reoxygenation. In conclusion, these results indicate that OT treatment evokes MSC protection through both intrinsic pathways and secretion of cytoprotective factors. Ex vivo cellular treatment with OT represents an attractive strategy aimed to maximize the biological and functional properties of effector cells.

Downregulation in GATA4 and Downstream Structural and Contractile Genes in the db/db Mouse Heart.

Reduced expression of GATA4, a transcriptional factor for structural and cardioprotective genes, has been proposed as a factor contributing to the development of cardiomyopathy. We investigated whether the reduction of cardiac GATA4 expression reported in diabetes alters the expression of downstream genes, namely, atrial natriuretic peptide (ANP), B-type natriuretic, peptide (BNP), and α- and ß-myosin heavy chain (MHC). db/db mice, a model of type 2 diabetes, with lean littermates serving as controls, were studied. db/db mice exhibited obesity, hyperglycemia, and reduced protein expression of cardiac GLUT4 and IRAP (insulin-regulated aminopeptidase), the structural protein cosecreted with GLUT4. Hearts from db/db mice had reduced protein expression of GATA4 (~35%) with accompanying reductions in mRNA expression of ANP (~40%), BNP (~85%), and α-MHC mRNA (~50%) whereas expression of ß-MHC mRNA was increased by ~60%. Low GATA4 was not explained by an increased ligase or atrogin1 expression. CHIP protein content was modestly downregulated (27%) in db/db mice whereas mRNA and protein expression of the CHIP cochaperone HSP70 was significantly decreased in db/db hearts. Our results indicate that low GATA4 in db/db mouse heart is accompanied by reduced expression of GATA4-regulated cardioprotective and structural genes, which may explain the development of cardiomyopathy in diabetes.

Changes in cardiac structure in hypertension produced by placental ischemia in pregnant rats: effect of tumor necrosis factor blockade.

OBJECTIVES: Chronic reduction of uteroplacental perfusion pressure (RUPP) in pregnant rats leads to placental ischemia, maternal endothelial cell dysfunction, hypertension and elevated levels of tumor necrosis factor-alpha (TNF-α). In this study we investigated the hypothesis that placental ischemia in pregnant rat, a model of preeclampsia, stimulates cardiac hypertrophy and fibrosis via a TNF-α-dependent mechanism. METHODS: Normal pregnant Sprague-Dawley rats and RUPP rats were evaluated on day 19 of gestation. To test the role of TNF-α in mediating change in the RUPP rat heart, a TNF-α inhibitor, etanercept, was administered on day 18 of gestation at a dose of 0.8 mg/kg, s.c. RESULTS: In comparison to normal pregnant rats, RUPP animals display enlarged cardiomyocytes, microvascular rarefaction, fibrosis, apoptosis as well as increased expression of markers of heart hypertrophy and fibrosis. Etanercept (E) treatment prevented enlargement of cardiomyocytes, fibrosis and apoptosis and this was accompanied by significantly lowered blood pressure in RUPP rats. Etanercept treatment lowered expression of mRNA for brain natriuretic peptide, a marker of cardiac hypertrophy. It also heightened expression of endothelial nitric oxide synthase and its phosphorylation as well as oxytocin receptor identified in cardiac microvessels. TNF-α inhibition prevented microvascular rarefaction in the heart as indicated by augmented CD31, a marker of angiogenesis. CONCLUSIONS: These results suggest that RUPP leads to microvascular rarefaction in the heart, exaggerated cardiomyocyte size, apoptosis, fibrosis, and the alteration of cardiac gene expression that are modulated by the inflammatory cytokine TNFα.

Oxytocin-Gly-Lys-Arg: a novel cardiomyogenic peptide.

BACKGROUND: Oxytocin (OT), synthesized in the heart, has the ability to heal injured hearts and to promote cardiomyogenesis from stem cells. Recently, we reported that the OT-GKR molecule, a processing intermediate of OT, potently increased the spontaneous formation of cardiomyocytes (CM) in embryonic stem D3 cells and augmented glucose uptake in newborn rat CM above the level stimulated by OT. In the present experiments, we investigated whether OT-GKR exists in fetal and newborn rodent hearts, interacts with the OT receptors (OTR) and primes the generation of contracting cells expressing CM markers in P19 cells, a model for the study of early heart differentiation. METHODOLOGY/PRINCIPAL FINDINGS: High performance liquid chromatography of newborn rat heart extracts indicated that OT-GKR was a dominant form of OT. Immunocytochemistry of mouse embryos (embryonic day 15) showed cardiac OT-GKR accumulation and OTR expression. Computerized molecular modeling revealed OT-GKR docking to active OTR sites and to V1a receptor of vasopressin. In embryonic P19 cells, OT-GKR induced contracting cell colonies and ventricular CM markers more potently than OT, an effect being suppressed by OT antagonists and OTR-specific small interfering (si) RNA. The V1a receptor antagonist and specific si-RNA also significantly reduced OT-GKR-stimulated P19 contracting cells. In comparison to OT, OT-GKR induced in P19 cells less α-actinin, myogenin and MyoD mRNA, skeletal muscle markers. CONCLUSIONS/SIGNIFICANCE: These results raise the possibility that C-terminally extended OT molecules stimulate CM differentiation and contribute to heart growth during fetal life.

Nitric oxide signaling in oxytocin-mediated cardiomyogenesis.

Oxytocin (OT), a hormone recently identified in the heart, induces embryonic and cardiac somatic stem cells to differentiate into cardiomyocytes (CM), possibly through nitric oxide (NO). We verified this hypothesis using P19 cells and P19 Clone 6 derivatives expressing a green fluorescent protein (GFP) reporter linked to cardiac myosin light chain-2v promoter. OT treatment of these cells induced beating cell colonies that were fully inhibited by N,G-nitro-L-arginine-methyl-ester (L-NAME), an inhibitor of NO synthases (NOS), partially reduced by 1400W, an inhibitor of inducible NOS, and ODQ, an inhibitor of NO-sensitive guanylyl cyclases. The NO generator S-nitroso-N-acetylpenicillamine (SNAP) reversed the L-NAME inhibition of cell beating and GFP expression. In OT-induced cells, L-NAME significantly decreased transcripts of the cardiac markers Nkx2.5, MEF2c, alpha-myosin heavy chain, and less, GATA4, endothelial NOS, and atrial natriuretic peptide, as well as the skeletal myocyte (SM) marker myogenin. Image analysis of OT-induced P19Cl6-GFP cells revealed ventricular CM coexpressing sarcomeric alpha-actinin and GFP, with some cells exclusively expressing alpha-actinin, most likely of the SM phenotype. The OT-mediated production of CM, but not SM, was diminished by L-NAME. In P19 cells, exogenously added OT stimulated the expression of its own transcript, which was reduced in the presence of L-NAME. Surprisingly, L-NAME alone decreased the expression of anti-stage specific embryonic antigen-1 marker of the undifferentiated state and induced some beating colonies as well as GFP in P19Cl6-GFP cells. Collectively, our data suggest that the pleiotropic action of NO is involved in the initiation of CM differentiation of P19 cells and maintenance of their undifferentiated state.

Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes.

We recently discovered the existence of the oxytocin/oxytocin receptor (OT/OTR) system in the heart. Activation of cardiac OTR stimulates the release of atrial natriuretic peptide (ANP), which is involved in regulation of blood pressure and cell growth. Having observed elevated OT levels in the fetal and newborn heart at a stage of intense cardiomyocyte hyperplasia, we hypothesized a role for OT in cardiomyocyte differentiation. We used mouse P19 embryonic stem cells to substantiate this potential role. P19 cells give rise to the formation of cell derivatives of all germ layers. Treatment of P19 cell aggregates with dimethyl sulfoxide (DMSO) induces differentiation to cardiomyocytes. In this work, P19 cells were allowed to aggregate from day 0 to day 4 in the presence of 0.5% DMSO, 10(-7) M OT and/or 10(-7) M OT antagonist (OTA), and then cultured in the absence of these factors until day 14. OT alone stimulated the production of beating cell colonies in all 24 independently growing cultures by day 8 of the differentiation protocol, whereas the same result was obtained in cells induced by DMSO only after 12 days. Cells induced with OT exhibited increased ANP mRNA, had abundant mitochondria (i.e., they strongly absorbed rhodamine 123), and expressed sarcomeric myosin heavy chain and dihydropyridine receptor-alpha 1, confirming a cardiomyocyte phenotype. In addition, OT as well as DMSO increased OTR protein and OTR mRNA, and OTA completely inhibited the formation of cardiomyocytes in OT- and DMSO-supplemented cultures. These results suggest that the OT/OTR system plays an important role in cardiogenesis by promoting cardiomyocyte differentiation.