Rainer Meyer

17β-Estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo

Objectives: NO production has been attributed to play a major role in cardiac diseases such as cardiac hypertrophy and cardiac remodeling after myocardial infarction which display significant gender-based differences. Therefore we assessed the effect of 17β-estradiol (E2) on estrogen receptor (ER) α and β and endothelial and inducible NO synthase in neonatal and adult rat cardiomyocytes. Methods: The presence of ERα and ERβ was demonstrated by immunofluorescence and western blot analysis as well as the expression pattern of inducible NO synthase (iNOS) and endothelial NOS (eNOS) in isolated cardiomyocytes from neonatal and adult rats. Furthermore, regulation of myocardial iNOS and eNOS expression by estrogen was evaluated in the myocardium from ovariectomized or sham-operated adult Wistar-Kyoto rats. Results: Incubation with E2 led to translocalization of the ER into the nucleus and increased receptor protein expression. E2 stimulated expression of iNOS and eNOS in both neonatal and adult cardiac myocytes. Coincubation with the pure anti-estrogen ICI 182.780 inhibited upregulation of ER and NOS expression. In ovariectomized rats myocardial iNOS and eNOS protein levels were significantly lower compared to sham-operated female animals. Conclusion: Taken together, these results show that E2 stimulates the expression of iNOS/eNOS in neonatal and adult cardiomyocytes in-vivo and in-vitro. These novel findings provide a potential mechanism of how estrogen may modulate NOS expression and NO formation in the myocardium.

Estrogenic hormone action in the heart: regulatory network and function.

Cardiovascular diseases are the leading cause of death in the industrialised countries and display significant gender-based differences. Estrogen plays an important role in the pathogenesis of heart disease and is able to modulate the progression of cardiovascular disease. The focus on the beneficial influence of estrogen is gradually shifting from the vascular system to the myocardium. The presence of functional estrogen receptors in the myocardium has been demonstrated. Estrogen is important for cardiovascular baseline physiology and modulates the myocardial response under pathological conditions. Here we summarise the current knowledge of the regulatory network of estrogenic action in the myocardium and its effects on cardiovascular function.

Activation of estrogen receptor β is a prerequisite for estrogen-dependent upregulation of nitric oxide synthases in neonatal rat cardiac myocytes

Physiological effects of estrogen on myocardium are mediated by two intracellular estrogen receptors, ERα and ERβ, that regulate transcription of target genes through binding to specific DNA target sequences. To define the role of ERβ in the transcriptional activation of both endothelial (eNOS) and inducible nitric oxide synthase (iNOS) in cardiac myocytes, we used the complete ERβ‐specific antagonist R,R‐tetrahydrochrysene (R,R‐THC). R,R‐THC inhibited activation of iNOS/eNOS promoter‐luciferase reporter constructs (iNOS/eNOS‐Luc) in a dose‐dependent fashion in COS7 cells selectively transfected with ERβ, but failed to influence ERα‐mediated increase of iNOS/eNOS‐Luc. In neonatal rat cardiomyocytes transfected with eNOS‐Luc or iNOS‐Luc, incubation with 17β‐estradiol (E2, 10−8 M) for 24 h stimulated expression of eNOS and iNOS. R,R‐THC (10−5 M) completely inhibited this effect. Furthermore, eNOS and iNOS protein expression in cardiac myocytes induced by E2 was completely blocked by R,R‐THC as shown by immunoblot analysis. Taken together, these results show that ERβ mediates transcriptional activation of eNOS and iNOS by E2.

TOLL-LIKE RECEPTOR 4, NITRIC OXIDE, AND MYOCARDIAL DEPRESSION IN ENDOTOXEMIA

The molecular mechanisms that mediate gram-negative sepsis-associated myocardial dysfunction remain elusive. Myocardial expression of inflammatory mediators is Toll-like receptor 4 (TLR4) dependent. However, it remains to be elucidated whether TLR4, expressed on cardiac myocytes, mediates impairment of cardiac contractility after lipopolysaccharide (LPS) application. Cardiac myocyte contractility, measured as sarcomere shortening of isolated cardiac myocytes from C3H/HeJ (with nonfunctional TLR4) and C3H/HeN (control), were recorded at stimulation frequencies between 0.5 and 10 Hz and after incubation with 1 and 10 μg/mL LPS for up to 8 h. Control cells treated with LPS were investigated with and without a competitive LPS inhibitor (E5564) and a specific inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea. In control mice, LPS reduced sarcomere shortening amplitude and prolonged duration of relaxation, whereas sarcomere shortening of C3H/HeJ cells was insensitive to LPS. NFκB and iNOS were upregulated after LPS application in control mice compared with C3H/HeJ. Inhibition of TLR4 by E5564 as well as inhibition of iNOS prevented the influence of LPS on contractile activity in control myocytes. LPS-dependent suppression of cardiac myocyte contractility was significantly blunted in C3H/HeJ mice. Competitive inhibition of functional TLR4 with E5564 protects cardiac myocyte contractility against LPS. These findings suggest that TLR4, expressed on cardiac myocytes, contributes to sepsis-induced myocardial dysfunction. E5564, currently under investigation in two clinical phase II trials, seems to be a new therapeutic option for the treatment of myocardial dysfunction in sepsis associated with endotoxemia.

Control of L-type calcium current during the action potential of guinea-pig ventricular myocytes

1 During an action potential the L‐type Ca2+ current (ICa,L) activates rapidly, then partially declines leading to a sustained inward current during the plateau phase. The reason for the sustained part of ICa,L has been investigated here. 2 In the present study the mechanisms controlling the ICa,L during an action potential were investigated quantitatively in isolated guinea‐pig ventricular myocytes by whole‐cell patch clamp. To measure the actual time courses of ICa,L and the corresponding L‐type channel inactivation (fAP) during an action potential, action potential‐clamp protocols combined with square pulses were applied. 3 Within the first 10 ms of the action potential the ICa,L rapidly inactivated by about 50 %; during the plateau phase inactivation proceeded to 95 %. Later, during repolarization, the L‐type channels recovered up to 25 %. 4 The voltage‐dependent component of inactivation during an action potential was determined from measurements of L‐type current carried by monovalent cations. This component of inactivation proceeded rather slowly and contributed only a little to fAP. ICa,L during an action potential is thus mainly controlled by Ca2+‐dependent inactivation. 5 In order to investigate the source of the Ca2+ controlling fAP, internal Ca2+ homeostasis was manipulated by the use of Ca2+ buffers (EGTA, BAPTA), by blocking Na+−Ca2+ exchange, or by blocking Ca2+ release from the sarcoplasmic reticulum (SR). Internal BAPTA markedly reduced the L‐type channel inactivation during the entire action potential, whereas EGTA affected fAP only during the middle and late plateau phases. Inhibition of Na+−Ca2+ exchange markedly increased inactivation of L‐type channels. Although blocking SR Ca2+ release decreased the fura‐2‐measured cytoplasmic Ca2+ concentration ([Ca2+]i) transient by about 90 %, it reduced L‐type channel inactivation only during the initial 50 ms of the action potential. Thus, it is Ca2+ entering the cell through the L‐type channels that controls the inactivation process for the majority of the action potential. Nevertheless, SR Ca2+‐release contributes 40–50 % to L‐type channel inactivation during the initial period of the action potential. However, the maximum extent of inactivation reached during the plateau is independent of Ca2+ released from the SR. 6 For the first time, the actual time course of L‐type channel inactivation has been directly determined during an action potential under various defined [Ca2+]i conditions. Thereby, the relative contribution to ICa,L inactivation of voltage, Ca2+ entering through L‐type channels, and Ca2+ being released from the SR could be directly demonstrated.

Estrogen Receptor β Protects the Murine Heart Against Left Ventricular Hypertrophy

Background—Left ventricular hypertrophy (LVH) displays significant gender-based differences. 17&bgr;-estradiol (E2) plays an important role in this process because it can attenuate pressure overload hypertrophy via 2 distinct estrogen receptors (ERs): ER&agr; and ER&bgr;. However, which ER is critically involved in the modulation of LVH is poorly understood. We therefore used ER&agr;-deficient (ER&agr;−/−) and ER&bgr;-deficient (ER&bgr;−/−) mice to analyze the respective ER-mediated effects. Methods and Results—Respective ER-deficient female mice were ovariectomized and were given E2 or placebo subcutaneously using 60-day release pellets. After 2 weeks, they underwent transverse aortic constriction (TAC) or sham operation. In ER&agr;−/− animals, TAC led to a significant increase in ventricular mass compared with sham operation. E2 treatment reduced TAC induced cardiac hypertrophy significantly in wild-type (WT) and ER&agr;−/− mice but not in ER&bgr;−/− mice. Biochemical analysis showed that E2 blocked the increased phosphorylation of p38–mitogen-activated protein kinase observed in TAC-treated ER&agr;−/− mice. Moreover, E2 led to an increase of ventricular atrial natriuretic factor expression in WT and ER&agr;−/− mice. Conclusions—These findings demonstrate that E2, through ER&bgr;-mediated mechanisms, protects the murine heart against LVH.

Rapid modulation of L‐type calcium current by acutely applied oestrogens in isolated cardiac myocytes from human, guinea‐pig and rat

Gender‐based differences in cardiovascular mortality may be due to a cardio‐protective effect of oestrogens on the myocardium. However, mRNA expression of oestrogen receptors in myocardial tissue of the adult heart has yet to be demonstrated. Furthermore, a calcium antagonistic action of 17beta‐oestradiol on myocardial tissue has been discussed. Therefore, two subjects were investigated in atrial myocytes of the human, and ventricular myocytes of guinea‐pig and rat in this study. (1) Are oestrogen receptors expressed in adult myocardial cells? (2) Is there an influence of oestrogens on the L‐type calcium current of cardiac myocytes? Expression of oestrogen receptors was investigated by reverse polymerase chain reaction. L‐type calcium current was usually measured by the patch‐clamp technique in whole‐cell recording mode under selective recording conditions, i.e. overlapping currents were blocked. One series of experiments was performed in perforated patch configuration to avoid internal perfusion. 17beta‐oestradiol inhibited L‐type calcium current reversibly in all three species. At 10(−5) M, the inhibition was 15–20%. This inhibition was independent of the sex and the species. A full concentration response curve of 17beta‐oestradiol on basal L‐type current was recorded from female guinea‐pig myocytes. The inhibition increased from 2% at 10(−7) M to about 30% at 10(−4) M 17beta‐oestradiol. The values could be fitted by a sum of two sigmoidal functions with log EC50 values of −6.5 and −4.9 M and Hill slopes of 2.5 for both. The specificity of the 17beta‐oestradiol action was tested by recording the L‐type current in the presence of 17alpha‐oestradiol and oestrone. 17alpha‐oestradiol also inhibited the current, but with a maximal inhibition of only 17%. The concentration‐response curve could be fitted by a single sigmoidal function (log EC50 −6−3 M; Hill slope 0.55). Oestrone did not influence the current at all. The decrease in L‐type current after the application of 17beta‐oestradiol via a rapid perfusion system developed with a time constant of 3–4 s, which was in the same range as that for the influence of isoprenaline. The isoprenaline‐stimulated L‐type current was much more susceptible to the inhibition by 17beta‐oestradiol, i.e. in pre‐stimulated cells (1) the inhibitory effect is significantly higher (e.g. at 10(−5) M, inhibition was 36.3% compared with 11.2% in untreated cells) and (2) an inhibitory effect can be seen with oestradiol concentrations as low as 10(−9) M. Although the concentrations needed to gain a calcium antagonistic influence on the basal current were much too high to explain a cardio‐protective influence of oestrogens, the presence of oestrogen receptors in cardiac myocytes of all three species, together with the shift in concentration dependence following pre‐stimulation by isoprenaline, suggest that myocytes are a potential target for oestrogen.

Profile and kinetics of L-type calcium current during the cardiac ventricular action potential compared in guinea-pigs, rats and rabbits

Abstract. The substantial interspecies differences in mammalian cardiac action potentials (APs) are attributed primarily to variations in K+ currents. In a comparative study on isolated ventricular myocytes from guinea-pigs, rabbits and rats, we investigated the influence of the species-based AP differences on the time course of the L-type Ca2+ current (ICa,L). In addition, we tested whether also species-dependent properties of the ICa,L contribute to its time course during the AP. In patch-clamp measurements, ICa,L was characterised using conventional square pulses and digitised APs as command voltages. Special interest was directed to the analysis of the actual time courses of ICa,L, and L-type channel activation and inactivation during APs. Although species-specific differences in AP shape strongly influence the amplitude and the time course of ICa,L, divergence in L-type channel inactivation was found as well. In each species ICa,L inactivation was related to Ca2+ influx via L-type channels. However, while L-type channels showed similar Ca2+ dependency in the rabbit and the rat, a 2-times higher Ca2+ influx was necessary to achieve a given degree of inactivation in the guinea-pig. Thus, inactivation of ICa,L is delayed in the guinea-pig, thereby contributing to the prolonged AP plateau in this species. Comparing the actual time course of ICa,L, and L-type channel activation and inactivation revealed that, in each species, fading of ICa,L during final repolarisation was caused by deactivation (i.e. closure of the channels d-gate) rather than inactivation.

Differential effects of 17β-estradiol on mitogen-activated protein kinase pathways in rat cardiomyocytes

Cardiac myocytes contain functional estrogen receptors, however, the effect of estrogen on growth-related signaling pathways such as mitogen-activated protein kinases (MAPK) in the pathogenesis of cardiac disease is unclear. MAPKs are critically involved in regulatory signaling pathways which ultimately lead to cardiac hypertrophy. Here we show that 17beta-estradiol (E2) activates extracellular signal-regulated kinase (ERK1/2), c-Jun-NH2-terminal protein kinase (JNK) and p38 in rat cardiomyocytes in a distinctive pattern. As shown by immunoblot analysis and phosphorylation assays, E2 (10(-9) M) induced a rapid and transient activation of ERK1/2 and a rapid but sustained increase of JNK phosphorylation. In contrast, E2 had only a marginal effect on p38 activation. Furthermore, MAPK phosphatase expression was induced by E2 and E2-stimulated expression of endothelial and inducible NO synthase was inhibited by PD 98059, an inhibitor of the ERK pathway. These novel observations may help to explain the role of estrogen in gender-based differences found in cardiac disease.

Toll-like receptor 4 deficiency: Smaller infarcts, but nogain in function

It has been reported that Toll-like receptor 4 (TLR4) deficiency reduces infarct size after myocardial ischemia/reperfusion (MI/R). However, measurement of MI/R injury was limited and did not include cardiac function. In a chronic closed-chest model we assessed whether cardiac function is preserved in TLR4-deficient mice (C3H/HeJ) following MI/R, and whether myocardial and systemic cytokine expression differed compared to wild type (WT). Infarct size (IS) in C3H/HeJ assessed by TTC staining after 60 min ischemia and 24h reperfusion was significantly smaller than in WT. Despite a smaller infarct size, echocardiography showed no functional difference between C3H/HeJ and WT. Left-ventricular developed pressure measured with a left-ventricular catheter was lower in C3H/HeJ (63.0 ± 4.2 mmHg vs. 77.9 ± 1.7 mmHg in WT, p < 0.05). Serum cytokine levels and myocardial IL-6 were higher in WT than in C3H/HeJ (p < 0.05). C3H/HeJ MI/R showed increased myocardial IL-1β and IL-6 expression compared to their respective shams (p < 0.05), indicating TLR4-independent cytokine activation due to MI/R. These results demonstrate that, although a mutant TLR4 signaling cascade reduces myocardial IS and serum cytokine levels, it does not preserve myocardial function. The change in inflammatory response, secondary to a non-functional TLR-4 receptor, may contribute to the observed dichotomy between infarct size and function in the TLR-4 mutant mouse.BackgoundIt has been reported that Toll-like receptor 4 (TLR4) deficiency reduces infarct size after myocardial ischemia/reperfusion (MI/R). However, measurement of MI/R injury was limited and did not include cardiac function. In a chronic closed-chest model we assessed whether cardiac function is preserved in TLR4-deficient mice (C3H/HeJ) following MI/R, and whether myocardial and systemic cytokine expression differed compared to wild type (WT).ResultsInfarct size (IS) in C3H/HeJ assessed by TTC staining after 60 min ischemia and 24h reperfusion was significantly smaller than in WT. Despite a smaller infarct size, echocardiography showed no functional difference between C3H/HeJ and WT. Left-ventricular developed pressure measured with a left-ventricular catheter was lower in C3H/HeJ (63.0 ± 4.2 mmHg vs. 77.9 ± 1.7 mmHg in WT, p < 0.05). Serum cytokine levels and myocardial IL-6 were higher in WT than in C3H/HeJ (p < 0.05). C3H/HeJ MI/R showed increased myocardial IL-1β and IL-6 expression compared to their respective shams (p < 0.05), indicating TLR4-independent cytokine activation due to MI/R.ConclusionThese results demonstrate that, although a mutant TLR4 signaling cascade reduces myocardial IS and serum cytokine levels, it does not preserve myocardial function. The change in inflammatory response, secondary to a non-functional TLR-4 receptor, may contribute to the observed dichotomy between infarct size and function in the TLR-4 mutant mouse.