Performance in Behavioral Testing in an Animal Model of Post-Surgical Hypoparathyroidism.

BACKGROUND: Hypoparathyroidism (HypoPT) is characterized by hypocalcemia and undetectable/inappropriately low PTH. Post-surgical HypoPT (PS-HypoPT) is the most common cause. Patients with PS-HypoPT present neuropsychological symptoms, probably due to the PTH deprivation in the central nervous system (CNS). However, these mechanisms are still not elucidated. The aim of this study was to evaluate the effects of PTH deprivation on CNS in an animal model of PS-HypoPT via a cognitive/behavioral assessment approach. METHODS: A surgical rat model of PS-HypoPT was obtained and treated with calcium to maintain normocalcemia. Twenty PS-HypoPT rats and twenty sham-operated controls (Crl) underwent behavioral testing in a Morris Water Maze (MWM), Open Field (OF), and Elevated Plus Maze (EPM). RESULTS: In the MWM, PTx rats showed a higher Escape Latency Time compared to Crl rats (p < 0.05); we observed a statistically significant improvement in the performance (day 1 to 8 p < 0.001), which was less pronounced in PTx group. In the OF test, the time and distance spent in the zone of interest were significantly lower in the PTx group compared with the Crl (p < 0.01 and p < 0.01). In the EPM experiment, the time spent in the close arm was significantly higher in the PTx group compared with the Crl (p < 0.01). CONCLUSIONS: This animal model of PS-HypoPT shows an impairment in spatial memory, which improved after training, and a marked anxiety-like behavior, resembling the condition of patients with PS-HypoPT. Further studies are needed to elucidate mechanisms.

Effect of Combined Levothyroxine (L-T4) and 3-Iodothyronamine (T1AM) Supplementation on Memory and Adult Hippocampal Neurogenesis in a Mouse Model of Hypothyroidism.

Mood alterations, anxiety, and cognitive impairments associated with adult-onset hypothyroidism often persist despite replacement treatment. In rodent models of hypothyroidism, replacement does not bring 3-iodothyronamine (T1AM) brain levels back to normal. T1AM is a thyroid hormone derivative with cognitive effects. Using a pharmacological hypothyroid mouse model, we investigated whether augmenting levothyroxine (L-T4) with T1AM improves behavioural correlates of depression, anxiety, and memory and has an effect on hippocampal neurogenesis. Hypothyroid mice showed impaired performance in the novel object recognition test as compared to euthyroid mice (discrimination index (DI): 0.02 ± 0.09 vs. 0.29 ± 0.06; t = 2.515, p = 0.02). L-T4 and L-T4+T1AM rescued memory (DI: 0.27 ± 0.08 and 0.34 ± 0.08, respectively), while T1AM had no effect (DI: -0.01 ± 0.10). Hypothyroidism reduced the number of neuroprogenitors in hippocampal neurogenic niches by 20%. L-T4 rescued the number of neuroprogenitors (mean diff = 106.9 ± 21.40, t = 4.99, pcorr = 0.003), while L-T4+T1AM produced a 30.61% rebound relative to euthyroid state (mean diff = 141.6 ± 31.91, t = 4.44, pcorr = 0.004). We performed qPCR analysis of 88 genes involved in neurotrophic signalling pathways and found an effect of treatment on the expression of Ngf, Kdr, Kit, L1cam, Ntf3, Mapk3, and Neurog2. Our data confirm that L-T4 is necessary and sufficient for recovering memory and hippocampal neurogenesis deficits associated with hypothyroidism, while we found no evidence to support the role of non-canonical TH signalling.

3-Iodothyronamine and 3,5,3'-triiodo-L-thyronine reduce SIRT1 protein expression in the HepG2 cell line.

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.

Exogenous 3-Iodothyronamine Rescues the Entorhinal Cortex from ß-Amyloid Toxicity.

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 amine-associated receptor 1 (TAAR1), and induces pro-learning and anti-amnestic effects in mice. Dysregulation of TH signaling has long been hypothesized to play a role in Alzheimer's disease (AD). In the present investigation, we explored the neuroprotective role of T1AM in beta amyloid (Aß)-induced synaptic and behavioral impairment, focusing on the entorhinal cortex (EC), an area that is affected early by AD pathology. Methods: Field potentials were evoked in EC layer II, and long-term potentiation (LTP) was elicited by high frequency stimulation (HFS). T1AM (5 µM) and/or Aß(1-42) (200 nM), were administered for 10 minutes, starting 5 minutes before HFS. Selective TAAR1 agonist RO5166017 (250 nM) and TAAR1 antagonist EPPTB (5 nM) were also used. The electrophysiological experiments were repeated in EC-slices taken from a mouse model of AD (mutant human amyloid precursor protein [mhAPP], J20 line). We also assessed the in vivo effects of T1AM on EC-dependent associative memory deficits, which were detected in mhAPP mice by behavioral evaluations based on the novel-object recognition paradigm. TAAR1 expression was determined by Western blot, whereas T1AM and its metabolite 3-iodothyroacetic acid (TA1) were assayed by high-performance liquid chromatography coupled to mass spectrometry. Results: We demonstrate the presence of endogenous T1AM and TAAR1 in the EC of wild-type and mhAPP mice. Exposure to Aß(1-42) inhibited LTP, and T1AM perfusion (at a concentration of 5 µM, leading to an actual concentration in the perfusion buffer ranging from 44 to 298 nM) restored it, whereas equimolar amounts of 3,5,3'-triiodo-L-thyronine (T3) and TA1 were ineffective. The response to T1AM was abolished by the TAAR1 antagonist EPPTB, whereas it was mimicked by the TAAR1 agonist RO5166017. In the EC of APPJ20 mice, LTP could not be elicited, but it was rescued by T1AM. The intra-cerebro-ventricular administration of T1AM (0.89 µg/kg) also restored recognition memory that was impaired in mhAPP mice. Conclusions: Our results suggest that T1AM and TAAR1 are part of an endogenous system that can be modulated to prevent synaptic and behavioral deficits associated with Aß-related toxicity.

Ultrastructural Localization of Histidine-rich Glycoprotein in Skeletal Muscle Fibers: Colocalization With AMP Deaminase.

Histidine-rich glycoprotein (HRG) is a plasma protein synthesized by the liver. We have given the first evidence of a tissue localization of HRG demonstrating its presence in skeletal muscle, associated with the zinc enzyme AMP deaminase (AMPD1). Moreover, we have shown that muscle cells do not synthesize HRG, but they can internalize it from plasma. We have recently demonstrated by confocal laser scanning microscopy that in human skeletal muscle, HRG is mainly localized in the myofibrils, preferentially at the I-band of the sarcomere, in the sarcoplasm, and in the nuclei. Using transmission electron microscopy and immunogold analysis, we carried out this study on human and rat normal skeletal muscles with the purpose to deepen the ultrastructural localization of HRG in skeletal muscle fibers. The immunogold analysis evidenced the presence of HRG in the sarcomeres, mainly in the I-band and to a less extent in the A-band, in the heterochromatin of nuclei, and in the sarcoplasmic reticulum. The colocalization of HRG and skeletal muscle AMPD1 was also analyzed. A colabeling of HRG and AMPD1 was evident at sarcomeric, sarcoplasmic reticulum, and nuclear levels. The significance of these interesting and new results is discussed in this article.

Vitamin D measurement and effect on outcome in a cohort of patients with heart failure

Objectives: The aims of this paper were to evaluate the levels of Vitamin D (VitD) in patients with heart failure (HF), compared to a control group, to assess the effects of VitD on HF outcome and to compare VitD measurement between LIAISON immunoassay and HPLC-MS-MS methods in this population. Design and Methods: We collected clinical, biochemical and outcome data from 247 patients with HF and in a subgroup of 151 patients, we measured VitD both with LIAISON and HPLC-MS-MS. Results: HF patients had statistically lower 25OHD levels (45.2 ± 23.7 nmol/L vs 58.2 ± 24.0 nmol/L, P < 0.001) and a statistically higher prevalence of VitD insufficiency (61.1% vs 39.5%, P < 0.001) and deficiency (24.7% vs 6.6%, P < 0.001), compared to healthy controls. There was a significant inverse relationship between baseline 25OHD and risk of HF-related death, with a HR of 0.59 (95% CI 0.37­0.92, P = 0.02), confirmed in a multivariate adjusted analysis. Kaplan­Meier survival analyses showed that VitD insufficiency was associated with reduced survival in HF patients (log rank P = 0.017). There was a good agreement between LIAISON and HPLC-MS-MS (Cohen's kappa coefficient 0.70), but the prevalence of VitD insufficiency was significantly higher with the former compared to the latter method (58.3%, n = 88 vs 55.6%, n = 84, P < 0.001). LIAISON underestimated the 25OHD levels and showed a mean relative bias of −0.739% with 95% of limits of agreement (−9.00 to +7.52%), when compared to HPLC-MS-MS. Conclusions: 25OHD levels adequately measured by HPLC-MS-MS showed to be low in HF population and to be correlated with HF-related risk of death.

3,5-Diiodo-l-Thyronine Increases Glucose Consumption in Cardiomyoblasts Without Affecting the Contractile Performance in Rat Heart.

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 < 0.05). Crystal violet staining confirmed a reduction of cell viability only upon treatment with 10 µM T2, while equimolar T3 and T4 did not share this effect. Glucose consumption was also significantly affected as indicated by glucose uptake being increased by 24 or 35% in cells exposed to 0.1 or 1.0 µM T2 (P < 0.05 in both cases). On the contrary, T3 did not affect glucose consumption which, in turn, was significantly reduced by 1 and 10 µM T4 (-24 and -41% vs control, respectively, P < 0.05 and P < 0.01). In the isolated perfused rat heart, 10 µM T2 produced a slight and transient reduction in cardiac output, while T3 and T4 did not produce any hemodynamic effect. Our findings indicate that T2 is taken up by cardiomyoblasts, and at 0.1-1.0 µM concentration it can modulate cardiac energy metabolism by increasing glucose consumption. Some evidence of toxicity and a transient impairment of contractile performance are observed only at 10 µM concentration. These effects appear to be specific for T2, since they are not reproduced by T3 or T4.

Quantification of dehydroepiandrosterone in human serum on a routine basis: development and validation of a tandem mass spectrometry method based on a surrogate analyte.

In the clinical laboratories, dehydroepiandrostenedione (DHEA) is usually quantified by immunoassay-based methods, which are often affected by cross-reactivity with endogenous interferences, such as 4-androsten-3ß-ol-17-one. The interfering compounds lead to a poor accuracy of the measurements, mainly at a low concentration level. The present paper describes a validated method based on tandem mass spectrometry coupled to liquid chromatography, for the accurate quantification of DHEA in serum. The peculiarity of this method is the use of calibrators and quality controls prepared by adding measured amounts of DHEA-D5, a stable isotope-labeled analogue of DHEA, to real serum from healthy subjects. DHEA-D5 is used in place of DHEA, which is usually present in unstripped serum at physiological levels, as it has the same basic structure, provides an equivalent instrumental response, and can be easily distinguish by DHEA by mass spectrometry due to its different m/z value. The method proved to be sensitive, with a LLOD of 0.09 ng/mL and a LLOQ of 0.23 ng/mL, and selective, with overall performances that allow its use on a routine basis.

Hypovitaminosis D in patients with heart failure: effects on functional capacity and patients' survival.

Chronic heart failure is a major cause of morbidity and mortality, but its prognosis remains poor. Vitamin D hormone has many extra-skeletal functions including a positive impact on the cardiovascular system, and has been proposed for mortality risk evaluation in heart failure patients. The aim of the present study was to evaluate vitamin D status in heart failure patients, measured by high performance liquid chromatography coupled with mass spectrometry and to correlate serum 25 hydroxy-vitamin D (25OHD) levels with functional (peak VO2%) and mortality (Metabolic Exercise Cardiac Kidney Index) heart failure parameters. We enrolled 261 consecutive patients diagnosed with heart failure; all patients underwent a comprehensive clinical and biochemical characterization, and serum 25OHD levels were measured by high performance liquid chromatography coupled with mass spectrometry. Cardiopulmonary test parameters and Metabolic Exercise Cardiac Kidney Index of mortality risk were measured in all patients. Serum 25OHD levels ranged between 2 and 45 ng/ml (mean 17 ± 9 ng/ml); most patients (87%) showed hypovitaminosis D, and 25% showed severe vitamin D deficiency (serum 25OHD < 10 ng/ml). Patients with 25OHD < 10 ng/ml had significantly lower cardiopulmonary test VO2/kg, peak VO2% and significantly higher N-terminalproBrain natriuretic peptide and Metabolic Exercise Cardiac Kidney Index, than patients with 25OHD > 10 ng/ml. Patients with peak VO2% < 50% showed significantly lower 25OHD compared to those with peak VO2% > 50%. There was a significant, positive correlation (r = 0.16, p = 0.008) between 25OHD levels and peak VO2%, and an inverse correlation with Metabolic Exercise Cardiac Kidney Index (r = -0.21, p < 0.001), even when adjusted for age, Body Mass Index, MDRD, N-terminalproBrain natriuretic peptide. In conclusion, our findings show that vitamin D levels are associated with functional and mortality heart failure prognosis parameters.

Design, Synthesis, and Evaluation of Thyronamine Analogues as Novel Potent Mouse Trace Amine Associated Receptor 1 (mTAAR1) Agonists.

Trace amine associated receptor 1 (TAAR1) is a G protein coupled receptor (GPCR) expressed in brain and periphery activated by a wide spectrum of agonists that include, but are not limited to, trace amines (TAs), amphetamine-like psychostimulants, and endogenous thyronamines such as thyronamine (T0AM) and 3-iodothyronamine (T1AM). Such polypharmacology has made it challenging to understand the role and the biology of TAAR1. In an effort to understand the molecular basis of TAAR1 activation, we rationally designed and synthesized a small family of thyronamine derivatives. Among them, compounds 2 and 3 appeared to be a good mimic of the parent endogenous thyronamine, T0AM and T1AM, respectively, both in vitro and in vivo. Thus, these compounds offer suitable tools for studying the physiological roles of mouse TAAR1 and could represent the starting point for the development of more potent and selective TAAR1 ligands.

Cardioprotection by ranolazine in perfused rat heart.

: 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.

Modulation of gene expression by 3-iodothyronamine: genetic evidence for a lipolytic pattern.

3-Iodothyronamine (T1AM) is an endogenous biogenic amine, structurally related to thyroid hormone, which is regarded as a novel chemical messenger. The molecular mechanisms underlying T1AM effects are not known, but it is possible to envisage changes in gene expression, since delayed and long-lasting phenotypic effects have been reported, particularly with regard to the modulation of lipid metabolism and body weight. To test this hypothesis we analysed gene expression profiles in adipose tissue and liver of eight rats chronically treated with T1AM (10 mg/Kg twice a day for five days) as compared with eight untreated rats. In vivo T1AM administration produced significant transcriptional effects, since 378 genes were differentially expressed in adipose tissue, and 114 in liver. The reported changes in gene expression are expected to stimulate lipolysis and beta-oxidation, while inhibiting adipogenesis. T1AM also influenced the expression of several genes linked to lipoprotein metabolism suggesting that it may play an important role in the regulation of cholesterol homeostasis. No effect on the expression of genes linked to toxicity was observed. The assay of tissue T1AM showed that in treated animals its endogenous concentration increased by about one order of magnitude, without significant changes in tissue thyroid hormone concentration. Therefore, the effects that we observed might have physiological or pathophysiological importance. Our results provide the basis for the reported effectiveness of T1AM as a lipolytic agent and gain importance in view of a possible clinical use of T1AM in obesity and/or dyslipidaemia.

Uptake and metabolic effects of 3-iodothyronamine in hepatocytes.

3-Iodothyronamine (T1AM) is an endogenous relative of thyroid hormone with profound metabolic effects. In different experimental models, T1AM increased blood glucose, and it is not clear whether this effect is entirely accounted by changes in insulin and/or glucagone secretion. Thus, in the present work, we investigated the uptake of T1AM by hepatocytes, which was compared with the uptake of thyroid hormones, and the effects of T1AM on hepatic glucose and ketone body production. Two different experimental models were used: HepG2 cells and perfused rat liver. Thyronines and thyronamines (T0AMs) were significantly taken up by hepatocytes. In HepG2 cells exposed to 1 µM T1AM, at the steady state, the cellular concentration of T1AM exceeded the medium concentration by six- to eightfold. Similar accumulation occurred with 3,5,3'-triiodothyronine and thyroxine. Liver experiments confirmed significant T1AM uptake. T1AM was partly catabolized and the major catabolites were 3-iodothyroacetic acid (TA1) (in HepG2 cells) and T0AM (in liver). In both preparations, infusion with 1 µM T1AM produced a significant increase in glucose production, if adequate gluconeogenetic substrates were provided. This effect was dampened at higher concentration (10 µM) or in the presence of the amine oxidase inhibitor iproniazid, while TA1 was ineffective, suggesting that T1AM may have a direct gluconeogenetic effect. Ketone body release was significantly increased in liver, while variable results were obtained in HepG2 cells incubated with gluconeogenetic substrates. These findings are consistent with the stimulation of fatty acid catabolism, and a shift of pyruvate toward gluconeogenesis. Notably, these effects are independent from hormonal changes and might have physiological and pathophysiological importance.

Distribution of exogenous [125I]-3-iodothyronamine in mouse in vivo: relationship with trace amine-associated receptors.

3-Iodothyronamine (T1AM) is a novel chemical messenger, structurally related to thyroid hormone, able to interact with G protein-coupled receptors known as trace amine-associated receptors (TAARs). Little is known about the physiological role of T1AM. In this prospective, we synthesized [125I]-T1AM and explored its distribution in mouse after injecting in the tail vein at a physiological concentration (0.3 nM). The expression of the nine TAAR subtypes was evaluated by quantitative real-time PCR. [125I]-T1AM was taken up by each organ. A significant increase in tissue vs blood concentration occurred in gallbladder, stomach, intestine, liver, and kidney. Tissue radioactivity decreased exponentially over time, consistent with biliary and urinary excretion, and after 24 h, 75% of the residual radioactivity was detected in liver, muscle, and adipose tissue. TAARs were expressed only at trace amounts in most of the tissues, the exceptions being TAAR1 in stomach and testis and TAAR8 in intestine, spleen, and testis. Thus, while T1AM has a systemic distribution, TAARs are only expressed in certain tissues suggesting that other high-affinity molecular targets besides TAARs exist.

LTI models for 3-iodothyronamine time dynamics: a multiscale view.

3-Iodothyronamine (T(1)AM) is a novel relative of thyroid hormone that plays a role in critical body regulatory processes such as glucose metabolism, thermal regulation, and heart beating. This paper was aimed at characterizing time dynamics of T(1)AM and its catabolite 3-iodothyroacetic acid (TA(1)) in different biological scales with linear time-invariant models. Culture medium samples coming from culture of H9c2 murine cells and perfusion liquid samples from perfused rat heart were collected after the injection of a T(1)AM bolus. T(1)AM and TA(1) concentrations in the samples were assayed with high-performance liquid chromatography coupled to tandem mass spectrometry. Kinetic constants relative to T(1)AM transport and conversion were estimated with weighted least-squares method. We found that these constants can be related with an allometric power law depending on mass, with a negative exponent of -0.27 ± 0.19, implying that the velocity of conversion and internalization of T(1)AM decreases with increasing of system mass.

Cardioprotective effect of 3-iodothyronamine in perfused rat heart subjected to ischemia and reperfusion.

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.

Effect of acute and chronic zofenopril administration on cardiac gene expression.

We investigated whether acute and chronic administration of zofenopril, an angiotensin converting enzyme inhibitor, may modulate the expression of genes which are involved in the pathophysiology of myocardial ischemia and heart failure. We used an acute and a chronic model. In the former isolated rat hearts were perfused for 120 min in the presence or in the absence of 10 µM zofenoprilat, the active metabolite of zofenopril. In the chronic model one group of rats was treated with zofenopril (15.2 mg/Kg die per os) for 15 days, while control rats were treated with the same diet, except that zofenopril was omitted. Total RNA was extracted from hearts, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to evaluate the expression of α myosin heavy chain, superoxide dismutase, heat shock protein 70 (HSP70), nitric oxide synthase 2 and 3 (NOS2, NOS3), heme oxygenase 1, atrial natriuretic peptide (ANP), muscle phosphofructokinase. Acute or chronic zofenopril administration did not produce any change in hemodynamic variables. qRT-PCR experiments showed that in the acute model ANP expression was slightly although not significantly increased. In the chronic model, significant changes in gene expression were detected: in particular, HSP70 was upregulated (1.06 ± 0.38 vs. 0.72 ± 0.20 arbitrary units, P = 0.025), while NOS3 was downregulated (0.66 ± 0.06 vs. 0.83 ± 0.18 arbitrary units, P = 0.007). In the chronic model, liver samples were also assayed, but no significant change in the expression of any gene was detected. We conclude that zofenopril can produce heart-specific effects on gene expression. Persistent changes were detected with regard to specific heat shock protein and nitric oxide synthase subtypes. This action might contribute to the therapeutical response, and particularly to the increased resistance to ischemia.

Tissue distribution and cardiac metabolism of 3-iodothyronamine.

3-iodothyronamine (T1AM) is a novel relative of thyroid hormone, able to interact with specific G protein-coupled receptors, known as trace amine-associated receptors. Significant functional effects are produced by exogenous T1AM, including a negative inotropic and chronotropic effect in cardiac preparations. This work was aimed at estimating endogenous T1AM concentration in different tissues and determining its cardiac metabolism. A novel HPLC tandem mass spectrometry assay was developed, allowing detection of T1AM, thyronamine, 3-iodothyroacetic acid, and thyroacetic acid. T1AM was detected in rat serum, at the concentration of 0.3±0.03 pmol/ml, and in all tested organs (heart, liver, kidney, skeletal muscle, stomach, lung, and brain), at concentrations significantly higher than the serum concentration, ranging from 5.6±1.5 pmol/g in lung to 92.9±28.5 pmol/g in liver. T1AM was also identified for the first time in human blood. In H9c2 cardiomyocytes and isolated perfused rat hearts, significant Na+-dependent uptake of exogenous T1AM was observed, and at the steady state total cellular or tissue T1AM concentration exceeded extracellular concentration by more than 20-fold. In both preparations T1AM underwent oxidative deamination to 3-iodothyroacetic acid. T1AM deamination was inhibited by iproniazid but not pargyline or semicarbazide, suggesting the involvement of both monoamine oxidase and semicarbazide-sensitive amine oxidase. Thyronamine and thyroacetic acid were not detected in heart. Finally, evidence of T1AM production was observed in cardiomyocytes exposed to exogenous thyroid hormone, although the activity of this pathway was very low.

Effects of zofenopril on cardiac sarcoplasmic reticulum calcium handling.

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.