Trace Amine Receptors and Their Ligands

1

Tissue Thyroid Hormones and Thyronamines

Alice Accorroni*1, Federica Saponaro*1, Riccardo Zucchi2

1Scuola Superiore Sant’Anna, Pisa, Italy

2University of Pisa, Pisa, Italy

*These authors contributed equally to the present paper

Address for correspondence:

Riccardo Zucchi, MD PhD

Laboratory of Biochemistry

Department of Pathology

University of Pisa

via Roma 55

56126 Pisa

Italy

Abstract

It has been known for a long time that changes in cardiac function are a major component of the clinical presentation of thyroid disease. Increased heart rate and hyperdynamic circulation are hallmarks of hyperthyroidism, while bradycardia and decreased contractility characterize hypothyroidism. Recent findings have provided novel insights in the physiology and pathophysiology of heart regulation by thyroid hormones. In this review we summarize the present knowledge on thyroxine (T4) transport and metabolism, and on the biochemical pathways leading to genomic and nongenomic effects produced by 3,5,3’-triiodothyronine (T3) and by its active metabolites, particularly 3,5-diiodothyronine (T2) and 3-iodothyronamine (T1AM). On this basis, specific issues of special interest for cardiology are discusses, namely: 1) Relevance of the regulation of proteins involved in the control of calcium homeostasis and in pacemaker cell activity, due to nongenomic as well as to classical genomic effects. 2) Stimulation of fatty acid oxidation by T2 and T1AM, the latter also causing a negative inotropic and chronotropic action at micromolar concentrations. 3) Induction of D3 deiodinase in heart failure, potentially causing selective cardiac hypothyroidism, whose clinical implications are still controversial. 4) Cardioprotective effect of T1AM, possibly occurring at physiological concentrations, and relevance of T3 and of thyroid hormone receptor 1 in post-infarction repair.

Keywords: thyroid hormone, 3,5-didiotothyronine, 3-iodothyronamine, heart failure, ischemia

Introduction

The heart is a major target of thyroid hormone (TH) and cardiac effects are important components of the clinical picture of hypo- and hyperthyroidism. Experimental and clinical endocrinology has long relied on two assumptions: i) TH acts by interacting with nuclear TH receptors (TRs), which represent transcription factors modulating the expression of a wide range of target genes; ii) TH availability can be evaluated by assaying serum 3,5,3’-trioiodothyronine (T3), that has the highest affinity for TRs and is therefore considered as the active form of TH, and its precursor thyroxine (T4), that has a lower affinity for TR and is regarded as a pro-hormone. The term “thyroid hormones” (THs) is often used to refer to both T3 and T4.

These assumptions turned out to be simplistic. It has been ascertained that many effects of TH do not involve the modulation of gene expression [1]. On the other hand, evidence has been provided suggesting that cellular TH concentration is not directly related to serum T3 and T4, because of the complexity of their transmembrane transport and tissue metabolism [2, 3]. In addition, putative TH metabolites, namely 3,5-didiodothyronine (T2) and 3-iodothyronamine (T1AM) have been shown to be independent chemical messengers, whose effects may be different from those produced by TH [4–7].

In this review we will summarize some consequences of this paradigm shift. In particular, we will discuss cardiac TH transport and metabolism, the putative production of active metabolites, cardiac genomic and non-genomic responses to TH and TH-related messengers, and the potential pathophysiological implications of the novel acquisitions.

Thyroid hormone distribution and metabolism in heart

Membrane transport

THs have to cross the plasma membrane to reach their receptors. Since THs have a lipophilic skeleton, it was initially believed that their passage through the cell membrane was occurring by passive diffusion [8, 9]. In 1954, Christensen et al [3] first suggested the presence of an energy-dependent mechanisms for the entry of THs in the intracellular space. Subsequent investigations addressed the possibility of a plasma membrane transport [3, 4] and the existence of a saturable and stereospecific TH uptake was eventually demonstrated [5–8]. However, only recently TH transporters have been identified at the molecular level, and their importance has been highlighted by the identification of a neurological syndrome, Allan-Herndon-Dudley syndrome, characterized by severe neurological involvement and reduced cerebro-spinal fluid (CSF) T4 concentration [14–16], which is associated with genetic mutation of a specific TH transporters.

TH transporters belong to the solute-carrier gene (SLC) gene family and include members of the following groups: monocarboxylate transporters (MCT), Na+/taurocholate-cotransporting polypeptide (NTCP), large neutral amino acid transporters (LAT), and organic anion-transporting polypeptide (OATP) family [17–21]. Apart from MCT8, all transporters are also involved in the translocation of other substances, mainly steroids and amino acids whose tissue concentrations exceed by far TH concentrations.

Monocarboxylate transporters involved in THs entry in the intracellular space are MCT8 (encoded by SLCA16A2 gene) and MCT10 (encoded by SLCA16A10 gene). They belong to a family of protein with 12 transmembrane domains and act in a sodium- and proton-independent fashion [22]. MCT8 is involved in the transport of T4, T3 but also of 3,3’,5’-triiodothyronine (rT3) and 3,5-diiodothyronine (T2) [23]. In humans, MCT8 mutations are responsible for Allan-Herndon-Dudley syndrome. MCT10 is a transporter for aromatic acids. When compared to MCT8, it has lower affinity for T4, and slightly higher affinity for T3 [24].

NTCPs are glycoproteins with seven transmembrane domains that belong to the solute carrier gene family 10A (SCL101) [25]. They are involved in the enterohepathic circulation of bile acids and transport the sulfate derivatives of T4 and T3 [26].

LAT are heterodimeric proteins formed by a 12 transmembrane domain light chain and a glycosylated heavy chain with one transmembrane domain [27, 28]. They carry neutral amino acids inside the cell. LAT1 (encoded by SLC7A5) and LAT2 (encoded by SLC7A8) are involved in the transport of THs, and LAT1 has a higher affinity than LAT2 for T3 [29].

OATPs are 12-transmembrane-domain proteins, involved in transport of amphipatic organic compounds like steroids, bile salts, drugs and anionic olygopeptides [21, 30]. Oatp1c1, Oatp1a4, Oatp3a1, Oatp2b1 are able to transport THs. All of them transport T4 [31–33], Oatp1a4 also transports T3 [26, 34], and Oatp1c1 transports T4, T3, rT3 and T4 sulfate [26].

With regard to cardiomyocytes, most investigations have been focused on mRNA or protein expression of specific TH transporters. As for other tissues, early studies in the ‘50s confirmed that cardiomyocytes show energy- and Na+-dependent carriers for both T4 and T3 [35, 36], and the main classes of TH transporters turned out to be OATPs and MCTs.

MCT8 is prominently expressed in the heart [37], while MCT10 is expressed only at low levels [33-35]. As to OATPs, the prevailing subtypes are OATP2B1 [40], OATP2A1, and OATP4A1 [41].

At the present stage, the lack of comprehensive functional studies does not permit to build a complete and throughout picture of the mechanisms of TH transport inside cardiomyocytes, and to understand the specific role of the different classes of transporters expressed at the cardiac level.

Metabolism

Once inside the cell, TH undergo tissue-specific metabolism that includes deiodination, sulfonation, conjugation, deamination, and decarboxylation. Cardiac metabolism consists of deiodination, that is discussed below, and possibly decarboxylation and deamination, that are discussed in the following paragraphs.

Deiodinases are the major enzymes involved in the regulation of TH tissue availability [42, 43], since they modulate T3 concentration removing specific iodine atoms from T4 and other iodothyronines. Deiodination of the phenolic ring (5’-deiodination or outer ring deiodination) causes T4 activation into T3, whereas deiodination at the level of the tyrosyl ring (5-deiodination or inner ring deiodination), inactivates T4 yielding 3,3’,5’-triiodothyronine, also known as reverse T3 (rT3) [44].

Three types of deiodinases have been identified and named as type I (D1), II (D2) and III (D3) deiodinase. They include a selenocysteine residue at the catalytic domain, and have different specificity. Indeed, D1 can function as either an outer or inner ring iodothyronine deiodinase; D2 only acts as an outer ring deiodinase; whereas D3 is the major T3 and T4 inactivating enzyme, catalysing inner ring deiodination [44, 45].

D1 was the first selenocysteine deiodinase to be discovered. It is a transmembrane protein, but its specific location on the plasma membrane or in the endoplasmic reticulum is still controversial [39, 40]. In any case, D1 brings a limited contribution to the intracellular levels of T3 when compared to D2 [48, 49], possibly due to the fact that D2 has a 1000-fold lower Km for T4 than D1, although TH sulfatation facilitates deiodination by D1 [50, 51].

D1 expression is predominant in liver and kidneys; however, low levels have also been identified in the heart, spleen and lungs [43]. Using RT-PCR, Yonemoto et al [52] found that D1 mRNA is expressed exclusively in cardiomyocytes but not in cardiac fibroblasts. In cultured rat myocardial cells, D1 mRNA levels and activity were regulated by both T3 and angiotensin II [52, 53]. T3 upregulated D1 gene transcription through a TR-mediated mechanism, while angiotensin II produced the same effects through a mechanism possibly involving voltage sensitive Ca2+ channel and protein kinase C.

D2 is an obligate outer ring selenodeiodinase that is therefore involved in the activation of T4 to T3 and in the conversion of rT3 to T2 [45]. D2 is located in the endoplasmic reticulum where it is an integral membrane protein with the catalytic component in the cytosol [54]. D2 has been identified in several tissues. With regard to cardiac expression, there are important differences between humans and rodents. Human heart expresses high levels of D2 mRNA, whereas only low levels are present in rodent hearts [55], and D2 overexpression in mouse myocardium leads to a mild chronic thyrotoxicosis [56]. The mechanism underlying this difference has been clarified by Dentice et al [57]. They demonstrated that human D2 expression needs the interaction between the transcription factors Nkx-2.5 and GATA-4 and a binding site in the dio2 gene. In rat heart, however, the corresponding binding site is lacking, accounting for the low D2 mRNA levels. This observation suggests that extrapolations from rodent models to human physiology and pathophysiology should be considered with great care.

D2 is subjected to regulation by T3; indeed, in rodents, T3 administration decreased D2 mRNA levels, while methimazole-induced hypothyroidism was associated with increased D2 activity [58]. D2 expression is lower in atria than in ventricles, but the functional implications of this difference have not been explored yet.

D3 causes inner ring deiodination and converts THs into their biologically inactive counterparts [59]. It metabolizes T4, T3 and T2, while the corresponding sulfated iodothyronines do not represent substrates for this enzyme [60]. D3 is mainly expressed in embryonic and foetal tissues where its expression is strictly regulated in a tissue-and time-specific pattern suggesting that exposure of tissues to the correct TH levels plays a major role in cellular proliferation and differentiation. It has been identified also in adult tissues, but its reactivation seems to be correlated with pathological conditions, like cancer, chronic inflammation and critical illness [45]. In the heart, increased D3 expression and activity has been associated with ventricle hypertrophy and myocardial infarction [61, 62], as detailed below.

Active metabolites

As discussed above, T4 deiodination yields either T3 or rT3. Since both T3 and rT3 are still substrates for deiodinases, diiodothyronines can also be produced, namely 3,5-diiodothyonine (T2), 3,3’-diiodothyronine, and 3’,5’-diiodothyonine.

T2 has been detected in human blood by immunological methods (reviewed in ref [63]), and recently a novel chemiluminescence-linked immunoassay has been developed [64]. Average T2 concentrations were close to 0.2 nM, although in about one third of patients T2 levels were below the detection limit of the assay. On the basis of these findings, it is likely that T2 is also present in tissues, but so far it has not been possible to detect endogenous T2 in heart. However, administration of exogenous T2 has produced significant functional effects on energy metabolism, as discussed below. 3,3’-T2 has also been detected both by radio-immuno assay [65] and by mass spectrometry coupled to HPLC [66, 67], and its serum concentration was in the picomolar range.

Besides deiodination, the classical pathway of TH catabolism/inactivation is thought to involve deamination (through transamination or direct oxidation) followed by decarboxylation, yielding thyroacetic acid derivatives that can undergo further deiodination steps. In the absence of deamination, decarboxylation would produce primary amines named thyronamines. In 2004, Scanlan et al [68] observed that a specific thyronamine, 3-iodothyronamine (T1AM), was present in mouse brain homogenate. T1AM was found to interact with a specific G protein-coupled receptor (now known as trace amine-associated receptor 1, or TAAR1) and to produce functional effects, and so it was considered as a novel chemical messenger. This hypothesis has been confirmed in subsequent investigations, and the picture has grown more complex [6]: besides TAAR1, T1AM may interact with other TAAR subtypes, with adrenergic receptors, and with monoamine transporters; its functional effects are now known to include stimulation of lipid vs carbohydrate catabolism, neurological responses, and several cardiac actions, that will be discussed below.

After the original detection in brain, T1AM has been identified in many rodent tissues, including the heart, through assays based on HPLC coupled to tandem mass spectrometry. In cardiac homogenates, T1AM concentration averaged a few pmoles per gram of tissue, and was on the same order as tissue T3 and T4 [4]. T1AM biosynthetic pathway must still be clarified. Although it is usually believed that the enzyme aromatic amino acid decarboxylase can decarboxylate THs and their derivatives, this hypothesis was not confirmed in a recent investigation [69]. In a rat gut preparation, T1AM has been obtained after administration of exogenous T4, through a pathway involving deiodination to T2, decarboxylation to T2AM by ornithine decarboxylase, and further deiodination to T1AM [70]. It is still unclear whether a similar pathway may exist in other tissues, and whether cardiac T1AM is produced in situ or is obtained from the circulation. As a matter of fact, both H9c2 cardiomyocytes and isolated rat hearts have been shown to accumulate exogenous T1AM, and at the steady state cellular concentration exceeded extracellular concentration by over 10-fold. The mechanism of T1AM uptake has not been ascertained, but it appears to be Na+-driven [4]. Cellular accumulation might reflect the existence of membrane or intracellular binding sites. While TAAR1 is expressed in heart [71], its density and/or the presence of additional cardiac receptors have not been determined so far.

Functional effects

Genomic effects of TH

Our understanding of TH mechanisms of action has evolved in the last decades; indeed, the picture that, at the beginning, only included genomic regulation, has now been broaden to include non-transcriptional actions mediated by TH receptors (TRs) and non-transcriptional and transcriptional effects mediated by other receptors.

TRs have been cloned for the first time in 1986 [72, 73].They belong to a larger family of intracellular receptors that counts over 50 members and also includes the receptors for steroid hormones, retinoic acid, vitamin D, and peroxisomal proliferators [74]. The core common structural characteristic of these receptors is represented by a central DNA-binding domain that contains two zinc-fingers and by a carboxyl-terminal ligand-binding domain. TR α and β are encoded by two genes, TR A on chromosome 17 and TR B on chromosome 3, respectively. Through alternative splicing, an array of various forms of TR proteins is produced, of which only TRα1, β1, β2, and β3 are functional [75]. TRs are expressed in all tissues with a different and finely regulated pattern of expression. With regard to the myocardium, TRα1 is predominant and is expressed alongside with TRβ1 and TRβ2. Indeed, in rats TRα1 covers 40% of T3 heart-binding capacity [76] and, in mice, TRα mRNA represents 70% of TR transcripts [77, 78].

TRs are transcription factors that produce their effects on gene regulation through the interaction with specific co-activators, co-repressors and DNA sequences, the so-called TH response elements (TREs) that are located in the regulatory regions of a wide variety of target genes and comprise two or more receptor-binding half-sites arranged as direct, inverted or palindromic repeats [79–81]. Depending on the nature of TREs (positively or negatively-regulated TREs), the hormonal status and the cellular environment, TR can repress or enhance gene transcription [79]. In the absence of T3, TR bind positively-regulated TREs and repress their transcription through the interaction with a corepressor complex [82, 83]. On the contrary, when interacting with negatively-regulated TREs, TR induce constitutive gene expression of the involved gene. T3 binding to the ligand binding domain induces major conformational changes, the subsequent release of the corepressor and the recruitment of an array of coactivators that induce, in genes containing positively-regulated TREs, gene transcription. While the complex of specific steps in this process and the variety of coactivators and repressors has been extensively determined [84], the specific mechanisms involved in T3 induction of gene repression in negatively-regulated TREs-containing genes have not been fully elucidated.

In the myocardium, TH regulation concerns many genes (Table 1), including those encoding for myofibril, sarcoplasmic reticulum and sarcolemma proteins.

With regard to the first group, TH is involved in a fine regulation of myosin heavy chain (MHC) isoenzyme predominance. The combination of two MHCs and four light chains represent the structure of myosin holoenzyme. Skeletal and cardiac muscles have a distinct pattern of expression, with the heart expressing three isoforms: V1, V2, and V3. V1 is the more efficient isoenzyme in terms of contraction speed, and it is composed of two MHCα [85]. Whereas, V3, which is composed of two MHCβ and V2, which is formed by a heavy chain heterodimer of α and β, are less efficient than V1. T3 administration has been demonstrated to increase the levels of MHCα while decreasing those of MHCβ mRNA [86]. This shift in MHC expression is associated to an increased maximal velocity of shortening in isolated ventricular muscle [87]. In addition, thyroid status regulates MHC conversion from embryonic to adult isoforms during heart development. Indeed, this shift is delayed by hypothyroidism while it is accelerated by hyperthyroidism [88, 89].

Troponin (Tn) expression is also under the control of TH. Troponin is a complex of three proteins (TnI, TnC, TnT), located on the thin filament and mediating the interactions with the thick filament. TnI has two main isoforms, known as slow skeletal TnI (ssTnI) and cardiac TnI (cTnI), whose cardiac expression is strictly regulated during development. Indeed, ssTnI is expressed during embryonic life while, cTnI is expressed in the heart from embryonic day 10 and represents the predominant TnI isoform in adult hearts. Conditions of altered TH availability influence the time course of the switching from ssTnI to cTnI. Indeed, isoform switching is delayed in hypothyroidism [90], while it is anticipated in hyperthyroidism [91].

TH has also been implicated in the transcriptional regulation of sarcoplasmic reticulum Ca2+-ATPase (SERCA) that plays a pivotal role in the control of intracytoplasmic Ca2+ levels, which in turn influence both cardiac diastolic and systolic function. SERCA represent a family of three transmembrane enzymes located at the level of sarcoplasmic reticulum (SR); SERCA1 is expressed only in fast twitch skeletal muscles, SERCA2 has been found in both slow twitch skeletal muscles and myocardium, while SERCA3 is expressed in a variety of muscular and non-muscular cells. These enzymes are finely regulated by phospholamban, another transmembrane protein that inhibits SERCA function and blocks SR Ca2+ uptake. This inhibition is removed by phospholamban phosphorylation, which is induced by beta adrenergic stimulation and other transduction pathways leading to cAMP production.

The genes encoding for SERCA2 and phospholamban contain TREs and are subject to TH transcriptional regulation; indeed, T3 and T4 induce an increase in SERCA2 mRNA levels while reducing phospholamban mRNA levels [92–95]. In addition, T3 also affects phospholamban phosphorylation, with a time course that suggest the presence of non-genomic mechanisms [96]. These effects accelerate SR calcium uptake improving cardiomyocyte relaxation and overall cardiac diastolic function [97]. They also increase the total calcium load of the SR. Since more calcium is available for release upon sarcolemmal depolarization, the calcium transient in increased and systolic function is also enhanced. The latter effect may be potentiated by the induction of an increased number of SR calcium channels/ ryanodine receptors [98].

Results regarding TH regulation of sarcoplasmic Na/Ca exchanger (NCX-1) gene expression are still controversial. Hojo et al demonstrated that T3 increases both NCX-1 mRNA and protein content [99]; however, a later report suggested that TH negatively regulate the expression of this exchanger [100].

With regard to the third group, TH modulate the expression of β-adrenergic receptors and enzymes involved in the regulation of ion fluxes through the membrane.

The similarities of TH cardiovascular effects to those produced by sympathoadrenal stimulation suggested the possibility that TH could act modulating the number and/or the sensitivity of cardiovascular adrenergic receptors. However, no clear-cut evidence has been obtained up to now. Indeed, several studies demonstrated that in isolated hearts or cultured cardiomyocytes, T3 administration significantly increased the levels of beta 1 adrenergic receptor mRNA and the sensitivity to adrenergic stimulation [101, 102]. However, in vivo studies in primates and humans have failed to confirm these results [103].

Moreover, T3 affects Na/K ATPase expression, inducing an increase in α2 and β1 subunit mRNA in rat neonatal cardiomyocytes [104, 105]. T3 also increases the ventricular expression of 2 voltage-gated potassium channel isoforms (Kv4.2 and Kv4.3), mediating the Ca2+-independent transient outward current (It) [106].

Interestingly, TH downregulate TRα1 expression [107] and this effect plays a role in the progression of post-ischemic heart failure.

Non genomic effects of TH

As already mentioned in the previous section, the effect of TH relies on two principal mechanisms, genomic and non-genomic ones, that can be differentiated on the basis of their time course and of the sensitivity to transcription and translation inhibitors. TH genomic actions require de novo protein synthesis and are therefore inhibited by compounds that interfere with translation and transcription. They are typically identifiable only after a time lapse of at least 1 h, whereas, non-genomic actions are likely to occur in a time frame of seconds or minutes.

TH rapid effects appear to be initiated at specific binding sites located at the plasma membrane but also in the cytoplasm and intracellular organelles. A putative extranuclear binding site is the heteromeric integrin αVβ3 [89], a structural protein of the plasma membrane. TH interaction with integrin αVβ3 can produce effects at the extracellular level, e.g. through the modulation of growth factor receptors [108]. Intracellular effects can also occur, either through translocation of proteins from the cytoplasm to the nucleus, or by activation of specific kinases involved in genomic regulation, namely Akt or mitogen-activated protein kinases (MAPKs), particularly extracellular regulated kinase 1/2 (ERK1/2) [109–113].

Other putative binding sites are represented by cytosolic TRs and specific TR isoforms. As an example, cytosolic TRα1 is involved in the maintenance of the actin cytoskeleton [90], while p30TRα1 isoform present at the plasma membrane might be involved in the regulation of the proliferation of non-malignant cells via the activation of MAPK and Akt pathways [114].

Most non-genomic actions produced in the heart by THs are attributable to their ability to modulate ion transporters and ion channels. In rabbit myocardium, the administration of 10-10 M T3 and T4 increased SERCA activity with a calmodulin-mediated mechanism [115]. However, the non-genomic effects of TH on Ca2+ intracellular levels are still controversial. In particular, Lomax et al [116] demonstrated that T3 application (10-9- 10-7 M) increased intracellular Ca2+ concentration in rat ventricular myocytes, whereas Zinman et al [117] proved the contrary in newborn rat cardiocytes.

Modulation of other ionic currents might also contribute to regulate intracellular Ca2+ content. Indeed, Wang et al. [100] demonstrated that the acute exposure of atrial cardiomyocytes to T3 induces an increase in INa that, in turns, activates Na+-Ca2+ exchange and ultimately leads to an increase in intracellular Ca2+. The effects of TH on INa had already been described previously in cultured rat myocytes, where acute exposure to 10 nM T3 slowed the inactivation of INa [118, 119]. Other reports also identified an increased INa after acute exposure to T3, but this effect was attributed to an increase of burst activity [120]. It is still unclear whether TH interact directly with the specific ion channels or the final effect is mediated by the activation of specific intracellular pathways.

Moreover, it has been demonstrated that TH modulate Na+/H+ exchanger in L-6 myoblasts by extranuclear mechanism and that the regulation of Na+/K+ ATPase also represents a non genomic action [121]. Reciprocal regulation of cardiac Na+/K+ ATPase and Na+/H+ exchanger has been hypothesized, but needs further evaluation [122].

TH stimulate inward rectifying K+ currents (IKI). Indeed, in guinea pig ventricle myocytes, the acute administration of T3 at a concentration ranging from 1 nM to 1 μM increased IKI by reducing the interbust interval [123] with the result of shortening action potential duration.

An important question is whether non genomic effects play a significant physiological or pathophysiological role in vivo in humans. The first evidence that non genomic actions of TH were identifiable in humans came from the work of Bernhard et al. [124], who demonstrated that the administration of 100 μg of T3 to healthy euthyroid male volunteers increased cardiac output and reduced systemic vascular resistance within a time frame reflecting non genomic action.

Even though a fixed distinction between non-genomic and genomic actions of TH in the heart has been used to simplify the description of TH mode of action, it has to be underlined that the two pathways might share common targets producing the overall effects in cardiovascular diseases that will be considered and discussed in the next sections.

Cardiac effects of T2

Administration of exogenous T2 has been reported to modulate energy metabolism. Rats subjected to high fat diet, and treated with T2 at the dosage of 0.25 g/g body weight intraperitoneally for one month, showed a significant reduction of body weight, liver fatty acid oxidation rate, and serum triglyceride and cholesterol [125]. Hepatic fatty acid accumulation was also reduced [126]. A reduction of body mass was confirmed in Wistar rats fed with a normal diet, which were treated with 0.25-0.75 g/g T2 by subcutaneous injection for 90 days [127]. Parenteral administration was required to elicit this response, since no effect was observed when similar doses were administered per os [128]. It is likely that T2 bioavailability is reduced after oral administration, but this hypothesis has not been directly tested.

The metabolic response to T2 has been attributed to the stimulation of mitochondrial fatty acid oxidation [129–132], possibly due to mitochondrial translocation of the fatty acid transporter FAT/CD36, which was observed in skeletal muscle mitochondria [133]. Direct interference with respiratory chain enzymes, particularly cytochrome oxidase, has also been suggested (reviewed in refs [63] and [134]), and partial mitochondrial uncoupling might occur [132], but no specific molecular target has been identified so far. While the emphasis has usually been placed on nongenomic actions, transcriptional effects have also been described, particularly increased expression of the genes coding for the alpha and beta subunits of the ATP-synthase [135].

In the investigation by Lanni e al (2005) [125], chronic T2 administration (0.25 g/g) did not produce significant changes in serum FT3, FT4 and TSH, although a nonsignificant 20% decrease in FT4 was reported. So it was claimed that no alteration of the hypothalamus-pituitary-thyroid axis was produced. Consistently, no evidence of systemic thyrotoxicosis was observed, and the heart rate was unchanged. On the other hand, according to Padron et al (2014) [127], treatment with 0.25-0.75 g/g T2 produced a dose-dependent decrease in total serum T3 and T4. Serum TSH was unchanged in the 0.25 g/g group and slightly reduced in the groups receiving higher dosages. The heart weight was unchanged, but the ratio of heart weight to body weight was significantly increased at the highest dosage. Recently, Jonas et al (2015) [136] reported that T2 treatment was associated to significant suppression of the hypothalamus-pituitary-thyroid axis, significant cardiac hypertrophy and changes in the pattern of gene expression similar to those induced by T3. It was concluded that T2 produced thyromimetic effects. However, the dose-response relationship was at variance with the previous reports, since the responses described above were observed after 14 days of treatment with 2.5 g/g T2, i.e. at a dosage about one order of magnitude higher than previously used. At the more common dosage of 0.25 g/g, T2 produced only minor effects, namely no change in fat mass, no change in cardiac effects and a decrease in serum T4 but not in serum T3.

The concentration issue is critical to evaluate the potential physiological or pathophysiological relevance of endogenous T2. Initial assays with immunological methods provided estimated serum concentrations on the order of 10-20 pmol/l in humans [63], while the CLIA method recently developed by Lemphul et al (2014) yielded average values on the order of 200 pmol/l, that was close to the detection limit of the assay. Unfortunately, serum T2 could not be determined in most animal studies, except for the investigation by Jonas et al (2015) [136], in which treatment with 0.25 or 2.5 g/g exogenous T2 increased serum T2 by 25-fold and 120-fold over the baseline, respectively. Similar relative changes were also reported for liver T2 accumulation.

There is a single clinical investigation in which T2 was administered to two human volunteers at a very low dosage (300 g/day), and interestingly after 28 days body weight was decreased and resting metabolic rate was increased in both patients [137], with no changes in cardiac function.

On the whole, T2 appears to produce significant effects on fatty acid metabolism and on mitochondrial function, although their physiological relevance remains to be determined, and the development of more accurate assays is necessary to solve the question. Specific cardiac effects have not been described so far, but chronic high dose T2 administration might lead to myocardial hypertrophy. It is also unclear whether the response to T2 is mediated by specific receptors, which have not been identified yet, or whether it is related to thyromimetic effects. The latter are certainly possible at high dosages, because T2 has a low affinity for TR [138, 139]. Since recent evidence suggests that T2 might be a precursor of T1AM, an intriguing hypothesis is that some effects of exogenous T2 might actually be due to increased T1AM production.

Cardiac effects of T1AM

In the isolated rat heart, administration of exogenous T1AM produced within a few seconds a significant negative inotropic effect, as shown by reduced aortic flow, cardiac output and developed pressure [68]. The heart rate was also decreased, although the negative inotropic and chronotropic effect could be dissociated, and the former occurred at lower concentrations. Dose-response curved yielded IC50’s of 20 M and 40 M for the decrease in cardiac output and heart rate, respectively [71, 140]. These effects were reversible after washing out T1AM, and could be counteracted by positive inotropic and chronotropic agents, such as isoproterenol. Substantial bradycardia was also demonstrated in vivo, in mice treated with 50 mg/Kg T1AM i.p. [68].

Cell culture experiments performed with H9c2 cardiomyocytes showed that the negative chronotropic effect was due to a decrease of the calcium transient, which was probably accounted for by increased diastolic calcium leak from the SR, leading to a reduction of the SR calcium pool [141]. In fact, in SR vesicles loaded in vitro with 45Ca, the kinetics of calcium-induced calcium release was normal, but passive calcium efflux was significantly increased under conditions promoting closure of the SR calcium release channel. Electrophysiological experiments did not reveal any significant effect on the L-type calcium channel, while a delayed rectifier potassium channels was inhibited [141]. The latter effect, if confirmed also in the pacemaker cells of the sinus node, might contribute to the observed negative chronotropic response.

TAAR1 is expressed in heart [71], but it is unclear if it is involved in the observed functional effects, since the potency of other trace amines was not consistent with their affinity for TAAR1 [140]. Other TAARs are also expressed in rat heart, particularly TAAR8, but it remains to be determined whether they are activated by T1AM and can modulate contractile function. Interestingly, recent investigations suggest that T1AM might be an inverse agonist at TAAR5 [142], and that it can also interact with adrenergic receptors, particularly 2A receptors [143].

The negative inotropic response was accentuated by genistein, a tyrosine kinase inhibitor, and counteracted by vanadate, a tyrosine phosphatase inhibitor. In addition, Western blot experiments with anti-phosphotyrosine antibodies showed reduced tyrosine phosphorylation of as-yet unidentified proteins in the microsomal and cytosolic fraction [71]. Therefore, it has been suggested that the transduction pathway activated by T1AM may reduce the phosphorylation of tyrosine residues on protein targets regulating ionic homeostasis.

T1AM has also been tested in a model of ischemia-reperfusion injury, represented by the isolated perfused rat subjected to 30 min of ischemia and 120 min of retrograde reperfusion. A significant reduction of infarct size was observed, but the dose-response curve was bell-shaped, since effective concentrations ranged from 125 nM to 12.5 M, while higher doses were not protective [144]. So cardioprotection was dissociated from the negative inotropic effect, and was actually observed in the absence of any change in pre-ischemic contractility or heart rate. A different transduction pathway was probably activated, since protection was abolished by chelerythrine, a protein kinase inhibitor, which did not affect the inotropic and chronotropic effects of T1AM [71]. The putative mitochondrial KATP channel glybenclamide also antagonized the response to T1AM. It was therefore speculated that T1AM might activate the transduction pathways involved in ischemic preconditioning, which are known to involve protein kinase C and mitochondrial effectors [144]. Interestingly, mitochondrial effects of T1AM have also been reported, since in isolated mitochondria T1AM concentrations in the nanomolar range affected ATP synthase activity, oxygen consumption and free radical production [145, 146].

A crucial question is the potential physiological or pathophysiological relevance of endogenous cardiac T1AM. So far this issue has been addressed only by comparing the effective doses of exogenous T1AM with endogenous T1AM levels. As discussed above, cardiac T1AM concentration lies in the nanomolar range (i.e. pmol/g), and so it is about three orders of magnitude lower than the IC50 for the negative inotropic effect produced by exogenous T1AM. Therefore it is hard to believe that endogenous T1AM may modulate contractile function, while it might affect the susceptibility to ischemic injury. However, it must be acknowledged that we still do not know the extent of T1AM compartmentation and the rate of its metabolic clearance [4]. Therefore, it is difficult to compare the endogenous concentrations occurring at receptor level under physiological conditions with those achieved when exogenous T1AM is administered in experimental models. Understanding the physiological role of cardiac T1AM will eventually require a more direct approach, which could be made possible by the development of specific receptor antagonists and appropriate transgenic models. This will in turn depend on further insight in T1AM biosynthesis, catabolism, and signaling.

With regard to T1AM metabolism, the heart has been shown to oxidize T1AM to 3-iodothyroacetic acid [4]. In other tissues, deiodination to thyronamine (T0AM), N-acetylation, and conjugation to sulfate and glucuronate have also been reported [147]. Interestingly, T0AM shares the negative inotropic and chronotropic properties of T1AM, although it is about 10-fold less potent [140], while N-acetyl-T1AM has been recently reported to produce a positive chronotropic effect in vivo [148]. The potential cardiac effects of T1AM derivatives represent therefore another interesting research field.

Pathophysiological implications

The heart in hyperthyroidism

In hyperthyroidism, TH effects on the heart are amplified, eventually leading to a hyperdynamic cardiocirculatory state. In hyperthyroid patients, cardiac output is higher than in normal subjects, as the result of different factors: increased resting heart rate, increased ejection fraction, increased blood volume and decreased systemic vascular resistance. It is still controversial if the high cardiac output condition is mainly due to the direct actions of T3 on cardiomyocytes (myocardial hypothesis) or to indirect hemodynamic changes (vasculature hypothesis), or maybe to a balanced interaction between these two mechanisms [149]. Early in 1986, Klein et al. tried to separate the two different mechanisms using an animal model in which a heterotopically transplanted heart coexisted with the recipient’s in situ heart. In this model, TH administration caused a larger increase in weight and protein content in the in situ working heart than in the non-working heart [150], supporting the concept that cardiac work and hemodynamic changes play a major role in the cardiac effects of hyperthyroidism.

The direct effects of high levels of TH on cardiomyocytes are consistent with the physiological responses described above, namely increased transcription of the genes for myosin heavy chain α and SERCA2, while phospholamban gene expression is reduced and phospholamban phosphorylation is decreased. Specific effects on the electrical activity of specialized myocytes in the sinus node have also been reported [151]. In rats, TH administration increased the expression of hyperpolarization-activated cyclic nucleotide gated channel gene (HCN2) [152] that is involved in pacemaker activity. Other ion channel proteins in the sinus node of the left atrium were also modulated by either genomic or non-genomic mechanisms, namely SR calcium channel, sodium/calcium exchanger protein, and sarcolemmal L and T type calcium channels [151].

Elevated levels of TH can reduce peripheral vascular resistance, through either direct or indirect actions. Indeed, T3 directly reduces the expression of angiotensin II type 1 receptor on peripheral vasculature and the contractile response to angiotensin II, while it stimulates the signaling pathway leading to nitric oxide (NO) production, that decreases calcium concentration and calcium sensitivity [153]. Data obtained in rat aortic rings suggest that the vasodilatory effect of TH is mediated by the endothelium [154]. However, T3 can also act on vascular smooth muscle cell (VSMCs) in a rapid non-genomic way, causing NO-dependent relaxation, through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/A) pathway [155].

The eventual functional effects include relaxation of arterial smooth muscle fibers and reduction in peripheral resistance. The latter is also supported by the increased metabolic rate that is produced in most tissues as a consequence of the metabolic effects of TH. As a result of peripheral vasodilatation, vasopressor reflex responses lead to tachycardia, while the renin angiotensin aldosterone system is activated, with increased sodium resorption, leading to increased preload [153].

These mechanisms explain why increased cardiac output is the typical feature of hyperthyroidism, and account for the symptoms and signs of hyperthyroidism: tachycardia, palpitation, systolic hypertension with decreased diastolic arterial pressure, increased left ventricular mass, and systolic murmurs. Sinus tachycardia is the most common arrhythmia in hyperthyroidism, but atrial fibrillation has a rather high prevalence (2-20%), increasing with age. In the presence of pre-existing coronary artery or cardiac muscle disease, angina pectoris may be triggered by the increased energy demand, while heart failure can develop as a consequence of decreased diastolic filling and impaired systolic function.

The heart in hypothyroidism

Hypothyroidism is characterized by low levels of T3 and T4, with compensatory high levels of TSH (except in the case of central hypothyroidism). In general, the cardiac effect of hypothyroidism are opposite to those due to hyperthyroidism, although symptoms and signs may be milder, at least in the early phase.

When T3 concentration is low, TH-dependent transcriptional effects are attenuated, leading to significant changes in contractile function. Delayed diastolic relaxation is a common feature in hypothyroidism, and it has been attributed to increased transcription of phospholamban, that inhibits cardiomyocyte SERCA2. The relevance of this target was confirmed using transgenic mice in which the SERCA2 gene was not under TH control, since hypothyroid transgenic mice had a normal contractile phenotype [156]. In hypothyroid animals, mRNA and proteins levels of myosin heavy chain β were decreased and altered myosin composition may contribute to functional abnormalities. Non-genomic actions of TH on cardiomyocytes can also be relevant in hypothyroidism, leading to modifications of sodium, calcium and potassium channels, as described above, and these effects may eventually reduce cytosolic calcium availability [97].

The impairment of contractile function in hypothyroidism appears to be mainly mediated by thyroid hormones receptor α (TRα), since TRα knockout mice showed the typical features consisting in delayed relaxation, decreased tension development and lower heart rate, while TRβ knockout mice had normal contractile function [78].

These experimental results are consistent with the clinical findings reported in hypothyroidism, namely bradycardia, subnormal cardiac output, prolongation of the pre-ejection period and reduction in left ventricular ejection period. On the whole, mechanical efficiency is impaired, despite a reduced myocardial oxygen demand. Mixedema can occur as a result of a longstanding undertreated hypothyroidism, and the term refers to the thickened, nonpitting edematous changes to the soft tissues of patients in a markedly hypothyroid state.

Hypertension is another feature of the hypothyroid state: blood pressure is higher, not easily controlled by conventional therapies and reversible after reaching euthyroidism. The pathophysiological mechanisms seem to be an increase in peripheral resistance [157], and in arterial stiffness secondary to myxedema of the arterial wall [158]. Heart function may be further compromised by the development of pericardial effusion, which occurs with severe, long-standing overt hypothyroidism [159]. Moreover, in long standing hypothyroidism left ventricular hypertrophy can develop, due to interstitial myxedema. Finally, hypercholesterolemia and hypertension cause an increased risk of atherogenesis and coronary artery disease.

Some cardiac abnormalities have been described even in subclinical hypothyroidism, particularly slow diastolic relaxation with impairment of early ventricular filling at rest and after exercise. Recently, a large cross-sectional survey of postmenopausal women [160] revealed a strong association between subclinical hypothyroidism and atherosclerotic cardiovascular disease, independent of the traditional risk factors.

In the context of subclinical hypothyroidism, it was recently argued that the crucial variable is represented by TH tissue concentration, which may be altered is spite of quite normal serum TH concentrations. Liu et al. created an animal model in which thyroidectomized rats were implanted with slow-release pellets containing T4, enabling the analysis of graded levels of TH function. At low T4 dose, a dissociation was observed between serum and cardiac TH concentrations: serum T3 and T4 were normalized, but cardiac T3 and T4 remained subnormal. Moreover, low-dose T4 failed to prevent cardiac atrophy or restore cardiac function and arteriolar density to normal values [161]. Consistently with these data, Saba et al applied a novel liquid chromatography-tandem mass spectrometry method to the quantification of cardiac tissue THs in a rat model, and confirmed that, after administration of exogenous T3 or T4, similar tissue T4 values were obtained within a relatively large range of plasma T4 [162].

Occurrence of tissue hypothyroidism in heart failure

The interaction between TH and heart is bidirectional, since altered TH metabolism has been reported in patients with heart insufficiency and a normal thyroid gland. Typically, T3 is low, despite initially normal TSH and T4 levels. In heart failure patients, the decrease in T3 was proportional to the severity of the disease suggesting that a sort of “low-T3 syndrome” is an independent predictor of a poor prognosis in heart failure [163].

In general, failing cardiomyocytes show a pattern of gene expression similar to that found in hypothyroidism, with regard to key proteins like contractile proteins, sodium-calcium exchanger, and β-adrenergic receptors. Apart from changes in serum THs, the molecular mechanisms might be: i) modulation TH receptors and co-receptors, ii) reduction of transporter activity and consequently of TH uptake, iii) changes in local THs metabolism.

In explanted hearts from patients undergoing transplantation, a significant decrease in TRα1 was observed, while the expression TRα2, that is unable to bind TH and is therefore considered as a suppressor of gene transcription, was increased [164]. In a mouse model of heart failure induced by ascending aortic constriction, reduced expression of TRα1 and TRβ1 was also reported [165]. More recent experimental investigations have opened a new scenario, suggesting that TRα1 expression could play a role as a regulator of dedifferentiation/redifferentiation in cardiac stress, depending on the presence or absence of TH. In particular, TRα1 may trigger remodeling and reparative processes, possibly by nongenomic mechanisms involving the PI3K/Akt/mTOR (mammalian target of rapamycin) signaling pathway or the p38MAPK cascade. The latter has also been related to TRα1-specific cardiac hypertrophy. These findings have potential clinical relevance, since these molecules can be targets of new therapeutic approaches in heart failure [166].

While changes in cardiac TH transporters have not been reported in heart failure, the activity of deiodinases is another crucial issue. In particular, D3 induction appears to be involved in the progression of cardiac hypertrophy, from an adaptive to a maladaptive stage, precipitating heart failure. In a model of post-infarction heart failure in mouse, Pol et al (2011) reported increased D3 activity and a significant reduction of tissue T3, with normal serum T3 [167]. Wassen et al. created a mouse model of heart failure in which chronic pulmonary arterial hypertension was induced by a single dose of pyrrolizidine alkaloid monocrotaline (MCT): part of these mice developed congestive heart failure (CHF) and died within 4-5 weeks. In CHF mice, the patter of gene expression resembled hypothyroidism, plasma T4, FT4 and T3 were significantly reduced (40-60%) with normal TSH, and there was a five-fold increase in cardiac D3 activity [62].

These investigations suggest that the increase of D3 in heart hypertrophy and failure could lead to a local reduction of T3, independently from the serum levels, and contribute to a “hypothyroid” condition in the affected myocardium. The trigger for the increased D3 activity seems to be the mismatch of oxygen delivery and consumption in the hypertrophic cardiomyocytes, with subsequent intracellular stabilization of the transcription factor HIF1α, that directly stimulates D3 gene expression [62].

The hypothesis that the failing heart is in a “local hypothyroid state” arises the question if treatment with T3 could be beneficial. This issue is further described in this issue by Pingitore et al.

TH and the ischemic heart

Low levels of T3 are commonly observed in patients in the first hours after acute myocardial infarction (AMI), and seem to correlate with the prognosis. Independently from prompt therapies and primary percutaneous coronary intervention, T3 levels were inversely related to recovery time and ejection fraction (EF%) [168].

Since THs are crucial for embryonic heart development, they have been suggested to play a key role in the reactivation of foetal phenotype after myocardial injury and in damage reparation. A central role in this shift to embryonic signalling is the re-expression of TRα1, which is also involved in the progression to heart failure, as discussed above. After acute myocardial infarction, cardiac hypertrophy occurs as a compensatory effect: in this condition, TRα1 is redistributed from the cytosol to the nucleus, in a process regulated by ERK and mTOR signalling. In cell culture experiments, TRα1 exposure to low levels of TH induced cardiomyocytes de-differentiation, while in the presence of adequate TH concentration differentiation was induced, possibly leading to reparation of injured myocardium. This question is extensively discusses in this issue by Cokkinos et al.

Conclusions and open questions

The heart is a major target organ of THs and cardiac abnormalities play an important role in the phenotypes of both hyperthyroidism and hypothyroidism. The clinical features can be understood on the basis of the genomic and nongenomic actions of THs on the myocardium and blood vessels, but the recent paradigm shift in described in this review has opened new perspectives and introduced new questions.

Subtle cardiac abnormalities have been observed in subclinical hypothyroidism. Basic science investigations have provided an explanation for these observations, by showing that tissue and serum TH concentrations are not identical, because of the complexity of transmembrane TH transport and tissue metabolism. Similar considerations suggest that the failing heart may actually been hypothyroid, because tissue T3 concentration may be reduced in spite of normal circulating THs. This possibility has been clearly demonstrated in specific experimental models of heart failure and attributed to the induction of D3 deiodinase, favoring T4 inactivation to rT3 over T3 production. Changes in TR1 expression might also contribute to the phenotype of the failing heart.

The actual occurrence of tissue hypothyroidism in human heart failure is still uncertain, and its implications are controversial. Since TH increase cardiac contractility, reduced T3 availability might be a causal mechanism contributing to contractile failure. On the other hand, it has been argued that reduced oxygen consumption linked to tissue hypothyroidism might be a compensatory mechanism, delaying the progression of cardiac insufficiency, particularly in ischemic heart disease. The ongoing clinical trials will hopefully provide new evidence to solve this controversy.

Experimental investigations have also shown that exposure to THs is able to affect the susceptibility to ischemia and the long term recovery after myocardial infarction. This is consistent with the role played by THs in other cases of regeneration and repair, and points to another possible subject for clinical investigations.

Further complexity has been introduced by the discovery of the functional effects of TH derivatives, like T2 and T1AM. While no specific cardiac effects have yet been described for T2, T1AM produces a negative chronotropic and inotropic effect in different experimental models. However, the physiological importance of these actions is uncertain, since they were observed in vitro at doses that turned out to be much higher than the endogenous levels. Notably, T1AM affects ischemic injury at much lower dosages, and might contribute to the modulation of cardiac function in ischemic heart disease.

Since selective TR agonist and synthetic T2 and T1AM analogs are available, a deeper understanding of the role of TH and TH derivatives in cardiac disease might open new therapeutic perspectives.

Disclosures

There is no conflict of interest for any author.

References

1. Davis, P. J., Leonard, J. L., & Davis, F. B. (2008). Mechanisms of nongenomic actions of thyroid hormone. Frontiers in neuroendocrinology, 29(2), 211–8. doi:10.1016/j.yfrne.2007.09.003

2. Hennemann, G., Docter, R., Friesema, E. C., de Jong, M., Krenning, E. P., & Visser, T. J. (2001). Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocrine reviews, 22(4), 451–76. doi:10.1210/edrv.22.4.0435

3. Bianco, A. C., & Kim, B. W. (2006). Deiodinases: implications of the local control of thyroid hormone action. The Journal of clinical investigation, 116(10), 2571–9. doi:10.1172/JCI29812

4. Saba, A., Chiellini, G., Frascarelli, S., Marchini, M., Ghelardoni, S., Raffaelli, A., … Zucchi, R. (2010). Tissue distribution and cardiac metabolism of 3-iodothyronamine. Endocrinology, 151(10), 5063–5073. doi:10.1210/en.2010-0491

5. Piehl, S., Hoefig, C. S., Scanlan, T. S., & Köhrle, J. (2011). Thyronamines—Past, Present, and Future. Endocrine Reviews, 32(1), 64–80. doi:10.1210/er.2009-0040

6. Zucchi, R., Accorroni, A., & Chiellini, G. (2014). Update on 3-iodothyronamine and its neurological and metabolic actions. Frontiers in physiology, 5, 402. doi:10.3389/fphys.2014.00402

7. Moreno, M., Silvestri, E., De Matteis, R., de Lange, P., Lombardi, A., Glinni, D., … Goglia, F. (2011). 3,5-Diiodo-L-thyronine prevents high-fat-diet-induced insulin resistance in rat skeletal muscle through metabolic and structural adaptations. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 25(10), 3312–24. doi:10.1096/fj.11-181982

8. Sorimachi, K., & Robbins, J. (1978). Uptake and metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells. Effects of potassium cyanide and dinitrophenol. Biochimica et biophysica acta, 542(3), 515–26. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/567494

9. ROBBINS, J., & RALL, J. E. (1957). The interaction of thyroid hormones and protein in biological fluids. Recent progress in hormone research, 13, 161–202; discussion 202–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/13477808

10. Rao, G. S., Eckel, J., Rao, M. L., & Breuer, H. (1976). Uptake of thyroid hormone by isolated rat liver cells. Biochemical and biophysical research communications, 73(1), 98–104. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/999706

11. Hillier, A. P. (1968). The uptake and release of thyroxine and triiodothyronine by the perfused rat heart. The Journal of physiology, 199(1), 151–60. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1365349&tool=pmcentrez&rendertype=abstract

12. Hillier, A. P. (1969). The uptake of thyroxine and tri-iodothyronine by perfused hearts. The Journal of physiology, 203(3), 665–74. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1351536&tool=pmcentrez&rendertype=abstract

13. Singh, S. P., Carter, A. C., Kydd, D. M., & Costanzo, R. R. (1976). Interaction between thyroid hormones and erythrocyte membranes: competitive inhibition of binding 131 I-L-triiodothyronine and 131 I-L-thyroxine by their analogs. Endocrine research communications, 3(2), 119–31. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/182449

14. Kakinuma, H., Itoh, M., & Takahashi, H. (2005). A Novel Mutation in the Monocarboxylate Transporter 8 Gene in a Boy with Putamen Lesions and Low Free T4 Levels in Cerebrospinal Fluid. The Journal of Pediatrics, 147(4), 552–554. doi:10.1016/j.jpeds.2005.05.012

15. Dumitrescu, A. M., & Refetoff, S. (2013). The syndromes of reduced sensitivity to thyroid hormone. Biochimica et biophysica acta, 1830(7), 3987–4003. doi:10.1016/j.bbagen.2012.08.005

16. Heuer, H., Maier, M. K., Iden, S., Mittag, J., Friesema, E. C. H., Visser, T. J., & Bauer, K. (2005). The monocarboxylate transporter 8 linked to human psychomotor retardation is highly expressed in thyroid hormone-sensitive neuron populations. Endocrinology, 146(4), 1701–6. doi:10.1210/en.2004-1179

17. Friesema, E. C. H., Jansen, J., & Visser, T. . (2005). Thyroid hormone transporters. Biochemical Society Transactions, 33(1), 228–232. doi:10.1042/BST0330228

18. Friesema, E. C. H., Docter, R., Moerings, E. P. C. M., Stieger, B., Hagenbuch, B., Meier, P. J., … Visser, T. J. (1999). Identification of Thyroid Hormone Transporters. Biochemical and Biophysical Research Communications, 254(2), 497–501. doi:10.1006/bbrc.1998.9974

19. Abe, T., Suzuki, T., Unno, M., Tokui, T., & Ito, S. (2002). Thyroid hormone transporters: recent advances. Trends in endocrinology and metabolism: TEM, 13(5), 215–20. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12185668

20. Hagenbuch, B. (2007). Cellular entry of thyroid hormones by organic anion transporting polypeptides. Best practice & research. Clinical endocrinology & metabolism, 21(2), 209–21. doi:10.1016/j.beem.2007.03.004

21. Hagenbuch, B., & Meier, P. J. (2004). Organic anion transporting polypeptides of the OATP/ SLC21 family: phylogenetic classification as OATP/ SLCO superfamily, new nomenclature and molecular/functional properties. Pfl�gers Archiv European Journal of Physiology, 447(5), 653–665. doi:10.1007/s00424-003-1168-y

22. Halestrap, A. P. (2012). The monocarboxylate transporter family--Structure and functional characterization. IUBMB life, 64(1), 1–9. doi:10.1002/iub.573

23. Friesema, E. C. H., Ganguly, S., Abdalla, A., Manning Fox, J. E., Halestrap, A. P., & Visser, T. J. (2003). Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. The Journal of biological chemistry, 278(41), 40128–35. doi:10.1074/jbc.M300909200

24. Friesema, E. C. H., Jansen, J., Jachtenberg, J.-W., Visser, W. E., Kester, M. H. A., & Visser, T. J. (2008). Effective cellular uptake and efflux of thyroid hormone by human monocarboxylate transporter 10. Molecular endocrinology (Baltimore, Md.), 22(6), 1357–69. doi:10.1210/me.2007-0112

25. Anwer, M. S., & Stieger, B. (2014). Sodium-dependent bile salt transporters of the SLC10A transporter family: more than solute transporters. Pflügers Archiv : European journal of physiology, 466(1), 77–89. doi:10.1007/s00424-013-1367-0

26. Visser, W. E., Friesema, E. C. H., & Visser, T. J. (2011). Minireview: thyroid hormone transporters: the knowns and the unknowns. Molecular endocrinology (Baltimore, Md.), 25(1), 1–14. doi:10.1210/me.2010-0095

27. Ritchie, J. W., Peter, G. J., Shi, Y. B., & Taylor, P. M. (1999). Thyroid hormone transport by 4F2hc-IU12 heterodimers expressed in Xenopus oocytes. The Journal of endocrinology, 163(2), R5–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10556789

28. Friesema, E. C. H., Docter, R., Moerings, E. P. C. M., Verrey, F., Krenning, E. P., Hennemann, G., & Visser, T. J. (2001). Thyroid Hormone Transport by the Heterodimeric Human System L Amino Acid Transporter. Endocrinology, 142(10), 4339–4348. doi:10.1210/endo.142.10.8418

29. Morimoto, E., Kanai, Y., Kim, D. K., Chairoungdua, A., Choi, H. W., Wempe, M. F., … Endou, H. (2008). Establishment and characterization of mammalian cell lines stably expressing human L-type amino acid transporters. Journal of pharmacological sciences, 108(4), 505–16. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19075510

30. Hagenbuch, B., & Meier, P. J. (2003). The superfamily of organic anion transporting polypeptides. Biochimica et biophysica acta, 1609(1), 1–18. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12507753

31. Huber, R. D., Gao, B., Sidler Pfändler, M.-A., Zhang-Fu, W., Leuthold, S., Hagenbuch, B., … Stieger, B. (2007). Characterization of two splice variants of human organic anion transporting polypeptide 3A1 isolated from human brain. American journal of physiology. Cell physiology, 292(2), C795–806. doi:10.1152/ajpcell.00597.2005

32. Zhang, Y., Chen, K., Sloan, S. A., Bennett, M. L., Scholze, A. R., O’Keeffe, S., … Wu, J. Q. (2014). An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex. The Journal of neuroscience : the official journal of the Society for Neuroscience, 34(36), 11929–47. doi:10.1523/JNEUROSCI.1860-14.2014

33. Leuthold, S., Hagenbuch, B., Mohebbi, N., Wagner, C. A., Meier, P. J., & Stieger, B. (2009). Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters. American journal of physiology. Cell physiology, 296(3), C570–82. doi:10.1152/ajpcell.00436.2008

34. Abe, T., Kakyo, M., Sakagami, H., Tokui, T., Nishio, T., Tanemoto, M., … Yawo, H. (1998). Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2. The Journal of biological chemistry, 273(35), 22395–401. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9712861

35. Rosic, M., Pantovic, S., Lucic, A., Ribarac-Stepic, N., & Andjelkovic, I. (2001). Kinetics of thyroxine (T(4)) and triiodothyronine (T(3)) transport in the isolated rat heart. Experimental physiology, 86(1), 13–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11429614

36. Everts, M. E., Verhoeven, F. A., Bezstarosti, K., Moerings, E. P., Hennemann, G., Visser, T. J., & Lamers, J. M. (1996). Uptake of thyroid hormones in neonatal rat cardiac myocytes. Endocrinology, 137(10), 4235–42. doi:10.1210/endo.137.10.8828482

37. Bonen, A., Heynen, M., & Hatta, H. (2006). Distribution of monocarboxylate transporters MCT1-MCT8 in rat tissues and human skeletal muscle. Applied physiology, nutrition, and metabolism = Physiologie appliquée, nutrition et métabolisme, 31(1), 31–9. doi:10.1139/h05-002

38. Kim, D. K., Kanai, Y., Matsuo, H., Kim, J. Y., Chairoungdua, A., Kobayashi, Y., … Endou, H. (2002). The human T-type amino acid transporter-1: characterization, gene organization, and chromosomal location. Genomics, 79(1), 95–103. doi:10.1006/geno.2001.6678

39. Kim, D. K., Kanai, Y., Chairoungdua, A., Matsuo, H., Cha, S. H., & Endou, H. (2001). Expression cloning of a Na+-independent aromatic amino acid transporter with structural similarity to H+/monocarboxylate transporters. The Journal of biological chemistry, 276(20), 17221–8. doi:10.1074/jbc.M009462200

40. Grube, M., Köck, K., Oswald, S., Draber, K., Meissner, K., Eckel, L., … Kroemer, H. K. (2006). Organic anion transporting polypeptide 2B1 is a high-affinity transporter for atorvastatin and is expressed in the human heart. Clinical pharmacology and therapeutics, 80(6), 607–20. doi:10.1016/j.clpt.2006.09.010

41. Fujiwara, K., Adachi, H., Nishio, T., Unno, M., Tokui, T., Okabe, M., … Abe, T. (2001). Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner. Endocrinology, 142(5), 2005–12. doi:10.1210/endo.142.5.8115

42. Oppenheimer, J. H., Schwartz, H. L., Mariash, C. N., Kinlaw, W. B., Wong, N. C., & Freake, H. C. (1987). Advances in our understanding of thyroid hormone action at the cellular level. Endocrine reviews, 8(3), 288–308. doi:10.1210/edrv-8-3-288

43. Bianco, A. C., Salvatore, D., Gereben, B., Berry, M. J., & Larsen, P. R. (2002). Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocrine reviews, 23(1), 38–89. doi:10.1210/edrv.23.1.0455

44. Kuiper, G. G. J. M., Kester, M. H. A., Peeters, R. P., & Visser, T. J. (2005). Biochemical mechanisms of thyroid hormone deiodination. Thyroid : official journal of the American Thyroid Association, 15(8), 787–98. doi:10.1089/thy.2005.15.787

45. Gereben, B., Zeöld, A., Dentice, M., Salvatore, D., & Bianco, A. C. (2008). Activation and inactivation of thyroid hormone by deiodinases: local action with general consequences. Cellular and molecular life sciences : CMLS, 65(4), 570–90. doi:10.1007/s00018-007-7396-0

46. Fekkes, D., van Overmeeren-Kaptein, E., Docter, R., Hennemann, G., & Visser, T. J. (1979). Location of rat liver iodothyronine deiodinating enzymes in the endoplasmic reticulum. Biochimica et biophysica acta, 587(1), 12–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/226168

47. Baqui, M. M., Gereben, B., Harney, J. W., Larsen, P. R., & Bianco, A. C. (2000). Distinct subcellular localization of transiently expressed types 1 and 2 iodothyronine deiodinases as determined by immunofluorescence confocal microscopy. Endocrinology, 141(11), 4309–12. doi:10.1210/endo.141.11.7872

48. Silva, J. E., & Larsen, P. R. (1978). Contributions of plasma triiodothyronine and local thyroxine monodeiodination to triiodothyronine to nuclear triiodothyronine receptor saturation in pituitary, liver, and kidney of hypothyroid rats. Further evidence relating saturation of pituitary nuclea. The Journal of clinical investigation, 61(5), 1247–59. doi:10.1172/JCI109041

49. Larsen, P. R., Silva, J. E., & Kaplan, M. M. (1981). Relationships between circulating and intracellular thyroid hormones: physiological and clinical implications. Endocrine reviews, 2(1), 87–102. doi:10.1210/edrv-2-1-87

50. Otten, M. H., Mol, J. A., & Visser, T. J. (1983). Sulfation preceding deiodination of iodothyronines in rat hepatocytes. Science (New York, N.Y.), 221(4605), 81–3. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6857270

51. Mol, J. A., & Visser, T. J. (1985). Rapid and selective inner ring deiodination of thyroxine sulfate by rat liver deiodinase. Endocrinology, 117(1), 8–12. doi:10.1210/endo-117-1-8

52. Yonemoto, T., Nishikawa, M., Matsubara, H., Mori, Y., Toyoda, N., Gondou, A., … Inada, M. (1999). Type 1 iodothyronine deiodinase in heart --effects of triiodothyronine and angiotensin II on its activity and mRNA in cultured rat myocytes. Endocrine journal, 46(5), 621–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10670746

53. Iodothyronine 5′-Deiodinase Activity in Cultured Rat Myocardial Cells: Characteristics and Effects of Triiodothyronine and Angiotensin II: Endocrinology: Vol 128, No 6. (n.d.). Retrieved December 2, 2015, from http://press.endocrine.org/doi/pdf/10.1210/endo-128-6-3105

54. Maciel, R. M., Ozawa, Y., & Chopra, I. J. (1979). Subcellular localization of thyroxine and reverse triiodothyronine outer ring monodeiodinating activities. Endocrinology, 104(2), 365–71. doi:10.1210/endo-104-2-365

55. Croteau, W., Davey, J. C., Galton, V. A., & St Germain, D. L. (1996). Cloning of the mammalian type II iodothyronine deiodinase. A selenoprotein differentially expressed and regulated in human and rat brain and other tissues. Journal of Clinical Investigation, 98(2), 405–417. doi:10.1172/JCI118806

56. Pachucki, J., Hopkins, J., Peeters, R., Tu, H., Carvalho, S. D., Kaulbach, H., … Larsen, P. R. (2001). Type 2 iodothyronin deiodinase transgene expression in the mouse heart causes cardiac-specific thyrotoxicosis. Endocrinology, 142(1), 13–20. doi:10.1210/endo.142.1.7907

57. Dentice, M., Morisco, C., Vitale, M., Rossi, G., Fenzi, G., & Salvatore, D. (2003). The different cardiac expression of the type 2 iodothyronine deiodinase gene between human and rat is related to the differential response of the Dio2 genes to Nkx-2.5 and GATA-4 transcription factors. Molecular endocrinology (Baltimore, Md.), 17(8), 1508–21. doi:10.1210/me.2002-0348

58. Wagner, M. S., Morimoto, R., Dora, J. M., Benneman, A., Pavan, R., & Maia, A. L. (2003). Hypothyroidism induces type 2 iodothyronine deiodinase expression in mouse heart and testis. Journal of molecular endocrinology, 31(3), 541–50. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/14664714

59. Huang, H., Marsh-Armstrong, N., & Brown, D. D. (1999). Metamorphosis is inhibited in transgenic Xenopus laevis tadpoles that overexpress type III deiodinase. Proceedings of the National Academy of Sciences of the United States of America, 96(3), 962–7. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=15333&tool=pmcentrez&rendertype=abstract

60. Santini, F., Chopra, I. J., Hurd, R. E., Solomon, D. H., & Teco, G. N. (1992). A study of the characteristics of the rat placental iodothyronine 5-monodeiodinase: evidence that it is distinct from the rat hepatic iodothyronine 5’-monodeiodinase. Endocrinology, 130(4), 2325–32. doi:10.1210/endo.130.4.1547744

61. Olivares, E. L., Marassi, M. P., Fortunato, R. S., da Silva, A. C. M., Costa-e-Sousa, R. H., Araújo, I. G., … Carvalho, D. P. (2007). Thyroid function disturbance and type 3 iodothyronine deiodinase induction after myocardial infarction in rats a time course study. Endocrinology, 148(10), 4786–92. doi:10.1210/en.2007-0043

62. Wassen, F. W. J. S., Schiel, A. E., Kuiper, G. G. J. M., Kaptein, E., Bakker, O., Visser, T. J., & Simonides, W. S. (2002). Induction of thyroid hormone-degrading deiodinase in cardiac hypertrophy and failure. Endocrinology, 143(7), 2812–5. doi:10.1210/endo.143.7.8985

63. Senese, R., Cioffi, F., de Lange, P., Goglia, F., & Lanni, A. (2014). Thyroid: biological actions of “nonclassical” thyroid hormones. The Journal of endocrinology, 221(2), R1–12. doi:10.1530/JOE-13-0573

64. Lehmphul, I., Brabant, G., Wallaschofski, H., Ruchala, M., Strasburger, C. J., Köhrle, J., & Wu, Z. (2014). Detection of 3,5-diiodothyronine in sera of patients with altered thyroid status using a new monoclonal antibody-based chemiluminescence immunoassay. Thyroid : official journal of the American Thyroid Association, 24(9), 1350–60. doi:10.1089/thy.2013.0688

65. Pinna, G., Hiedra, L., Meinhold, H., Eravci, M., Prengel, H., Brödel, O., … Baumgartner, A. (1998). 3,3’-Diiodothyronine concentrations in the sera of patients with nonthyroidal illnesses and brain tumors and of healthy subjects during acute stress. The Journal of clinical endocrinology and metabolism, 83(9), 3071–7. doi:10.1210/jcem.83.9.5080

66. Soldin, O. P., & Soldin, S. J. (2011). Thyroid hormone testing by tandem mass spectrometry. Clinical biochemistry, 44(1), 89–94. doi:10.1016/j.clinbiochem.2010.07.020

67. Jonklaas, J., Sathasivam, A., Wang, H., Finigan, D., Soldin, O. P., Burman, K. D., & Soldin, S. J. (2014). 3,3’-diiodothyronine concentrations in hospitalized or thyroidectomized patients: results from a pilot study. Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists, 20(8), 797–807. doi:10.4158/EP13453.OR

68. Scanlan, T. S., Suchland, K. L., Hart, M. E., Chiellini, G., Huang, Y., Kruzich, P. J., … Grandy, D. K. (2004). 3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone. Nature medicine, 10(6), 638–42. doi:10.1038/nm1051

69. Hoefig, C. S., Renko, K., Piehl, S., Scanlan, T. S., Bertoldi, M., Opladen, T., … Köhrle, J. (2012). Does the aromatic L-amino acid decarboxylase contribute to thyronamine biosynthesis? Molecular and cellular endocrinology, 349(2), 195–201. doi:10.1016/j.mce.2011.10.024

70. Hoefig, C. S., Wuensch, T., Rijntjes, E., Lehmphul, I., Daniel, H., Schweizer, U., … Köhrle, J. (2015). Biosynthesis of 3-iodothyronamine from L-thyroxine in murine intestinal tissue. Endocrinology, en20141499. doi:10.1210/en.2014-1499

71. Chiellini, G., Frascarelli, S., Ghelardoni, S., Carnicelli, V., Tobias, S. C., DeBarber, A., … Zucchi, R. (2007). Cardiac effects of 3-iodothyronamine: a new aminergic system modulating cardiac function. The FASEB Journal, 21(7), 1597–1608. doi:10.1096/fj.06-7474com

72. Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Gruol, D. J., & Evans, R. M. The c-erb-A gene encodes a thyroid hormone receptor. Nature, 324(6098), 641–6. doi:10.1038/324641a0

73. Sap, J., Muñoz, A., Damm, K., Goldberg, Y., Ghysdael, J., Leutz, A., … Vennström, B. The c-erb-A protein is a high-affinity receptor for thyroid hormone. Nature, 324(6098), 635–40. doi:10.1038/324635a0

74. Flamant, F., Baxter, J. D., Forrest, D., Refetoff, S., Samuels, H., Scanlan, T. S., … Samarut, J. (2006). International Union of Pharmacology. LIX. The pharmacology and classification of the nuclear receptor superfamily: thyroid hormone receptors. Pharmacological reviews, 58(4), 705–11. doi:10.1124/pr.58.4.3

75. Chiamolera, M. I., Sidhaye, A. R., Matsumoto, S., He, Q., Hashimoto, K., Ortiga-Carvalho, T. M., & Wondisford, F. E. (2012). Fundamentally Distinct Roles of Thyroid Hormone Receptor Isoforms in a Thyrotroph Cell Line Are due to Differential DNA Binding. Molecular Endocrinology, 26(6), 926–939. doi:10.1210/me.2011-1290

76. Schwartz, H. L., Lazar, M. A., & Oppenheimer, J. H. (1994). Widespread distribution of immunoreactive thyroid hormone beta 2 receptor (TR beta 2) in the nuclei of extrapituitary rat tissues. The Journal of biological chemistry, 269(40), 24777–82. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7929155

77. Johansson, C., Vennström, B., & Thorén, P. (1998). Evidence that decreased heart rate in thyroid hormone receptor-alpha1-deficient mice is an intrinsic defect. The American journal of physiology, 275(2 Pt 2), R640–6. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9688704

78. Gloss, B., Trost, S., Bluhm, W., Swanson, E., Clark, R., Winkfein, R., … Dillmann, W. (2001). Cardiac ion channel expression and contractile function in mice with deletion of thyroid hormone receptor alpha or beta. Endocrinology, 142(2), 544–50. doi:10.1210/endo.142.2.7935

79. Yen, P. M. (2001). Physiological and molecular basis of thyroid hormone action. Physiological reviews, 81(3), 1097–142. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11427693

80. McKenna, N. J., & O’Malley, B. W. (2002). Combinatorial control of gene expression by nuclear receptors and coregulators. Cell, 108(4), 465–74. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11909518

81. Cheng, S. Y. (2000). Multiple mechanisms for regulation of the transcriptional activity of thyroid hormone receptors. Reviews in endocrine & metabolic disorders, 1(1-2), 9–18. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11704997

82. Hu, X., & Lazar, M. A. (1999). The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature, 402(6757), 93–6. doi:10.1038/47069

83. Rosenfeld, M. G., & Glass, C. K. (2001). Coregulator codes of transcriptional regulation by nuclear receptors. The Journal of biological chemistry, 276(40), 36865–8. doi:10.1074/jbc.R100041200

84. Wu, Y., & Koenig, R. J. (2000). Gene regulation by thyroid hormone. Trends in endocrinology and metabolism: TEM, 11(6), 207–11. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10878749

85. Reiser, P. J., Moss, R. L., Giulian, G. G., & Greaser, M. L. (1985). Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition. The Journal of biological chemistry, 260(16), 9077–80. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/4019463

86. Gustafson, T. A., Markham, B. E., & Morkin, E. (1986). Effects of thyroid hormone on alpha-actin and myosin heavy chain gene expression in cardiac and skeletal muscles of the rat: measurement of mRNA content using synthetic oligonucleotide probes. Circulation research, 59(2), 194–201. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/3742743

87. Morkin, E., Flink, I. L., & Goldman, S. Biochemical and physiologic effects of thyroid hormone on cardiac performance. Progress in cardiovascular diseases, 25(5), 435–64. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6221355

88. Gambke, B., Lyons, G. E., Haselgrove, J., Kelly, A. M., & Rubinstein, N. A. (1983). Thyroidal and neural control of myosin transitions during development of rat fast and slow muscles. FEBS letters, 156(2), 335–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6852266

89. Butler-Browne, G. S., Herlicoviez, D., & Whalen, R. G. (1984). Effects of hypothyroidism on myosin isozyme transitions in developing rat muscle. FEBS letters, 166(1), 71–5. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6692924

90. Averyhart-Fullard, V., Fraker, L. D., Murphy, A. M., & Solaro, R. J. (1994). Differential regulation of slow-skeletal and cardiac troponin I mRNA during development and by thyroid hormone in rat heart. Journal of molecular and cellular cardiology, 26(5), 609–16. doi:10.1006/jmcc.1994.1073

91. Huang, X., Lee, K. J., Riedel, B., Zhang, C., Lemanski, L. F., & Walker, J. W. (2000). Thyroid hormone regulates slow skeletal troponin I gene inactivation in cardiac troponin I null mouse hearts. Journal of molecular and cellular cardiology, 32(12), 2221–8. doi:10.1006/jmcc.2000.1249

92. Zarain-Herzberg, A., Marques, J., Sukovich, D., & Periasamy, M. (1994). Thyroid hormone receptor modulates the expression of the rabbit cardiac sarco (endo) plasmic reticulum Ca(2+)-ATPase gene. The Journal of biological chemistry, 269(2), 1460–7. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8166809

93. Hartong, R., Wang, N., Kurokawa, R., Lazar, M. A., Glass, C. K., Apriletti, J. W., & Dillmann, W. H. (1994). Delineation of three different thyroid hormone-response elements in promoter of rat sarcoplasmic reticulum Ca2+ATPase gene. Demonstration that retinoid X receptor binds 5’ to thyroid hormone receptor in response element 1. The Journal of biological chemistry, 269(17), 13021–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8175722

94. Rohrer, D., & Dillmann, W. H. (1988). Thyroid hormone markedly increases the mRNA coding for sarcoplasmic reticulum Ca2+-ATPase in the rat heart. The Journal of biological chemistry, 263(15), 6941–4. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/2966798

95. Nagai, R., Zarain-Herzberg, A., Brandl, C. J., Fujii, J., Tada, M., MacLennan, D. H., … Periasamy, M. (1989). Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone. Proceedings of the National Academy of Sciences of the United States of America, 86(8), 2966–70. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=287041&tool=pmcentrez&rendertype=abstract

96. Ojamaa, K., Kenessey, A., & Klein, I. (2000). Thyroid hormone regulation of phospholamban phosphorylation in the rat heart. Endocrinology, 141(6), 2139–44. doi:10.1210/endo.141.6.7514

97. Kahaly, G. J., & Dillmann, W. H. (2005). Thyroid hormone action in the heart. Endocrine reviews, 26(5), 704–28. doi:10.1210/er.2003-0033

98. Arai, M., Otsu, K., MacLennan, D. H., Alpert, N. R., & Periasamy, M. (1991). Effect of thyroid hormone on the expression of mRNA encoding sarcoplasmic reticulum proteins. Circulation research, 69(2), 266–76. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1830516

99. Hojo, Y., Ikeda, U., Tsuruya, Y., Ebata, H., Murata, M., Okada, K., … Shimada, K. (1997). Thyroid hormone stimulates Na(+)-Ca2+ exchanger expression in rat cardiac myocytes. Journal of cardiovascular pharmacology, 29(1), 75–80. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9007674

100. Reed, T. D., Babu, G. J., Ji, Y., Zilberman, A., Ver Heyen, M., Wuytack, F., & Periasamy, M. (2000). The expression of SR calcium transport ATPase and the Na(+)/Ca(2+)Exchanger are antithetically regulated during mouse cardiac development and in Hypo/hyperthyroidism. Journal of molecular and cellular cardiology, 32(3), 453–64. doi:10.1006/jmcc.1999.1095

101. Bahouth, S. W. (1991). Thyroid hormones transcriptionally regulate the beta 1-adrenergic receptor gene in cultured ventricular myocytes. The Journal of biological chemistry, 266(24), 15863–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1651924

102. Das, D. K., Bandyopadhyay, D., Bandyopadhyay, S., & Neogi, A. (1984). Thyroid hormone regulation of beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in foetal heart. Acta endocrinologica, 106(4), 569–76. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6089483

103. J. D. Rutherford, S. F. Vatner, E. B. (1979). Adrenergic control of myocardial contractility in conscious hyperthyroid dogs. American Journal of Physiology - Heart and Circulatory Physiology, 237(5), H590–H596.

104. Huang, F., He, H., & Gick, G. (1994). Thyroid hormone regulation of Na,K-ATPase alpha 2 gene expression in cardiac myocytes. Cellular & molecular biology research, 40(1), 41–52. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7804325

105. Liu, B., Huang, F., & Gick, G. (1993). Regulation of Na,K-ATPase beta 1 mRNA content by thyroid hormone in neonatal rat cardiac myocytes. Cellular & molecular biology research, 39(3), 221–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8293039

106. Shimoni, Y., Fiset, C., Clark, R. B., Dixon, J. E., McKinnon, D., & Giles, W. R. (1997). Thyroid hormone regulates postnatal expression of transient K+ channel isoforms in rat ventricle. The Journal of physiology, 500 ( Pt 1, 65–73. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9097933

107. Pantos, C., Mourouzis, I., Galanopoulos, G., Gavra, M., Perimenis, P., Spanou, D., & Cokkinos, D. V. (2010). Thyroid hormone receptor alpha1 downregulation in postischemic heart failure progression: the potential role of tissue hypothyroidism. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et métabolisme, 42(10), 718–24. doi:10.1055/s-0030-1255035

108. Mousa, S. A., Bergh, J. J., Dier, E., Rebbaa, A., O’Connor, L. J., Yalcin, M., … Davis, P. J. (2008). Tetraiodothyroacetic acid, a small molecule integrin ligand, blocks angiogenesis induced by vascular endothelial growth factor and basic fibroblast growth factor. Angiogenesis, 11(2), 183–90. doi:10.1007/s10456-007-9088-7

109. Davis, P. J., Lin, H.-Y., Tang, H.-Y., Davis, F. B., & Mousa, S. A. (2013). Adjunctive input to the nuclear thyroid hormone receptor from the cell surface receptor for the hormone. Thyroid : official journal of the American Thyroid Association, 23(12), 1503–9. doi:10.1089/thy.2013.0280

110. Davis, F. B., Mousa, S. A., O’Connor, L., Mohamed, S., Lin, H.-Y., Cao, H. J., & Davis, P. J. (2004). Proangiogenic action of thyroid hormone is fibroblast growth factor-dependent and is initiated at the cell surface. Circulation research, 94(11), 1500–6. doi:10.1161/01.RES.0000130784.90237.4a

111. Cohen, K., Flint, N., Shalev, S., Erez, D., Baharal, T., Davis, P. J., … Ashur-Fabian, O. (2014, July 13). Thyroid hormone regulates adhesion, migration and matrix metalloproteinase 9 activity via αvβ3 integrin in myeloma cells. Oncotarget. Impact Journals. Retrieved from http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path%5B%5D=2205&path%5B%5D=3560

112. Shih, A., Lin, H.-Y., Davis, F. B., & Davis, P. J. (2001). Thyroid Hormone Promotes Serine Phosphorylation of p53 by Mitogen-Activated Protein Kinase †. Biochemistry, 40(9), 2870–2878. doi:10.1021/bi001978b

113. Lin, H.-Y., Sun, M., Tang, H.-Y., Lin, C., Luidens, M. K., Mousa, S. A., … Davis, P. J. (2009). L-Thyroxine vs. 3,5,3’-triiodo-L-thyronine and cell proliferation: activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. American journal of physiology. Cell physiology, 296(5), C980–91. doi:10.1152/ajpcell.00305.2008

114. Kalyanaraman, H., Schwappacher, R., Joshua, J., Zhuang, S., Scott, B. T., Klos, M., … Pilz, R. B. (2014). Nongenomic thyroid hormone signaling occurs through a plasma membrane-localized receptor. Science signaling, 7(326), ra48. doi:10.1126/scisignal.2004911

115. Rudinger, A., Mylotte, K. M., Davis, P. J., Davis, F. B., & Blas, S. D. (1984). Rabbit myocardial membrane Ca2+-adenosine triphosphatase activity: stimulation in vitro by thyroid hormone. Archives of biochemistry and biophysics, 229(1), 379–85. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6230996

116. Lomax, R. B., Cobbold, P. H., Allshire, A. P., Cuthbertson, K. S., & Robertson, W. R. (1991). Tri-iodothyronine increases intra-cellular calcium levels in single rat myocytes. Journal of molecular endocrinology, 7(1), 77–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1654054

117. Zinman, T., Shneyvays, V., Tribulova, N., Manoach, M., & Shainberg, A. (2006). Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes. Journal of cellular physiology, 207(1), 220–31. doi:10.1002/jcp.20562

118. Huang, C. J., Geller, H. M., Green, W. L., & Craelius, W. (1999). Acute effects of thyroid hormone analogs on sodium currents in neonatal rat myocytes. Journal of molecular and cellular cardiology, 31(4), 881–93. doi:10.1006/jmcc.1998.0930

119. Harris, D. R., Green, W. L., & Craelius, W. (1991). Acute thyroid hormone promotes slow inactivation of sodium current in neonatal cardiac myocytes. Biochimica et biophysica acta, 1095(2), 175–81. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1657196

120. Dudley, S. C., & Baumgarten, C. M. (1993). Bursting of cardiac sodium channels after acute exposure to 3,5,3’-triiodo-L-thyronine. Circulation research, 73(2), 301–13. Retrieved from