Hypotension in hereditary cardiomyopathy.

It is well accepted that hypertension may lead to the development of heart failure (HF). However, little is known about the development of hypotension that may contribute to the onset of hereditary cardiomyopathy (HCM), thus promoting heart failure and early death. The purpose of this study is to verify whether a decrease in blood pressure takes place during different phases of HCM (asymptomatic, necrosis, hypertrophy, and heart failure). Using the well-known animal model, the UM-X7.1 hamster strain of HCM (HCMH), our results showed the absence of a change in mean arterial pressure (MAP) during the asymptomatic phase preceding the development of necrosis in HCMHs when compared to age-matched normal hamster (NH). However, there was a progressive decrease in MAP that reached its lowest level during the heart failure phase. The MAP during the development of the necrosis phase of HCM was accompanied by a significant increase in the level of the sodium-hydrogen exchanger, NHE1. Treatments with the potent NHE1 inhibitor, EMD 87580 (rimeporide), did not affect MAP of NH. However, treatments with EMD 87580 during the three phases of the development of HCM significantly reversed the hypotension associated with HCM.Our results showed that the development of HCM is associated with hypotension. These results suggest that a decrease in blood pressure could be a biomarker signal for HCM leading to HF and early death. Since the blockade of NHE1 significantly but partially prevented the reduction in MAP, this suggests that other mechanisms can contribute to the development of hypotension in HCM.

Taurine and cardiac disease: state of the art and perspectives.

Taurine is a nonessential amino acid that has received much attention. Two organs, the heart and the brain, are known to produce their own taurine, but in very limited quantities. It is for this reason that supplementation with this amino acid is necessary. Today, taurine is present in almost all energy drinks. A very vast literature reported beneficial effects of taurine in hepatic dysfunction, gastrointestinal injury, kidney diseases, diabetes, and cardiovascular diseases. Most of its effects were attributed to its modulation of Ca2+ homeostasis as well as to its antioxidant properties. In this review, we will focus on the current status of taurine modulation of the cardiovascular system and discuss future avenues for its use as a supplement therapy in a specific cardiovascular disease, namely hypertrophy, and heart failure.

Angiotensin II induces apoptosis of human right and left ventricular endocardial endothelial cells by activating the AT2 receptor 1.

Endocardial endothelial cells (EECs) form a monolayer lining the ventricular cavities. Studies from our laboratory and the literature have shown differences between EECs isolated from the right and left ventricles (EECRs and EECLs, respectively). Angiotensin II (Ang II) was shown to induce apoptosis of different cell types mainly via AT1 receptor activation. In this study, we verified whether Ang II induces apoptosis of human EECRs and EECLs (hEECRs and hEECLs, respectively) and via which type of receptor. Using the annexin V labeling and in situ TUNEL assays, our results showed that Ang II induced apoptosis of both hEECRs and hEECLs in a concentration-dependent manner. Our results using specific AT1 and AT2 receptor antagonists showed that the Ang-II-induced apoptosis in both hEECRs and hEECLs is mediated mainly via the AT2 receptor. However, AT1 receptor blockade partially prevented Ang-II-induced apoptosis, particularly in hEECRs. Hence, our results suggest that mainly AT2 receptors mediate Ang-II-induced apoptosis of hEECRs and hEECLs. The damage of EECs would affect their function as a physical barrier between the blood and cardiomyocytes, thus affecting cardiomyocyte functions.

Difference in the response to angiotensin II between left and right ventricular endocardial endothelial cells.

Previous studies focused on the right ventricular endocardial endothelial cells (EECRs) and showed that angiotensin II (Ang II) induced increase in cytosolic and nuclear calcium via AT1 receptor activation. In the present study, we verified whether the response of left EECs (EECLs) to Ang II is different than that of EECRs. Our results showed that the EC50 of the Ang II-induced increase of cytosolic and nuclear calcium in EECLs was 10× higher (around 2 × 10-13 mol/L) than in EECRs (around 8 × 10-12 mol/L). The densities of both AT1 and AT2 receptors were also higher in EECLs than those previously reported in EECRs. The effect of Ang II was mediated in both cell types via the activation of AT1 receptors. Treatment with Ang II induced a significant increase of cytosolic and nuclear AT1 receptors in EECRs, whereas the opposite was found in EECLs. In both cell types, there was a transient increase of cytosolic and nuclear AT2 receptors following the Ang II treatment. In conclusion, our results showed that both AT1 and AT2 receptors densities are higher in both EECLs compared to what was reported in EECRs. The higher density of AT1 receptors in EECLs compared to REECs may explain, in part, the higher sensitivity of EECLs to Ang II.

Intracellular Free Calcium Measurement Using Confocal Imaging.

Confocal microscopy, coupled to high-performance hardware and software systems, has provided scientists with the capability of overcoming some of the limitations of standard microscopic imaging measurements of intracellular ions. The technique for loading of ion fluorescent probes is easily achieved; however, the quality of calcium measurements depends on the way of using the confocal system. In order to optimize this technique, scientists need to be familiar with the basic approaches and limitations of confocal microscopy. In this chapter, we will describe sample preparation, fluorescent probe loading, labeling of intracellular compartments, and the setting of parameters as well as protocols for measurements and limitations of the technique.

Na(+)-H(+) exchanger inhibitor prevents early death in hereditary cardiomyopathy.

Using the UM-X7.1 hereditary cardiomyopathic and muscular dystrophy hamsters (HCMH), we tested the effects of lifelong preventive or curative treatments during the heart failure phase with the NHE-1 inhibitor EMD 87580 (EMD) or with the angiotensin-converting enzyme inhibitor cilazapril on the intracellular Na(+) and Ca(2+) overloads, elevated level of NHE-1, necrosis, hypertrophy, heart failure, and early death. Our results showed that 310-day pretreatment of 30-day-old HCMHs with EMD significantly prevented cardiac necrosis, cardiomyocyte hypertrophy, and reduced the heart to body mass ratio. This treatment significantly prevented Na(+) and Ca(2+) overloads and the increase in NHE-1 protein level observed in HCMHs. Importantly, this lifelong preventive treatment significantly decreased the levels of creatine kinase and prevented early death of HCMHs. Curative treatment of hypertrophic 275-day-old HCMHs for 85 days with EMD significantly prevented hypertrophy and early death of HCMHs. However, treatments with cilazapril did not have any significant effects on the cardiac parameters studied or on early death of HCMHs. Our results suggest that the increase in the NHE-1 level and the consequent Na(+) and Ca(2+) overloads are implicated in the pathological process leading to heart failure and early death in HCMHs, and treatment with the NHE-1 inhibitor is promising for preventing early death in hereditary cardiomyopathy.

Nuclear Membranes ETB Receptors Mediate ET-1-induced Increase of Nuclear Calcium in Human Left Ventricular Endocardial Endothelial Cells.

In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane (PM) ET(A)R and ET(B)R were reported to mediate ET-1-induced increase of intracellular calcium [Ca](i); however, this effect was mediated by ET(A)R in right EECs (EECRs). In this study, we verified whether, as for the PM, nuclear membranes (NMs) ET-1 receptors activation in EECLs and EECRs induce an increase of nuclear calcium ([Ca](n)) and if this effect is mediated through the same receptor type as in PM. Using a plasmalemma-perforated technique and 3D confocal microscopy, our results showed that, as in PM intact cells, superfusion of nuclei of both cell types with cytosolic ET-1 induced a concentration-dependent sustained increase of [Ca](n). In EECRs, the ET(A)R antagonist prevented the effect of ET-1 on [Ca](n) without affecting EECLs. However, in both cell types, the effect of cytosolic ET-1 on [Ca](n) was prevented by the ETBR antagonist. In conclusion, both NMs' ET(A)R and ET(B)R mediated the effect of cytosolic ET-1 on [Ca](n) in EECRs. In contrast, only NMs' ET(B)R activation mediated the effect of cytosolic ET-1 in EECLs. Hence, the type of NMs' receptors mediating the effect of ET-1 on [Ca](n) are different from those of PM mediating the increase in [Ca](i).

Nuclear membrane R-type calcium channels mediate cytosolic ET-1-induced increase of nuclear calcium in human vascular smooth muscle cells.

The objective of this work was to verify whether, as in the case of the plasma membrane of human vascular smooth muscle cells (hVSMCs), cytosolic ET-1-induced increase of nuclear calcium is mediated via the activation of calcium influx through the steady-state R-type calcium channel. Pharmacological tools to identify the R-type calcium channels, as well as real 3-D confocal microscopy imaging techniques coupled to calcium fluorescent probes, were used to study the effect of cytosolic ET-1 on nuclear calcium in isolated nuclei of human hepatocytes and plasma membrane perforated hVSMCs. Our results showed that pre-treatment with pertussis toxin (PTX) or cholera toxin (CTX) prevented cytosolic ET-1 (10(-9) mol/L) from inducing a sustained increase in nuclear calcium. Furthermore, the L-type calcium channel blocker nifedipine did not prevent cytosolic ET-1 from inducing an increase in nuclear calcium, as opposed to the dual L- and R-type calcium channel blocker isradipine (PN200-110) (in the presence of nifedipine). In conclusion, the preventative effect with PTX and CTX, and the absence of an effect with nifedipine, as well as the blockade by isradipine on cytosolic ET-1-induced increase in nuclear calcium, suggest that this nuclear calcium influx in hVSMCs is due to activation of the steady-state R-type calcium channel. The sarcolemmal and nuclear membrane R-type calcium channels in hVSMCs are involved in ET-1 modulation of vascular tone in physiology and pathology.

Nuclear membranes GPCRs: implication in cardiovascular health and diseases.

G-protein coupled receptors (GPCRs) are commonly present at the plasma membrane and their signaling modulates excitation-contraction coupling and excitation-secretion coupling of excitable and non-excitable cells of the cardiovascular system. Their effect on excitation-gene expression coupling was attributed, in part, to the nuclear translocation of their signaling and/or to the entry into the nuclear membrane of the internalized GPCRs. However, the recently established paradigm showed that, in addition to plasma membrane G-proteins, GPCRs exist as native nuclear membranes receptors and they modulate nuclear ionic homeostasis and function. These nuclear membrane GPCRs could function independently of plasma membrane GPCRs. Growing evidence also shows that these nuclear membrane GPCRs contribute to protein synthesis and also undergo changes in pathological conditions. The presence of a GPCR at both the plasma and nuclear membranes and/or only at the nuclear membranes represents a new challenge to better understand their contribution to cell physiology and pathology and, consequently, to the development of new therapeutic drugs targeting this category of receptors.

Whole-cell and nuclear NADPH oxidases levels and distribution in human endocardial endothelial, vascular smooth muscle, and vascular endothelial cells.

The results of our study show that whole-cell and nuclear levels of NADPH oxidase-1 (NOX1) are similar in human vascular endothelial cells (hVECs) and smooth muscle cells (hVSMCs), but lower in human endocardial endothelial cells (hEECs). NOX2 levels were higher in hVECs and lower in hVSMCs. NOX3 levels were the same in hVECs and hVSMCs, but lower in hEECs. NOX4 levels were similar in all of the cell types. NOX4 levels were higher in hVECs than in hVSMCs. NOX5 was also present throughout the 3 cell types, including their nuclei, in the following order: hEECs > hVSMCs > hVECs. The level of basal reactive oxygen species (ROS) was highest in hVECs and lowest in hVSMCs. However, the Ca(2+) level was highest in hVSMCs and lowest in hVECs. These findings suggest that all types of NOXs exist in hEECs, hVECs, and hVSMCs, although their density and distribution are cell-type dependent. The density of the different NOXs correlated with the ROS level, but not with the Ca(2+) level. In conclusion, NOXs, including NOX3, exist in cardiovascular cells and their nuclei. The nucleus is a major source of ROS generation. The nuclear NOXs may contribute to ROS and Ca(2+) homeostasis, which may affect cell remodeling, including the formation of nuclear T-tubules in vascular diseases and aging.

Receptors and ionic transporters in nuclear membranes: new targets for therapeutical pharmacological interventions.

Work from our group and other laboratories showed that the nucleus could be considered as a cell within a cell. This is based on growing evidence of the presence and role of nuclear membrane G-protein coupled receptors and ionic transporters in the nuclear membranes of many cell types, including vascular endothelial cells, endocardial endothelial cells, vascular smooth muscle cells, cardiomyocytes, and hepatocytes. The nuclear membrane receptors were found to modulate the functioning of ionic transporters at the nuclear level, and thus contribute to regulation of nuclear ionic homeostasis. Nuclear membranes of the mentioned types of cells possess the same ionic transporters; however, the type of receptors is cell-type dependent. Regulation of cytosolic and nuclear ionic homeostasis was found to be dependent upon a tight crosstalk between receptors and ionic transporters of the plasma membranes and those of the nuclear membrane. This crosstalk seems to be the basis for excitation-contraction coupling, excitation-secretion coupling, and excitation - gene expression coupling. Further advancement in this field will certainly shed light on the role of nuclear membrane receptors and transporters in health and disease. This will in turn enable the successful design of a new class of drugs that specifically target such highly vital nuclear receptors and ionic transporters.

Nuclear membrane receptors for ET-1 in cardiovascular function.

Plasma membrane endothelin type A (ET(A)) receptors are internalized and recycled to the plasma membrane, whereas endothelin type B (ET(B)) receptors undergo degradation and subsequent nuclear translocation. Recent studies show that G protein-coupled receptors (GPCRs) and ion transporters are also present and functional at the nuclear membranes of many cell types. Similarly to other GPCRs, ET(A) and ET(B) are present at both the plasma and nuclear membranes of several cardiovascular cell types, including human cardiac, vascular smooth muscle, endocardial endothelial, and vascular endothelial cells. The distribution and density of ET(A)Rs in the cytosol (including the cell membrane) and the nucleus (including the nuclear membranes) differ between these cell types. However, the localization and density of ET-1 and ET(B) receptors are similar in these cell types. The extracellular ET-1-induced increase in cytosolic ([Ca](c)) and nuclear ([Ca](n)) free Ca(2+) is associated with an increase of cytosolic and nuclear reactive oxygen species. The extracellular ET-1-induced increase of [Ca](c) and [Ca](n) as well as intracellular ET-1-induced increase of [Ca](n) are cell-type dependent. The type of ET-1 receptor mediating the extracellular ET-1-induced increase of [Ca](c) and [Ca](n) depends on the cell type. However, the cytosolic ET-1-induced increase of [Ca](n) does not depend on cell type. In conclusion, nuclear membranes' ET-1 receptors may play an important role in overall ET-1 action. These nuclear membrane ET-1 receptors could be targets for a new generation of antagonists.

Nonpeptidic antagonists of ETA and ETB receptors reverse the ET-1-induced sustained increase of cytosolic and nuclear calcium in human aortic vascular smooth muscle cells.

Our previous work showed that ET-1 induced a concentration-dependent increase of cytosolic Ca2+ ([Ca]c) and nuclear Ca2+ ([Ca]n) in human aortic vascular smooth muscle cells (hVSMCs). In the present study, using hVSMCs and 3-dimensional confocal microscopy coupled to the Ca2+ fluorescent probe Fluo-3, we showed that peptidic antagonists of ETA and ETB receptors (BQ-123 (10(-6) mol/L) and BQ-788 (10(-7) mol/L), respectively) prevented, but did not reverse, ET-1-induced sustained increase of [Ca]c and [Ca]n. In contrast, nonpeptidic antagonists of ETA and ETB (respectively, BMS-182874 (10(-8)-10(-6) mol/L) and A-192621 (10(-7) mol/L)) both prevented and reversed ET-1-induced sustained increase of [Ca]c and [Ca]n. Furthermore, activation of the ETB receptor alone using the specific agonist IRL-1620 (10(-9) mol/L) induced sustained increases of [Ca]c and [Ca]n, and subsequent administration of ET-1 (10(-7) mol/L) further increased nuclear Ca2+. ET-1-induced increase of [Ca]c and [Ca]n was completely blocked by extracellular application of the Ca2+ chelator EGTA. Pretreatment with the G protein inhibitors pertussis toxin (PTX) and cholera toxin (CTX) also prevented the ET-1 response; however, strong membrane depolarization with KCl (30 mmol/L) subsequently induced sustained increase of [Ca]c and [Ca]n. Pretreatment of hVSMCs with either the PKC activator phorbol-12,13-dibutyrate or the PKC inhibitor bisindolylmaleimide did not affect ET-1-induced sustained increase of intracellular Ca2+. These results suggest that both ETA- and ETB-receptor activation contribute to ET-1-induced sustained increase of [Ca]c and [Ca]n in hVSMCs. Moreover, in contrast to the peptidic antagonists of ET-1 receptors, the nonpeptidic ETA-receptor antagonist BMS-182874 and the nonpeptidic ETB-receptor antagonist A-192621 were able to reverse the effect of ET-1. Nonpeptidic ETA- and ETB-receptor antagonists may therefore be better pharmacological tools for blocking ET-1-induced sustained increase of intracellular Ca2+ in hVSMCs. Our results also suggest that the ET-1-induced sustained increase of [Ca]c and [Ca]n is not mediated via activation of PKC, but via a PTX- and CTX-sensitive G protein calcium influx through the R-type Ca2+ channel.

ETB receptor dependent alteration in aortic responses to ET-1 in the cardiomyopathic hamster.

The aim of this study was to verify whether an alteration in the aortic endothelin-1 (ET-1) response takes place in UM-X7.1 cardiomyopathic hamsters. Our results showed that ET-1 (10(-12) - 10(-5) mol/L) induces dose-dependent sustained increases in tension in the intact and endothelium denuded aortas from both normal and cardiomyopathic hamsters. The EC50 values of ET-1 of both intact and endothelium denuded aortas of normal hamsters were similar (2.2 x 10(-9) mol/L and 1.8 x 10(-9) mol/L, respectively). However, in cardiomyopathic hamsters, the EC50 of ET-1 in intact aortas was higher (1.5 x 10(-8) mol/L) than that of the endothelium denuded preparations (2.7 x 10(-9) mol/L). The EC50 of ET-1 in normal and cardiomyopathic hamster denuded aortas were similar. However, the EC50 of ET-1 in intact aortas of cardiomyopathic hamster was higher (1.5 x 10(-8) mol/L) than that of normal hamsters (2.2 x 10(-9) mol/L). Pre-treatment with the ETA receptor antagonist ABT-627 (10(-5)mol/L) of intact and endothelium denuded aortas from both normal and cardiomyopathic hamsters significantly prevented ET-1 (10(-7) mol/L) from inducing an increase in tension. Pre-treatment with the ETB receptor antagonist A-192621 (10(-5) mol/L) had no effect on the ET-1-induced increase in tension in endothelium denuded aortas of both normal and cardiomyopathic hamsters, as well as in intact preparations of normal animals. However, blockade of the ETB receptors in intact aortas of cardiomyopathic hamsters significantly (p < 0.001) potentiated the ET-1-induced increase in tension. In summary, an attenuation of the contraction response to ET-1 was found in UM-X7.1 cardiomyopathic hamsters when compared with normal age-matched hamsters. This alteration of the ET-1 effect in the aortas of cardiomyopathic hamsters seems to be dependent on the presence of the endothelium and could be due, in part, to an increase in the contribution of endothelial ETB receptors to relaxation, which in turn acts as a physiological depressor of ET-1 vasoconstriction. Our results suggest that an increase in the endothelium ETB receptor density may play a role in the development of hypotension in UM-X7.1 cardiomyopathic hamsters.

G-protein-coupled receptors, channels, and Na+-H+ exchanger in nuclear membranes of heart, hepatic, vascular endothelial, and smooth muscle cells.

The action of several peptides and drugs is thought to be primarily dependent on their interactions with specific cell surface G-protein-coupled receptors and ionic transporters such as channels and exchangers. Recent development of 3-D confocal microscopy allowed several laboratories, including ours, to identify and study the localization of receptors, channels, and exchangers at the transcellular level of several cell types. Using this technique, we demonstrated in the nuclei of several types of cells the presence of Ca(2+) channels as well as Na(+)-H(+) exchanger and receptors such as endothelin-1 and angiotensin II receptors. Stimulation of these nuclear membrane G-protein-coupled receptors induced an increase of nuclear Ca(2+). Our results suggest that, similar to the plasma membrane, nuclear membranes possess channels, exchangers and receptors such as those for endothelin-1 and angiotensin II, and that the nucleus seems to be a cell within a cell. This article will emphasize these findings.

Chronic ethanol consumption enhances phenylephrine-induced contraction in the isolated rat aorta.

Changes in reactivity to phenylephrine in aortas isolated from 2-, 6-, and 10-week ethanol-treated rats and their age-matched control and isocaloric rats were investigated. Chronic ethanol consumption enhances the contractile response of endothelium-intact and -denuded rat aortic rings to phenylephrine, a response that is time-independent. Pretreatment with indomethacin reduced E(max) for phenylephrine in denuded aortas from ethanol-treated rats but not control or isocaloric rats. After indomethacin treatment, no differences in E(max) from phenylephrine were observed among the groups. SQ29548 ([1S-[1alpha-2alpha(Z)3alpha,4alpha]]-7-[3-[[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid), an antagonist of prostaglandin H(2)/thromboxane A(2) (TXA(2)) receptors, did not alter phenylephrine-induced contraction in control or isocaloric aortas. However, in ethanol-treated aortas, E(max) was reduced to control level. Moreover, phenylephrine-stimulated release of thromboxane B(2), a stable metabolite of TXA(2), was higher in tissues from ethanol-treated rats. Simultaneous measurement of the changes in [Ca(2+)](i) and contraction induced by phenylephrine showed that both parameters are higher in the rat aorta from ethanol-treated rats. CaCl(2)-induced contraction in free Ca(2+) solution containing phenylephrine was increased in ethanol-treated aortas. Additionally, the enhancement in CaCl(2)-induced contraction was prevented by SQ29548. The major contribution of the present study is that it demonstrates a detailed description of the mechanisms involved in the enhancement of phenylephrine-induced contraction in rat aorta from ethanol-treated rats. We provided evidence that this response was not different among the three periods of treatment employed in this study and that it is maintained by two mechanisms: an increased release of vascular smooth muscle-derived vasoconstrictor prostanoids (probably TXA(2)) and an enhanced extracellular Ca(2+) influx.

NHE-1-dependent intracellular sodium overload in hypertrophic hereditary cardiomyopathy: prevention by NHE-1 inhibitor.

The purpose of this study was to verify whether myocardial intracellular Na(+) overload may take place in the hereditary cardiomyopathic hamster (CMH), as a result of an increased activity of the Na(+)-H(+) exchanger isoform-1 (NHE-1). Our results showed that simultaneous intracellular Na(+) and Ca(2+) overloads as well as an increase of NHE-1 protein level took place during the development of necrosis and hypertrophy in the CMH. Treatment of 30-day-old CMHs during the development of necrosis and in the absence of hypertrophy with the specific NHE-1 inhibitor EMD87580 (EMD) for 50 days significantly prevented the increase of NHE-1 protein level and intracellular Na(+) and Ca(2+) overloads as well as necrosis. Treatment of CMHs during the development of hypertrophy with EMD for 198 days prevented the development of both necrosis and hypertrophy. In conclusion, our results suggest that NHE-1 overexpression as well as Na(+) and Ca(2+) overloads do take place during the development of necrosis and hypertrophy in hereditary CMHs. Moreover, our results suggest that the blockade of NHE-1 by EMD87580 prevents these diseases by preventing the increase of Na(+) influx through the NHE-1.