The Relationship Between Perfluoroalkyl Substances Concentrations and Thyroid Function in Early Childhood: A Prospective Cohort Study.

Background: Exposure to perfluoroalkyl substances (PFAS) has been suggested to affect thyroid function; however, data on early-life exposure and thyroid function in early childhood are scarce. We investigated the cross-sectional and longitudinal relationships of early-life exposure to PFAS with thyroid function at 2, 4, and 6 years of age. Methods: This study used data on PFAS exposure and thyroid function from the Environment and Development of Children (EDC) cohort study. A total of 660 children who visited at least once at 2, 4, or 6 years of age (381 children aged 2 years, 569 children aged 4 years, and 511 children aged 6 years) were included in this study. Serum thyrotropin (TSH) levels were measured at 2, 4, and 6 years of age. The relationship of serum PFAS (sPFAS) concentrations with TSH levels at the three time points was assessed by repeated-measure analysis using linear mixed models. The serum levels of free thyroxine (fT4) and triiodothyronine (T3) were measured once (at 6 years of age). The relationship of sPFAS with fT4 and T3 levels at 6 years of age was investigated by linear regression analyses. Results: None complained of hyper- or hypothyroid symptoms with normal fT4 and T3 levels. Repeated-measure analysis showed that TSH levels at 2, 4, and 6 years of age were inversely associated with serum perfluorononanoic acid (sPFNA), after adjusting for age, sex, and/or dietary iodine intake (p < 0.05). When stratified by sex, TSH levels were inversely associated with serum perfluorooctanoic acid (sPFOA) in boys and sPFNA in girls (p < 0.05 for both). fT4 levels at 6 years of age were positively related to sPFNA and serum perfluorohexane sulfonic acid at 2 years of age and sPFOA at 6 years of age, and T3 levels at 6 years of age showed positive relationships with serum perfluorodecanoic acid and serum perfluorooctane sulfonic acid at 6 years of age (p < 0.05 for all). When stratified by sex, similar positive relationships for sPFAS with fT4 and T3 levels were significant among boys only. Conclusions: A significant relationship was found between early-life exposure to PFAS and thyroid function. Early-life exposure to PFAS was associated with decreased TSH and increased fT4 or T3 levels among preschool-age children.

Di-(2-ethylhexyl) phthalate inhibits expression and internalization of transthyretin in human placental trophoblastic cells.

During pregnancy, fetal thyroid hormones (THs) are dependent on maternal placental transport and their physiological level is crucial for normal fetal neurodevelopment. Earlier research has shown that Di-(2-ethylhexyl) phthalate (DEHP) disrupts thyroid function and THs homeostasis in pregnant women and fetuses, and affects placental THs transport. However, the underlying mechanisms are poorly understood. The present study, therefore, aimed to systematically investigate the potential mechanisms of DEHP-induced disruption in the placental THs transport using two human placental trophoblastic cells, HTR-8/SVneo cells and JEG-3 cells. While the exposure of DEHP at the doses of 0-400 µM for 24 h did not affect cell viability, we found reduced consumption of T3 and T4 in the culture medium of HTR-8/Svneo cells treated with DEHP at 400 µM. DEHP treatment did not affect T3 uptake and the expression of monocarboxylate transporters 8 (MCT8) and organic anion transporters 1C1 (OATP1C1). However, DEHP significantly inhibited transthyretin (TTR) internalization, down-regulated TTR, deiodinase 2 (DIO2), and thyroid hormone receptors mRNA expression and protein levels, and up-regulated deiodinase 3 (DIO3) protein levels in a dose-dependent manner. These results indicate that DEHP acts on placental trophoblast cells, inhibits its TTR internalization, down-regulates TTR expression and affects the expression of DIO2, DIO3, and thyroid hormone receptor. These may be the mechanisms by which PAEs affects THs transport through placental.

The effect of thyroid dysfunction and treatment on adropin, asprosin and preptin levels in rats.

OBJECTIVES: Thyroid hormones have important roles in normal development and energy regulating mechanisms as well as signaling mechanisms that affect energy consumption through central and peripheral pathways. The aim of this study was to determine the effects of thyroid dysfunction on adropin, asprosin and preptin levels in rat. METHODS: The study was performed on the 38 male Wistar-albino rats. Experiment groups were designed as follows. 1-Control, 2-Hypothyroidism; To induce hypothyroidism PTU was applied by intraperitoneal as 10 mg/kg/day for 2 weeks. 3-Hypothyroidism + Thyroxine; Previously animals were made with hypothyroidism by 1 week PTU application and then 1 week l-thyroxine was given by intraperitoneal as 1.5 mg/kg/day. 4-Hyperthyroidism; Rats were made with hyperthyroidism by 3 weeks l-thyroxine (0.3 mg/kg/day). 5-Hyperthyroidism + PTU; Animals were made hyperthyroisim by l-thyroxine as groups 4, then 1 week PTU was applied to treatment of hiperthyrodism. At the end of supplementation animals were sacrificed and blood samples were collected for FT3, FT4, adropin, asprosin, preptin analysis. RESULTS: FT3 ve FT4 levels were reduced significantly in hypothyroidism while increased in hyperthyroidism (p<0.001). Hipothyrodism led to reduces adropin, asprosin and preptin levels. And also hyperthyroidism reduced adropin and preptin levels (p<0.001). CONCLUSIONS: The results of study show that experimental hypothyroidism and hyperthyroidism lead to significantly change to adropin, asprosin and preptin levels. However, correction of thyroid function caused to normals levels in asprosin and preptin.

Integrated thyroid endocrine disrupting effect on zebrafish (Danio rario) larvae via simultaneously repressing type II iodothyronine deiodinase and activating thyroid receptor-mediated signaling following waterborne exposure to trace azocyclotin.

As a widely used organotin acaricide nowadays, azocyclotin (ACT) could induce thyroidal endocrine disruption in fishes and amphibians, but its dominant disrupting mode remains unknown. In this study, zebrafish were firstly exposed to ACT (0.18-0.36 ng/mL) from 2 hpf (hours post fertilization) to 30 dpf (days post fertilization), and a series of developmental toxicological endpoints and thyroid hormones were measured. Result showed that no developmental toxicity to zebrafish was found in 0.18 and 0.24 ng/mL groups except decreased body weight (30 dpf, 0.24 ng/mL). However, exposed to 0.36 ng/mL ACT led to reductions in heartbeat (48 hpf), hatching rate (72 hpf) and bodyweight (30 dpf). General tendencies of decreases in free T3 but increases in free T4 and reductions in ratio of free T3/T4 were also found, inferring that type II deiodinase (Dio2) was repressed. This inference was confirmed by Western analysis that Dio2 expression reduced by 42.7% after 0.36 ng/mL ACT treatment. Moreover, RNA-Seq analysis implied that exposed to 0.36 ng/mL ACT altered the genome-wide gene expression profiles of zebrafish. Totally 5660 genes (involving 3154 down-regulated and 2596 up-regulated genes) were differentially expressed, and 13 deferentially expressed genes including down-regulated dio2 were significantly enriched in thyroid hormone signaling pathway. Subsequently, an in vitro thyroid receptor-reporter gene assay using GH3 cells was performed to further explore the potential disrupting mechanism. Result showed that luciferase activity slightly increased after exposure to ACT alone or ACT combined with low level T3, but was suppressed when combined with high level T3. It indicted there probably existed a competitive relationship in some extent between ACT and T3 in vivo. Overall, the present study provided preliminary evidences that long-term exposure to trace ACT repressed Dio2 expression, declined T3 and then activated thyroid receptor-mediated signaling, thereby leading to integrated thyroid endocrine disruption in zebrafish larvae.

The role of thyroglobulin in thyroid hormonogenesis.

In humans, the thyroid hormones T3 and T4 are synthesized in the thyroid gland in a process that crucially involves the iodoglycoprotein thyroglobulin. The overall structure of thyroglobulin is conserved in all vertebrates. Upon thyroglobulin delivery from thyrocytes to the follicular lumen of the thyroid gland via the secretory pathway, multiple tyrosine residues can become iodinated to form mono-iodotyrosine (MIT) and/or di-iodotyrosine (DIT); however, selective tyrosine residues lead to preferential formation of T4 and T3 at distinct sites. T4 formation involves oxidative coupling between two DIT side chains, and de novo T3 formation involves coupling between an MIT donor and a DIT acceptor. Thyroid hormone synthesis is stimulated by TSH activating its receptor (TSHR), which upregulates the activity of many thyroid gene products involved in hormonogenesis. Additionally, TSH regulates post-translational changes in thyroglobulin that selectively enhance its capacity for T3 formation - this process is important in iodide deficiency and in Graves disease. 167 different mutations, many of which are newly discovered, are now known to exist in TG (encoding human thyroglobulin) that can lead to defective thyroid hormone synthesis, resulting in congenital hypothyroidism.

Low free triiodothyronine levels predict symptomatic intracranial hemorrhage and worse short-term outcome of thrombolysis in patients with acute ischemia stroke.

The aim of the study was to determine whether thyroid hormones level on admission in patients with ischemic stroke, treated with intravenous recombinant tissue type plasminogen activator (rtPA), was associated with symptomatic intracranial hemorrhage (sICH) and worse outcomes at 3 months.Patients with acute ischemic stroke (AIS) receiving intravenous rtPA thrombolytic treatment on our stroke unit between January 2015 and June 2016 were included in this study. Serum-free triiodothyronine (fT3), free thyroxine (fT4), total triiodothyronine (tT3), total thyroxine (tT4), and thyroid-stimulating hormone (TSH) were detected on admission. The endpoints were sICH, and poor functional outcomes at 3 and 6 months.In all, 159 patients (106 males; mean age 65.36 ±â€Š10.02 years) were included. FT3 was independently associated with sICH (odds ratio [OR] 0.204, 95% confidence interval [CI] 0.065-0.642) and poor outcomes at 3 months (OR 0.396, 95% CI 0.180-1.764). The cut-off values of fT3 for sICH was 3.54 pg/mL (sensitivity 83%; specificity 83%; area under the curve 0.88). FT3 values ≤3.54 pg/mL increased risk for sICH by 3.16-fold (95% CI 0.75-1.0) compared with fT3 values >3.54 pg/mL.Low fT3 levels at admission were independently associated with sICH and worse outcomes at 3 months in AIS patients receiving rtPA thrombolytic therapy.

De novo triiodothyronine formation from thyrocytes activated by thyroid-stimulating hormone.

The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Under normal physiological circumstances, only one-fifth of circulating T3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating hormone receptors (TSHRs), patients develop a syndrome of relative T3 toxicosis. Thyroidal T4 production results from iodination of thyroglobulin (TG) at residues Tyr5 and Tyr130, whereas thyroidal T3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T3 is formed de novo independently of deiodination from T4 We found that upon iodination in vitro, de novo T3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibody. We present data suggesting that TSH-stimulated TG phosphorylation contributes to enhanced de novo T3 formation. These effects were reversed within a few days after removal of the hyperstimulating conditions. Indeed, direct exposure of PCCL3 cells to human serum from two patients with Graves' disease, but not control sera, led to secretion of TG with an increased intrinsic ability to form T3 upon in vitro iodination. Furthermore, TG secreted from human thyrocyte cultures hyperstimulated with TSH also showed an increased intrinsic ability to form T3 Our data support the hypothesis that TG processing in the secretory pathway of TSHR-hyperstimulated thyrocytes alters the structure of the iodination substrate in a way that enhances de novo T3 formation, contributing to the relative T3 toxicosis of Graves' disease.

Thyroid hormone biosynthesis and release.

Thyroid hormones (TH) 3,5,3',5'- tetraiodothyronine or thyroxine (T4) and 3,5,3'- triiodothyronine (T3) contain iodine atoms as part of their structure, and their synthesis occur in the unique structures called thyroid follicles. Iodide reaches thyroid cells through the bloodstream that supplies the basolateral plasma membrane of thyrocytes, where it is avidly taken up through the sodium/iodide symporter (NIS). Thyrocytes are also specialized in the secretion of the high molecular weight protein thyroglobulin (TG) in the follicular lumen. The iodination of the tyrosyl residues of TG preceeds TH biosynthesis, which depends on the interaction of iodide, TG, hydrogen peroxide (H2O2) and thyroid peroxidase (TPO) at the apical plasma membrane of thyrocytes. Thyroid hormone biosynthesis is under the tonic control of thyrotropin (TSH), while the iodide recycling ability is very important for normal thyroid function. We discuss herein the biochemical aspects of TH biosynthesis and release, highlighting the novel molecules involved in the process.

Sepsis leads to thyroid impairment and dysfunction in rat model.

Sepsis was a systemic response to a local infection. Apoptosis was observed in the experimental sepsis. In this study, cecal ligation and puncture (CLP)-induced sepsis was established in rats. We found that sepsis decreased thyroid hormone levels, including triiodothyronine (T3), thyroxine (T4), free T3 (fT3), and free T4 (fT4). Besides, we detected the increasing expression level of Caspase-3 and increasing ratio of TUNEL positive cells in the thyroid after sepsis. Furthermore, a series of pathological ultrastructural changes were observed in thyroid follicular epithelial cells by CLP-induced sepsis. This study established a sepsis animal model and provided the cellular and molecular basis for decoding the pathological mechanism in thyroid with the occurrence of sepsis.

Neuro-Modulation of Immuno-Endocrine Response Induced by Kaliotoxin of Androctonus Scorpion Venom.

Kaliotoxin (KTX), a specific blocker of potassium channels, exerts various toxic effects due to its action on the central nervous system. Its use in experimental model could help the understanding of the cellular and molecular mechanisms involved in the neuropathological processes related to potassium channel dysfunctions. In this study, the ability of KTX to stimulate neuro-immuno-endocrine axis was investigated. As results, the intracerebroventricular injection of KTX leads to severe structural-functional alterations of both hypothalamus and thyroid. These alterations were characterized by a massive release of hormones' markers of thyroid function associated with damaged tissue which was infiltrated by inflammatory cell and an imbalanced redox status. Taken together, these data highlight that KTX is able to modulate the neuro-endocrine response after binding to its targets leading to the hypothalamus and the thyroid stimulation, probably by inflammatory response activation and the installation of oxidative stress in these organs.

Chemistry and Biology in the Biosynthesis and Action of Thyroid Hormones.

Thyroid hormones (THs) are secreted by the thyroid gland. They control lipid, carbohydrate, and protein metabolism, heart rate, neural development, as well as cardiovascular, renal, and brain functions. The thyroid gland mainly produces l-thyroxine (T4) as a prohormone, and 5'-deiodination of T4 by iodothyronine deiodinases generates the nuclear receptor binding hormone T3. In this Review, we discuss the basic aspects of the chemistry and biology as well as recent advances in the biosynthesis of THs in the thyroid gland, plasma transport, and internalization of THs in their target organs, in addition to the deiodination and various other enzyme-mediated metabolic pathways of THs. We also discuss thyroid hormone receptors and their mechanism of action to regulate gene expression, as well as various thyroid-related disorders and the available treatments.

Rapid preoperative blockage of thyroid hormone production / secretion in patients with Graves' disease.

PRINCIPLES: Preoperative management of hyperthyroid patients with Graves' disease who are unable to tolerate thionamides or have poor adherence to therapy is a challenging clinical problem. The goal of our study was to demonstrate the clinical efficacy of a rapid preoperative thyroid hormone blocking protocol and to assess specific surgical and treatment-related complications. METHODS: Ten patients with thyrotoxicosis due to Graves' disease were treated with a rapid thyroid hormone blocking protocol of Lugol's solution, dexamethasone and a beta-blocker. Two patients continued to receive antithyroid therapy with carbimazole. Adrenal function was assessed 4-6 weeks postoperatively with a low dose (1 µg) adrenocorticotrophic hormone-stimulation test. RESULTS: Before treatment, all patients had severe hyperthyroidism. Baseline median and interquartile range (IQR) of fT4 was 68.9 (45.7-92.1) pmol/l, and baseline median fT3 and IQR, 30 (19.1-40.9) pmol/l. After 10 days of treatment, the levels of free hormones were significantly reduced with fT4 concentrations slightly elevated (fT4, 26.7 [17-36.4] pmol/l, p <0.001 compared with corresponding pretreatment values), and the fT3 concentration was normal in 8/10 patients (fT3, 6.1 [4.6-7.6] pmol/l, p <0.001 compared with corresponding pretreatment values). All patients were clinically euthyroid with a heart rate of <80/min. Drug tolerability was excellent, and there were no side effects or exacerbation of hyperthyroidism. The peri- and postoperative course was uneventful in all cases. Adrenal function was normal in 7 out of 10 patients 4-6 weeks postoperatively. Three patients showed prolonged secondary adrenal insufficiency with normalisation of adrenal function after 3 to 6 months. CONCLUSION: Rapid and effective preoperative preparation of patients with Graves' disease is achievable with Lugol's solution, dexamethasone and a beta-blocker. The risk of temporary hypothalamic-pituitary-adrenal axis suppression has to be taken into account.

In vivo effect of insulin on the hormone production of immune cells in mice - gender differences.

The immune cells of rat and man synthesize, store and secrete hormones, characteristic to the endocrine glands. In the present experiments female and male CD1 mice were treated with 10 IU/kg insulin sc. (the controls with normal saline) and after 30 min peritoneal fluid was gained. The cells of the peritoneal fluid (lymphocytes and the monocyte-granulocyte group) were studied by immunocytochemical flow-cytometry to adrenocorticotropic hormone (ACTH), triiodothyronine (T3), histamine and serotonin content. In the female mice each hormone level was significantly lower in the insulin-treated animals, except histamine in the monocyte-granulocyte group. In the insulin-treated male animals, the hormone levels were similar to the control. The results 1) support the previously hypothesized hormonal network in the immune system, 2) justify that the insulin effect is not species dependent and 3) call attention to the sex, species and organ differences in the response.

Hormonogenic donor Tyr2522 of bovine thyroglobulin. Insight into preferential T3 formation at thyroglobulin carboxyl terminus at low iodination level.

A tryptic fragment (b5TR,NR), encompassing residues 2515-2750, was isolated from a low-iodine (0.26% by mass) bovine thyroglobulin, by limited proteolysis with trypsin and preparative, continuous-elution SDS-PAGE. The fragment was digested with Asp-N endoproteinase and analyzed by reverse-phase HPLC electrospray ionization quadrupole time-of-flight mass spectrometry, revealing the formation of: 3-monoiodotyrosine and dehydroalanine from Tyr2522; 3-monoiodotyrosine from Tyr2555 and Tyr2569; 3-monoiodotyrosine and 3,5-diiodotyrosine from Tyr2748. The data presented document, by direct mass spectrometric identifications, efficient iodophenoxyl ring transfer from monoiodinated hormonogenic donor Tyr2522 and efficient mono- and diiodination of hormonogenic acceptor Tyr2748, under conditions which permitted only limited iodination of Tyr2555 and Tyr2569, in low-iodine bovine thyroglobulin. The present study thereby provides: (1) a rationale for the preferential synthesis of T3 at the carboxy-terminal end of thyroglobulin, at low iodination level; (2) confirmation for the presence of an interspecifically conserved hormonogenic donor site in the carboxy-terminal domain of thyroglobulin; (3) solution for a previous uncertainty, concerning the precise location of such donor site in bovine thyroglobulin.

Changes in thyroid status of rats after prolonged exposure to low dose dichlorodiphenyltrichloroethane.

The effect of low dose dichlorodiphenyltrichloroethane (DDT), omnipresent ecotoxicant and endocrine disruptor, on the functioning of the endocrine system is an urgent problem. We studied the effect of low dose DDT on thyroid status in rats. Rats receiving DDT in a dose of 1.890±0.086 µg/kg for 6 weeks showed increased concentrations of thyroid hormones, particularly triiodothyronine, and reduced level of thyrotropin. Longer exposure reduced the production of thyroid hormones. The dynamics of thyroid status parameters during DDT treatment in a low dose was similar to changes observed during the development of hypothyroidism induced by iodine deficiency.

Regulatory feedback loop between T3 and microRNAs in renal cancer.

microRNAs, short non-coding RNAs, influence key physiological processes, including hormonal regulation, by affecting the expression of genes. In this study we hypothesised that the expression of microRNAs targeting thyroid hormone pathway genes may be in turn regulated by thyroid hormone signalling. It is known that the expression of DIO1, a gene contributing to triiodothyronine (T3) signalling, is regulated by miR-224. Thus, we analysed mutual regulation between triiodothyronine pathway and miR-224/miR-452/GABRE cluster. Firstly, we found that miR-452 directly regulates the expression of thyroid hormone receptor TRß1 in renal cancer cells. In turn, the expression of miR-224/452/GABRE cluster and other microRNAs targeting TRß1 was influenced by T3 treatment and/or TR silencing. miR-452 expression correlated with intracellular T3 concentrations in renal tumours. In conclusion, we propose a new mechanism of feedback regulation, by which in renal cancer microRNAs regulate the expression of T3 pathway genes, while T3 in turn regulates expression of microRNAs.

Effects of perchlorate on BDE-47-induced alteration thyroid hormone and gene expression of in the hypothalamus-pituitary-thyroid axis in zebrafish larvae.

To investigate the effects of perchlorate on thyroid hormone disturbances induced by 2,2',4',4-tetrabromodiphenyl ether (BDE-47) via thyroid hormone (TH)-mediated pathways, zebrafish embryos were exposed to a combination of BDE-47 and PER from the time of fertilisation to 14 d (dpf). The whole-body content of TH and the expression of genes and proteins related to the hypothalamic-pituitary-thyroid (HPT) axis were analysed. Co-exposure to BDE-47 and PER decreased the body weight and increased malformation rates relative to the effects of exposure to only BDE-47. Compared with the exposure to BDE-47 alone, the exposure to a combination of BDE-47 (10 µg/L) and PER (3.5 mg/L) significantly up-regulated the expression of genes involved in TH synthesis (NIS and Nkx2.1a) and significantly down-regulated the expression of genes related to the regulation of the HPT axis (CRH and TSHß). The expression of TG at the gene and protein levels was significantly up-regulated, but the expression of TTR was significantly down-regulated in the co-exposures relative to BDE-47 treated alone. In addition, the larger reduction in the T4 level resulting from exposure to the mixture of BDE-47 and PER demonstrated that PER enhanced the thyroid-disruptive effects of BDE-47. These results help to elucidate the complicated chemical interactions and the molecular mechanism of action of these two TH disruptors.

[Nonthyroidal illness (NTI)].

Thyroxine (T4), a major secretory product of thyroid gland, needs to be converted to 3,5,3'-triiodothyronine (T3) by iodothyronine deiodinases to exert its biological effect. Nonthyroidal illness, also known as low T3 syndrome, is associated with low serum T3 concentrations, which are inversely correlated to the severity of the illness. The patients with nonthyroidal illness do not show compensatory rise in serum TSH concentrations, and sometimes develop low serum T4 and TSH concentrations. It has been postulated that decreased extrathyroidal conversion of T4 to T3 is a responsible mechanism underlying low T3 syndrome. The roles of three types of iodothyronine deiodinases (D1, D2, D3) in the pathophysiology of nonthyroidal illness are discussed.

Distribution of histone3 lysine 4 trimethylation at T3-responsive loci in the heart during reversible changes in gene expression.

Expression in the adult heart of a number of cardiac genes, including the two genes comprising the cardiac myosin heavy chain locus (Myh), is controlled by thyroid hormone (T3) levels, but there is minimal information concerning the epigenetic status of the genes when their expressions change. We fed mice normal chow or a propyl thio uracil (PTU, an inhibitor of T3 production) diet for 6 weeks, or the PTU diet for 6 weeks followed by normal chow for a further 2 weeks. Heart ventricles from these groups were then used for ChIP-seq analyses with an antibody to H3K4me3, a well-documented epigenetic marker of gene activation. The resulting data show that, at the Myh7 locus, H3K4me3 modifications are induced primarily at 5' transcribed region in parallel with increased expression of beta myosin heavy chain (MHC). At the Myh6 locus, decreases in H3K4me3 modifications occurred at the promoter and 5' transcribed region. Extensive H3K4me3 modifications also occurred at the intergenic region between the two Myh genes, which extended into the 3' transcribed region of Myh7. The PTU-induced changes in H3K4me3 levels are, for the most part, reversible but are not invariably complete. We found full restoration of Myh6 gene expression upon PTU withdrawal; however, the H3K4me3 pattern was only partially restored at Myh6, suggesting that full reexpression of Myh6 does not require that the H3K4me3 modifications return fully to the untreated conditions. Together, our data show that the H3K4me3 modification is an epigenetic marker closely associated with changes in Myh gene expression.