Indapamide accentuates cardiac chronotropic responses to epidermal growth factor in chick cardiomyocytes

Tissue & Cell, 1996 28 (4) 469-472 © 1996 Pearson Professional Ltd.

Indapamide accentuates cardiac chronotropic responses to epidermal growth factor in chick cardiomyocytes S. W. Rabkin

Abstract. We have previously shown that indapamide [chloro-4-N-(methyl-2-indolinyl-1)-sulfamoyal-3benzamide] has a direct action on the heart to alter ion fluxes. This study sought to examine the potential interaction between indapamide and epidermal growth factor (EGF). Cardiomyocytes were prepared as primary culture from 7-day-old chick embryo hearts as aggregates that have a pattern of consistent spontaneous contraction. Indapamide enhanced the positive chronotropic response to EGF observed in chick embryonic ventricular myocyte aggregates while indapamide itself did not alter cardiac contractile frequency. Taken in conjunction with data that calcium channel blockade, inhibition of sodium entry or Na+-Ca 2+ exchange in the cardiomyocyte opposes the positive chronotropic action of EGF on the cardiomyocyte, this study has identified an agent, indapamide, that accentuates the cardiomyocyte response to EGF. Keywords" Epidermalgrowthfactor, cardiac contractilefrequency, indapamide

Introduction Epidermal growth factor (EGF), one of the earliest recognized growth factors (Carpenter and Cohen, 1979; Moolenoaar et al., 1983; Soltoff and Cantley, 1988), is a member of a family of polypeptide growth factors (Pimentel, 1994) that act on many cell types including the cardiomyocyte element of the heart (Rabkin et al., 1987; Nair et al., 1989; Rabkin, 1990; Lau 1993; Nair et al., 1993). Our laboratory was the first to demonstrate an action of EGF on the myocyte element of the heart. Based on the data that EGF alters ion fluxes across membranes (Carpenter and Cohen, 1979) and the dependency of cardiomyocytes for the generation of their contractile function on changes in membrane ionic movement (Carmeliet and Vereecke, 1979; Sperelakis et al., 1975), we postulated that EGF would affect spontaneous generation of cardiac contraction University of British Columbia, Vancouver, British Columbia, Canada. Received 28 February 1995 Accepted 20 February 1996 Correspondence to: Dr Simon W. Rabkin, University of British Columbia, Room 240, 2660 Oak Street, Vancouver, B,C., V6H 2Z6, Canada. Tel: (604) 875-5847 Fax: (604) 875-5849.

and, indeed, verified this in the chick cardiomyocyte (Rabkin et al., 1987). The mechanism of the EGF effect on cardiac automaticity involves alteration of ion fluxes across the cardiac membrane (Rabkin, 1990) but EGF-induced increases of intracellular cAMP in the heart have also been suggested (Nair et al., 1989; Nair et al., 1993). The factors that modulate the action of EGF on this important aspect of cardiac function remain unclear. Indapamide, [chloro-4-N-(methyl2-indolinyl-1)-sulfamoyal-3-benzamide], an indoline derivative of chlorsulphonamide is clinically useful as a diuretic and/or antihypertensive agent (Chaffman et al., 1984; Campbell and Brackman, 1990). Indapamide, like other diuretics (Benos, 1982), may prove to be a useful probe for the understanding of cardiac cellular events. Indapamide's direct action(s) on the heart has seen limited investigation until recently (Hemvael and Dipalma, 1980; Rabkin t993; Rabkin 1994). Indapamide antagonizes the effect of increases in extracellular potassium or calcium on cardiac contractile frequency with distinct differences from the diuretics hydrochlorothiazide and amiloride (Rabkin, 1989; Rabkin, 1993). Whether indapamide is able to modulate the action of EGF on the heart is uncertain. The 469

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importance of growth factors for the heart is based on the data that various growth factors are involved in cardiac hypertrophy, a major predictor of cardiovascular morbidity and mortality (Schneider and Parker, 1990). The objectives of this study were to test the hypothesis that indapamide modulates EGF's action on the heart.

Materials and methods Cell cultures Chick embryonic ventricular cells were cultured following the method of DeHaan (1967) as previously described (Rabkin & Sunga, 1987). Briefly, white Leghorn eggs were incubated in an automatic incubator (Marsh Rollex, San Diego, California, USA) for 7 days at 37.8°C and 87% humidity. Hearts were then isolated under sterile conditions from the 7-day chick embryo. Blood and connective tissue were removed, disaggregation was carried out by digestions in 0.005% trypsin (Gibco Laboratories, Burlington, Ontario, Canada), 0.1% BSA and DNAase 1* 107 Dornase units/mL, DMS8 (Worthington Biochemicals, Frederic, NJ, USA) at 37.8°C. After three digestions, the digests were diluted 1:5 in culture medium and the cells centrifuged for three minutes at 1000 x g. Cell aggregates Cell aggregates were made following previously described methods (Rabkin et al., 1987; Rabkin, 1990). Cells were maintained in medium 818A (20% M199, 73% DBSK buffer, 6% fetal bovine serum, penicillin at 100u/mL, streptomycin at 10 l.tg/mL plus fungizone 0.25 gg/mL. DBSK buffer had the following composition (raM): NaC1 116, NaH2PO4 1.0, MgSO4 0.8, Na2HPO 4 1.0, dextrose 5.6, CaC12 1.8 and NaHCO3 26. After 48 to 72 h, as needed, flasks with aggregates were emptied into a 35 x 10 mm Petri dish and a pair of Petri dishes were placed in larger dishes with intake for 5% CO2 in air, bubbled through water, on the stage of a Wilovert inverted microscope (Wild Leitz, Germany) in a specially designed plexiglass enclosure with a constant temperature of 37°C. The pH of the media was 7.43. When the beating rate of an aggregate was constant, an alliquot of medium was withdrawn and placed in a Petri dish within the incubator. EGF or indapamide, or both or their diluent were added to the withdrawn medium, mixed well and returned to the aggregate dish. Contractile frequencies were recorded for each aggregate at regular intervals. EGF, indapamide and the combination of indapamide and EGF were done concurrently, whenever possible, to reduce variability of response. The concentrations of indapamide and EGF were selected from previous experiments (Rabkin et al., 1987; Rabkin, 1990; Rabkin, 1993).

Drugs and chemicals Epidermal growth factor was obtained from Collaborative Research Inc. (Lexington, MA, USA). Indapamide was from Servier (Montreal, Canada) and was made into solution with either DMSO or ethanol which also served as the diluent for EGF experiments. Culture media was obtained from Gibco (Burlington, Ontario, Canada). Data analysis The data are presented as the mean __ 1SEM. Hypothesis testing used analysis of variance. The null hypothesis was rejected if the probability of a Type I error was less than 5% (P<0.05).

Results Under the conditions of the experiment, contractile frequency of myocardial cell aggregates is relatively constant and stable for periods in excess of 2 h (Rabkin et al., 1987). EGF, 20ng/ml, (with the diluent for indapamide) produced a small but definite increase in contractile frequency within the first 30 min of exposure (Figure 1). This response was markedly and significantly (P<0.05) enhanced by indapamide 10-6M. This increase was almost maximum at 10 rain but increased slightly thereafter. Indapamide, 10 .6 M, in the absence of EGF, did not alter cardiac contractile frequency. The chronotropic responses to EGF are usually noted after 20 min (Rabkin et al., 1987) so the data herein, by focusing on the initial responses, suggest an acceleration of EGF effects.

Discussion This study found that indapamide accentuated, or magnified, the effect of EGF to increase contractile frequency of cardiomyocytes. Indapamide alone did not increase cardiac contractile frequency, neither in the dose that accentuated the response to EGF nor across a wide range of other concentrations (Rabkin, 1994). The mechanism of this synergistic effect may be through the action of both EGF and indapamide on cardiac ion fluxes. Of the potential ion fluxes, the least likely is an interaction through calcium entry into the cardiomyocyte. EGF increases calcium entry into the cell (Soltoff and Cantley, 1988; Oettgen et al., 1985) and further increases intracellular calcium ([Ca2+]i) by mechanisms that include release of calcium from the endoplasmic reticulum via increases in inositol 1,4,5 triphosphate (IP3) (Hepler et al., 1987). Increases in calcium entry, that occur with increases in extracellular calcium ([Ca2--]o), accentuate the effect of EGF while calcium channel antagonists that decrease calcium entry, blunt the chronotropic response to EGF (Rabkin, 1990). In contrast, we have demonstrated that indapamide antag-

INDAPAMIDE AND EGF EFFECTS ON CARDIOMYOCYTES

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onises the effect of increasing [Ca2+]o (Rabkin, 1993). Furthermore, data in vascular smooth muscle suggest that indapamide either decreases or does not change calcium influx (Mironneau et al., 1981; DeWidt et al., 1984). The action of indapamide to accentuate the effect of EGF is the opposite of the effect of amiloride, which inhibits Na+-H + exchange (Benos, 1982; Frelin et al., 1984), on the chronotropic response to EGF. Similarly, dichlorobenzamil which preferentially inhibits Na÷-Ca 2+ exchange (Siegl et al., 1984; Rabkin, 1989) or diltiazem which inhibits voltage-dependent calcium influx (Kojima and Sperelakis, 1983), antagonized EGF-induced increases in cardiac contractile frequency (Rabkin, 1990). By inference one can exclude inhibition of each of these three pathways (Ca 2+ or Na ÷ entry or Na+-Ca 2÷ exchange), in the mechanism of action of indapamide on the cardiomyocyte response to EGF. The accentuation of EGF by indapamide might act through an action of indapamide and EGF on cardiac potassium fluxes (Rabkin, 1990). The ability of indapamide to alter K ÷ currents is currently being investigated (Calder et al., 1994; Lefez et al., 1994). While cardiac contractile frequency is only one aspect of cardiac function, it is nevertheless a fundamentally important aspect of the heart whose automaticity is the basis of cardiac contraction. Ventricular myocytes from embryonic chick heart have limitations in the extrapolation to adult myocytes from other species; however, these data can be interpreted within the context of extensive previous information on chick cardiomyocytes (Sperelakis et al., 1975). Myocardial cell aggregates

have properties more closely analogous to older intact hearts (Sperelakis et al., 1975; MacDonald and Sachs, 1975). Another advantage of this system is that while myocardial cell cultures confront the problem of concomitant growth of fibroblasts, the assessment of contractile frequency obviates this problem. A direct effect on the myocytes can be ascertained as only myocytes contract. EGF increases growth of embryonic chick cardiomyocytes (Lau, 1993) and cardiac mesenchymal (nonmyocyte) cells (Balk et al., 1982). The process of cell growth and differentiation in embryonic cardiac cells in culture are dependent on neighbouring cells similarly differentiating perhaps due to the transmission of factor(s) from one cell to another (Gurdon, 1988). EGF, which is localized to the perinuclear region of chick cardiomyocytes (Lau, 1994), might be secreted from the cells and act as a growth signal for other cardiomyocytes (Lau, 1993). Whether indapamide accentuates cardiomyocyte growth responses to EGF remains to be determined. In summary, this study identified a previously unrecognized effect of indapamide to amplify EGF's action on a functional aspect of cardiac function, spontaneous contractile frequency. Because neither calcium channel blockade nor inhibition of sodium entry nor inhibition of Na ÷-Ca 2÷ exchange in the cardiomyocyte opposes the action of EGF, this study suggests another mechanism of action of EGF, perhaps a synergistic interaction of indapamide and EGF on potassium rectifier currents in the heart.

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