Rational promoter selection for gene transfer into cardiac cells

doi:10.1016/S0022-2828(03)00140-8

Journal of Molecular and Cellular Cardiology

Volume 35, Issue 7, July 2003, Pages 823-831

https://doi.org/10.1016/S0022-2828(03)00140-8 Get rights and content

Abstract

Cardiomyocytes (CMCs) are extremely difficult to transfect with non-viral techniques, but they are efficiently infected by adenoviruses. The most commonly used promoters to drive protein expression in cardiac myocytes are of viral origin, since they are believed to be constitutively active and minimally regulated by physiological or pharmacological challenge of cells. In recombinant adenoviruses, we systematically compared three different promoters: the cytomegalovirus (CMV), the Rous sarcoma virus (RSV), and a synthetic promoter with three MEF2 transcription factor-binding sites upstream of the heat-shock protein 68 minimal promoter. We determined their basal activity in primary cardiac cells as well as their possible stimulation by commonly used agonists. The CMV promoter was activated up to 60-fold by the phorbol ester phorbol myristate acetate (PMA) and/or forskolin in neonatal rat CMCs and cardiac fibroblasts. Primary adult rat CMCs had higher basal expression from the CMV promoter that was not activated by PMA or forskolin. The RSV promoter was less affected by agonists and was more active in cardiac myocytes compared to cardiac fibroblasts. The MEF2-responsive promoter showed high basal expression in both myocytes and fibroblasts, and minimal induction by phorbol esters and forskolin. The relevance of reporter gene induction was confirmed with a contractile protein, troponin T (TnT). The CMV promoter driving TnT could be induced more than 15-fold with phenylephrine or forskolin to replace the endogenous protein almost to completion at a multiplicity of infection of 10. These results suggest the following use of the tested promoters: an inducible system (CMV), a myocyte-enriched system (RSV), or a stable control system (MEF2).

Introduction

Cardiomyocytes (CMCs) are generally considered to be one of the most difficult cell types to transfect with DNA constructs. Adenovirus vectors have facilitated gene transfer into both adult and neonatal rat CMCs in vitro as well as neonatal and adult myocardium in vivo [1], [2], [3], [4], [5]. To drive high-level expression of proteins of interest in cardiovascular cells, most investigators use viral promoters in recombinant adenovirus vectors. Previous experiments have shown that the cytomegalovirus (CMV) promoter can be activated up to 23-fold by forskolin and phorbol ester in human vascular smooth muscle cells [6]. Regulation of other viral promoters has been less extensively studied in cardiac myocytes. Different viral promoters have never been compared directly in isolated cardiac cells with regard to (1) their specificity for myocyte expression vs. cardiac fibroblasts, (2) the amount of protein produced, and (3) their regulation by physiological or pharmacological intervention.

We constructed adenovirus vectors with either the luciferase or β-galactosidase cDNA, driven by three different promoters, while maintaining the termination and polyadenylation signals constant (Fig. 1). The first is the 800 bp CMV immediate-early promoter, the second is the Rous sarcoma virus (RSV) long-terminal repeat (LTR), both extensively used for gene transfer experiments [7]. The third vector contains an artificial promoter consisting of a heat-shock protein 68 (hsp68) minimal promoter and three binding sites from the desmin gene for the transcription factor MEF2. MEF2 is a transcription factor that is expressed primarily in developing muscle and brain tissue. A transgenic mouse expressing this construct showed expression in embryonic muscle and brain tissues [8]. In the adult, MEF2 activity was very low in the heart and increased with pathologic conditions such as hypertrophy associated with overexpression of a constitutive calmodulin-dependent protein kinase [9]. Our study demonstrates differential basal activity and stimulation of the tested promoters that should lead to differential use of these promoters depending on the experimental design.

Section snippets

Isolation and culture of neonatal rat CMCs and cardiac fibroblasts

Hearts from several litters of 1–3-d-old Wistar rats were excised and cells were isolated by trypsin digestion (0.1%) as previously described [10]. To enrich for CMCs, the cell suspension was preplated on regular polystyrene dishes for 30–45 min. CMCs were cultured in serum-free MEM supplemented with transferrin (1 mg/ml), insulin (1 μg/ml), and bovine serum albumin (BSA) (1 mg/ml) for 24–48 h before experiments were carried out. Dishes from preplating were cultured in DMEM supplemented with

Results

In order to develop the optimal promoter system for use in different applications in primary cardiac myocytes, we tested three promoters with respect to their basal activity and response to agonist stimulation. Using a replication-deficient adenovirus vector containing the luciferase cDNA under control of the CMV promoter (Ad-CMV-Luc), we tested inducers of two different signal transduction pathways for their effects on the CMV promoter in neonatal and adult rat CMCs. Phorbol myristate acetate

Discussion

This study was designed to compare two commonly used viral promoters and a synthetic promoter in an adenoviral context with regard to their expression in primary cardiac cells and their regulation by pharmacological intervention that are commonly used in cardiac myocytes.

The CMV promoter, thought to be maximally active at basal conditions, has been chosen for many gene transfer experiments. However, it has more recently been shown to be regulated by many stimuli, such as α interferon

Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ma 2185/1-1 to A. Maass and TPB6, SFB355 to L. Neyses) and by NIH grant HL50560 to. L.A. Leinwand. The authors would like to thank R.C. Thompson and M. Buvoli for continuous discussion and support.

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Modulation of gene expression is a biomedical powerful strategy to investigate the regulatory mechanisms of structure, function, contractile apparatus, cytoskeleton and myocyte signaling pathways. Viral vectors are very efficient tools for gene transfer into cardiomyocytes, a cell type that is not efficiently transfected, overcoming the use of traditional transfection protocols based on electroporation or lipofection (Bauer et al., 2005; Djurovic et al., 2004; Louch et al., 2011; Maass et al., 2003). The lentivirus belongs to the family of retroviruses and can integrate into the host genome and it is transmitted to cell progeny in the case of division.
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This strong enhancer has little cell type preference and contains consensus binding sites for transcription factors such as nuclear factor 1 (NF1) (Niller and Hennighausen, 1991), and 90 (NF90) (Reichman et al., 2002), NF-kappaB/Rel (Sun et al., 2001), CREB/ATF (Stamminger et al., 1990), AP-1 (Rideg et al., 1994), SP-1 (Luu and Flores, 1997), ELK-1 (Chan et al., 1996), retinoic acid receptor and RAR-RXR family members (Angulo et al., 1996). Hence, the CMV-IE promoter can be stimulated by a wide range of different stimuli, such as phorbol myristate acetate, forskolin (Maass et al., 2003) and dibutyryl-cAMP (Zhang et al., 2003), prostaglandin E2, tumor necrosis factor alfa and other cytokines (Kline et al., 1998; Prosch et al., 1995) and lipopolysaccharide (Lee et al., 2004). In addition to the pharmacological stimulation, it has been shown that heat shock (HS) enhanced CMV-IE promoter driven gene expression transduced by adenovirus (Lee et al., 2002).
In CHO-K1 cells, heat shock strongly activated reporter-gene expression driven by the cytomegalovirus immediate-early (CMV-IE) promoter from adenoviral and plasmid vectors. Heat shock treatment (2 h at 42.5 °C) significantly enhanced the promoter DNA-binding activity in nuclear extracts. In CHO cells expressing mGluR1a and mGluR5a receptors under the control of the CMV promoter, heat shock increased receptor protein expression, mRNA levels and receptor function estimated by measurement of PI hydrolysis, intracellular Ca²⁺ and cAMP. Hyperthermia increased average amplitudes of Ca²⁺ responses, the number of responding cells, and revealed the toxic properties of mGluR1a receptor. Heat shock also effectively increased the expression of EGFP. Hence, heat shock effects on mGluR expression and function in CHO cells may be attributed to the activation of the CMV promoter. Moreover, this effect was not limited to CHO cells as heat shock also increased EGFP expression in PC-12 and HEK293 cells. Heat shock treatment may be a useful tool to study the function of proteins expressed in heterologous systems under control of the CMV promoter. It may be especially valuable for increasing protein expression in transient transfections, for enhancing receptor expression in drug screening applications and to control the expression of proteins endowed with toxic properties.
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¹: Present address: Department of Medicine, West German Cancer Center, University of Essen Medical School, Germany.

²: Present address: Department of Medicine, University of Manchester, Manchester, UK.

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Original ArticleRational promoter selection for gene transfer into cardiac cells

Abstract

Introduction

Section snippets

Isolation and culture of neonatal rat CMCs and cardiac fibroblasts

Results

Discussion

Acknowledgements

J Mol Cell Cardiol

Gene

Biochem Biophys Res Commun

Meth Cell Biol

Cell

Gene

J Mol Cell Cardiol

J Mol Cell Cardiol

J Biol Chem

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus

J Clin Invest

Widespread long-term gene transfer to mouse skeletal muscles and heart

J Clin Invest

Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo

Proc Natl Acad Sci USA

Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus

Gene Ther

Enhancer stimulation unmasks latent gene transfer after adenovirus-mediated gene delivery into human vascular smooth muscle cells

Circ Res

Transcriptional activity of MEF2 during mouse embryogenesis with a MEF2-dependent transgene

Development

CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo

J Clin Invest

Original Article
Rational promoter selection for gene transfer into cardiac cells