Modulation of cardiac ionic homeostasis by 3-iodothyronamine.

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.

Cardiac effects of trace amines: pharmacological characterization of trace amine-associated receptors.

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.

Short-term effects of pressure overload on the expression of genes involved in calcium homeostasis.

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.

Cardiac effects of 3-iodothyronamine: a new aminergic system modulating cardiac function.

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.

Modulation of cardiac sarcoplasmic reticulum calcium release by aenosine: a protein kinase C- dependent pathway.

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.

Effects of A1 adenosine receptor stimulation on the expression of genes involved in calcium homeostasis.

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.

3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone.

Thyroxine (T(4)) is the predominant form of thyroid hormone (TH). Hyperthyroidism, a condition associated with excess TH, is characterized by increases in metabolic rate, core body temperature and cardiac performance. In target tissues, T(4) is enzymatically deiodinated to 3,5,3'-triiodothyronine (T(3)), a high-affinity ligand for the nuclear TH receptors TR alpha and TR beta, whose activation controls normal vertebrate development and physiology. T(3)-modulated transcription of target genes via activation of TR alpha and TR beta is a slow process, the effects of which manifest over hours and days. Although rapidly occurring effects of TH have been documented, the molecules that mediate these non-genomic effects remain obscure. Here we report the discovery of 3-iodothyronamine (T(1)AM), a naturally occurring derivative of TH that in vitro is a potent agonist of the G protein-coupled trace amine receptor TAR1. Administering T(1)AM in vivo induces profound hypothermia and bradycardia within minutes. T(1)AM treatment also rapidly reduces cardiac output in an ex vivo working heart preparation. These results suggest the existence of a new signaling pathway, stimulation of which leads to rapid physiological and behavioral consequences that are opposite those associated with excess TH.

Cardioprotective effect of zofenopril in perfused rat heart subjected to ischemia and reperfusion.

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.

S-nitrosothiol detection in isolated perfused rat heart.

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.

Effect of ghrelin and synthetic growth hormone secretagogues in normal and ischemic rat heart.

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.

Ca2+ channel remodeling in perfused heart: Effects of mechanical work and interventions affecting Ca2+ cycling on sarcolemmal and sarcoplasmic reticulum Ca2+ channels.

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<0.01), with unchanged Kd. Such modifications were reversible. Similar results were obtained in the nifedipine and cardioplegia groups. Low preload perfusion produced similar contractile effects as hypocalcemic perfusion, but it had no effect on radioligand binding. After hypocalcemic perfusion, DHPR and RyR gene expression, evaluated by RT-PCR, were not modified. Chelerythrine (protein kinase C inhibitor) and lavendustin C (Ca2+/calmodulin-dependent protein kinase II inhibitor), but not H-89 (protein kinase A inhibitor), abolished the effects of hypocalcemic perfusion on [3H]-PN 200-110 and [3H]-ryanodine binding. We conclude that reduced Ca2+ entry and/or intracellular Ca2+ cycling determines DHPR and RyR remodeling through posttranslational protein modifications. Both protein kinase C and Ca2+/calmodulin-dependent protein kinase II appear to play a role in this phenomenon.

Effect of cardiac A(1) adenosine receptor overexpression on sarcoplasmic reticulum function.

OBJECTIVE: We investigated the effect of A(1) adenosine receptor overexpression, which has been reported to increase myocardial tolerance to ischemia-reperfusion injury, on sarcoplasmic reticulum (SR) Ca(2+) handling. METHODS: Transgenic mouse hearts (approximately 300-fold A(1) adenosine receptor overexpression) and wild-type mouse hearts were perfused in the Langendorff mode and subjected either to 80 min of aerobic perfusion or to 30 min of aerobic perfusion, 20 min of global ischemia and 30 min of reperfusion. The hearts were then homogenized and used to assay SR oxalate-supported 45Ca(2+) uptake and [3H]-ryanodine binding. RESULTS: Transgenic hearts showed increased resistance to ischemia-reperfusion, as shown by lower diastolic tension (1.5 +/- 0.2 vs. 2.6 +/- 0.1 g, P<0.05) and higher recovery of developed tension (45 +/- 3 vs. 30 +/- 4% of the baseline, P<0.05) following ischemia-reperfusion. Under baseline conditions, oxalate-supported 45Ca(2+) uptake was lower in transgenic hearts, owing to reduced V(max) (10.6 +/- 2.0 vs. 17.8 +/- 2.7 nmol/min per mg of protein, P<0.05), and the difference was preserved after ischemia-reperfusion (10.0 +/- 1.0 vs. 15.7 +/- 2.5 nmol/min per mg of protein, P<0.05). No significant difference in [3H]-ryanodine binding was observed. CONCLUSIONS: A(1) adenosine receptor overexpression is associated with a decreased rate of active Ca(2+) transport into the SR. We hypothesize that changes in SR function may cause a depletion of the SR Ca(2+) pool, which might protect from ischemic injury by delaying the development of cytosolic Ca(2+) overload during ischemia.