Possible regulatory roles of promoter g-quadruplexes in cardiac function-related genes - human TnIc as a model.

G-quadruplexes (G4s) are four-stranded DNA secondary structures, which are involved in a diverse range of biological processes. Although the anti-cancer potential of G4s in oncogene promoters has been thoroughly investigated, the functions of promoter G4s in non-cancer-related genes are not well understood. We have explored the possible regulatory roles of promoter G4s in cardiac function-related genes using both computational and a wide range of experimental approaches. According to our bioinformatics results, it was found that potential G4-forming sequences are particularly enriched in the transcription regulatory regions (TRRs) of cardiac function-related genes. Subsequently, the promoter of human cardiac troponin I (TnIc) was chosen as a model, and G4s found in this region were subjected to biophysical characterisations. The chromosome 19 specific minisatellite G4 sequence (MNSG4) and near transcription start site (TSS) G4 sequence (-80 G4) adopt anti-parallel and parallel structures respectively in 100 mM KCl, with stabilities comparable to those of oncogene G4s. It was also found that TnIc G4s act cooperatively as enhancers in gene expression regulation in HEK293 cells, when stabilised by a synthetic G4-binding ligand. This study provides the first evidence of the biological significance of promoter G4s in cardiac function-related genes. The feasibility of using a single ligand to target multiple G4s in a particular gene has also been discussed.

G-quadruplexes-novel mediators of gene function.

Since the famous double-helix model was proposed, chromosomal DNA has been regarded as a rigid molecule containing the genetic information of an organism. It is clear now that DNA can adopt many transient, complex structures that can perform different biological functions. The G4 DNA (also called DNA G-quadruplex or G-tetraplex), a four-stranded DNA structure composed of stacked G-tetrads (guanine tetrads), has attracted much attention during the past two decades due to its ability to adopt a variety of structures and its possible biological functions. This review gives a glimpse on the structural diversity and biophysical properties of these fascinating DNA structures. Common methods that are widely used in investigating biophysical properties and biological functions of G4 DNA are described briefly. Next, bioinformatics studies that indicate evidence of evolutionary selection and potential functions of G4 DNA are discussed. Finally, examples of various biological functions of different G4 DNA are given, and potential roles of G4 DNA in respect of cardiovascular science are discussed.

Clenbuterol induces cardiac myocyte hypertrophy via paracrine signalling and fibroblast-derived IGF-1.

The ß(2)-selective adrenoreceptor agonist clenbuterol promotes both skeletal and cardiac muscle hypertrophy and is undergoing clinical trials in the treatment of muscle wasting and heart failure. We have previously demonstrated that clenbuterol induces a mild physiological ventricular hypertrophy in vivo with normal contractile function and without induction of α-skeletal muscle actin (αSkA), a marker of pathological hypertrophy. The mechanisms of this response remain poorly defined. In this study, we examine the direct action of clenbuterol on cardiocyte cultures in vitro. Clenbuterol treatment resulted in increased cell size of cardiac myocytes with increased protein accumulation and myofibrillar organisation characteristic of hypertrophic growth. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed elevated mRNA expression of ANP and brain natriuretic peptide (BNP) but without change in αSkA, consistent with physiological hypertrophic growth. Clenbuterol-treated cultures also showed elevated insulin-like growth factor I (IGF-1) mRNA and activation of the protein kinase Akt. Addition of either IGF-1 receptor-blocking antibodies or LY294002 in order to inhibit phosphatidylinositol 3-kinase, a downstream effector of the IGF-1 receptor, inhibited the hypertrophic response indicating that IGF-1 signalling is required. IGF-1 expression localised primarily to the minor population of cardiac fibroblasts present in the cardiocyte cultures. Together these data show that clenbuterol acts to induce mild cardiac hypertrophy in cardiac myocytes via paracrine signalling involving fibroblast-derived IGF-1.

Analysis of cardiac myocyte biology in transgenic mice: a protocol for preparation of neonatal mouse cardiac myocyte cultures.

We describe a method of isolating and maintaining primary cultures of mouse neonatal cardiac myocytes (NCM). This is derived from the well-established procedure for making NCM cultures from rat neonates by sequential digestion of rat ventricular myocardial pieces using a collagenase/pancreatin mixture. One-day-old mouse neonates are taken and the heart excised. The great vessels, atria, and top section of the ventricular chambers are cut away and the remaining ventricular myocardium is cut into small cubes (about 1-2 mm(3)). Heart pieces from at least 30 animals are then subjected to short (15-25 min) digestion in a shaking water bath in the presence of collagenase and pancreatin. Cell supernatants are taken and pooled together for a total of five digestion steps. The cells are then plated on gelatinized culture dishes and allowed to attach overnight. Myocyte cultures were inspected microscopically for up to 4 days, revealing that many myocytes beat throughout this period. This protocol may be of use for making primary cardiac myocyte cultures from transgenic mice and for investigating gene transcription and cell signalling.

Evaluation of frequency, type, and function of gap junctions between skeletal myoblasts overexpressing connexin43 and cardiomyocytes: relevance to cell transplantation.

Cell transplantation of skeletal myoblasts (SMs) is one possible treatment for repairing cardiac tissue after myocardial injury. However, inappropriate electrical coupling between grafted SMs and host cardiomyocytes may be responsible for the arrhythmias observed in clinical trials of SM transplantation. Whether functional gap junctions occur between the two cell types remains controversial. We have studied the ability of SMs to electrically couple with isolated adult rat cardiomyocytes (CMs) and assessed whether connexin43 (Cx43) overexpression enhanced gap junctional conductance (Gj). C2C12 myoblast lines overexpressing Cx43 were generated by gene transfection and clonal selection. CMs were cocultured with either SMs overexpressing Cx43 (CM-SM(Cx43)) or control SMs (CM-SM(WT)) in vitro. Gj between pairs of SMs and CMs was quantified with dual whole cell patch clamping. Formation of Gj occurred between 22% of CM-SM(WT) pairs (n=73) and 48% of CM-SM(Cx43) pairs (n=71, P<0.001). The Gj of CM-SM(Cx43) pairs (29.7+/-4.3 nS, n=21) was greater than that of CM-SM(WT) pairs (14.8+/-2.0 nS, n=12, P<0.05). The overexpression of Cx43 in SMs increased the formation of electrical communication and the steady-state conductance between SMs and CMs. Enhanced gap junctional conductance may be useful to promote the integration of transplanted SMs into the myocardium.

Cultured interstitial cells from human heart valves express both specific skeletal muscle and non-muscle markers.

Cardiac valve interstitial cells are a phenotypically diverse and dynamic population, comprising myofibroblasts, fibroblasts and smooth muscle cells. To understand how these contribute to valve function and to optimize the choice of cells for seeding tissue-engineered valves, we are fingerprinting interstitial cells from all four human heart valves for useful phenotypic markers. We have begun by selecting markers indicated as of interest from previous work on myofibroblast-like cell lines. We show that interstitial cells express a variety of skeletal muscle contractile proteins and the skeletal muscle transcription factor myogenin, but not the related factors MyoD, myf-5 and MRF4, suggesting partial activation of the muscle programme in these cells. Expression of non-muscle isoforms of creatine kinase (CK-B) and AMP deaminase (AMPD2 and AMPD3) was found in contrast to muscle-restricted isoforms. Non-muscle isoforms of alpha- and beta-tropomyosins were detected specifically in contrast to skeletal muscle-specific isoforms. Several members of the Frizzled (FZD) family of Wnt receptors were also detected. In addition, intact cusps of all four valves from pig were capable of contacting to non-receptor and receptor-mediated stimulation in vitro. We conclude that interstitial cells from human heart valves express various sarcomeric proteins, and suggest that these cells have contractile potential due to a unique pattern of expression of both muscle-specific and non-muscle isoforms of metabolic and structural proteins. This may be under the control of myogenin, activated through specific Wnt/FZD signaling. Identifying such molecular markers could prove useful for engineering allogenic non-valve cell sources for seeding the synthetic valve.

Phenylephrine requires the TATA box to activate transcription of GLUT1 in neonatal rat cardiac myocytes.

Cardiac hypertrophy and heart failure occur in association to alterations in glucose uptake and metabolism. Phenylephrine, among other hypertrophic agonists, has been reported to increase expression of GLUT1 in neonatal rat cardiac myocytes by activating transcription. However, the specific cis- or trans-acting factors in the GLUT1 gene that are targeted by this agonist remain elusive. Here we describe that the activity of the -99/+134 basal promoter of rat GLUT1 is increased by phenylephrine. Nevertheless, this is not mediated by previously described binding sites (GC-box, MG1E) in the promoter. Rather, the TATA box is required by the agonist to activate transcription from the promoter. Interestingly, The Ras-ERK mitogen-activated protein (MAP) kinase pathway is involved in the actions of phenylephrine on GLUT1 transcription, and the effects of Ras on the activity of the promoter depend on the integrity of the TATA box. Our data indicate that phenylephrine induces the expression of the TBP-associated factor TAF(II)250 mRNA, which increases in parallel to the expression of GLUT1, suggesting that altering the expression of basal transcription factors could be one mechanism by which phenylephrine may regulate the activity of the GLUT1 promoter.

The slow skeletal muscle troponin T gene is expressed in developing and diseased human heart.

Cardiac muscle development is characterised by the activation of contractile protein genes and subsequent modulation of expression resulting, ultimately, in the formation of a mature four-chambered organ. Myocardial gene expression is also altered in the adult in response to pathological stimuli and this is thought to contribute to the altered contractile characteristics of the diseased heart. We have examined the expression of the slow skeletal troponin T (TnT) gene in the human heart during development and in disease using whole mount in situ hybridisation and real-time quantitative (TaqMan) polymerase chain reaction (PCR). Slow skeletal TnT mRNA shows transitory and regional expression in the early foetal heart, which occurs at different times in atria and ventricles. In ventricular myocardium, expression is seen in the outer epicardial layer at a time when the coronary circulation is being established. Expression was detected at low levels in the adult human heart and was significantly increased in end-stage heart failure. Similarly, expression was readily detectable during early rat heart development and was up-regulated in pressure overload hypertrophy in adult. Together these data show for the first time that slow skeletal TnT mRNA is readily detectable during early human heart development. They further suggest that slow skeletal TnT may be responsive to myocardial stress and that elevated levels may contribute to myocardial dysfunction in adult disease.

Role of interleukin-1beta in acute inflammation and graft death after cell transplantation to the heart.

BACKGROUND: Poor survival of grafted cells is a major factor hindering the therapeutic effect of cell transplantation; however, the causes of cell death remain unclear. We hypothesized that interleukin-1beta (IL-1beta) might play a role in the acute inflammatory response and graft death after cell transplantation and that inhibition of IL-1beta might improve graft survival. METHODS AND RESULTS: 14C-labeled male skeletal muscle precursor cells were implanted into female mouse hearts by direct intramuscular injection. The amount of 14C-label provides an estimate of the surviving cell number, whereas the amount of male-specific Smcy gene measured by polymerase chain reaction indicates the total (surviving+proliferated) number of donor-derived cells. At 10 minutes after implantation, 44.8+/-2.4% of the grafted cells survived and this steadily decreased to 14.6+/-1.1% by 24 hours, and to 7.9+/-0.6% by 72 hours (n=6 in each point). Proliferation of the surviving cells, which began after 24 hours, resulted in an increase in the total cell number from 15.5+/-0.8% at 24 hours to 24.4+/-1.6% at 72 hours. Acute inflammation was prominent at 24 hours and was reduced by 72 hours, in parallel with IL-1beta expression. Administration of anti-IL-1beta antibody improved graft survival at both 24 (25.6+/-1.6%) and 72 hours (14.8+/-1.1%) and resulted in a 2-fold increase in the total cell number at 72 hours (45.8+/-2.4%). The effects of IL-1beta inhibition corresponded with a reduced inflammatory response. CONCLUSIONS: IL-1beta is involved in acute inflammation and graft death after direct intramyocardial cell transplantation. Targeted inhibition of IL-1beta may be a useful strategy to improve graft survival.

Enhanced effect of myocardial gene transfection by VP22-mediated intercellular protein transport.

One of the major issues in myocardial gene therapy is poor transfection efficiency. The herpes simplex virus protein VP22 is known to facilitate intercellular protein transport. Not only VP22 but also VP22-linked protein are exported from the cytoplasm of cells, in which it is synthesised endogenously, and transferred to surrounding cells, where it is translocated into the nuclei. However, the feasibility and efficiency of the intercellular trafficking properties of VP22-linked protein in the myocardium has not been clarified. Rat hearts were transfected by direct intramyocardial injection of naked plasmid vectors encoding either lacZ or VP22-linked lacZ. At day 5 following transfection, similar numbers of cardiomyocytes surrounding the injection sites showed beta-galactosidase (beta-gal) expression in the cytoplasm in both groups. In addition to this, following transfection of VP22-linked lacZ, most of the cardiomyocytes adjacent to the cytoplasmic-positive cells demonstrated nuclear-localised beta-gal expression. The number of these nuclear-positive cardiomyocytes, which are thought to be secondary protein-transported cells, was 4.3-fold greater than that of primary transfected, cytoplasmic-positive cells. Western blot analysis demonstrated that the amount of targeted protein expression is 2.9-fold greater following VP22-lacZ transfection (VP22-linked beta-gal; approximately 40 kDa bigger than wild-type beta-gal) compared with lacZ transfection (wild-type beta-gal). This data highlights the efficiency of the VP22-mediated intercellular protein delivery in the myocardium following in vivo gene transfection and suggests that the VP22-mediated effect is useful in enhancing the efficacy of myocardial gene therapy.

The slow skeletal muscle troponin T gene is expressed in developing and diseased human heart.

Cardiac muscle development is characterised by the activation of contractile protein genes and subsequent modulation of expression resulting, ultimately, in the formation of a mature four-chambered organ. Myocardial gene expression is also altered in the adult in response to pathological stimuli and this is thought to contribute to the altered contractile characteristics of the diseased heart. We have examined the expression of the slow skeletal troponin T (TnT) gene in the human heart during development and in disease using whole mount in situ hybridisation and real-time quantitative (TaqMan) polymerase chain reaction (PCR). Slow skeletal TnT mRNA shows transitory and regional expression in the early foetal heart, which occurs at different times in atria and ventricles. In ventricular myocardium, expression is seen in the outer epicardial layer at a time when the coronary circulation is being established. Expression was detected at low levels in the adult human heart and was significantly increased in end-stage heart failure. Similarly, expression was readily detectable during early rat heart development and was up-regulated in pressure overload hypertrophy in adult. Together these data show for the first time that slow skeletal TnT mRNA is readily detectable during early human heart development. They further suggest that slow skeletal TnT may be responsive to myocardial stress and that elevated levels may contribute to myocardial dysfunction in adult disease. (Mol Cell Biochem 263: 91-97, 2004).

Hypertrophic agonists induce the binding of c-Fos to an AP-1 site in cardiac myocytes: implications for the expression of GLUT1.

OBJECTIVES: Serum is among the agents known to induce hypertrophy of cardiac myocytes, which occurs concomitant with an increase in AP-1-mediated transcription. We have examined if this effect correlates with changes in the relative abundance of particular AP-1 heterodimers, as their exact composition under these conditions is unknown. Furthermore, we obtained insight on the specific role of c-Fos from studying the induction of the glucose transporter GLUT1 by serum in fibroblasts. METHODS: We characterised the AP-1 heterodimers expressed in neonatal cardiac myocytes by supershift electrophoretic mobility shift assay (EMSA) analysis. Quantitative changes in transcription were measured using a luciferase reporter vector, and we examined the expression of the glucose transporter GLUT1 in cardiac myocytes and a c-Fos knockout-derived fibroblast cell line by western blotting. RESULTS: Transcriptionally active AP-1 in combinations of c-Jun, JunD and JunB with Fra1, Fra2 and possibly FosB, are expressed in cardiac myocytes. Hypertrophic stimuli transiently induced AP-1 dimers containing c-Fos, and this was dependent on the ERK mitogen-activated protein kinase pathway and coincided with the activation of AP-1-mediated transcription and the induction of GLUT1 in cardiac myocytes. In fibroblasts, the induction of GLUT1 by serum required the specific expression of c-Fos. CONCLUSION: Our data suggest that induction of c-Fos containing AP-1 heterodimers may partly activate AP-1-mediated transcription in cardiac myocytes treated with hypertrophic agonists under conditions known to induce GLUT1. Data obtained in fibroblasts treated with serum lead us to hypothesise that c-Fos might play a major role in the regulation of GLUT1 expression.

Differential regulation of the muscle-specific GLUT4 enhancer in regenerating and adult skeletal muscle.

We have reported a novel functional co-operation among MyoD, myocyte enhancer factor-2 (MEF2), and the thyroid hormone receptor in a muscle-specific enhancer of the rat GLUT4 gene in muscle cells. Here, we demonstrate that the muscle-specific enhancer of the GLUT4 gene operates in skeletal muscle and is muscle fiber-dependent and innervation-independent. Under normal conditions, both in soleus and in extensor digitorum longus muscles, the activity of the enhancer required the integrity of the MEF2-binding site. Cancellation of the binding site of thyroid hormone receptor enhanced its activity, suggesting an inhibitory role. Muscle regeneration of the soleus and extensor digitorum longus muscles caused a marked induction of GLUT4 and stimulation of the enhancer activity, which was independent of innervation. During muscle regeneration, the enhancer activity was markedly inhibited by cancellation of the binding sites of MEF2, MyoD, or thyroid hormone receptors. Different MEF2 isoforms expressed in skeletal muscle (MEF2A, MEF2C, and MEF2D) and all members of the MyoD family had the capacity to participate in the activity of the GLUT4 enhancer as assessed by transient transfection in cultured cells. Our data indicate that the GLUT4 enhancer operates in muscle fibers and its activity contributes to the differences in GLUT4 gene expression between oxidative and glycolytic muscle fibers and to the GLUT4 up-regulation that occurs during muscle regeneration. The activity of the enhancer is maintained in adult muscle by MEF2, whereas during regeneration the operation of the enhancer depends on MEF2, myogenic transcription factors of the MyoD family, and thyroid hormone receptors.

The cardiac valve interstitial cell.

Cardiac valve interstitial cells (ICs) are a heterogeneous and dynamic population of specific cell types that have many unique characteristics. They are responsible for maintaining the extracellular scaffold that provides the mechanical characteristics vital for sustaining the unique dynamic behaviour of the valve. A number of cellular phenotypes can be distinguished: some are sparsely arranged throughout the valve leaflets, whilst others are arranged in thin bundles. These cells express molecular markers similar to those of skeletal, cardiac and smooth muscle cells (SMCs) and in particular, many ICs express smooth muscle (SM) alpha-actin, a marker of myofibroblasts. In this respect, these cells exhibit a profile unlike skin fibroblasts, which may allude to their role in valve function.