Neuroendocrine properties of intrinsic cardiac adrenergic cells in fetal rat heart.

Intrinsic cardiac adrenergic (ICA) cells in developing rat heart constitute a novel adrenergic signaling system involved in cardiac regulation. Regulatory mechanisms of ICA cells remain to be defined. Immunohistochemical study of fetal rat hearts demonstrated ICA cells with catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). The mRNA of TH and PNMP was also detected in fetal rat hearts before sympathetic innervation. Immunoreactivity of norepinephrine transporter (NET) was localized to ICA cells in rat heart tissue and primary cell culture. For the functional study, the activity of intracellular Ca2+ concentration ([Ca2+]i) transients was quantified by a ratio fluorescent spectrometer in cultured ICA cells and myocytes. ICA cells generated spontaneous [Ca2+]i transients that were eliminated by tetrodotoxin or Ca(2+)-free solutions and showed greatly reduced amplitude with the addition of L-type Ca2+ channel blocker nifedipine. [3H]norepinephrine studies demonstrate release and uptake of norepinephrine. Functional interaction between catecholamines produced by the ICA cells and cocultured myocytes was evident by the effect of the beta-adrenergic blocker atenolol eliciting a dose-dependent reduction in the amplitude and frequency of [Ca2+]i transients of beating myocytes. Hypoxia inhibited [Ca2+]i transient activity of ICA cells, which subsequently produced a reoxygenation-mediated rebound augmentation of [Ca2+]i transients. We conclude that ICA cells are capable of catecholamine synthesis, release, and uptake. They generate spontaneous [Ca2+]i transient activity that can be regulated by oxygen tension. ICA cells may provide an alternative adrenergic supply to maintain cardiac contractile and pacemaker function at rest and during stress in the absence of sympathetic innervation.

Modulation of the inflammatory response in the cardiomyocyte and macrophage.

Numerous cardiac disease processes have been linked to the overproduction of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) in the cardiovascular system. Chronic and acute exposure to hyperphysiologic levels of NO has been suggested as an agent in chronic transplant rejection, various cardiomyopathies, reperfusion injury, and the inflammatory state following cardiopulmonary bypass. Proinflammatory cytokines and inflammatory cell types, such as macrophage and neutrophils, have also been implicated in the pathophysiology associated with the previously mentioned syndromes. Previous work by this group has shown that lipopolysaccharide (LPS) in combination with tumor necrosis factor-alpha (TNF-alpha) can increase iNOS expression and the production of NO in macrophage. With this in mind, we hypothesized that increased iNOS expression and NO production generated by LPS and TNF-alpha in the macrophage could be mimicked in the cardiomyocyte and potentially account for some aspect of the cardiac dysfunction attributed to NO. Furthermore, this increased expression of iNOS and NO production could be returned to control using the glucocorticoid, dexamethasone, a known iNOS transcription blocker. Using fetal rat cardiomyocytes in primary culture cell line and a murine macrophage cell line, RAW 264.7, the expression of iNOS was quantified with specific FITC-conjugated antibodies using fluorescence activated cell sorter (FACS) and NO production with a Bioxytech nitric oxide spectrophotometric assay. The myocytes and macrophage were separated into three groups, Control, TNF + LPS, and (+) Dexamethasone. The control groups received no TNF or LPS or dexamethasone, TNF + LPS groups received TNF-alpha and LPS for 8 hours with no dexamethasone, and the (+) Dexamethasone groups were pretreated with dexamethasone for 8 hours and stimulated with TNF-alpha and LPS along with a second 8-hour treatment of dexamethasone. The macrophage cell groups treated with TNF-alpha and LPS showed a 335% increase over control in iNOS expression, and NO production was increased 494% from control. Macrophage treated with dexamethasone experienced an attenuation of iNOS expression of 200% toward control from stimulated levels and 202% decrease in NO production from stimulated levels toward control. Cardiomyocytes exhibited no statistically significant change in the expression of iNOS or NO production with stimulation or dexamethasone treatments. In conclusion, iNOS and NO were elevated in macrophage, which can be blunted in the presence of dexamethasone in the macrophage. Curiously, iNOS could not be stimulated in the cardiomyocyte, suggesting inflammatory cells may be largely responsible for the elevated iNOS and NO experienced in some cardiovascular diseases. The clinical relevance of this study is the introduction of specific iNOS inhibitors into the cardiopulmonary bypass circuit could serve as a potential mechanism for modulating the inflammatory response surrounding cardiopulmonary bypass. Likewise, therapeutic glucocorticoid administration could improve outcomes for patients with inflammatory cardiovascular disease states related to elevated NO production.

Cloning and characterization of P110, a novel small nucleolar U3 ribonucleoprotein, expressed in early development.

We report the cloning of a BrdU-sensitive transcript of 4.1 kb from an immortalized quail heart cell line containing an open reading frame of 940 amino acids (110 kDa, pI approximately 5.18). The mRNA encoding P110 appears in the heart and neural tube by 36 h of avian development, at a time when these organs are rapidly developing. Analysis of the DNA-deduced protein sequence revealed a bipartite nuclear localization signal, and a highly charged domain rich in both acidic and basic residues. Immunofluorescent staining with polyclonal antibodies raised against a P110 peptide localized the protein to the nucleolus of avian and mammalian cells. Although database search showed significant homology with an uncharacterized cDNA from human brain and several human and mouse Expressed Sequence Tags, there was no close homology to known nucleolar proteins. Immunoprecipitation of P110 from cell sonicates revealed it contained U3 small nucleolar RNA, but no significant amounts of other box C/D small nucleolar RNAs. These data suggest that P110 is one of the U3 small nucleolar ribonucleoproteins that are involved in rRNA processing.

Expression of alpha and beta integrins during terminal differentiation of cardiomyocytes.

BACKGROUND: In the myocardium, myocyte cell division is irreversibly blocked shortly after birth. The signal that initiates cell cycle withdrawal is unknown. The purpose of this study was to relate changes in expression of beta1 integrin and its associated alpha subunits to cardiomyocyte cell cycle progression during the fetal-to-neonatal developmental transition in rat. METHODS AND RESULTS: The developmental expression pattern and function of beta 1 integrin and several of its associated alpha subunits were examined using reverse transcription (RT) polymerase chain reaction (PCR) and beta 1 blocking antibodies. During the fetal to neonatal transition, a dramatic shift occurred in the levels of beta1 and alpha isoforms. At the 17-day fetal stage only beta 1A was present, which remained relatively constant until immediately after birth then decreased by 30% at the adult stage. By contrast, beta 1D appeared at fetal day 18, increased at neonatal day 2, and afterwards remained constant. This resulted in a ratio of beta 1A to beta 1D of about 1:1 in the adult heart. The integrin beta 1-associated subunits, alpha 3, alpha 6, and alpha 7, were expressed at extremely low levels in 17-day fetal cardiomyocytes. After birth alpha 3 and alpha 6 transiently increased at the 2-day neonatal stage, while alpha 7 isoforms B, C, and X2 progressively increased to the adult stage. Unlike skeletal muscle cells, fluorescence-activated cell sorting analysis (FACS) showed no down regulation of the alpha 5 beta 1 fibronectin receptor during cell cycle withdrawal. Treatment of cultured cardiomyocytes with beta1 blocking antibody inhibited the cell cycle in fetal but not in neonatal cells. CONCLUSION: These results suggest that progression through the cardiomyocyte cell cycle may be dependent upon cell attachment via integrin beta1 and correlate with changes that occur in beta1 spliced variants and their respective alpha isoforms.

Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocytes.

Cardiomyocyte hypertrophy and cell death are often observed in the end stages of heart failure. The triggers of these two cellular processes are not known under most pathological conditions. Oxidants are by-products of aerobic metabolism. The level of oxidants increases as a result of ischemic reperfusion. Using H9C2 and primary cultured neonatal rat cardiomyocytes, we found that a 2-h pulse treatment with H(2)O(2) at 250 microM or lower caused activation of DEVD sequence specific caspases. The activity of DEVD-ase peaked with 200 microM H(2)O(2) at 24 h. While a fraction of the cells detached and showed nuclear condensation, the majority of the cells (>55%) survived the treatment and appeared to enlarge when cultured for 5 days. These cells showed increases in cell surface area, cell volume, and protein content. With 200 microM H(2)O(2), treated cells appeared to be six times bigger in volume and contained three times more protein per cell than untreated cells. The enlarged cells showed enhanced actin stress fibers and disrupted myofibrils. Our data indicate that while H(2)O(2) can cause cell death, the surviving cardiomyocytes undergo hypertrophy.

IGF I induction of p53 requires activation of MAP kinase in cardiac muscle cells.

The aim of this study was to investigate whether IGF I induction of p53 expression and p21 promoter require activation of MAP kinase in cardiac muscle cells. Compared to cardiomyocytes transfected with control vector, activation of MAP kinase by IGF I was decreased by approximately 60-70% in the cells transfected with dominant negative MAP kinase Y185. Transfection with Y185 also resulted in decreased induction of p53 mRNA by IGF I (70% reduction). In the cells transfected with a wildtype p21WAF1/CIP1 promoter construct, activation of luciferase reporter gene by IGF I was decreased in the cells co-transfected with Y185. To further confirm these findings, cells were preincubated with PD98059, a specific MAP kinase kinase inhibitor. As expected, PD98059 inhibited induction of p53 mRNA and p21WAF1/CIP1 promoter by IGF I. These data indicate that transcriptional activation of p53 and p21WAF1/CIP1 by IGF I involves MAP kinase pathway in cardiomyocytes, and thus link MAP kinase to negative modulation of the cell cycle in cardiac muscle cells.

Structure, regulation and function of avian glypican.

Glypicans are a group of membrane-bound heparan sulfate proteoglycans (HSPG) that are tissue specific and developmentally regulated. Transcripts for avian glypican are found in endocardial cushions, limb buds, somites and forebrain of early chick embryos. Since avian glypican is not well characterized, the cellular localization, regulation of expression, and possible function during cardiac development have been studied. A polyclonal antibody was raised against a 20-amino acid peptide corresponding to an antigenic sequence within avian glypican core protein. The antibody recognized the expressed core protein in bacterial lysates and the endogenous HSPG in the proteoglycan fraction from chick forebrain. Immunolocalization studies indicated that the core protein is associated with cell membranes. The level of mRNA for avian glypican in MEQC (myc embryonic quail cardiomyocytes) grown in medium containing 10% fetal calf serum was compared to the message levels in cells grown without serum for 3 days. By Northern analysis, glypican transcripts were increased markedly after serum starvation. Up-regulation of glypican transcripts by serum withdrawal was partially prevented by addition of TGFbeta-1 and bFGF, suggesting that these growth factors may regulate its expression. MEQC cells deprived of serum migrated into clumps that could be blocked by an antisense OND (oligodeoxynucleotide) to the mRNA encoding the avian glypican. The same antisense OND inhibited the migration of endothelial cells from chick tubular heart explants over the surface of collagen gels. These results indicate that avian glypican may play a role in cell migration during development of endocardial cushions.

Changes in E2F complexes containing retinoblastoma protein family members and increased cyclin-dependent kinase inhibitor activities during terminal differentiation of cardiomyocytes.

Cardiomyocyte terminal differentiation was examined by studying the interaction of retinoblastoma protein (pRb) family members with E2F during the developmental transition from 17-day fetal to 2-day neonatal. Additionally, the expression pattern of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors responsible for modulating the phosphorylation of pRb were studied. p107, pRb, and p130 are regulators of cellular proliferation, differentiation, and cell cycle exit and entry, respectively. The active, underphosphorylated form of these proteins targets the E2F family of transcriptional factors that play a critical role in the control of genes associated with DNA synthesis. Electromobility shift analyses demonstrated E2F complexed with p107 in proliferating fetal cardiomyocytes, whereas in 2-day neonatal cells, E2F was principally associated with p130 and a low level of pRb. At the 2-day neonatal stage, decreased protein levels were observed for cyclins D2, D3, and E, and CDK2 and CDK4. No changes were observed in the mRNA levels of the D-cyclins in neonatal cells; however, the transcripts for cyclins A and E and CDK4 were diminished. In skeletal myoblasts, differentiation is associated with induction of p21, a CDK inhibitor, by a MyoD-dependent pathway. Although heart cells lack MyoD, CDK assays demonstrated that the activity of CDKs 2, 4, and 6 were downregulated in 2-day neonatal cells, and CDC2 was increased. RT-PCR indicated that p21 mRNA was induced 1.4-, 2.0-, and 3.1-fold in the 2-day neonatal, 7-day neonatal, and adult stages, respectively, compared to the 17-day fetal stage. At the protein level, p21 also increased at the 2-day neonatal stage. Kinase inhibitory immunodepletion assays showed that CDK inhibitory activity was markedly increased in the 2-day neonate. Although mRNA levels of the p27 CDK inhibitor were unchanged, its protein level and inhibitory effect on CDK2 and CDK4 were increased. Thus, cardiomyocytes retain the capacity to proliferate until the early neonatal period when a series of changes occur, including a switch in pRb partners, a decrease in CDK levels and induction of CDK inhibitory activity, which is associated with terminal differentiation.

In vitro and in vivo suppression of gluconeogenesis by inhibition of pyruvate carboxylase.

The mechanism of inhibition of gluconeogenesis by phenylalkanoic acids was studied in vitro and in vivo. In vitro production of 14CO2 from labeled glucose or palmitate was not inhibited at 4 mM, a concentration of phenylacetic acid that inhibited gluconeogenesis from lactate/pyruvate. In vitro studies with isolated mitochondria showed that the CoA ester of phenylacetic acid was formed. The parent phenylalkanoic acid had no effect on purified pyruvate carboxylase activity, but phenylacetyl CoA ester decreased pyruvate carboxylation in a concentration-dependent manner. Phenylacetic acid inhibited gluconeogenesis in isolated rat liver cells from 10 mM lactate/1 mM pyruvate (decreased 39%, P < 0.05), but not 10 mM L-glutamine or [14C]aspartate, showing that the inhibition of gluconeogenesis occurred at the level of pyruvate carboxylase. A 20 mg bolus with infusion of 1 mg/min of phenylpropionic acid decreased blood glucose levels of normal [110 +/- 12 to 66 +/- 11 mg/dL, N = 7, P < 0.05 (unpaired Student's t-test vs control)] and streptozocin diabetic rats [295 +/- 14 to 225 +/- 12 mg/dL, N = 7, P < 0.01 (paired t-test vs basal)]. Hepatic glucose production in control and diabetic rats was suppressed under conditions where liver glycogen was depleted, indicating that gluconeogenesis had been inhibited in vivo. The results suggest the possibility that the inappropriate overproduction of glucose can be controlled by inhibitors of pyruvate carboxylase. This class of inhibitors may be useful in the treatment of non-insulin-dependent diabetes mellitus.

Development of a thyroid hormone analogue for the treatment of congestive heart failure.

The possibility that thyroid hormone or a thyroid hormone analogue that improves cardiac performance might be useful in the treatment of heart failure has-been examined. In the rat postinfarction model of heart failure, treatment with low doses (1.5 micrograms/100 g) of thyroxine (T4) for 3 days produced a positive inotropic response, including an increase in left ventricular (LV) dP/dt and a decrease in LV end-diastolic pressure (LVEDP). When treatment with T4 was continued at the same or higher doses (3 to 15 micrograms/100 g) for 10-12 days, heart rate was increased and improvement in LVEDP was not sustained. To identify an analogue with a more favorable hemodynamic profile, single- and double-ring compounds related to T4 were screened for thyromimetic activity in heart cell cultures and for their ability to bind thyroid hormone receptors. One of the analogues selected, 3,5-diiodothyropropionic acid (DITPA), was found to have inotropic selectivity in hypothyroid rats. When administered (375 micrograms/100 g) to rats with ventricular dysfunction after myocardial infarction in combination with captopril, there was improvement of the resting and stressed cardiac index and LV filling pressure. Similar improvement in cardiac performance was obtained when DITPA was administered to rabbits after infarction. Thus a thyroid hormone analogue with inotropic selectivity may be a useful adjunct to other measures in the treatment of heart failure.

Thyroid hormone influences beta myosin heavy chain (beta MHC) expression.

3,5,3'-Triiodo-L-thyronine (T3) regulation of beta-MHC expression was studied in rat fetal heart cells using deletion mutants of both the rat and human promoter regions fused to a CAT expression vector. T3 inhibited the expression of human and rat beta-MHC constructs with an IC50 of about 1nM, which was similar to the EC50 for beta MHC-mRNA observed in cardiomyocytes. Deletion analysis of beta MHC promoter constructs suggested that a T3 response element (TRE) is located near the start of transcription. Possibly, T3-receptor binding at this site interferes with formation of the transcriptional initiation complex.

Identification of simple substituted phenols with thyromimetic activity: cardiac effects of 3,5-diiodo-4-hydroxyphenylpropionic acid.

To define the minimal structural requirements for cardiac activity of thyroid hormone analogs, a series of substituted phenols were screened for their ability to bind bacterially expressed thyroid hormone receptors. Compounds with binding activity then were tested for their ability to induce expression of alpha-myosin heavy chain mRNA in primary cultures of fetal rat cardiomyocytes, a sensitive marker for potential inotropic activity. 3,5-Diiodo-4-hydroxyphenylpropionic acid (DIHPA) was found to bind specifically to bacterially expressed alpha-1 and beta-1 thyroid hormone receptors (Kaff approximately 1 to 2 x 10(5) M-1) and to induce alpha-myosin heavy chain (EC50 approximately 5 x 10(-7)). To assess the effects of DIHPA on cardiac performance in vivo, hemodynamic measurements were made in three groups of hypothyroid rats treated for 5 days with s.c. doses of DIHPA (15 mg/100 g), L-thyroxine (T4, 1.5 micrograms/100 g) or saline. Compared to controls, DIHPA and T4 produced increases in heart rate, left ventricular +dP/dtmax, -dP/dtmax, and isovolumic relaxation. In isometric papillary muscles preparations, DIHPA and T4 shortened time-to-peak tension and time-from-peak tension to 50% decline as compared with saline-treated controls. Muscles from both drug-treatment groups showed similar responses to graded doses of isoproterenol (10(-8) to 10(-3) M) and to variations in Ca++ concentration of the muscle bath (0.3125 to 3.75 x 10(-3) M). Thus, DIHPA is a novel thyromimetic compound with effects on myocardial function similar to those observed with T4.

Combination treatment with captopril and the thyroid hormone analogue 3,5-diiodothyropropionic acid. A new approach to improving left ventricular performance in heart failure.

BACKGROUND: An agent that improves left ventricular (LV) performance by non-cAMP-mediated mechanisms would be valuable in the treatment of chronic heart failure. We have shown earlier that the thyroid hormone analogue 3,5-diiodothyropropionic acid (DITPA) binds to nuclear receptors, alters transcription of T3-responsive genes, and increases +dP/dtmax in hypothyroid rats with substantially less effect on heart rate and metabolism than thyroid hormone, which makes it a selective cardiotonic agent. METHODS AND RESULTS: To determine whether DITPA might be useful in treating heart failure, we compared chronic treatment with normal saline, captopril (2 g/L), or the combination of DITPA (375 micrograms/100 g) and captopril (2 g/L) in Sprague-Dawley rats beginning 3 weeks after coronary artery ligation. Both DITPA/captopril and captopril treatment decreased LV end-diastolic pressure compared with controls (21 +/- 2 and 26 +/- 2 mm Hg, respectively, vs 34 +/- 3 mm Hg, P < .05 for each). The addition of DITPA to captopril produced a 36% increase in resting cardiac index (P < .05) and shifted the cardiac function curve upward and to the left, indicative of enhanced myocardial performance. Also, DITPA/captopril compared with captopril treatment or control produced an increase in the rate of LV relaxation, as manifested by a decrease in tau, the time constant of LV pressure decline (17.5 +/- 1.0 vs 22.2 +/- 1.7 milliseconds, P < .05) and a larger absolute value for -dP/dtmax (-4561 +/- 361 vs -3346 +/- 232 mm Hg/s, P < .05). These changes occurred without changes in heart rate, LV mass, LV systolic pressure, or peripheral resistance relative to captopril treatment (P > .05). CONCLUSIONS: The combination of DITPA and captopril improved cardiac output, increased -dP/dtmax, and increased the rate of LV relaxation to a greater extent than captopril treatment in the rat postinfarction model of heart failure. Use of a cardiotonic analogue of thyroid hormone represents a new approach to improving LV performance and may be a useful adjunct to afterload reduction for the treatment of heart failure.

Studies on the use of thyroid hormone and a thyroid hormone analogue in the treatment of congestive heart failure.

In heart failure, cardiac output is insufficient to meet the needs of the body for oxygen delivery. Available data suggest that alterations in thyroid hormone metabolism may contribute to defective myocardial performance. Accordingly, thyroid hormone or a thyroid hormone analogue that improves cardiac performance might be useful in the treatment of heart failure and has been studied. Experimental and theoretical results of these studies are reviewed and indicate that thyroid hormone increases cardiac output by a combination of effects on the heart and peripheral circulation, specifically by increasing myocardial contractile performance and decreasing venous compliance. In the rat postinfarction model of heart failure, treatment with low doses of thyroxine (1.5 micrograms/100 g) for 3 days produced a positive inotropic response, including an increase in rate of change of left ventricular pressure and a decrease in left ventricular end-diastolic pressure. These changes could be attributed to conversion to triiodothyronine, the active intracellular form of thyroid hormone. When treatment with thyroxine was continued at the same or higher doses (3 to 15 micrograms/100 g) for 10 to 12 days, heart rate increased and improvement in left ventricular end-diastolic pressure was not sustained. More favorable results were obtained with 3,5-diiodothyropropionic acid, a cardiotonic thyroid hormone analogue administered at doses of 375 microgram/100 g, given in combination with captopril. Thus, triiodothyronine or a thyroid hormone analogue may be a useful adjunct to other measures in the treatment of heart failure.

A repressor region in the human beta-myosin heavy chain gene that has a partial position dependency.

Expression of the human beta-myosin heavy chain (beta MHC) gene was studied by transient assay in culture and in situ by direct injection of plasmids into adult rat hearts. In this report we describe a unique repressor region located -326/-309 (5'-TTGGTGGTCGTGGTCAGT-3') of the human beta MHC gene that is conserved among the rat, rabbit, and human beta MHC genes. This sequence conferred repression onto heterologous promoters when the sequence was located 5' but not 3' to the promoters. This partial positional dependency suggests that the factor may act by limiting the binding of enhancers, located more proximally, to their DNA binding sites.

Cardiac effects of 3,5-diiodothyropropionic acid, a thyroid hormone analog with inotropic selectivity.

Thyroid hormone exerts a strong positive inotropic action on the heart and induces alpha-myosin heavy chain (MHC) gene expression. 3,5-Diiodothyropropionic acid (DITPA), a carboxylic acid analog with low metabolic activity, was observed to induce alpha-MHC mRNA in heart cell culture with EC50 approximately 5 x 10(-7) M. To determine if the compound has positive inotropic actions, the effects of DITPA and L-thyroxine on heart rate, left ventricular pressures, left ventricular dP/dt, myosin isoenzymes and hepatic alpha-glycerolphosphate dehydrogenase activity were compared in hypothyroid rats. Binding affinities of DITPA and triiodothyronine for bacterially expressed alpha-1 and beta-1 thyroid hormone receptors (TRs) also were determined. Over the dosage range of 150 to 1500 micrograms/100 g, DITPA produced increases in left ventricular dP/dt comparable to those obtained with L-thyroxine at dosages of 1.5 to 15 micrograms/100 g, but with significantly less tachycardia. The increase in alpha-MHC mRNA was about the same with both compounds whereas alpha-MHC protein content and GPDH activity increased less with DITPA. These differences could not be explained by preferential binding of DITPA to TR subtypes. Because heart rate is a major determinant of myocardial oxygen consumption, DITPA is able to achieve increased cardiac performance at lower myocardial oxygen costs.

Characterization of a strong positive cis-acting element of the human beta-myosin heavy chain gene in fetal rat heart cells.

A strong positive element within the proximal promoter region of the human beta-myosin heavy chain (beta-MHC) gene that is required for high level expression in primary cultures of fetal rat heart cells was localized by transient assays and DNase I footprinting to positions- 277/-298. Using gel shift studies, this sequence was found to bind specifically at high affinity (Kd approximately 4 x 10(-9) M) to a transcriptional factor (beta F1) found in nuclear extracts from rabbit heart. Dimethyl sulfate interference studies suggested that beta F1 may bind as a dimer to two hexameric imperfect direct repeats containing the consensus sequence 5'-(C/G)-T-G-(T/A)-G-G-3'. Gel shift analyses suggested that beta F1 is related to the M-CAT factor, which is known to control muscle-specific expression of the cardiac troponin T gene. A clustered mutation of the region between the putative binding half-sites and within the "M-CAT"-like domain abolished beta-MHC promoter activity. The sequence of the positive element also contains binding motifs for several transcriptional factors that regulate viral and cellular genes, including AP4, AP5, TEF-1, and MyoD-like proteins. When multiple copies of the beta-MHC element were inserted downstream from the transcriptional initiation site of the thymidine kinase gene, it did not act as a classical enhancer, showing some dependence upon orientation.

An anti-sense c-erbA clone inhibits thyroid hormone-induced expression from the alpha-myosin heavy chain promoter.

The effects of thyroid hormone on expression of cardiac myosin heavy chain genes generally are thought to be mediated by nuclear 3,5,3'-triiodo-L-thyronine (T3) receptors that have been identified as the products of the protooncogene, c-erbA. This hypothesis has been tested by transfection of cardiomyocytes in primary culture with a plasmid, pRSVhEACAT-, expressing anti-sense c-erbA mRNA. Because only a low percentage of cells (20%) could be transfected in primary culture an alpha-myosin heavy chain-chloramphenicol acetyltransferase fusion construct was used as a reporter gene. The results indicate that the anti-sense plasmid almost completely blocks T3-induced activity of the reporter gene (less than 1% control) while transfection of a similar amount of the sense construct, pRSVhEACAT+, has no effect. When the c-erbA plasmids were cotransfected with constructs containing T3-independent promoters, no effects on expression were observed. The combined use of an anti-sense construct and a report gene provides a means of studying the role of c-erbA products in intracellular signal transduction even in differentiated, nondividing cells like those of the heart.