alpha-MHC Gene Research Articles

Expression of Cardiac Specific Cell Marker in Ex Vivo Differentiated Canine iPSC

Induced Pluripotent stem cells (iPSC) have a high ability to renew and differentiate themselves into various lineages and as vehicles of cell based therapy. Stem cell can differentiate under appropriate in vitro and in vivo conditions into different cell types. This study described the establishment of condition for in vitro expression of alpha MHC gene in cardiac differentiated canine iPSC (ciPSC). In vitro differentiation of canine iPSCs via embryoid bodies (EBs) were produced by ‘Hanging Drop’ method. EB’s were differentiated by using IMDM differentiation media: FBS – 10%, NEAA (100X) – 0.5%, Â-Mercaptoethanol- 100mM, Gentamycin- 5µg/ml supplemented with Azacytidine- 0.5µM. During differentiation, EBs were collected on day 4, 6, 8, 12, 16, 20 and 24 for characterization of cardiomyocytes specific marker expression. Total RNA from EBs were extracted by using Trizol method and subsequently cDNA were synthesized. The differentiated cells expressed cardiac specific gene (Alpha MHC) which started from day 6 of differentiation upto day 24 Immunocytochemistry and relative expression of cardiac specific genes revealed that ciPSC have the potential to differentiate into cardiomyocytes which can be used for cardiac tissue regeneration and as disease models for pharmaceutical testing.

Carfilzomib-induced cardiotoxicity mitigated by dexrazoxane through inhibition of hypertrophic gene expression and oxidative stress in rats

Carfilzomib (CFZ) is an inhibitor of proteasome that is generally used in the treatment of multiple myeloma but due to its cardiotoxicity clinical use may be limited. Dexrazoxane (DZR), an inhibitor of topoisomerase-II, prevents cardiac damage by reducing the formation of reactive oxygen species and hypertrophic gene expression. This study evaluated the protective effect of DZR on CFZ-induced cardiotoxicity. Thirty-two male Albino rats were randomly divided into four groups (n = 8). Group I received DMSO, Group II received CFZ (4 mg/kg, intraperitoneally [i.p.]) twice weekly up to day 16, Group III received DZR (20 mg/kg, i.p.) for 16 days and CFZ twice weekly for 16, Group IV received DZR (40 mg/kg, i.p.) for 16 days and CFZ twice weekly for 16. CFZ-induced cardiotoxicity was assessed by hematological, biochemical, mRNA expression, oxidative stress and histopathological studies. CFZ-induced significant changes have been observed in blood parameters including red blood cells, white blood cells, hemoglobin and hematocrit concentrations which were associated with increase in cardiac enzymes markers like creatine kinase (CK), CK-MB and lactate dehydrogenase. Treatment with DZR reversed the hematological statistics and the biochemical markers of CFZ-induced cardiotoxicity. Furthermore, DZR also attenuated the effects of CFZ-induced toxic effect on redox markers such as malondialdehyde and reduced glutathione. Above findings were further confirmed by beta-myosin heavy chain (β-MHC) and alpha-MHC (α-MHC) gene expression. Histopathological reports suggested that DZR ameliorates CFZ-induced changes in cardiac cellular architecture in rats. These results confirm that DZR protects heart from CFZ-induced cardiotoxicity.

Abstract 309: Two SoxD Family Transcription Factors Influence Heart Function at Baseline and After Cardiac Overload

Background: Sox6, a member of the SoxD family of transcription factors, has recently been established as a mediator of the actions of MyomiRs miR-208b and 499 in skeletal muscle. Sox6 suppresses the expression of slow skeletal muscle genes and its deletion promotes skeletal muscle fiber switching in mice. However, the role of Sox6 in the heart remains elusive. Given the importance of MyomiRs in regulating cardiac stress responses and the regulation of Sox6 by MyomiRs in the heart, we postulate that Sox6 is a central regulator of cardiac hypertrophy and heart failure. Results: Because of functional redundancy of Sox6 and Sox5 in multiple tissues, Sox6 fl/fl mice were crossbred with Sox5 fl/fl mice and mice expressing CRE recombinase under the control of the alphaMHC gene promoter to obtain Sox5 fl/fl / Sox6 fl/fl /CRE+ ( Sox5/6 cKO) animals or CRE- controls. Sox5/6 cKO animals were viable and born at expected Mendelian ratios. At baseline, Sox5/6 cKO mice did not show any signs of hypertrophy or cardiac failure as assessed by echocardiography at the age of 9 weeks. While cardiac size and heart function measured by echocardiography was unaltered in mice aged 4-5 months, cardiac catheterization revealed significant decreases in contractility in Sox5/6 cKO animals. In addition, qPCR analysis of hearts showed increased expression of slow myosin Myh7 and Nppa , a marker of cardiac stress, in Sox5/6 cKO animals compared to controls. Following cardiac pressure overload for 3 weeks by transverse aortic constriction (TAC), significant decreases in cardiac function, as measured by echocardiography, were apparent in Sox5/6 cKO compared to control mice. Sox5/6 cKO mice subjected to TAC also showed signs of cardiac failure such as increased wet lung weight. Conclusion: Cardiac-specific deletion of Sox6 and Sox5 lead to reduction of cardiac function at baseline and exacerbated susceptibility to the development of cardiac failure after cardiac pressure overload. The contribution of each individual transcription factor to the observed phenotypes is currently being investigated.

The CAAT‐binding transcription factor 1/nuclear factor 1 binding site is important in β‐myosin heavy chain antisense promoter regulation in rats

The rat heart expresses two myosin heavy chain (MHC) isoforms, beta and alpha; these genes are arranged in tandem on the same chromosome. We have reported that an antisense (AS) beta RNA starts in the intergenic (IG) region between beta and alpha genes and extends to overlap the beta gene. We propose that in adult rats, both the alpha sense and IG betaAS RNA expression are activated by an IG bidirectional promoter and that the transcription of betaAS RNA interferes with the sense beta, resulting in low levels of beta mRNA and high levels of alpha, a phenotype seen in a typical rat heart. A previous report examined the activity of the betaAS promoter and showed that a 559 bp fragment of the betaAS promoter (-2285 to -1726; relative to alphaMHC gene start site) injected into rat ventricle was activated in control heart, and decreased significantly in response to hypothyroidism (propylthiouracil induced) and diabetes (streptozotocin induced) and increased in hyperthyroid rats (T(3) induced), similar in pattern to the endogenous betaAS RNA. In the present paper, we demonstrate with electrophoretic mobility shift analyses that ventricular nuclear proteins are interacting with a nuclear factor 1/CAAT-binding transcription factor 1 (NF1/CTF1) binding site, and a supershift assay indicates that the protein binding at this site is antigenetically related to the CTF1/NF1 factor. Moreover, a mutation of the CTF1/NF1 site within the 559 bp promoter region nearly abolished promoter activity in vivo in control, STZ- and PTU-treated rats. Based on these findings, we conclude that the NF1 site is critical to betaAS promoter regulation.

Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies

Various types of embryoid body (EB) that were formed from mouse embryonic stem (ES) cells under various culture conditions were characterized in terms of gene expression pattern to estimate the differentiation status of the bodies. The gene expression of typical markers (i.e., GATA-4, GATA-6, transthyretin [TTR], alpha-fetoprotein [AFP], Nkx2.5, and alpha-myosin heavy chain [alpha-MHC]) was quantitatively analyzed in various types of EB, and the gene expression pattern of those marker genes was graphically shown for each EB. The gene expression pattern accurately represented the differentiation status of the EBs. The gene expression pattern indicated that the Nkx2.5 and alpha-MHC genes were highly expressed in the EBs formed from 1000 ES cells in a low-adherence 96-well plate. By transferring the EBs into an attachment culture, cardiomyocytes were more efficiently generated in the outgrowth of the EBs. When we increased the seeding cell number from 1000 to 4000 ES cells, the gene expression pattern changed, that is, the expression levels of the TTR and AFP genes increased, whereas those of the Nkx2.5 and alpha-MHC genes decreased, and the trend of differentiation changed from cardiomyogenesis to visceral yolk-sac-like structure formation.

Developmental expression and comparative genomic analysis of Xenopus cardiac myosin heavy chain genes

Myosin heavy chains (MHC) are cytoskeletal motor proteins essential to the process of muscle contraction. We have determined the complete sequences of the Xenopus cardiac MHC genes, alpha-MHC and ventricular MHC (vMHC), and have characterized their developmental expression profiles. Whereas alpha-MHC is expressed from the earliest stages of cardiac differentiation, vMHC transcripts are not detected until the heart has undergone chamber formation. Early expression of vMHC appears to mark the cardiac conduction system, but expression expands to include the ventricle and outflow tract myocardium during subsequent development. Sequence comparisons, transgenic expression analysis, and comparative genomic studies indicate that Xenopus alpha-MHC is the true orthologue of the mammalian alpha-MHC gene. On the other hand, we show that the Xenopus vMHC gene is most closely related to chicken ventricular MHC (vMHC1) not the mammalian beta-MHC. Comparative genomic analysis has allowed the detection of a mammalian MHC gene (MyH15) that appears to be the orthologue of vMHC, but evidence suggests that this gene is no longer active.

Single-stranded DNA-binding Proteins PURα and PURβ Bind to a Purine-rich Negative Regulatory Element of the α-Myosin Heavy Chain Gene and Control Transcriptional and Translational Regulation of the Gene Expression: IMPLICATIONS IN THE REPRESSION OF α-MYOSIN HEAVY CHAIN DURING HEART FAILURE

The alpha-myosin heavy chain is a principal molecule of the thick filament of the sarcomere, expressed primarily in cardiac myocytes. The mechanism for its cardiac-restricted expression is not yet fully understood. We previously identified a purine-rich negative regulatory (PNR) element in the first intron of the gene, which is essential for its cardiac-specific expression (Gupta, M., Zak, R., Libermann, T. A., and Gupta, M. P. (1998) Mol. Cell. Biol. 18, 7243-7258). In this study we cloned and characterized muscle and non-muscle factors that bind to this element. We show that two single-stranded DNA-binding proteins of the PUR family, PURalpha and PURbeta, which are derived from cardiac myocytes, bind to the plus strand of the PNR element. In functional assays, PURalpha and PURbeta repressed alpha-myosin heavy chain (alpha-MHC) gene expression in the presence of upstream regulatory sequences of the gene. However, from HeLa cells an Ets family of protein, Ets-related protein (ERP), binds to double-stranded PNR element. The ERP.PNR complex inhibited the activity of the basal transcription complex from homologous as well as heterologous promoters in a PNR position-independent manner, suggesting that ERP acts as a silencer of alpha-MHC gene expression in non-muscle cells. We also show that PUR proteins are capable of binding to alpha-MHC mRNA and attenuate its translational efficiency. Furthermore, we show robust expression of PUR proteins in failing hearts where alpha-MHC mRNA levels are suppressed. Together, these results reveal that (i) PUR proteins participate in transcriptional as well as translational regulation of alpha-MHC expression in cardiac myocytes and (ii) ERP may be involved in cardiac-restricted expression of the alpha-MHC gene by preventing its expression in non-muscle cells.

The myocardial protein S100A1 plays a role in the maintenance of normal gene expression in the adult heart.

S100A1 and S100B are members of a family of 20 kDa Ca2+-binding homodimers that play a role in signal transduction in mammalian cells. S100A1 is the major isoform in normal heart and S100B, normally a brain protein, is induced in hypertrophic myocardium and functions as an intrinsic negative modulator of the hypertrophic response. In order to examine the function of S100A1, we first showed that, in contrast to S100B, S100A1 was downregulated in rat experimental models of myocardial hypertrophy following myocardial infarction or pressure overload. Second, in co-transfection experiments in cultured neonatal rat cardiac myocytes, S100A1 inhibited the alpha1-adrenergic activation of promoters of genes induced during the hypertrophic response including the fetal genes skeletal alpha actin (skACT), and beta-myosin heavy chain (MHC) and S100B, but not the triiodothyronine (T3) activation of the promoter of the alpha-MHC gene, that is normally expressed in adult myocardium. These results suggest that S100A1 is involved in the maintenance of the genetic program that defines normal myocardial function and that its downregulation is permissive for the induction of genes that underlie myocardial hypertrophy.

TNFalpha decreases alphaMHC expression by a NO mediated pathway: role of E-box transcription factors for cardiomyocyte specific gene regulation.

Tumor necrosis factor alpha(TNFalpha) is thought to play a key role in the pathogenesis of cardiac failure. In the myocardium, TNFalpha enhances the expression of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) has been shown to affect beta-agonist-dependent cardiac contractility and relaxation. It is not clear, however, whether TNFalpha mediated NO release has sustained cardiac effects, by altering expression of cardiomyocyte specific genes such as alpha-myosin heavy chain (alphaMHC). Neonatal rat ventricular cardiomyocytes (CM) were stimulated with TNFalpha and/or the NOS inhibitor nitro-L-arginine (L-NNA). Protein binding to the E-box enhancer element in the alphaMHC promoter was evaluated by electrophoretic mobility shift assay (EMSA) and transcriptional activity of the E-box consensus motif was determined by luciferase assay. mRNA levels of the endogenous alphaMHC gene were assessed by RT-PCR. In vivo studies were performed in transgenic mice with cardiac specific over-expression of TNFalpha. CM treated with TNFalpha exhibited decreased levels of alphaMHC transcripts (69 +/- 8% of control), the effect of TNFalpha was reversed by L-NNA (94 +/- 14% of control). As shown by EMSA, TNFalpha reduced protein binding to the alphaMHC E-box enhancer motif via NO dependent pathways. Addition of the NO-donor sodium nitroprusside (SNP) to CM nuclear extracts dose dependently disrupted protein binding to the alphaMHC E-box. Furthermore, exposure of CM to TNFalpha or SNP decreased transcription from an E-box luciferase-reporter construct (TNFalpha: 74 +/- 12%; SNP 250 microM: 72 +/- 10%; SNP 500 microM: 66 +/- 11% of control). In myocardial tissue of TNFalpha transgenic mice, increased nitrotyrosine staining, decreased protein binding to the alphaMHC E-box motif and reduced expression of alphaMHC (62 +/- 26%) were observed. The present study shows that TNFalpha reduces alphaMHC transcript levels in cardiomyocytes. Our data obtained in cultured CM and in TNFalpha transgenic mice support the notion that TNFalpha exerts these effects by NO and E-box dependent mechanisms in vitro and possibly in vivo.

Pulse Electrophoresis of Muscle Myosin Heavy Chains in Sodium Dodecyl Sulfate–Polyacrylamide Gels

We have developed a new method that provides enhanced resolution of myosin heavy chain (MHC) isoforms by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.

Protein kinase-A dependent phosphorylation of transcription enhancer factor-1 represses its DNA-binding activity but enhances its gene activation ability.

The cAMP-dependent signaling pathway has been implicated in cardiac cell growth/differentiation and muscle gene transcription. Previously, we have identified a cAMP-inducible E-box/M-CAT hybrid motif in the cardiac alpha-myosin heavy chain (alpha-MHC) gene promoter. The two factors, TEF-1 and Max, that bind to this motif are found to physically associate with each other and exert a positive cooperative effect for gene regulation. Here we show that TEF-1, but not Max, is a substrate for protein kinase-A (PK-A)-dependent phosphorylation. TEF-1 is phosphorylated by PK-A at residue serine-102. This post-translational modification of TEF-1 repressed its DNA-binding activity, but not its ability to interact with the Max protein. Replacement of serine-102 in TEF-1 by a neutral or a charged amino acid did not abolish its DNA-binding ability, suggesting that changing a charge at the 102 amino-acid position of TEF-1 was not sufficient to inhibit its DNA-binding activity. We also show that PK-A response of the alpha-MHC gene is stimulated by the presence of wild-type TEF-1 but not by mutant TEF-1 having serine-102 replaced by alanine, suggesting that phosphorylation at this residue accounts for the cAMP/PK-A response of the gene. Thus, these data demonstrate that TEF-1 is a direct target of cAMP/PK-A signaling in cardiac myocytes.

Dexamethasone induced cardiac hypertrophy in newborn rats is accompanied by changes in myosin heavy chain phenotype and gene transcription.

Cardiac hypertrophy has been observed in newborn infants treated with dexamethasone (DEX). This study was undertaken to examine whether DEX-induced hypertrophy in newborn rats is associated with redistribution of cardiac myosin heavy chain (MHC) isoforms and, if so, the effects involve transcriptional regulation. Newborn rats were injected with either DEX (1 mg/kg/day; s.c.) or equivalent volume normal saline for 1, 3, 5, 7 or 9 days. Hypertrophy was quantified by heart dry/wet wt ratios, heart/body wt ratios, and total protein content of the myocardium. Changes in the expression of cardiac MHC mRNA were characterized by northern blot and slot blot analyses, using isoform specific probes for alpha- and beta-MHC genes. DEX effect on alpha-MHC gene transcription was analyzed by transiently transfecting various alpha-MHC promoter/CAT reporter constructs into primary cultures of cardiac myocytes derived from one day old rat pups. DEX administration into newborn rats produced significant cardiac hypertrophy ranging from 23% at day 1 to 59% at 9 days. The hypertrophy was accompanied by immediate increase (83%) in steady state level of the alpha-MHC mRNA within one day and a maximum increase (148%) at 7 days of treatment. The steady state level of beta-MHC mRNA declined by 25% at day 1 and a maximum decrease of 54% at day 7 of DEX treatment. The changes in MHC mRNA were also reflected in their protein levels as determined by V1 and V3 isozyme analysis. DEX treatment of primary cultures of cardiomyocytes following transfection with alpha-MHC promoter/CAT reporter constructs resulted in increased CAT expression in a dose dependent manner. The minimum alpha-MHC gene sequences responding to DEX treatment were located between the -200 to -74-bp region of the gene, resulting in 2-fold and 6-fold activation of CAT reporter after 0.05 and 0.1 mM doses of DEX, respectively. Our data indicate that DEX induced cardiac hypertrophy in newborn rats is accompanied by increased expression of alpha-MHC and decreased expression of beta-MHC. The alpha-MHC effects are mediated in part through transcriptional mechanisms.

In vivo regulation of beta-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer.

Cardiac beta-myosin heavy chain (beta-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length beta-MHC promoter fragments in normal control and in altered thyroid states. The test beta-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control alpha-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that beta- and alpha-MHC genes were individually and reciprocally regulated by thyroid hormone. The beta-to-alpha ratios of reporter gene expression demonstrated an almost threefold larger beta-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the -408-bp beta-MHC promoter construct caused transcriptional activity to drop to null. When studied in the -3, 500-bp beta-MHC promoter, construct activity was reduced ( approximately 100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the beta-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

Expression of MEF2 genes during human cardiac development.

To better understand the regulatory mechanisms in gene expression of human cardiomyocytes, we studied the expression of MEF2 genes encoding transcription factors during the course of cardiac development. Expression of all four MEF2 transcripts (MEF2A, MEF2B, MEF2C, and MEF2D) were detected in all developmental stage of the human heart, while Mef2b transcripts were down-regulated in mouse heart development. Although none of the MEF2 genes, besides mouse Mef2b, exhibited any remarkable quantitative change in their transcripts, qualitative changes in MEF2 transcripts were found during the course of cardiac development. In particular, MEF2D transcripts showed prominent changes by alternative splicing in the perinatal period. MEF2D transcripts containing the 21-base exon (exon b) were predominantly expressed after birth. At the same time, transcripts of the alpha myosin heavy chain (alphaMHC) gene increased after birth, as the splicing pattern in transcripts of the cardiac troponin T (cTnT) gene changed to decrease the transcripts of cTnT1 after birth. These changes seemed to be correlated with the alternative splicing changes of MEF2 genes, especially MEF2D. The alternative splicing as well as transcriptional regulation in MEF2 genes might be important for regulating the alphaMHC gene and the maturation of cardiomyocytes.

Myocardial retinoid X receptor, thyroid hormone receptor, and myosin heavy chain gene expression in the rat during adult aging.

Although previous studies have shown that cardiac myosin heavy chain (MHC) composition undergoes a switch from the alpha- to beta-isoform in the heart during adult aging, the underlying mechanisms responsible for this switch are unknown. Cardiac MHC gene expression is regulated, in part, by thyroid hormone responsive elements present in the regulatory control regions of the alpha- and beta-MHC genes. Age-associated changes in the expression of thyroid hormone receptors (THRs) and/or retinoid X receptors (RXRs), the heterodimeric partner for THRs, could explain the age-associated changes in MHC expression. Accordingly, we measured mRNA levels for the cardiac muscle MHCs and the rat THR and RXR genes in the left ventricles of Wistar rats at 2, 6, and 24 months of age. Although there were no significant changes in RXR alpha or RXR beta mRNA levels with age, both alpha 1 and alpha 2 THR mRNA levels decreased significantly between 2 and 6 months of age. During this same time period, the mRNA levels for alpha-MHC declined by more than half, whereas beta-MHC mRNA levels remained low and unchanged. On the other hand, between 6 and 24 months, when mRNA levels for beta-MHC increased and alpha-MHC continued to decrease, there was a significant decline in THR beta 1 and RXR gamma mRNA levels accompanied by a reduction in the THR beta 1 and RXR gamma protein levels. These data show a pattern of change that suggests that the decline in alpha-MHC gene expression may be biphasic and due to a decline in alpha 1 (and possibly alpha 2) THR levels between 2 and 6 months of age and a decline in THR beta 1 and RXR gamma levels at later stages. In contrast, the increase in beta-MHC gene expression was associated only with the changes in THR beta 1 and RXR gamma mRNA and protein levels.

Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, Max, for positive regulation of cardiac alpha-myosin heavy-chain gene expression.

The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac alpha-myosin heavy chain (alpha-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the alpha-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the alpha-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three- to fourfold transactivation of the alpha-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in alpha-MHC gene regulation.

Myocyte-specific enhancer factor 2 and thyroid hormone receptor associate and synergistically activate the alpha-cardiac myosin heavy-chain gene.

The muscle-specific regulatory region of the alpha-cardiac myosin heavy-chain (MHC) gene contains the thyroid hormone response element (TRE) and two A/T-rich DNA sequences, designated A/T1 and A/T2, the putative myocyte-specific enhancer factor 2 (MEF2) binding sites. We investigated the roles of the TRE and MEF2 binding sites and the potential interaction between thyroid hormone receptor (TR) and MEF2 proteins regulating the alpha-MHC promoter. Deletion mutation analysis indicated that both the A/T2 motif and TRE were required for muscle-specific expression of the alpha-MHC gene. The alpha-MHC enhancer containing both the A/T2 motif and TRE was synergistically activated by coexpression of MEF2 and TR in nonmuscle cells, whereas neither factor by itself activated the alpha-MHC reporters. The reporter construct containing the A/T2 sequence and the TRE linked to a heterologous promoter also showed synergistic activation by coexpression of MEF2 and TR in nonmuscle cells. Moreover, protein binding assays demonstrated that MEF2 and TR specifically bound to one another in vitro and in vivo. The MADS domain of MEF2 and the DNA-binding domain of TR were necessary and sufficient to mediate their physical interaction. Our results suggest that the members of the MADS family (MEF2) and steroid receptor superfamily (TR) interact with one another to synergistically activate the alpha-cardiac MHC gene expression.

Sympathetic control of cardiac myosin heavy chain gene expression.

Several neuroendocrine factors have been shown to influence the muscle phenotype. Various physiological reports have suggested the role of adrenergic nervous system for cardiac myosin heavy chain (MHC) expression. We have used cultured fetal rat heart myocytes to investigate the role of cAMP on the alpha- and beta-MHC gene expression. In low density cultures, addition of 1 mM 8 Br cAMP resulted in up regulation of alpha-MHC and down regulation of beta-MHC mRNA. This antithetic effect of cAMP depends on the basal expression of both expression of both MHC transcripts. In transient transfection analysis employing a series of alpha-MHC gene promoter/reporter constructs, we identified a 13 bp E-box M-CAT hybrid motif (EM element) which conferred a basal muscle specific and cAMP-inducible expression of the alpha-MHC gene. Data obtained from the mobility gel-shift analysis indicated that one of the factor(s) binding to the EM element is related to troponin T M-CAT binding factor (TEF-1). To test whether the protein binding to this sequence could be a substrate for cAMP-dependent phosphorylation, the cardiac nuclear proteins were preincubated in a kinase reaction buffer either with a catalytic subunit of PKA (CatPKA) or with cAMP, and binding activity of proteins to the EM element was evaluated by mobility gel shift assay. In a concentration dependent manner, a twofold increase in the intensity of the retarded band was observed. Furthermore, at 100 units of CatPKA, an additional band of faster mobility was observed which was not present either when phosphorylated nuclear extract was incubated with alkaline phosphatase or when ATP was absent in kinase reaction buffer. These results strongly suggest that factor(s) binding to the EM element is a substrate for cAMP dependent phosphorylation.

Catecholamines and cardiac growth.

The present knowledge concerning the alpha- and beta-adrenergic systems in the regulation of cardiac growth and gene expression is reviewed. To investigate the mechanism by which cAMP regulates the expression of cardiac genes we have used cultured myocytes derived from fetal rat hearts. We have shown previously that the addition of Br cAMP to the culture medium produced an increase in alpha-myosin heavy chain (alpha-MHC) mRNA level, in its rate of transcription as well as in the amount of V1 isomyosin. To characterize the promoter element(s) involved in cAMP responsive regulation of alpha-MHC expression we performed transient transfection analysis with a series of alpha-MHC gene promoter-CAT constructs. We have identified a 13 bp E-box/M-CAT hybrid motif (EM element) which conferred a basal muscle specific and cAMP inducible expression of the alpha-MHC gene. Using mobility shift assay we have documented that one of the EM element binding protein is TEF-1. Moreover, by incubating cardiac nuclear extracts with the catalytic subunit of PK-A we have found that factor(s) binding to the EM element is a substrate for cAMP dependent phosphorylation.

Identification of a Contractile-responsive Element in the Cardiac α-Myosin Heavy Chain Gene

The mechanisms by which the cardiac-specific alpha-myosin heavy chain (alpha-MHC) gene responds to contractile activity was studied in cultured cardiomyocytes and in vivo. Deletion analysis of the alpha-MHC promoter transiently transfected into neonatal rat cardiomyocytes localized the contractile-responsive element within -80 to -40 base pairs of the transcriptional start site. Mutational analysis of an E-box motif at position -47 showed that it was necessary for the contractile response both in cultured cardiomyocytes and in the intact heart. Competition gel mobility shift experiments indicated that the protein-DNA complex formed within the -39 to -59 base pair region could be competed by the E-box element at -309 of the alpha-MHC gene and that base substitutions within an E-box motif at -47 eliminated the protein-DNA complex. To identify the contractile-responsive nuclear protein, antibodies specific for E12/E47, an E-box binding basic-helix-loop-helix (bHLH) protein, and antibodies recognizing upstream stimulatory factor (USF), a widely expressed bHLH-leucine zipper transcription factor, were studied for their ability to inhibit cardiomyocyte nuclear protein binding to the E-box motif at -47. Anti-USF antibody abolished formation of the protein-DNA complex, thus identifying the protein as antigenically related to USF and demonstrating that bHLH-leucine zipper proteins are involved in the contractile-induced expression of the cardiac alpha-MHC gene.