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Sarcoidosis is a systematic granulomatous disorder. Genetic susceptibility could play a central role in the disease development and progression. In this study, we investigated whether caspase recruitment domain 15 (CARD15) gene haplotypes are associated with the onset or the clinical course of sarcoidosis. Three hundred Caucasian sarcoidosis patients and 381 matched controls were included. Eight haplotype-tagging single nucleotide polymorphisms (SNPs) in the CARD15 gene were examined by mass spectrometry-based SNP genotyping. By haplotype analysis, mutations located in between tested SNPs can also be identified. Therefore, we can conclude that there is no association between the CARD15 gene and the development or a special phenotype of sarcoidosis in our cohort.

Background Pressure overload induces the cardiac expression of parathyroid hormone-related protein (PTHrP). Plasma levels are elevated in patients with heart disease. It is unknown whether this represents an epiphenomenon or suggests involvement in hypertrophy. Aim To identify a potential role of PTHrP in pressure induced hypertrophy and heart failure. Methods and results Pressure load was produced via thoracic aortic constriction (TAC) and application of a PTHrP antagonist (PTHrP(7–34)) via osmotic minipumps in mice. Main findings were confirmed in vitro by exposing isolated adult ventricular mice cardiomyocytes to PTHrP(1–34) (100 nmol/l). TAC treated animals developed myocardial hypertrophy within 2 weeks. The heart weight to body weight ratio increased from 5.02±0.14 mg/g (sham/vehicle) and 5.16±0.19 mg/g (sham/antagonist) to 6.59±0.85 mg/g (TAC/vehicle) and 7.07±0.80 mg/g (TAC/antagonist) (each n=6–8; p

BACKGROUND ACE inhibitors are widely used to antagonize the biological activity of angiotensin II in hypertensive heart disease. Oestrogen reduces angiotensin type 1 receptor expression, and thereby modifies angiotensin signalling. AIM To investigate the interaction of oestrogen status and ACE inhibition on the development of left ventricular hypertrophy and expression of transforming growth factor (TGF)-beta(1) in female spontaneously hypertensive rats (SHR). METHODS AND RESULTS Intact female SHR, ovariectomised SHR, and ovariectomised SHR with 17beta-oestradiol (E2) replacement therapy were either treated with placebo or the ACE inhibitor moexiprilat. Blood pressure, left ventricular hypertrophy, and expression of TGF-beta(1) and TGF-beta(1)-regulated genes were investigated. ACE inhibition reduced blood pressure in all groups. When normalised to blood pressure, a significant reduction in hypertrophy was found in ovariectomised animals receiving E2. Expression of TGF-beta(1) was increased in all three groups treated with the ACE inhibitor, with top levels in ovariectomised animals. Moreover, expression of ornithine decarboxylase (ODC), an adrenoceptor dependent gene, downstream of TGF-beta(1), was up-regulated upon ACE inhibition, except in animals which were ovariectomised and oestrogen supplemented. Parathyroid hormone-related peptide, a growth factor negatively regulated by TGF-beta(1), was down-regulated in all animals receiving the ACE inhibitor. CONCLUSION ACE inhibition modulated TGF-beta(1) and TGF-beta(1) dependent genes. Oestrogen deficiency alone did not influence the progression of cardiac hypertrophy in this model of female SHR.

(1) Little is known about the interaction of 17beta-estradiol (E2) and the vasoactive endothelin system in the heart. Endothelin signaling is activated in a failing heart and may contribute to myocardial dysfunction and remodeling. Therefore, we investigated the regulation of proteins of the endothelin system (ppET-1, ECE and ETA-R and ETB-R) in the hearts of female spontaneously hypertensive rats (SHR) with respect to E2. (2) Relative expression levels of the respective cardiac mRNA obtained from sham-operated, ovariectomized and ovariectomized E2-substituted SHR were quantified by real-time PCR. Ovariectomy led to a significant upregulation of the ETB-R mRNA (2.6+/-0.8-fold) in the left ventricular myocardium, which was not attendant with an alteration of ETA-R, ECE and ppET-1 mRNA expression. (3) An upregulation of the relative expression level of ETB-R protein due to ovariectomy was also demonstrated by radioligand binding assay. (4) Upregulation of both ETB-R mRNA and ETB-R protein expression was completely inhibited by E2 replacement. (5) To confirm these results in in vitro experiments, we quantified the mRNA of ET-R subtypes from isolated cardiomyocytes in the presence and absence of E2 (10-8 m, 24 h). Our data showed a markedly downregulated level of ETB-R mRNA in cardiomyocytes stimulated with E2. ETB-R downregulation was not attendant with the alteration of ETA-R, ECE and ppET-1 mRNA expression. (6) Taken together, these data demonstrate that estrogen regulates the expression of ETB-R in rat ventricular myocardium in vivo and in vitro. These observations may help to understand gender-based differences found in cardiovascular disease.

The influence of radio frequency (RF) fields of 180, 900, and 1800 MHz on the membrane potential, action potential, L-type Ca(2+) current and potassium currents of isolated ventricular myocytes was tested. The study is based on 90 guinea-pig myocytes and 20 rat myocytes. The fields were applied in rectangular waveguides (1800 MHz at 80, 480, 600, 720, or 880 mW/kg and 900 MHz, 250 mW/kg) or in a TEM-cell (180 MHz, 80 mW/kg and 900 MHz, 15 mW/kg). Fields of 1800 and 900 MHz were pulsed according to the GSM-standard of cellular phones. The specific absorption rates were determined from computer simulations of the electromagnetic fields inside the exposure devices by considering the structure of the physiological test arrangement. The electrical membrane parameters were measured by whole cell patch-clamp. None of the tested electrophysiological parameters was changed significantly by exposure to RF fields. Another physical stimulus, lowering the temperature from 36 degrees C to 24 degrees C, decreased the current amplitude almost 50% and shifted the voltage dependence of the steady state activation parameter d(infinity) and inactivation parameter f(infinity) of L-type Ca(2+) current by about 5 mV. However, at this lower temperature RF effects (900 MHz, 250 mW/kg; 1800 MHz, 480 mW/kg) on L-type Ca(2+) current were also not detected.

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 17β-estradiol (E2) activates extracellular signal-regulated kinase (ERK1/2), e-Jun-NH 2 -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.

The L-type calcium current (ICa,L) plays an important role in excitation-contraction coupling of heart cells, as it forms the major trigger for Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) and provides Ca2+ for refilling of the SR (Callewaert, 1992; Barry & Bridge, 1993). The voltage relation of this current is bell shaped with an inward current maximum at about 10 mV and a reversal potential around 60 mV (McDonald et al. 1994). At the top of the action potential between 40 and 50 mV, when ICa,L is activated, ICa,L is already near its reversal potential and thus small. Moreover, the potential of maximum ICa,L is not reached before the repolarization of the cell. The obvious contradiction that ICa,L is important for excitation- contraction coupling on the one hand, but is small during the action potential on the other hand, leads to the idea of applying voltage-clamp pulses consisting of digitized action potentials to record the time course of ICa,L directly during the action potential. This so-called action potential clamp was introduced by Trautwein's group (e.g. Doerr et al. 1990). For every cell under investigation, Doerr et al. (1990) digitized its individual action potential together with the stimulus and applied this signal as the command voltage. Thus all current systems became activated as during the action potential elicited previously, resulting in zero membrane current. A specific current system became obvious as (inverted) compensation current after application of its specific blocker. Arreola et al. (1991) used one representative action potential as the command voltage for all investigated cells. In this case a specific current system can be directly recorded after block or inactivation of all overlapping currents. The method of Arreola et al. (1991) was later also applied by Bouchard et al. (1995) for the investigation of Na+−Ca2+ exchange in rat myocytes and by Grantham & Cannell (1996) for determination of the relative contribution of Na+-Ca2+ exchange and ICa,L to the Ca2+ influx during an action potential. All these investigations proved that the ICa,L during an action potential inactivates much more slowly with a complex time course compared with that elicited by rectangular voltage-clamp steps. The behaviour of the L-type channels can be described by the peak conductance, the driving force and two time- and voltage-dependent variables d and f (Reuter, 1973). While the activation parameter d can easily be determined from classical voltage-clamp experiments (Isenberg & Klockner, 1982), the inactivation parameter f depends on voltage and internal Ca2+ (Bechem & Pott, 1985; Lee et al. 1985; Hadley & Hume, 1987; Shirokov et al. 1993), and thus on Ca2+ fluxes through the sarcolemma and the SR membrane. However, the relative contribution of these different components to channel inactivation during an action potential remains uncertain (Campbell & Giles 1990; McDonald et al. 1994). Nevertheless, the time course of ICa,L has been reconstructed in models (e.g. DiFrancesco & Noble, 1985; Luo & Rudy, 1994; Noble et al. 1998). For this a number of parameters had to be extracted from experiments employing rectangular voltage pulses. Although the reconstruction of ICa,L matched the measured current time course rather well (see Luo & Rudy, 1994), the relative contributions of the different parameters have not yet been confirmed by experiments. However, such validation is thought to be necessary (Noble et al. 1998). The aim of the present study was to investigate the mechanisms controlling ICa,L during an action potential. The results presented here reveal a detailed description of the time course of L-type channel inactivation during an action potential. Moreover, we were able to demonstrate the relative contributions of voltage- and Ca2+-dependent processes to inactivation of L-type channels. In addition, the importance of Ca2+ from different sources (i.e. SR release or influx from the external solution) for L-type channel inactivation was estimated.

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.

The intracellular calcium concentration ([Ca{sup 2+}]{sub i}) of isolated ventricular cardiac myocytes of the guinea pig was measured during the application of pulsed high-frequency electromagnetic fields. The high-frequency fields were applied in a transverse electromagnetic cell designed to allow microscopic observation of the myocytes during the presence of the high-frequency fields. The [Ca{sup 2+}]{sub i} was measured as fura-2 fluorescence by means of digital image analysis. Both the carrier frequency and the square-wave pulse-modulation pattern were varied during the experiments (carrier frequencies: 900, 1,300, and 1,800 MHz pulse modulated at 217 Hz with 14% duty cycle; pulsation pattern at 900 MHz; continuous wave, 16 Hz,and 50 Hz modulation with 50% duty cycle and 30 kHz modulation with 80% duty cycle). The mean specific absorption rate (SAR) values in the solution were within one order of magnitude of 1 mW/kg. They varied depending on the applied carrier frequency and pulse pattern. The experiments were designed in three phases: 500 s of sham exposure, followed by 500 s of field exposure, then chemical stimulation without field. The chemical stimulation (K{sup +}-depolarization) indicated the viability of the cells. The K{sup +} depolarization yielded a significant increase in [Ca{sup 2+}]{sub i}. Significant differences betweenmore » sham exposure and high-frequency field exposure were not found except when a very small but statistically significant difference was detected in the case of 900 MHz/50 Hz. However, this small difference was not regarded as a relevant effect of the exposure.« less

The effect of various concentrations of 2-deoxy-D-glucose (2DG) and sodium fluoroacetate (SFA) on the Q of day-11 and 12 rat embryos was studied in vitro. The dose-response curves—% inhibition versus log concentration—were linear for 2DG between 500 μM and 50 mM; day-11 rat embryos showed a higher rate of inhibition than day-12 embryos and adult kidney tissue. The inhibitory effect of SFA on the embryonic Q in vitro was significantly lower than on adult kidney tissue slices. These in vitro effects are in agreement with recent investigations on the energy metabolism of rat embryos that revealed a change from glycolysis to an oxidative, respiratory metabolism during organogenesis. The administration of a single dose of 2DG and SFA to pregnant rats at day 9 or 10 of pregnancy significantly reduced the Q and dry weight of the embryos at day 11, whereas both parameters were unchanged at day 12. The same treatment was nonteratogenic, which is in contrast to the findings of other investigators.