TRH System Research Articles

Short-term doxorubicin cardiotoxic effects: involvement of cardiac Thyrotropin Releasing Hormone system

Doxorubicin is an antineoplastic in the anthracycline class widely used for the treatment of several solid tumors and blood cancers. Cardiotoxicity is the major dose-limiting adverse effect of the drug. Chronic and accumulated doxorubicin administration cause myocyte damage and myocardial fibrosis. Doxorubicin-associated cardiotoxicity can be also observed after a short-course drug treatment even without clinical evidence of cardiac disease. Nevertheless, acute underlying mechanisms involved in the initiation of drug-induced cardiotoxicity remain poorly explored despite their similarities with pathophysiological conditions where cardiac TRH (cTRH) plays a central role. We showed that cTRH mediates myocardial injury induced by hypertension, and angiotensin II. Further, cTRH overexpression induces cardiac apoptosis, hypertrophy and fibrosis. AimTo demonstrate that cTRH could mediate acute doxorubicin cardiotoxicity. Main methodA single injection of doxorubicin (10 mg kg/day i.p.) was used to evaluate acute cardiac damage in a short-term experimental model of doxorubicin-induced cardiotoxicity. While inhibiting cTRH by small interfering RNA (siRNA), we evaluated the progression of cardiotoxicity. Key findingsWe found a doxorubicin-induced TRH overexpression in the LV, which was associated with apoptosis, hypertrophy and fibrosis. siRNA-mediated cTRH suppression prevented the doxorubicin-associated cardiac histological lesions. Significancesdoxorubicin requires an active cardiac TRH system to promote heart injury.

Abstract 027: Participation of Cardiac Thyrotroping Releasing Hormone (cTRH) in the Doxorrubicin (D) Induced Cardiotoxicityin in Mice

We have demonstrated TRH hyperactivity in the hypertrophied Left ventricle (LV) of SHR. Its specific inhibition attenuates hypertrophy development in spite of the significant higher pressure observed (Schuman M, Hypertension 2011). LV-TRH over-expression induces several features of the hypertrophied heart, including increases in the apoptotic index Bax/Bcl2, and the activated caspase 3 observed by immunohystochemistry (Schuman M, AJPH&C 2014). In addition, we have found that TRH expression was induced by D in primary cultures of cardiac cells. Based on these results we hypothesized that cTRH could participate in the D cardiotoxicity effects. Indeed, we used C57 adult males (n=10) with a single D or saline injection (200uL,10 mg/kg ip) which previously (24 h, under anesthesia) received an intracardiac injection of a specific TRH-siRNA to inhibit LV-TRH expression or scrambled Con-siRNA. Mice were sacrificed 2,4 and 7 days post D injection and body weight was measured. Genes expression was measured by real time PCR and protein by immunohystochemistry (ANOVA and Tukey test). Body weight showed a mild but not significant decrease in D treated animals. D significantly increased TRH gene expression and TRH protein content reaching the maximum at 7 days post injection (2d: 145%, 4d:190% and 7d: 250%) (p& 0.05), which were not observed in the groups with D+TRH-siRNA indicating the effectiveness of the specific TRH inhibition. Also at this time TRH inhibition attenuates (p&0.05) D-induced increase in the apoptotic index Bax/Bcl2 and the augmented activated caspase 3 content pointing out the participation of the cardiac TRH in the D-induced apoptosis. Similar results were observed with hypertrophic and fibrotic markers gene expression (BNP, BMHC and col III) which showed a significant increase (p& 0.05) only in the groups with D and the intact TRH system (D+Con-siRNA). Fibrosis results were confirmed by Sirius Red and Masson techniques. On the whole, we demonstrated for the first time that LV-TRH system is required for both, Doxorrubicin induction of apoptosis and consecutively hypertrophy and fibrosis in the mouse heart. Even more, we found that cTRH inhibition attenuates doxorrubicin induced damage suggesting a novel mechanisms in the cardiotoxicity injury.

Abstract P141: Cardiac TRH Partly Mediates Angiotensin II-induced Fibrotic and Hypertrophy Effects in "in vivo" and "in vitro" Models

Cardiac TRH (cTRH) is overexpressed in the hypertrophied ventricle (LV) of the SHR. Additionally in vivo siRNA-TRH treatment induced downregulation of LV-TRH preventing cardiac hypertrophy and fibrosis demonstrating that TRH is involved in hypertrophic and fibrotic processes. Moreover, in a normal heart, the increase of LV TRH expression alone could induce structural changes where fibrosis and hypertrophy could be involved, independently of any other system alterations. Is well-known the cardiac hypertrophy/ fibrotic effects induced by AII, raising the question of whether specific LV cTRH inhibition might attenuates AII induced cardiac hypertrophy and fibrosis in mice. We challenged C57 mice with AII (osmotic pumps,14 days; 2 mg/kg) to induce cardiac hypertrophy vs saline. Groups were divided and , simultaneously to pump surgery, injected intracardiac with siRNA-TRH and siRNA-Con as its control. Body weight, water consume and SABP were measured daily. As expected, AII significantly increased SABP (p<0.05) in both groups treated , although cardiac hypertrophy (heart weight/body weight) was only evident in the group with the cardiac TRH system undamaged, suggesting that the cardiac TRH system function as a necessary mediator of the AII-induced hypertrophic effect. As hypothesized, we found an AII-induced increase of TRH (p<0.05) gene expression (real-t PCR) confirmed by immunofluorescence that was not observed in the group AII+siRNA-TRH demonstrating the specific siRNA treatment efficiency. Furthermore, AII significantly increase (p<0.05) BNP (hypertrophic marker), III collagen and TGFB (fibrosis markers) expressions only in the group with AII with the cardiac TRH system intact. On the contrary, the group with AII and the cTRH system inhibited, shows genes expressions similar to the saline control group. We confirmed these results by immunofluorescence. Similar fibrotic results were observed with NIH3T3 cell culture where we demonstrated that AII induced TRH gene expression (p<0.05) and its inhibition impedes AII-induced increase of TGFB and III/I collagens expressions telling us about the role of the cTRH in the AII fibrosis effects. Our results point out that the cardiac TRH is involved in the AII-induced hypertrophic and fibrotic effects.

Cardiac Delivery of Interference RNA for Thyrotropin-Releasing Hormone Inhibits Hypertrophy in Spontaneously Hypertensive Rat

See related article, pp 103–109 Thyrotropin-releasing hormone (TRH) is a tripeptide synthesized in the paraventricular nucleus of the hypothalamus and is best known for its classic role in stimulating the release of thyroid-stimulating hormone (TSH) and prolactin from the anterior pituitary gland. By stimulating the release of TSH, TRH regulates thyroxine (T4) and triiodothyronine (T3) production and secretion into the bloodstream. The presence of a TRH system in the paraventricular and preoptic nuclei of the hypothalamus suggests a direct role of TRH in cardiovascular physiology. Indeed, blood pressure (BP), heart rate, and contractility increase after intracerebroventricular1 or intravenous2 administration of TRH in rats. Although administration of exogenous TRH at pharmacological doses has helped to identify the potential roles of TRH, further elucidation of endogenous TRH function and mediation by its 2 receptors in rodents (in humans, only 1 TRH receptor has been identified thus far) have been hampered because of the lack of receptor subtype-selective antagonists. Furthermore, these observations are consistent with the fact that the heart is a major target organ for thyroid hormones, and that there are marked changes in cardiac function and structure in patients with hypothyroidism or hyperthyroidism.3 Spontaneously hypertensive rats (SHR) are used as a model of human essential hypertension. It presents an increase in both TRH content and TRH precursor mRNA abundance in the preoptic area, with a higher cerebrospinal fluid TRH concentration and TRH receptor number in this area.4 This model demonstrates a gradual progression of myocardial hypertrophy, fibrosis, left ventricular (LV) dysfunction, and heart failure. Persistent hypertension begins when rats reach ≈2 months of age and is followed by a relatively long period of stable, compensated hypertrophy. By 18 to 24 months, male SHR develop heart failure and exhibit a marked upregulation of genes that encode …

Thyrotropin-Releasing Hormone Receptor 1-Deficient Mice Display Increased Depression and Anxiety-Like Behavior

TRH is a neuropeptide with a variety of hormonal and neurotransmitter/neuromodulator functions. In particular, TRH has pronounced acute antidepressant effects in both humans and animals and has been implicated in the mediation of the effects of other antidepressive therapies. Two G protein-coupled receptors, TRH receptor 1 (TRH-R1) and TRH-R2, have been identified. Here we report the generation and phenotypic characterization of mice deficient in TRH-R1. The TRH-R1 knockout mice have central hypothyroidism and mild hyperglycemia but exhibit normal growth and development and normal body weight and food intake. Behaviorally, the TRH-R1 knockout mice display increased anxiety and depression levels while behaving normally in a number of other aspects examined. These results provide the first direct evidence that the endogenous TRH system is involved in mood regulation, and this function is carried out through TRH-R1-mediated neural pathways.

SiRNA-mediated silencing of the diencephalic thyrotropin-releasing hormone precursor gene decreases the arterial blood pressure in the obese agouti mice

Obesity is associated with increased cardiovascular morbidity and mortality, in part through development of hypertension. Leptin promotes weight loss by reducing food intake and increasing energy expenditure through sympathetic stimulation. It also counteracts the starvation-induced suppression of thyroid hormone by up-regulating the expression of TRH. On the other hand, it is known that the extrahypothalamic TRH system participates in cardiovascular function modulating sympathetic system activity. In order to challenge the testable hypothesis that obesity may raise arterial blood pressure (ABP) through TRH system activation, we herein analyze the participation of the TRH system in the elevation of ABP in the obese agouti yellow mice. These mice are characterized by resistance to the weight reducing effect of leptin although they show a preserved sympathetic response to leptin along with a mild hypertension compared with the control strain (121+/-2 vs 102+/-2 mmHg, p less than 0.001, n=10). We report here that hyperleptinemic agouti mice showed a 1.8-fold elevation of diencephalic TRH content compared with controls, and we demonstrate that a long lasting specific inhibition of TRH system by icv treatment with siRNA against preproTRH normalizes systolic ABP independently of the thyroid status. These results suggest that the interaction leptin-diencephalic TRH may be one of the mechanisms involved in the mild hypertension of the obese agouti mice.

Central Antisense Treatment Against Thyroliberin Normalizes Blood Pressure in Spontaneously Hypertensive Rats Without Affecting Thyroid Status.

P202 Thyroliberin (TRH) participates in central cardiovascular regulation and hypothalamic TRH precursor gene overexpression induces hypertension which can be reversed by antisense (AS) treatment. SHR show an increased central TRH synthesis and release and TRH receptor number. We show here that a phosphotioate AS against the preTRH injected icv decreased the augmented diencephalic TRH content (ng/mg protein) along with a normalization of systolic blood pressure (SABP) at 24-48 hs in SHR without effect in normotensive WKY rats (table). Saline or sense treatment had no effects. To investigate if changes in thyroid function may explain these results, we studied plasma TSH, T 3 (ng/ml) and T 4 (ug%) in control (CON) and AS-treated WKY and SHR. Although TSH was elevated in SHR compared to WKY rats in basal conditions and AS reverted it (table), no differences (n=5) were observed between strains with or without AS treatment in plasma T 4 (CON; WKY: 5.0±0.4 vs SHR: 5.3±0.5 and AS; WKY:5.4±0.3 vs SHR: 5.6±0.4) or T 3 (CON; WKY: 1.3±0.4 vs SHR: 1.1±0.2 and AS; WKY: 1.5±0.2 vs SHR: 1.4±0.3). To summarize, TRH AS decreases the higher diencephalic TRH content and changed arterial blood pressure to that of WKY rats even though SHR were basally and after AS treatment remained clinically euthyroid. These results suggest that the central TRH system is involved in the etiopathogenesis of hypertension in this model.

Central Antisense Treatment Against Thyroliberin Normalizes Blood Pressure in Spontaneously Hypertensive Rats Without Affecting Thyroid Status.

P202 Thyroliberin (TRH) participates in central cardiovascular regulation and hypothalamic TRH precursor gene overexpression induces hypertension which can be reversed by antisense (AS) treatment. SHR show an increased central TRH synthesis and release and TRH receptor number. We show here that a phosphotioate AS against the preTRH injected icv decreased the augmented diencephalic TRH content (ng/mg protein) along with a normalization of systolic blood pressure (SABP) at 24-48 hs in SHR without effect in normotensive WKY rats (table). Saline or sense treatment had no effects. To investigate if changes in thyroid function may explain these results, we studied plasma TSH, T 3 (ng/ml) and T 4 (ug%) in control (CON) and AS-treated WKY and SHR. Although TSH was elevated in SHR compared to WKY rats in basal conditions and AS reverted it (table), no differences (n=5) were observed between strains with or without AS treatment in plasma T 4 (CON; WKY: 5.0±0.4 vs SHR: 5.3±0.5 and AS; WKY:5.4±0.3 vs SHR: 5.6±0.4) or T 3 (CON; WKY: 1.3±0.4 vs SHR: 1.1±0.2 and AS; WKY: 1.5±0.2 vs SHR: 1.4±0.3). To summarize, TRH AS decreases the higher diencephalic TRH content and changed arterial blood pressure to that of WKY rats even though SHR were basally and after AS treatment remained clinically euthyroid. These results suggest that the central TRH system is involved in the etiopathogenesis of hypertension in this model.

Thyrotropin-Releasing Hormone Hyperactivity in the Preoptic Area of Spontaneously Hypertensive Rats

Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation through the activation of different neurotransmitter systems at distinct extrahypothalamic sites. To study possible alterations in the TRH system in the hypertensive state, we measured TRH concentration in cerebrospinal fluid and TRH content of the preoptic area in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) by radioimmunoassay. In addition, we also measured the density of the TRH receptor in this area by a rapid filtration technique using [3H]methyl-TRH. We found a significant increase in both the TRH content (634 +/- 61 versus 350 +/- 26 pg/mg protein, SHR versus WKY; P < .01, n = 5) and density of TRH receptors without changes in affinity (Bmax, 5.0 +/- 0.1 versus 3.3 +/- 0.1 fmol/mg protein, P < .01, n = 4). An increase in TRH concentration was also found in the cerebrospinal fluid of SHR (30 +/- 3 versus 21 +/- 2 pg/mL, P < .01, n = 5), suggesting increased TRH release in the central nervous system. Northern blot analysis indicated a threefold augmented abundance of TRH precursor mRNA in the preoptic area of SHR. A polyclonal antibody raised against TRH injected peripherally or intracerebroventricularly lowered arterial blood pressure in SHR but not in WKY. In addition, long-term treatment with enalapril (5 mg/kg twice daily), which was effective in inhibiting serum angiotensin-converting enzyme activity by more than 50%, decreased arterial blood pressure and preoptic area TRH content of SHR, whereas another vasodilator, diltiazem (10 mg/kg every 8 hours), failed to produce a similar change.(ABSTRACT TRUNCATED AT 250 WORDS)

Different regulation of thyrotropin releasing hormone content and release in paraventricular nucleus (PVN) and median eminence (ME) of rat hypothalamus during in vitro incubation.

TRH is present throughout the central nervous system possessing many different functions. Only TRH synthesized in the hypothalamic PVN and transported to the ME regulates anterior pituitary secretion of thyrotropin. To investigate this hypophysiotropic system, we have developed a method using dissected rat PVN and ME, representing mostly cell bodies and nerve terminals respectively, of the same TRH system. Tissues were incubated for four 30 min periods each in Locke's medium with alternatively normal and high KCl concentration. Repeated KCl-induced membrane depolarization resulted in significantly increased TRH release from both tissues (15% of TRH content) which was dependent on Ca2+ influx. Some important differences were found in the regulation of TRH in the PVN and ME: Comparison of TRH content in the tissue before and after incubation with the amount of secreted TRH showed new production of TRH during incubation in the ME, but not in the PVN. Frequent medium replacement during depolarization revealed that TRH secretion at the level of the ME, but not the PVN, is probably inhibited by some substance released during incubation. These data suggest that there is a different regulation of TRH in the PVN and ME and that TRH secreted by the isolated PVN may come mostly from the perikarya and represent paracrine neurohormone secretion.

Processing of Thyrotropin-Releasing Hormone (TRH) Prohormone in the Rat Olfactory Bulb Generates Novel TRH-Related Peptides*

Based on the deduced amino acid sequence of rat TRH prohormone (pro-TRH), proteolytic processing of this polyprotein precursor is expected to produce, beside TRH, several other novel peptides. These peptides should correspond to connecting segments flanking the repeated TRH progenitor sequence and to various C- and/or N-terminally extended forms of TRH. The profile of the endogenous products of the TRH system was studied in rat brain using multiple RIAs coupled to molecular sieve filtration and HPLC separations. In extracts from the rat hypothalamus, TRH and two pro-TRH-connecting peptides, prepro-TRH-(160-169) and prepro-TRH-(178-199) were detected in molar ratios corresponding to those expected for a nearly complete processing of the prohormone molecule. In the olfactory bulb, pro-TRH is processed differently, since peptides containing TRH at their N-termini, [pGlu172] prepro-TRH-(172-199) and [pGlu154]prepro-TRH-(154-169), were found to be major end products along with prepro-TRH-(160-169) and prepro-TRH-(178-199). The dissimilarity in tissue content suggests that differential processing of TRH precursor by various enzymatic pathways may act as a regulating mechanism for TRH and TRH-related activities. The cellular localization of C-terminally extended forms of TRH in rat olfactory bulb was examined by the indirect immunoperoxidase method, using antisera directed against prepro-TRH-(160-169) and pre-pro-TRH-(178-199). Cell bodies and nerve fibers were detected in the glomerular and external plexiform layers of the main olfactory bulb. The presence of extended forms of TRH in interneurons and middle tufted cells of the main olfactory bulb suggests that in light of the recent biological properties described for prepro-TRH-(160-169), these peptides may act as neuromodulators for olfactory epithelium inputs or neurotransmitters within more rostrally located olfactory areas in the forebrain.

Methamphetamine投与による脳内substance P系, TRH系の変化におよぼす選択的D-1, D-2遮断薬の効果

The effects of pretreatment with either SCH 23390, a selective D-1 antagonist or YM-09151-2, a selective D-2 antagonist, on methamphetamine (MAP)-induced changes in the substance P and TRH system were investigated. A single dose of 4 mg/kg of MAP reduced the striatal level of substance P 30 min after injection. This reduction was reversed by pretreatment with YM-09151-2 but not with SCH 23390. Repeated administration of 4 mg/kg of MAP for 14 days reduced specific substance P and TRH binding in the striatum at 1 h, but not at 48 h, after the last injection. The reduction of striatal substance P receptor binding was prevented by pretreatment YM-09151-2 but not SCH 23390 prior to each MAP administration. The reduction of striatal TRH receptor binding was prevented by pretreatment with either antagonist. These results indicate that changes in striatal substance P level and substance P receptor binding induced by subchronic MAP treatment are mediated via activation of D-2 but not D-1 receptors by MAP-released dopamine, while changes in striatal TRH receptor binding are mediated via both D-1 and D-2 receptors.

Psychosis of epilepsy--an approach to a biological basis for postictal and interictal psychoses.

Postictal and interictal changes in behavior and the corresponded neurochemical data in the kindling model of epilepsy are reviewed briefly to explore a biological base for epileptic psychosis. Kindling can be a potent animal model to study this problem. A possible participation of both TRH and opioid system in prolonged postictal behavior disorder and of TRH, mesolimbic system and/or PI response to excitatory amino acid in interictal behavior disorder are discussed. A further experimental study is required to disclose the basic mechanism of epileptic psychosis.

Effect of chronic desipramine treatment on neurotransmitter-thyrotropin releasing hormone--thyrotropin interactions in the rat.

Long-term administration of the antidepressant drug, desipramine (20 mg/kg/day, orally for 28 days), decreased the stimulatory effect of the alpha 2-adrenoceptor agonist, clonidine (250 micrograms/kg, i.p.) on thyrotropin (TSH) secretion in the rat, but did not alter basal TSH secretion. beta-Adrenoceptor-mediated inhibition of TSH secretion by isoproterenol (1 mg/kg, i.p.) was unaffected by chronic desipramine treatment, as were the stimulatory effect of TSH-releasing hormone (TRH, 5 micrograms/kg, i.v.) on TSH release and its inhibition by the alpha-adrenoceptor antagonist, phentolamine (2 mg/kg, i.p.). These findings suggest that chronic desipramine treatment induces subsensitivity of alpha 2-adrenoceptors which modulate TSH secretion in the rat while not affecting beta-adrenoceptor-mediated inhibition of TSH release. These findings suggest that pituitary TRH receptors are unchanged but that changes occurred at the hypothalamic level in alpha 2-adrenoceptor-mediated stimulation of TRH release. Although cerebral beta-adrenoceptors have been shown convincingly to be down-regulated after chronic desipramine treatment, their function in the hypothalamic TRH system after 28 days of treatment with desipramine appears to be unimpaired.

Thyroliberin action in pituitary cells is not inhibited by pertussis toxin.

The effects of pertussis toxin on the responses of rat pituitary-tumour (GH) cells to thyrotropin-releasing hormone (thyroliberin, TRH) were examined. Treatment of cells with pertussis toxin did not alter the affinity or concentration of TRH receptors, or the sensitivity of the TRH receptor to inhibition by guanine nucleotides. TRH caused an increase in low-Km GTPase activity in membrane-containing fractions from both control and pertussis-toxin-treated cells. TRH stimulation of inositol phosphate formation was insensitive to pertussis toxin. TRH caused a biphasic increase in the concentrations of cytosolic free Ca2+ as monitored by intracellularly trapped Quin 2, and this increase was the same in control and toxin-treated cultures. The toxin did not alter the increase in prolactin and growth-hormone (somatotropin) release stimulated by TRH or shift the TRH dose-response curve, and it did not affect the TRH-induced rise in prolactin synthesis measured over 24 h. However, pertussis toxin did block the ability of somatostatin and muscarinic agonists to inhibit prolactin and growth-hormone secretion stimulated by vasoactive intestinal peptide when analysed under the same conditions as those in which the TRH system was unaffected. These data indicate that the guanine nucleotide effects on TRH binding and activity are not mediated by Ni, but possibly by another member of the family of guanine-nucleotide-dependent regulatory proteins.

Thyrotropin secretory response to thyrotropin-releasing hormone in the hypothyroid perinatal rat: further evidence of thyrotroph independence of the hypothalamus during early ontogenesis.

Propylthiouracil fed to pregnant rats for the last week of gestation to induce maternal and fetal hypothyroidism induced a 3-fold rise in plasma TSH concentration in the newborn pups compared to a 4-fold rise in their mothers. Subcutaneous administration of 1 ng/g BW TRH caused a greater rise in plasma TSH in the hypothyroid pups than in their mothers. These results, in combination with published data, indicate that the apparent independence of pituitary-thyroid function from TRH control during early ontogenesis in the rat is primarily due to delayed maturation of the hypothalamic TRH system.