Stimulation of gene expression in neonatal cardiac myocytes by raised extracellular pH.

Abstract

In response to alpha adrenergic agonists cultured neonatal rat ventricular myocytes exhibit many of the features of hypertrophy in vivo including: (i) an increase in cell size in the absence of cell division, (ii) an increase in the rate of protein synthesis, (iii) an increase in the assembly of contractile proteins into sarcomeric units and (iv) characteristic changes in gene expression. These include the rapid induction of a programme of immediate-early genes, including the c-fos, c-jun and Egr-l genes, upregulation of a number of constitutively expressed proteins involved in muscle contraction such as ventricular myosin light chain-2 (vMLC2) and cardiac a-actin and reexpression of genes normally only expressed during the embryonic stage of ventricular development such as atrial natriuretic factor (ANF), B-myosin heavy chain and skeletal aactin [I]. In the perfused rat heart and in isolated adult cardiac myocytes we have shown that a-adrenergic agonists stimulate protein synthesis by binding to a, receptors [2,3]. We have also reported that an increase in extracellular pH @Ho) increases intracellular pH (pHi) and stimulates protein synthesis in perfused hearts [4]. Because a,-adrenergic agonists induce intracellular alkalinization in the heart, this suggests a role for increased pHi in the stimulation of translation by these agents [2]. To determine whether intracellular alkalinization may also be important in the regulation of transcription, cultured neonatal rat ventricular myocytes were exposed to an increase in pHo and the effect on gene expression assayed using luciferase (LUX) reporter gene constructs under the control of promoters for ANF and vMLC2. Ventricular myocytes were isolated from the hearts of 1-2 day old rats as described previously [5]. For transfection experiments, myocytes were plated on gelatin-coated 60mm tissue culture dishes at a density of 1 million cells per dish in plating medium (Maintenance medium DMEW199 (4:l) containing 10% horse serum and 5% FCS) and were maintained at 37°C in a humidified atmosphere of 5% CO,. Twenty hours after plating and 4 h before transfection, medium was changed to 4 mls maintenance medium containing 4% horse serum. Plasmids were diluted in 0.25 M CaCI, and an equal volume of 50 mM N,NBis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES, pH 6.9), 280 mM NaCI, 1.5 mM N%HPO, was added. After 20 min, 1 ml of this suspension (containing 15 pg LUX reporter plasmid and 4 pg of a CMV-driven P-galactosidase (P-gal) vector to control for transfection efficiency) was added to each plate. After overnight transfection, cells were washed once with 10% horse serum in maintenance medium and twice with maintenance medium before being incubated in maintenance medium. The pH of the medium was increased by addition of 54 mM NaHCO, where indicated. After 48 h, myocytes were washed three times with ice-cold phosphate-buffered saline (PBS) and extracted on ice with 0.1 M potassium phosphate (pH7.9), 0.5% (v/v) Triton X-100, 1 mM dithiothreitol (0.4 ml) for 15 min. Luciferase and P-gal activities in cell extracts were assayed by standard methods [5]. For protein synthesis experiments myocytes were plated at a density of 1 million cells per 35 mm dish. Protein synthesis was assayed by the incorporation of 'H phenylalanine into 0.56 M HCIO, insoluble material [3]. Results are presented as the mean 5 SEM of experiments on 4 or 5 separate preparations of myocytes. A. 3 . 0 1 -