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Novel stimulatory actions of the phytoestrogen genistein: effects on the gain of cardiac excitation-contraction coupling¹

National Heart and Lung Institute, Imperial College London, UK

²Correspondence: Department of Cardiac Medicine, National Heart and Lung Institute, Imperial College, Dovehouse St., London SW3 6LY, UK. E-mail: r.liew{at}imperial.ac.uk

SPECIFIC AIMS

Consumption of the phytoestrogen, genistein, a major component of soya beans, is thought to decrease the incidence of cardiovascular disease, although the precise mechanisms responsible remain obscure. Genistein can exert direct actions on the heart, including inhibiting the L-type Ca²⁺ current (I_Ca,L), which may play an important role in cardioprotection. We investigated the hypothesis that genistein exerts additional actions on cardiac excitation-contraction coupling and report on novel effects on isolated cardiac myocytes that culminate in an overall stimulation of cell contractility.

PRINCIPAL FINDINGS

1. Effects on cell shortening and the Ca²⁺ transient
Direct superfusion of field-stimulated guinea pig ventricular myocytes with genistein (10 and 40 µM) produced an acute increase in cell shortening by 24 ± 7% (mean±SE, P<0.01) and 47 ± 13% (P<0.01), respectively, compared with shortening in control, normal tyrode (NT) solution. The Ca²⁺ transient (measured using the Ca²⁺-sensitive fluorescent dye, indo-1) was similarly increased by 14 ± 3% (P<0.01) and 23 ± 5% (P<0.001), respectively. In contrast, the mammalian estrogen 17ß-estradiol (40 µM) had the opposite effect and decreased cell shortening and the Ca²⁺ transient by 57.4 ± 11.8% and 39.9 ± 15.2%, respectively. All these effects were reversible upon washout with NT. Genistein (10 and 40 µM) also significantly affected myocyte twitch kinetics by shortening the time-to-peak contraction and time-to-half relaxation (R50). The corresponding times for the Ca²⁺ transient were accelerated by 40 µM, but not 10 µM, genistein.

2. Contribution of tyrosine kinase inhibition to genistein action
We used the potent and specific phosphotyrosine phophatase inhibitor bpV(phen) to investigate whether the above actions of genistein were mediated through tyrosine kinase inhibition. Genistein (40 µM) continued to increase myocyte contractility in the presence of 1 µM bpV(phen), although the degree of stimulation was significantly less than that produced by genistein alone. The stimulatory effect of genistein on the Ca²⁺ transient was completely reversed in the presence of bpV(phen), with a lower indo-1 ratio observed. Taken together, these findings suggest that the positive inotropic effect of genistein can be subdivided into at least two distinct pathways: the first produces an increased Ca²⁺ transient and involves tyrosine kinase inhibition whereas the second increases cell contraction independent of changes in intracellular Ca²⁺.

3. Inhibition of the L-type Ca²⁺ current
Single electrode voltage clamp was used to determine the effects of genistein on the initial trigger to excitation-contraction coupling (ECC), I_Ca,L; 40 µM genistein markedly inhibited I_Ca,L by 71 ± 7% (P<0.001). This inhibition occurred very quickly (within seconds) and was evident throughout the voltage range tested.

The observation of increased cell shortening despite a simultaneous decrease in I_Ca,L implies that genistein increases the gain of cardiac ECC (defined as cell contraction relative to the I_Ca,L trigger). The distinct mechanisms underlying this increase in gain were further unraveled in relation to possible effects of genistein on the sarcoplasmic reticulum (SR), Na⁺/Ca²⁺ exchanger and/or myofilament Ca²⁺ sensitivity.

4. Effects on the SR Ca²⁺ load and Na⁺/Ca²⁺ exchanger
The SR Ca²⁺ load was assessed using rapid application of a 10 mM caffeine pulse in the presence of 0Na⁺/0Ca²⁺ solution (to prevent Ca²⁺ removal via the Na⁺/Ca²⁺ exchanger). The caffeine-induced rise in indo-1 ratio significantly increased after superfusion with 40 µM genistein and decreased back to baseline levels after a washout period in NT, Fig. 1A, B .

View larger version (34K): [in this window] [in a new window]   Figure 1. Effect of genistein on the SR Ca2+ load and Na+/Ca2+ exchanger. A) Bar chart showing increased SR load in the presence of 40 µM genistein (Gen) compared with initial baseline SR load in NT (NT1) and after washout with NT (NT2); n=9 from 3 hearts. B) Sample traces showing steady-state Ca2+ transients in NT and Gen and subsequent caffeine-induced contractions (CIC) in the presence of Na+-free/Ca2+-free solution (0Na+/0Ca2+). C) Bar chart showing increased time constant (tau) in the presence of Gen compared with NT1 and NT2; n=12 from 3 hearts. D) Sample traces of the decay of CICs in NT and Gen in the absence of 0Na+/0Ca2+. Monoexponential curves have been fitted and values of tau are shown in brackets. E) Mechanical alternans produced by 40 µM genistein. Steady-state cell contractions are shown before (NT1) and after (NT2) genistein application in the same cell. Alternans was seen only in the presence of genistein. (**P<0.01)

We next examined whether genistein exerted any inhibitory actions on the Na⁺/Ca²⁺ exchanger, which could account for the increased SR Ca²⁺ load. Na⁺/Ca²⁺ exchanger function was determined using a 10 mM caffeine pulse maintained throughout the relaxation phase (to prevent SR Ca²⁺ sequestration) in the absence of 0Na⁺/0Ca²⁺ solution. The time constant of relaxation (tau), determined from a single monoexponential fitted to the decline phase of the caffeine-induced Ca²⁺ transient, was taken as a measure of Na⁺/Ca²⁺ exchanger function. Genistein significantly increased tau compared with control values in NT (Fig. 1C, D) , suggesting an impairment of Na⁺/Ca²⁺ exchanger function.

In a small number of cells (<5%), genistein produced mechanical alternans in which the amplitude of cell contraction continuously alternated between two extremes, Fig. 1 E. This phenomenon was observed only in the presence of genistein and is consistent with a Ca²⁺ overloaded SR.

5. Tetanization studies to assess myofilament Ca²⁺ sensitivity
We used the technique of rapid, repetitive field stimulation (10 Hz) after thapsigargin treatment (which irreversibly inhibits the SR Ca²⁺-ATPase) to determine the effects of genistein on myofilament Ca²⁺ sensitivity. Stimulation of cells at 0.5 Hz after thapsigargin treatment revealed that only 43% (9 of 21) continued to exhibit increased contractions in the presence of genistein. There was no corresponding increase in the Ca²⁺ transient in these cells, suggesting that the increased shortening observed with genistein was mediated through a heightened myofilament response to Ca²⁺. This was subsequently confirmed during tetanic stimulation, which showed a greater change in cell shortening per indo-1 ratio change (sensitivity index) in the presence of genistein compared with NT at an external Ca²⁺ concentration ([Ca²⁺]_o) of 2 mM, Fig. 2 A. A higher [Ca²⁺]_o of 8 mM produced a rise in the sensitivity index, although genistein had no additional effects at this Ca²⁺ concentration. Sample recordings of tetanic cell shortening and the corresponding changes in indo-1 ratio in the presence and absence of genistein are shown in Fig. 2B .

View larger version (29K): [in this window] [in a new window]   Figure 2. Assessment of genistein effects on myofilament Ca2+ sensitivity during tetanic stimulation (10 Hz) in cardiac myocytes pretreated with 1 µM thapsigargin. A) Bar graph showing tetanic shortening per indo-1 unit change in the presence and absence of 40 µM genistein (Gen) using 2 and 8 mM [Ca2+]o (n=9 from 4 hearts). B) Representative examples of cell shortening and the corresponding indo-1 change during tetanic stimulation. Genistein markedly increased shortening at both concentrations of [Ca2+]o while producing little change in the indo-1 ratio (NT=normal tyrode; *P<0.05).

CONCLUSIONS AND SIGNIFICANCE

Our novel findings of increased cell shortening and Ca²⁺ transients in the presence of genistein, despite marked inhibition of I_Ca,L, point toward additional, previously unknown stimulatory actions of this widely consumed phytoestrogen. Thus, the overall cardiac action of genistein is one of stimulation rather than inhibition, as would be the case if its predominant action were that of Ca²⁺ antagonism. One component of this positive inotropic effect appears to be related to the unique ability of genistein to inhibit tyrosine kinase activity. The observation that 17ß-estradiol produces the opposite effect on myocyte contractility reinforces the notion that genistein exerts distinct mechanisms of action.

The increase in gain of cardiac ECC produced by genistein can be further related to actions on SR Ca²⁺ handling, Na⁺/Ca²⁺ exchanger function, and myofilament Ca²⁺ sensitivity. Although increased SR Ca²⁺ uptake and subsequent Ca²⁺ load may be a compensatory response to impaired Na⁺/Ca²⁺ exchanger function, it is likely that genistein also directly interacts with the SR, as evidenced by the accelerated relaxation and contraction times in the presence of genistein. If genistein only affected the Na⁺/Ca²⁺ exchanger, then the twitch relaxation time would be expected to increase. As far as we are aware, this study is the first to demonstrate that genistein increases myofilament Ca²⁺ sensitivity. The finding that thapsigargin treatment prevented the genistein-induced increase in Ca²⁺ transient during field stimulation can be attributed to inhibition of SR Ca²⁺ uptake by thapsigargin, thereby blocking the stimulatory effects of genistein on the SR Ca²⁺ load. An increased myofilament Ca²⁺ sensitivity in the proportion of cells stimulated by genistein was confirmed during tetanization, in which the sensitivity index at 2 mM [Ca²⁺]_o was increased in the presence of 40 µM genistein. The finding that genistein no longer increased the sensitivity index using at 8 mM [Ca²⁺]_o may be explained by an already maximal myofilament response to Ca²⁺ at this higher [Ca²⁺]_o. Figure 3 summarizes the relation of these novel stimulatory actions of genistein to cardiac ECC.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic summarizing the effects of genistein on excitation-contraction coupling. Genistein inhibits the L-type Ca2+ current, but simultaneously increases the sarcoplasmic reticulum (SR) Ca2+ load through direct actions on the SR and the sarcolemmal Na+/Ca2+ exchanger. Genistein increases myofilament Ca2+ sensitivity in a proportion of cells. The net effect of these interactions is an increase in gain of excitation-contraction coupling.

The growth of soy-based foods and health supplements has escalated in recent years mainly due to claims of beneficial effects on cardiovascular disease and certain cancers. Our current findings of stimulatory effects of genistein on myocyte contractility contrast with its known relaxing effects on the vasculature and cast some doubt on its safety in cardiovascular disease. This study shows that the acute cardiac actions of genistein are more complex than previously realized and suggests the need for further consideration before it is widely accepted and marketed as a cardioprotective/therapeutic agent.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0760fje; doi: 10.1096/fj.02-0760fje

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