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Aims Sodium-glucose cotransporter 2 inhibitors have beneficial effects on heart failure and cardiovascular mortality in diabetic and non-diabetic patients, with unclear mechanisms. Autophagy is a cardioprotective mechanism under acute stress conditions, but excessive autophagy accelerates myocardial cell death leading to autosis. We evaluated the protective role of empagliflozin (EMPA) against cardiac injury in murine diabetic cardiomyopathy. Methods and results Male mice, rendered diabetics by one single intraperitoneal injection of streptozotocin and treated with EMPA (30 mg/kg/day), had fewer apoptotic cells (4.9 ± 2.1 vs. 1 ± 0.5 TUNEL-positive cells %, P < 0.05), less senescence (10.1 ± 2 vs. 7.9 ± 1.2 β-gal positivity/tissue area, P < 0.05), fibrosis (0.2 ± 0.05 vs. 0.15 ± 0.06, P < 0.05 fibrotic area/tissue area), autophagy (7.9 ± 0.05 vs. 2.3 ± 0.6 fluorescence intensity/total area, P < 0.01), and connexin (Cx)-43 lateralization compared with diabetic mice. Proteomic analysis showed a down-regulation of the 5′ adenosine monophosphate-activated protein kinase (AMPK) pathway and upstream activation of sirtuins in the heart of diabetic mice treated with EMPA compared with diabetic mice. Because sirtuin activation leads to the modulation of cardiomyogenic transcription factors, we analysed the DNA binding activity to serum response elements (SRE) of serum response factor (SRF) by electromobility shift assay. Compared with diabetic mice [0.5 ± 0.01 densitometric units (DU)], non-diabetic mice treated with EMPA (2.2 ± 0.01 DU, P < 0.01) and diabetic mice treated with EMPA (2.0 ± 0.1 DU, P < 0.01) significantly increased SRF binding activity to SRE, paralleled by increased cardiac actin expression (4.1 ± 0.1 vs. 2.2 ± 0.01 target protein/β-actin ratio, P < 0.01). EMPA significantly reversed cardiac dysfunction on echocardiography in diabetic mice and inhibited excessive autophagy in high-glucose-treated cardiomyocytes by inhibiting the autophagy inducer glycogen synthase kinase 3 beta (GSK3β), leading to reactivation of cardiomyogenic transcription factors. Conclusion Taken together, our results describe a novel paradigm in which EMPA inhibits hyperactivation of autophagy through the AMPK/GSK3β signalling pathway in the context of diabetes.

Vascular progenitor cells are activated to repair and form a neointima following vascular damage such as hypertension, atherosclerosis, diabetes, trauma, hypoxia, primary cancerous lesions and metastases as well as catheter interventions. They play a key role not only in the resolution of the vascular lesion but also in the adult neovascularization and angiogenesis sprouting (i.e., the growth of new capillaries from pre-existing ones), often associated with carcinogenesis, favoring the formation of metastases, survival and progression of tumors. In this review, we discuss the biology, cellular plasticity and pathophysiology of different vascular progenitor cells, including their origins (sources), stimuli and activated pathways that induce differentiation, isolation and characterization. We focus on their role in tumor-induced vascular injury and discuss their implications in promoting tumor angiogenesis during cancer proliferation and migration.

© 2021 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.Gravity alterations elicit complex and mostly detrimental effects on biological systems. Among these, a prominent role is occupied by oxidative stress, with consequences for tissue homeostasis and development. Studies in altered gravity are relevant for both Earth and space biomedicine, but their implementation using whole organisms is often troublesome. Here we utilize planarians, simple worm model for stem cell and regeneration biology, to characterize the pathogenic mechanisms brought by artificial gravity alterations. In particular, we provide a comprehensive evaluation of molecular responses in intact and regenerating specimens, and demonstrate a protective action from the space-apt for nanotechnological antioxidant cerium oxide nanoparticles.

Background 3-Iodothyronamine (T1AM) is an endogenous messenger chemically related to thyroid hormone. Recent results indicate significant transcriptional effects of chronic T1AM administration involving the protein family of sirtuins, which regulate important metabolic pathways and tumor progression. Therefore, the aim of this work was to compare the effect of exogenous T1AM and 3,5,3′-triiodo-L-thyronine (T3) chronic treatment on mammalian sirtuin expression in hepatocellular carcinoma cells (HepG2) and in primary rat hepatocytes at micromolar concentrations. Materials and methods Sirtuin (SIRT) activity and expression were determined using a colorimetric assay and Western blot analysis, respectively, in cells treated for 24 h with 1–20 μM T1AM or T3. In addition, cell viability was evaluated by the MTTtest upon 24 h of treatment with 0.1–20 μM T1AM or T3. Results In HepG2, T1AM significantly reduced SIRT 1 (20 μM) and SIRT4 (10–20 μM) protein expression, while T3 strongly decreased the expression of SIRT1 (20 μM) and SIRT2 (any tested concentration). In primary rat hepatocytes, T3 decreased SIRT2 expression and cellular nicotinamide adenine dinucleotide (NAD) concentration, while on sirtuin activity it showed opposite effects, depending on the evaluated cell fraction. The extent of MTT staining was moderately but significantly reduced by T1AM, particularly in HepG2 cells, whereas T3 reduced cell viability only in the tumor cell line. Conclusions T1AM and T3 downregulated the expression of sirtuins, mainly SIRT1, in hepatocytes, albeit in different ways. Differences in mechanisms are only observational, and further investigations are required to highlight the potential role of T1AM and T3 in modulating sirtuin expression and, therefore, in regulating cell cycle or tumorigenesis.

Background: A novel form of thyroid hormone (TH) signaling is represented by 3-iodothyronamine (T1AM), an endogenous TH derivative that interacts with specific molecular targets, including trace am...

3,5-diiodo-L-thyronine (3,5-T2) is an endogenous derivative of thyroid hormone with potential metabolic effects. It has been detected in human blood by immunological methods, but a reliable assay based on mass spectrometry (MS), which is now regarded as the gold standard in clinical chemistry, is not available yet. Therefore, we aimed at developing a novel ad-hoc optimized method to quantitate 3,5-T2 and its isomers by MS in human serum. Serum samples were obtained from 28 healthy subjects. Two ml of serum were deproteinized with acetonitrile and then subjected to an optimized solid phase extraction-based procedure. To lower background noise, the samples were furtherly cleaned by hexane washing and acetonitrile precipitation of residual proteins. 3,5-T2 and its isomers 3,3′-T2 and 3′,5′-T2 were then analyzed by HPLC coupled to tandem MS. Accuracy and precision for T2 assay were 88–104% and 95–97%, respectively. Recovery and matrix effect averaged 78% and +8%, respectively. 3,5-T2 was detected in all samples and its concentration averaged (mean ± SEM) 41 ± 5 pg/ml, i.e., 78 ± 9 pmol/l. In the same samples the concentration of 3,3′-T2 averaged 133±15 pg/ml, i.e., 253±29 pmol/l, while 3′,5′-T2 was not detected. 3,5-T2 concentration was significantly related to 3,3′-T2 concentration (r = 0.540, P < 0.01), while no significant correlation was observed with either T3 or T4 in a subset of patients in which these hormones were assayed. In conclusion, our method is able to quantify 3,5-T2 and 3,3′-T2 in human serum. Their concentrations lie in the subnanomolar range, and a significant correlation was detected between these two metabolites in healthy individuals.

3,5-diiodo-l-thyronine (T2) is an endogenous derivative of thyroid hormone that has been suggested to regulate energy expenditure, resting metabolic rate and oxygen consumption with a mechanism that involves the activation of mitochondrial function. In this study, we focused on the cardiac effects of T2, which have been poorly investigated so far, by using both in vitro and ex vivo models. As a comparison, the response to T3 and T4 was also determined. Rat cardiomyoblasts (H9c2 cells) were used to determine T2, T3, and T4 uptake by high-performance liquid chromatography–tandem mass spectrometry. In the same experimental model, MTT test, crystal violet staining, and glucose consumption were investigated, using T2 concentrations ranging from 0.1 to 10 µM. To assess cardiac functional effects, isolated working rat hearts were perfused with T2, T3, or T4 in Krebs-Ringer buffer, and the hemodynamic variables were recorded. T2 was taken up by cardiomyoblasts, and in cell lysate T2 levels increased slowly over time, reaching higher concentrations than in the incubation medium. T2 significantly decreased MTT staining at 0.5–10 µM concentration (P

Obesity is a complex disease associated with environmental and genetic factors. 3-Iodothyronamine (T1AM) has revealed great potential as an effective weight loss drug. We used metabolomics and associated transcriptional gene and protein expression analysis to investigate the tissue specific metabolic reprogramming effects of subchronic T1AM treatment at two pharmacological daily doses (10 and 25 mg/kg) on targeted metabolic pathways. Multi-analytical results indicated that T1AM at 25 mg/kg can act as a novel master regulator of both glucose and lipid metabolism in mice through sirtuin-mediated pathways. In liver, we observed an increased gene and protein expression of Sirt6 (a master gene regulator of glucose) and Gck (glucose kinase) and a decreased expression of Sirt4 (a negative regulator of fatty acids oxidation (FAO)), whereas in white adipose tissue only Sirt6 was increased. Metabolomics analysis supported physiological changes at both doses with most increases in FAO, glycolysis indicators and the mitochondrial substrate, at the highest dose of T1AM. Together our results suggest that T1AM acts through sirtuin-mediated pathways to metabolically reprogram fatty acid and glucose metabolism possibly through small molecules signaling. Our novel mechanistic findings indicate that T1AM has a great potential as a drug for the treatment of obesity and possibly diabetes.

The present study was undertaken because no previous developmental studies exist on MCH neurons in any avian species. After validating a commercially-available antibody for use in chickens, immunohistochemical examinations first detected MCH neurons around embryonic day (E) 8 in the posterior hypothalamus. This population increased thereafter, reaching a numerical maximum by E20. MCH-positive cell bodies were found only in the posterior hypothalamus at all ages examined, restricted to a region showing very little overlap with the locations of hypocretin/orexin (H/O) neurons. Chickens had fewer MCH than H/O neurons, and MCH neurons also first appeared later in development than H/O neurons (the opposite of what has been found in rodents). MCH neurons appeared to originate from territories within the hypothalamic periventricular organ that partially overlap with the source of diencephalic serotonergic neurons. Chicken MCH fibers developed exuberantly during the second half of embryonic development, and they became abundant in the same brain areas as in rodents, including the hypothalamus (by E12), locus coeruleus (by E12), dorsal raphe nucleus (by E20) and septum (by E20). These observations suggest that MCH cells may play different roles during development in chickens and rodents; but once they have developed, MCH neurons exhibit similar phenotypes in birds and rodents.

Difficulties have been reported in quantitating 3-iodothyronamine (T1AM) in blood or serum, and tentatively attributed to problems in extraction or other pre-analytical steps. For this reason, even cell culture experiments have often be performed with unphysiological protein-free media. The aim of this study was to evaluate the recovery of exogenous T1AM added to a standard cell culture medium, namely Dulbecco's minimum essential medium (DMEM) supplemented with fetal bovine serum (FBS), and to other biological matrixes.Cell culture media (Krebs-Ringer buffer, DMEM, FBS, DMEM + FBS, used either in the absence or in the presence of NG108-15 cells) and other biological matrixes (rat brain and liver homogenates, human plasma, and blood) were spiked with T1AM and/or deuterated T1AM (d4-T1AM) and incubated for times ranging from 0 to 240 minutes. Samples were then extracted using a liquid/liquid method and analyzed using liquid chromatography coupled to mass spectrometry in order to assay T1AM and its metabolites, namely 3-iodothyroacetic acid (TA1), thyronamine, thyroacetic acid, N-acetyl-T1AM, and T1AM esters.In FBS-containing buffers, T1AM decreased exponentially over time, with a half-life of 6-17 minutes, depending on FBS content, and after 60 minutes, it averaged 0-10% of the baseline. T1AM metabolites were not detected, except for minimum amounts of TA1. Notably, d4-T1AM decreased over time at a much lower rate, reaching 50-70% of the baseline at 60 minutes. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide. In the presence of cells, T1AM concentration decreased virtually to 0 within 60 minutes, but TA1 accumulated in the incubation medium, with quantitative recovery. Spontaneous decrease in T1AM concentration with isotopic difference was confirmed in rat organ homogenates and human blood.These results suggest binding and sequestration of T1AM and/or its aldehyde derivative by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenous T1AM.

Interest in the potential therapeutic utility of topiramate for treating bipolar disorder was stimulated by published reports of investigator-initiated open label clinical studies. Because chronic lithium, carbamazepine and valproate decrease the turnover of arachidonic acid (AA) but not docosahexaenoic acid (DHA) in brain phospholipids of the awake rat, we tested if topiramate would produce similar results. Rats received either topiramate (20 mg/kg twice per day) or vehicle for 14 days and then while unanesthetized were infused intravenously with either [1-(14)C] AA or [1-(14)C] DHA for 5 min while blood was collected from the femoral artery at fixed times. Topiramate did not alter the incorporation rate of AA or DHA from their respective brain acyl-CoA pool into brain phospholipids, nor the turnover of AA and DHA in brain phospholipids. The results of our study indicate that topiramate does not possess a pharmacological property that three drugs with proven efficacy in treating bipolar disorder have in common.

Nitrogen monoxide (NO) has important cardiovascular actions, and it has been suggested that they may be partly mediated by the reaction with protein sulfhydryl groups to produce S-nitrosothiols. In this work we describe and test a method that allows S-nitrosothiol detection in crude membrane preparations obtained from isolated perfused rat hearts. Isolated rat hearts were perfused under control conditions or in the presence of the NO donors SIN-1 and isosorbide dinitrate. Additional hearts were subjected to 10-20 min of ischemia followed or not by 10-20 min of reperfusion. At the end of perfusion a crude membrane fraction was prepared, and S-nitrosothiol concentration was assayed fluorometrically, on the basis of 2,3-naphthotriazole production from 2,3-diaminonaphthylene. The sensitivity of the method, as evaluated using S-nitrosoalbumin, was on the order of 1-2 pmol/mg of protein. S-nitrosothiols were undetectable under control conditions, as well as after ischemia or ischemia-reperfusion. On the other hand, significant S-nitrosothiol formation was observed after infusion of SIN-1 or isosorbide dinitrate (26.4 +/- 7.4 and 19.9 +/- 5.6 pmol per mg of protein, respectively). In conclusion, S-nitrosothiol production was observed in rat heart membranes after exposure to NO donors, while S-nitrosothiol concentration was below the sensitivity limits of the assay either under baseline conditions or after acute ischemia and reperfusion.

We used the isolated working rat model to evaluate the effect of therapeutic concentrations (5-10 μM) of ranolazine on contractile performance, oxygen consumption, irreversible ischemic injury, and sarcoplasmic reticulum (SR) function. Ischemic injury was induced by 30 minutes of global ischemia followed by 120 minutes of Langendorff reperfusion and evaluated on the basis of triphenyltetrazolium chloride staining. SR function was determined on the basis of [H]-ryanodine binding, the kinetics of calcium-induced calcium release, measured by quick filtration technique, and oxalate-supported calcium uptake. In working hearts, ranolazine significantly reduced oxygen consumption (P = 0.031), in the absence of significant changes in contractile performance, and decreased irreversible ischemic injury (P = 0.011), if administered either before ischemia-reperfusion (25.4% ± 4.7% vs. 42.7% ± 6.0%) or only at the time of reperfusion (20.2% ± 5.2% vs. 43.7% ± 9.9%). In SR experiments, treatment with ranolazine determined a significant reduction in [H]-ryanodine binding (P = 0.029), because of decreased binding site density (369 ± 9 vs. 405 ± 12 fmol/mg), and in the kinetics of SR calcium release (P = 0.011), whose rate constant was decreased, whereas active calcium uptake was not affected. Ranolazine effectiveness at reperfusion and its ability to module SR calcium release suggest that this drug might be particularly useful to induce cardioprotection during coronary revascularization interventions, although the relevance of the effects on calcium homeostasis remains to be determined.

Objective: Stimulation of A3 adenosine receptors has been shown to protect cardiac myocytes from ischemic injury, but the mechanism of this action is unknown. We evaluated the effect of adenosine agonists and antagonists on the sarcoplasmic reticulum (SR) Ca2+ channels. Methods: Isolated rat hearts were perfused with control buffer or different adenosine agonists and antagonists. Hearts were then homogenized and used to determine SR Ca2+-induced Ca2+ release, assayed by quick filtration technique after loading with 45Ca2+, and the binding of [3H]ryanodine, a specific ligand of the SR Ca2+ release channel. In parallel experiments, hearts were challenged with 30 min of global ischemia and 120 min of reperfusion, and the extent of tissue necrosis was evaluated by triphenyltetrazolium chloride staining. Results: Perfusion with the A1

1. The use of anthraquinone antineoplastic agents is limited by their cardiac toxicity, which is largely due to activation of the sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor). MEN 10755 is a new disaccharide analogue of doxorubicin. We have evaluated its effects on SR function and its toxicity in isolated working rat hearts. 2. In rat SR vesicles, doxorubicin stimulated [(3)H]-ryanodine binding by increasing its Ca(2+)-sensitivity. At 1 microM Ca(2+), ryanodine binding increased by 15.3+/-2.5 fold, with EC(50)=20.6 microM. Epirubicin produced a similar effect, i.e. 9.7+/-0.6 fold stimulation with EC(50)=11.1 microM. MEN 10755 increased ryanodine binding by 1.9+/-0.3 fold (P

3-Iodothyronamine (T1AM) is regarded as a hormone-like substance thanks to its endogenous nature, its interaction with specific receptors trace amine-associated receptor 1 and its biological effects. We characterized T1AM transport and conversion in an in vitro culture of H9c2 murine cells, after a T1AM bolus injection. Samples of cell medium culture and cell lysate were assayed by high-performance liquid chromatography coupled to tandem mass spectrometry. We performed comparative experiments by adding to T1AM bolus amino oxidase inhibitors as iproniazid, pargyline (monoamine oxidase, MAO inhibitors), aminoguanidine, and semicarbazide (semicarbazide-sensitive amino oxidase, SSAO inhibitors). A mathematical model was developed, based on the assumption that T1AM is transported with a mechanism that is typical of hormone transport (i.e., EGF or insulin). We noticed that surface receptors downregulation could play a major role in T1AM dynamics. We also estimated that T1AM catabolism is mainly affected by MAO inhibitors, which produce a dramatic decrease in the kinetic constants related to T1AM degradation, while no significant changes were observed in experiments with SSAO inhibitors.

3-iodothyronamine (T(1)AM) is an endogenous compound which shares structural and functional features with biogenic amines and is able to interact with a specific class of receptors, designed as trace amine associated receptors. T(1)AM has significant physiological effects in mammals and produces a reversible, dose-dependent negative inotropic and chronotropic effect in heart. The aim of the present study was to investigate if T(1)AM is able to reduce irreversible tissue injury in isolated rat hearts subjected to ischemia and reperfusion, as evaluated by triphenyltetrazolium chloride staining. We observed that T(1)AM reduced infarct size at concentrations (125 nM to 12.5 μM) which did not produce any significant hemodynamic action. The dose-response curve was bell-shaped and peaked at 1.25 μM. T(1)AM-induced cardioprotection was completely reversed by the administration of chelerythrine and glibenclamide, suggesting a protein kinase C and K (ATP) (+) -dependent pathway, while it was not additive to the protection induced by cyclosporine A, suggesting modulation of mitochondrial permeability transition. At cardioprotective concentration, T(1)AM reduced the time needed for cardiac attest during ischemia, but it did not affect sarcoplasmatic reticulum Ca(2+) handling, as demonstrated by unaltered ryanodine receptor binding properties. In conclusion, in isolated rat heart T(1)AM produces a cardioprotective effect which is mediated by a protein kinase C and K (ATP) (+) -dependent pathway and is probably linked to modulation of mitochondrial permeability transition and/or ischemic arrest time.

3-iodothyronamine (T(1)AM) is a novel endogenous relative of thyroid hormone, able to interact with trace amine-associated receptors, a class of plasma membrane G protein-coupled receptors, and to produce a negative inotropic and chronotropic effect. In the isolated rat heart 20-25 microM T(1)AM decreased cardiac contractility, but oxygen consumption and glucose uptake were either unchanged or disproportionately high when compared to mechanical work. In adult rat cardiomyocytes acute exposure to 20 microM T(1)AM decreased the amplitude and duration of the calcium transient. In patch clamped cardiomyocytes sarcolemmal calcium current density was unchanged while current facilitation by membrane depolarization was abolished consistent with reduced sarcoplasmic reticulum (SR) calcium release. In addition, T(1)AM decreased transient outward current (I(to)) and I(K1) background current. SR studies involving 20 microM T(1)AM revealed a significant decrease in ryanodine binding due to reduced B(max), no significant change in the rate constant of calcium-induced calcium release, a significant increase in calcium leak measured under conditions promoting channel closure, and no effect on oxalate-supported calcium uptake. Based on these observations we conclude T(1)AM affects calcium and potassium homeostasis and suggest its negative inotropic action is due to a diminished pool of SR calcium as a result of increased diastolic leak through the ryanodine receptor, while increased action potential duration is accounted for by inhibition of I(to) and I(K1) currents.

Thyronamines are decarboxylated derivatives of thyroid hormone. 3-Iodothyronamine (T1AM) has been detected in blood and in several tissues. where it is likely produced from thyroid hormone by the consequent action of aromatic amino acid decarboxylase and deiodinases. In vitro, high-affinity interaction has been observed between T1AM and a novel G-protein-coupled receptor known as trace-amine-associated receptor 1 (TAAR1). TAAR1 and other receptors of this family are expressed in several tissues, including the heart. Functional effects have been observed after administration of exogenous T1AM: in the isolated heart, a negative inotropic and chronotropic action was produced, and the resistance to ischemic injury was increased, possibly as a consequence of an action on intracellular calcium homeostasis. Extracardiac effects include reduction of body temperature, increased lipid versus carbohydrate metabolism, modulation of insulin secretion, and inhibition of neuronal catecholamine reuptake. T1AM may play an important physiological or pathophysiological role, and this signaling system might allow the development of new therapeutic agents.

Isolated rat hearts were perfused for 120 minutes in the presence or in the absence of 10 microM zofenoprilat, the active metabolite of zofenopril. At the end of perfusion, cardiac tissue was used to assay sarcoplasmic reticulum (SR) (45)Ca uptake and SR calcium release, which was determined by automatized quick filtration technique after SR vesicle loading with (45)Ca. The expression of genes involved in the control of calcium homeostasis was evaluated by polymerase chain reaction after reverse transcription. In chronic experiments, SR (45)Ca uptake and gene expression were measured in hearts derived from rats treated with 15 mg*kg(-1)*day(-1) zofenopril for 15 days. Acute or chronic zofenopril administration did not produce any change in contractile performance. In acute experiments, SR (45)Ca uptake was significantly increased after exposure to zofenoprilat. The rate constant of calcium-induced calcium release was slightly although not significantly higher, and the calcium leak measured under conditions promoting SR channel closure was significantly increased. In the chronic model, significant increase in the rate of SR (45)Ca uptake was confirmed. Gene expression was not modified, except for decreased phospholamban expression, which is observed in the acute but not in the chronic model. In conclusion, zofenopril increases SR calcium cycling and stimulates active calcium uptake into the SR.

3-Iodothyronamine (T(1)AM) is an endogenous compound derived from thyroid hormone through decarboxylation and deiodination, which interacts with a novel G protein-coupled receptor, known as trace amine-associated receptor 1 (TAAR1). TAAR1 and other receptors of this family are expressed in several tissues, including the heart. Functional effects have been observed after administration of exogenous T(1)AM: in the isolated heart, a negative inotropic and chronotropic action was produced, and the resistance to ischemic injury was increased, possibly as a consequence of an action on intracellular calcium homeostasis. Extracardiac effects include reduction of body temperature, increased lipid versus carbohydrate metabolism, and modulation of insulin secretion. T(1)AM might play an important physiological or pathophysiological role, and this signaling system might allow the development of new therapeutical agents.

Trace amine-associated receptors, a novel class of G-protein coupled receptors which respond to trace amines but not to classical biogenic amines, have been found to be expressed in heart. Therefore, we investigated the cardiac effects of the trace amines p-tyramine, beta-phenylethylamine, octopamine, and tryptamine. Isolated rat hearts were perfused in the presence of trace amines, monitoring the hemodynamic variables. In addition, radioligand binding experiments with [3H]-p-tyramine and [125I]-3-iodothyronamine were performed in rat ventricular tissue. Octopamine, beta-phenylethylamine, and tryptamine produced a dose-dependent negative inotropic effect as shown by reduced cardiac output (IC(50)=109 microM, 159 microM, and 242 microM, respectively). In the same preparation a similar effect was produced by thyronamine and 3-iodothyronamine, with IC(50)=94 microM and 27 microM, respectively. The negative inotropic effect of octopamine was confirmed in a papillary muscle preparation. All trace amines except tryptamine increased the heart rate, but this action could be attributed to their sympathomimetic properties, since it was abolished by propranolol. The negative inotropic effect of trace amines was significantly increased by the tyrosine kinase inhibitor genistein. Specific and saturable binding of [(3)H]-p-tyramine and [125I]-3-iodothyronamine was observed in ventricular tissue. While [3H]-p-tyramine was displaced by 3-iodothyronamine, [(125)I]-3-iodothyronamine was not displaced by p-tyramine. In conclusion, trace amines and thyronamines are negative inotropic agents. Their effect appears to be mediated by a subtype of trace amine-associated receptor which is characterized by the rank of potency: 3-iodothyronamine > thyronamine = octopamine = beta-phenylethylamine, while tryptamine and p-tyramine are significantly less active.

We investigated whether in the isolated perfused rat heart acute pressure overload may affect the expression of genes involved in calcium homeostasis, namely sarcolemmal L-type Ca2+ channel, Na+/Ca2+ exchanger, sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and ryanodine receptor. Hearts were subjected to 210 min of perfusion under the following conditions: (i) standard working heart perfusion with preload and afterload set at 20 and 100 cm, respectively; (ii) working heart perfusion at high afterload (180 cm); (iii) retrograde infusion of St. Thomas' Hospital cardioplegic solution. In all models gene expression was determined by RT-PCR. Significant decrease in the expression of the sarcoplasmic reticulum Ca2+-ATPase gene was observed in the high afterload group. No significant change in the expression of any other gene was observed in any group. The reported effect was not detected after 60 min of perfusion, and it was blunted in the presence of the protein kinase C inhibitor chelerythrine, while the calcineurin inhibitor cyclosporin A was ineffective. In conclusion, the sarcoplasmic reticulum Ca2+-ATPase gene is downregulated after short-term (210 min) perfusion at high afterload, possibly through a protein kinase C-dependent pathway. This mechanism might play a relevant pathophysiological role in the response to pressure overload and in the development of hypertrophy.

Cardioprotective interventions are defined as interventions able to increase myocardial resistance to ischemia. The authors approach the issue of cardioprotection on the basis of the present knowledge about the biochemical mechanisms responsible for the injury produced by myocardial ischemia or ischemia-reperfusion. Reversible and irreversible injury are distinguished. The former is largely accounted for by the direct consequences of reduced ATP synthesis, which causes decreased ATP phosphorylation potential, acidosis and phosphate accumulation. The biochemical mechanisms leading to irreversible injury include osmotic overload, production of toxic lipid metabolites, cytosolic calcium overload, and generation of reactive oxygen species, which lead to membrane disruption, mitochondrial dysfunction and possibly to the activation of apoptotic pathways. The major effect of the classical cardioprotective agents (nitrates, beta adrenergic antagonists, calcium channel blockers) consists in affecting ATP demand/supply ratio in such a way as to delay the decrease in ATP phosphorylation potential. Other drugs have been introduced, which allegedly interfere directly with the mechanisms responsible for irreversible ischemic injury. These include 3-ketoacyl-CoA tiolase inhibitors, modulators of intracellular calcium channels, ionic exchanger inhibitors, free radical scavengers, caspase inhibitors, purinergic agonists, K(+)(ATP) channel openers, and modulators of mitochondrial permeability transition. The results obtained with these substances in experimental models and in the clinical setting are discussed. Special attention is devoted to angiotensin converting enzyme inhibitors, whose direct cardioprotective properties has recently been demonstrated.

3-Iodothyronamine T1AM is a novel endogenous thyroid hormone derivative that activates the G protein-coupled receptor known as trace anime-associated receptor 1 (TAAR1). In the isolated working rat heart and in rat cardiomyocytes, T1AM produced a reversible, dose-dependent negative inotropic effect (e.g., 27+/-5, 51+/-3, and 65+/-2% decrease in cardiac output at 19, 25, and 38 microM concentration, respectively). An independent negative chronotropic effect was also observed. The hemodynamic effects of T1AM were remarkably increased in the presence of the tyrosine kinase inhibitor genistein, whereas they were attenuated in the presence of the tyrosine phosphatase inhibitor vanadate. No effect was produced by inhibitors of protein kinase A, protein kinase C, calcium-calmodulin kinase II, phosphatidylinositol-3-kinase, or MAP kinases. Tissue cAMP levels were unchanged. In rat ventricular tissue, Western blot experiments with antiphosphotyrosine antibodies showed reduced phosphorylation of microsomal and cytosolic proteins after perfusion with synthetic T1AM; reverse transcriptase-polymerase chain reaction experiments revealed the presence of transcripts for at least 5 TAAR subtypes; specific and saturable binding of [125I]T1AM was observed, with a dissociation constant in the low micromolar range (5 microM); and endogenous T1AM was detectable by tandem mass spectrometry. In conclusion, our findings provide evidence for the existence of a novel aminergic system modulating cardiac function.

Ghrelin, the natural ligand of the GH secretagogue (GHS) receptor, was originally isolated from the stomach and detected in several tissues, but a systematic study of its tissue distribution has not been performed. In the present investigation, we evaluated ghrelin gene expression (by RT-PCR technique) and ghrelin protein concentration (by enzyme immunoassay technique) in tissues obtained from control rats as well as in rats subjected to 48-h fasting. The ghrelin gene was expressed in stomach, small intestine, brain, cerebellum, pituitary, heart, pancreas, salivary gland, adrenal, ovary and testis, with maximum expression occurring in the stomach, while no significant expression was detected by standard RT-PCR in liver, lung, kidney and skeletal muscle. Ghrelin protein was detected in stomach, small intestine, brain, cerebellum, pituitary, lung, skeletal muscle pancreas, salivary gland, adrenal, ovary and testis, at concentrations ranging from 0.05 to 1.43 ng/mg of homogenate protein (the highest concentration occurred in the lung, followed by the brain). Ghrelin was not detectable in the heart, liver and kidney. Therefore, gene and protein expression were dissociated. Fasting did not produce significant changes in ghrelin gene expression, while the distribution of ghrelin between different tissues was significantly modified: protein concentration increased in the brain, cerebellum, lung and salivary gland, while it decreased in the stomach.

We have already reported that A(3) adenosine receptor stimulation reduces [(3)H]-ryanodine binding and sarcoplasmic reticulum Ca(2+) release in rat heart. In the present work we have investigated the transduction pathway responsible for this effect. Isolated rat hearts were perfused for 20 min in the presence of the following substances: 100 nM N(6)-(iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA), an A(3) adenosine agonist; 10 muM U-73122, a phospholipase C inhibitor; 2 muM chelerythrine, a protein kinase C inhibitor. At the end of perfusion, the hearts were homogenized and [(3)H]-ryanodine binding was assayed. IB-MECA produced a significant decrease in ryanodine binding, which was abolished in the presence of chelerythrine but not in the presence of U-73122. RT-PCR experiments showed that ryanodine receptor gene expression was not affected by IB-MECA. In Western blot experiments, ryanodine receptor phosphorylation on serine 2809 was not modified after perfusion with IB-MECA. We conclude that modulation of SR Ca(2+) release channel by IB-MECA is dependent on protein kinase C activation. However, in this model protein kinase C activation is not due to phospholipase C activation. In addition, changes in ryanodine receptor gene expression or direct phosphorylation of the ryanodine receptor on serine 2809 residue do not appear to occur.

We investigated whether A 1 adenosine receptor stimulation affects expression of genes involved in calcium homeostasis, including sarcolemmal L-type Ca 2+ channel, Na + /Ca 2+ exchanger, sarcoplasmic reticulum (SR) Ca 2+ -ATPase, phospholamban, or ryanodine receptor. Three models of A 1 stimulation were used: i) an acute model, i.e. isolated perfused rat hearts treated for 120 min with 15 nM R-phenylisopropyladenosine (R-PIA), an A 1 receptor agonist; ii) a subacute model, i.e. rats treated with 1.5 mg/kg R-PIA e.v. and sacrificed after 24 h; iii) a transgenic model, i.e. mice overexpressing A 1 adenosine receptors. In all models gene expression was determined by RT-PCR, and oxalate-supported Ca 2+ uptake, representing SR Ca 2+ uptake, was measured in the crude homogenate. Significant increase in the expression of the phospholamban gene was observed in each model of A 1 stimulation, while the expression of the other four genes was not significantly modified. In the acute model, SR Ca 2+ uptake was unaffected, however in the subacute and transgenic models uptake rate was significantly reduced. In parallel experiments, hearts obtained from the subacute model demonstrated a significant reduction in irreversible tissue injury from 30 min of ischemia and 120 min of reperfusion. Increased resistance to ischemia has already been reported also in our transgenic model. In conclusion, A 1 adenosine receptor stimulation up-regulates phospholamban gene expression, which leads within 24 h to a reduced rate of SR Ca 2+ uptake. Changes in Ca 2+ homeostasis might contribute to the delayed cardioprotective effect of adenosine.

When administered chronically to rats, drugs that are effective in bipolar disorder-lithium and the anticonvulsants, valproic acid and carbamazepine-have been shown to downregulate the expression of certain enzymes involved in brain arachidonic acid (AA) release and cyclooxygenase (COX)-mediated metabolism. Phase II clinical trials with the anticonvulsant topiramate [2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate] suggest that this drug may also be effective for bipolar disorder.To see if topiramate has effects similar to those of the other three drugs, we administered topiramate to rats for 14 days at 20 mg/kg, p.o. twice daily.Compared with p.o. vehicle, topiramate treatment did not significantly affect the brain activity or protein level of cytosolic phospholipase A2, secretory PLA2, or Ca2+-independent iPLA2. Additionally, brain protein levels of COX-1, COX-2, 5-lipoxygenase, and cytochrome P450 epoxygenase were unchanged.These results suggest that topiramate does not modify expression of the enzymes involved in brain AA metabolism that have been shown to be targeted by lithium, valproic acid, or carbamazepine. If topiramate proves effective in bipolar disorder, it may not act by modulating brain AA metabolism. In view of the proven anticonvulsant effect of topiramate, our results also suggest that the AA cascade is not involved in the anti-seizure properties of the drug.

We investigated the effect of different ACE inhibitors on tissue injury in isolated rat hearts subjected to 30 minutes of ischemia followed by 120 minutes of reperfusion. Zofenoprilat (1-100 microM), but not enalaprilat or lisinopril, significantly reduced infarct size, as estimated on the basis of triphenyltetrazolium chloride staining. The protection was not reproduced by the angiotensin II receptor antagonist irbesartan, and it was partly abolished by the bradykinin receptor antagonist HOE 140. Zofenoprilat molecule contains a sulfhydryl group, and its administration, as compared with enalaprilat or lisinopril administration, was associated with better preservation of protein thiols at the end of ischemia. We conclude that zofenopril has a specific cardioprotective effect, which might be related either to interference with bradykinin metabolism or to preservation of protein sulfhydryl groups.

Nitrogen monoxide (NO) has important cardiovascular actions, and it has been suggested that they may be partly mediated by the reaction with protein sulfhydryl groups to produce S-nitrosothiols. In this work we describe and test a method that allows S-nitrosothiol detection in crude membrane preparations obtained from isolated perfused rat hearts. Isolated rat hearts were perfused under control conditions or in the presence of the NO donors SIN-1 and isosorbide dinitrate. Additional hearts were subjected to 10-20 min of ischemia followed or not by 10-20 min of reperfusion. At the end of perfusion a crude membrane fraction was prepared, and S-nitrosothiol concentration was assayed fluorometrically, on the basis of 2,3-naphthotriazole production from 2,3-diaminonaphthylene. The sensitivity of the method, as evaluated using S-nitrosoalbumin, was on the order of 1-2 pmol/mg of protein. S-nitrosothiols were undetectable under control conditions, as well as after ischemia or ischemia-reperfusion. On the other hand, significant S-nitrosothiol formation was observed after infusion of SIN-1 or isosorbide dinitrate (26.4 +/- 7.4 and 19.9 +/- 5.6 pmol per mg of protein, respectively). In conclusion, S-nitrosothiol production was observed in rat heart membranes after exposure to NO donors, while S-nitrosothiol concentration was below the sensitivity limits of the assay either under baseline conditions or after acute ischemia and reperfusion.

Receptors for growth hormone secretagogues have been identified in cardiac tissue, but their functional role is unknown. We have investigated the effect of different growth hormone secretagogues on contractile performance and on the susceptibility to ischemic injury, in isolated working rat hearts. In particular, we tested the endogenous secretagogue ghrelin and the synthetic secretagogues hexarelin and MK-0677. Under aerobic conditions, none of these substances produced any significant hemodynamic effects. In hearts subjected to 30 minutes of ischemia followed by 120 minutes of reperfusion, the synthetic peptidyl secretagogue hexarelin (1 microM) significantly reduced infarct size, as estimated on the basis of triphenyltetrazolium chloride staining, while the non-peptidyl secretagogue MK-0677 was ineffective. The endogenous peptidyl secretagogue ghrelin (20 nM) was also protective, while desacylated ghrelin, which is devoid of biological effects, did not modify ischemic injury. The protection provided by hexarelin was partly abolished by the protein kinase C inhibitor chelerythrine. We conclude that ghrelin and hexarelin have a specific cardioprotective effect, which is independent of growth hormone secretion, and might be related to protein kinase C activation.

We investigated whether changes in cardiac work or in Ca2+ fluxes may affect the expression of sarcolemmal or sarcoplasmic reticulum Ca2+ channels (DHPRs and RyRs, respectively). Isolated rat hearts were perfused at low Ca2+ concentration (0.8 mM instead of 1.5 mM), at low preload (5 cm instead of 20 cm), in the presence of 100 nM nifedipine or with a cardioplegic solution. After 60 min, hypocalcemic perfusion produced significant reduction in [3H]-PN 200-110 and [3H]-ryanodine binding, due to approximately 30% reduction in Bmax (P