High salt-induced morphological and glycocalyx remodeling of human vascular smooth muscle cells is reversible but induces a high sodium salt-like sensitive memory.

Our recent work showed that short-term treatment (1-2 days) with high sodium salt had no effect on the morphology of human vascular smooth muscle cells (hVSMCs). However, chronic (long-term treatment, 6-16 days) high sodium salt (CHSS) induced hypertrophy and decreased the relative density of the glycocalyx in hVSMCs. Whether this CHSS effect is reversible at both the morphological and the intracellular calcium and sodium levels is unknown. In the present study, we tested the hypothesis that the effect of CHSS on the morphological and functional levels of hVSMCs is reversible. However, it induced an irreversible increase in the sensitivity of the cells following short-term treatment with high extracellular Na+. We tested the effects of the removal of CHSS treatment on the morphology and intracellular sodium and calcium of hVSMCs. Our results showed that restoring average sodium concentration (145 mM) modeled back the relative density of the glycocalyx, the intracellular resting calcium and sodium levels, and the whole cell and nuclear volumes of hVSMCs. In addition, it induced a permanent remodeling of hVSMCs' response to a short-term increase in the extracellular level of sodium salt by developing spontaneous cytosolic and nuclear calcium waves. Our results showed that CHSS is reversible at both the morphological and basal intracellular ionic levels. However, it maintained a high sensitivity to short-term elevation of extracellular sodium. These results suggest that even if chronic high salt is corrected, it induces a high sodium salt-like sensitive memory.

Morphological and Functional Remodeling of Vascular Endothelium in Cardiovascular Diseases.

The vascular endothelium plays a vital role during embryogenesis and aging and is a cell monolayer that lines the blood vessels. The immune system recognizes the endothelium as its own. Therefore, an abnormality of the endothelium exposes the tissues to the immune system and provokes inflammation and vascular diseases such as atherosclerosis. Its secretory role allows it to release vasoconstrictors and vasorelaxants as well as cardio-modulatory factors that maintain the proper functioning of the circulatory system. The sealing of the monolayer provided by adhesion molecules plays an important role in cardiovascular physiology and pathology.

Calcium Homeostasis, Transporters, and Blockers in Health and Diseases of the Cardiovascular System.

Calcium is a highly positively charged ionic species. It regulates all cell types' functions and is an important second messenger that controls and triggers several mechanisms, including membrane stabilization, permeability, contraction, secretion, mitosis, intercellular communications, and in the activation of kinases and gene expression. Therefore, controlling calcium transport and its intracellular homeostasis in physiology leads to the healthy functioning of the biological system. However, abnormal extracellular and intracellular calcium homeostasis leads to cardiovascular, skeletal, immune, secretory diseases, and cancer. Therefore, the pharmacological control of calcium influx directly via calcium channels and exchangers and its outflow via calcium pumps and uptake by the ER/SR are crucial in treating calcium transport remodeling in pathology. Here, we mainly focused on selective calcium transporters and blockers in the cardiovascular system.

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.

Age-dependent effect of insulin in the regulation of intracellular calcium in ventricular cardiomyocytes.

In this study, we wanted to verify whether the effect of insulin on calcium homeostasis depends on the heart's development stage. Using a quantitative 3D confocal microscopy, we tested the effect of a high insulin concentration (100 µU) in freshly cultured ventricular cardiomyocytes from newborn and adult rats. Our results showed that the cytosolic basal level of calcium was higher in newborn cardiomyocytes with no change in the nuclear basal calcium level compared with the adult cardiomyocytes; in addition, insulin induced a slow increase of cytosolic and nuclear calcium in newborn ventricular cardiomyocytes, followed by two phases. However, the first phase of slow cytosolic and nuclear calcium increase was absent in adult rat ventricular cardiomyocytes. Furthermore, the time to the onset of increase of cytosolic and nuclear calcium was longer in newborn cardiomyocytes compared with adults. Moreover, the time to peak of the calcium transient was shorter in newborns than in adult cardiomyocytes. These results demonstrate that insulin differently regulates calcium homeostasis in newborns than in adult cardiomyocytes. Thus, newborn rat cardiomyocytes, commonly used in research as a model for adult cardiomyocytes, should be used with caution when dealing with insulin in normal and disease conditions.

Vascular smooth muscle remodeling in health and disease.

In blood vessels, vascular smooth muscle cells (VSMCs) generally exist in two major phenotypes: contractile and non-contractile (synthetic). The contractile phenotype is predominant and includes quiescent or differentiated VSMCs, which function as the regulators of blood vessel diameter and blood flow. According to some literature in the field, contractile VSMCs do not switch to the non-contractile phenotype due to the activation of specific transcription factors that are considered as guardians of the contractile phenotype. However, a vast amount of the literature uses the terms remodeling and phenotype switching of contractile VSMCs interchangeably based mainly on studies dealing with atherosclerosis. The use of the terms remodeling and switching to describe changes in phenotype based on morphological criteria can be confusing. The term remodeling was first used to describe morphological changes in the heart and was soon used to describe phenotype changes of contractile VSMCs based on morphological criteria. The latter were introduced in early studies, and new molecular criteria were later added, including changes in gene expression, which could be irreversible. In this review, we will discuss the different views concerning remodeling and possible switching of contractile VSMCs to a non-contractile phenotype. We conclude that only remodeling of contractile VSMCs may take place upon vascular injury and disease.

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.

Endocardial endothelial cell hypertrophy takes place during the development of hereditary cardiomyopathy.

Endocardial endothelial cells constitute a barrier between the circulating blood and ventricular cardiomyocytes. Although recently our group demonstrated the importance of this type of endothelial cells in excitation-secretion coupling, there is no information on whether this type of cells contributes to cardiac pathologies such as cardiac hypertrophy. Using the well-known model of human hypertrophy and heart failure, the UM-X7.1 hereditary cardiomyopathic hamster, our results showed that during the phase of necrosis and in the absence of cardiac hypertrophy, isolated endocardial endothelial cells underwent a significant increase in cell volume compared to cells isolated from age-matched normal hamsters. This increase of the volume of endocardial endothelial cells persisted during the development of cardiac hypertrophy in the hereditary cardiomyopathic hamster. These results demonstrate for the first time, that endocardial endothelial hypertrophy precedes the development of hypertrophy in hereditary cardiomyopathy and may, via its released factors, contribute to the development of cardiac hypertrophy. These results demonstrate the importance of endocardial endothelial cells in cardiac diseases such as hypertrophy. This type of cells constitutes a new target for understanding hypertrophy and heart failure.

Extracellular and intracellular tumor necrosis factor alpha modulates cytosolic and nuclear calcium in human cardiovascular cells 1.

Tumor necrosis factor alpha (TNFα) and its type 1 receptor (TNFR1) are implicated in several autoimmune diseases, including rheumatoid arthritis, and are associated with complications at the cardiovascular level. Using human cardiomyocytes, vascular smooth muscle, vascular endothelial, and endocardial endothelial cells coupled to indirect immunofluorescence, our results showed the presence of TNFR1 at the levels of the plasma membrane (including the cytosol) and mostly at the level of the nuclear membranes (including the nucleoplasm). The distribution of the receptor is different between cell types; however, the density is significantly higher at the nuclear level in all 4 cell types. The density of the receptor was the highest in contractile cells including the cardiomyocytes and vascular smooth muscle cells, compared with endothelial cells including endocardial endothelial and vascular endothelial cells. Using the Ca2+ probe Fluo-3 coupled to quantitative confocal microscopy, our results showed that the cytokine induced a sustained Ca2+ increase in both the cytosol and nucleoplasm of all 4 cell types. This increase was more significant at the nuclear level, mainly in endothelial cells. Our results demonstrated the presence of TNFR1 at both the cell and nuclear membranes of cardiovascular cells, and that its activation modulated both cytosolic and nuclear Ca2+.

Increase of NADPH oxidase 3 in heart failure of hereditary cardiomyopathy 1.

During the development of heart failure in humans and animal models, an increase in reactive oxygen species (ROS) levels was observed. However, there is no information whether this increase of ROS is associated with an increase in the density of specific isoforms of NADPH oxidases (NOXs) 1-5. The objective of this study was to verify whether the densities of NOXs 1-5 change during the development of heart failure. Using the well-known model of cardiomyopathic hamsters, the UM-X 7.1 line, a model that strongly resembles the pathology observed in humans from a morphological and functional point of view, our studies showed that, as in humans, NOXs 1-5 are present in both normal and UM-X7.1 hamster hearts. Even though the densities of NOXs 2 and 5 were unchanged, the levels of both NOXs 1 and 4 significantly decreased in UM-X7.1 hamster hearts during heart failure. These changes were accompanied with a significant increase in NOX3 level. These results suggest that, during heart failure, NOX3 plays a vital role in compensating the decrease of NOXs 1 and 4. This increase in NOX3 may also be responsible, at least in part, for the reported increase in ROS levels in 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.

High salt-induced hypertrophy of human vascular smooth muscle cells associated with a decrease in glycocalyx.

All health organizations agree that, presently, the average daily salt (sodium chloride) consumption per person has attained almost double the recommended amount. A chronic high salt diet contributes to the increase in blood pressure and to the development of cardiovascular disease. Although our knowledge of hypertension, in general, is abundant, little is known about salt-sensitive hypertension. Here we tested the hypothesis that acute and/or chronic high salt mimicking that present in high-salt sensitive hypertensive patients may induce hypertrophy of human vascular smooth muscle cells (hVSMCs) and their nuclei that are associated with damage to the plasma membrane glycocalyx. Using quantitative 3D confocal microscopy coupled to immunofluorescence techniques, we tested the effects of acute (2-4 days) and chronic (6-16 days) treatments of hVSMCs without (145 mM) or with high (149 mM) extracellular sodium chloride. Our results showed that acute treatment with high salt significantly decreased the relative density of membrane glycocalyx without affecting the whole cell and nuclear volumes of hVSMCs. However, chronic treatments with high salts induced significant decreases in the relative density of glycocalyx accompanied by significant increases in the whole cell and nuclear volumes as well as in the protein/DNA ratio. The high salt-induced hVSMC hypertrophy was associated with a sustained increase in intracellular sodium and calcium. Our results clearly showed that, increasing salt concentration by as little as 4 mM immediately induced damage to the cell membrane glycocalyx leading to chronic Na+ and Ca2+ overloads and hVSMC hypertrophy. The latter may reduce the lumen of arteries leading to an increase in blood pressure. Future identification of the mechanisms that are implicated in a high salt-induced remodeling of hVSMCs may permit the development of new therapeutic interventions for the treatment of high salt-sensitive hypertension and the prevention of the associated cardiovascular diseases.

Physical contact between human vascular endothelial and smooth muscle cells modulates cytosolic and nuclear calcium homeostasis.

The interaction between vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) plays an important role in the modulation of vascular tone. There is, however, no information on whether direct physical communication regulates the intracellular calcium levels of human VECs (hVECs) and (or) human VSMCs (hVSMCs). Thus, the objective of the study is to verify whether co-culture of hVECs and hVSMCs modulates cytosolic ([Ca2+]c) and nuclear calcium ([Ca2+]n) levels via physical contact and (or) factors released by both cell types. Quantitative 3D confocal microscopy for [Ca2+]c and [Ca2+]n measurement was performed in cultured hVECs or hVSMCs or in co-culture of hVECs-hVSMCs. Our results show that: (1) physical contact between hVECs-hVECs or hVSMCs-hVSMCs does not affect [Ca2+]c and [Ca2+]n in these 2 cell types; (2) physical contact between hVECs and hVSMCs induces a significant increase only of [Ca2+]n of hVECs without affecting the level of [Ca2+]c and [Ca2+]n of hVSMCs; and (3) preconditioned culture medium of hVECs or hVSMCs does not affect [Ca2+]c and [Ca2+]n of both types of cells. We concluded that physical contact between hVECs and hVSMCs only modulates [Ca2+]n in hVECs. The increase of [Ca2+]n in hVECs may modulate nuclear functions that are calcium dependent.

Na+-H+ exchanger and proton channel in heart failure associated with Becker and Duchenne muscular dystrophies.

Cardiomyopathy is found in patients with Duchenne (DMD) and Becker (BMD) muscular dystrophies, which are linked muscle diseases caused by mutations in the dystrophin gene. Dystrophin defects are not limited to DMD but are also present in mild BMD. The hereditary cardiomyopathic hamster of the UM-X7.1 strain is a particular experimental model of heart failure (HF) leading to early death in muscular dystrophy (dystrophin deficiency and sarcoglycan mutation) and heart disease (δ-sarcoglycan deficiency and dystrophin mutation) in human DMD. Using this model, our previous work showed a defect in intracellular sodium homeostasis before the appearance of any apparent biochemical and histological defects. This was attributed to the continual presence of the fetal slow sodium channel, which was also found to be active in human DMD. Due to muscular intracellular acidosis, the intracellular sodium overload in DMD and BMD was also due to sodium influx through the sodium-hydrogen exchanger NHE-1. Lifetime treatment with an NHE-1 inhibitor prevented intracellular Na+ overload and early death due to HF. Our previous work also showed that another proton transporter, the voltage-gated proton channel (Hv1), exists in many cell types including heart cells and skeletal muscle fibers. The Hv1 could be indirectly implicated in the beneficial effect of blocking NHE-1.

Angiotensin II receptors' modulation of calcium homeostasis in human vascular endothelial cells.

Angiotensin II (AngII) plays an important role in the regulation of vascular smooth muscle function. However, little is known about AngII and its receptors AT1 (AT1R) and AT2 (AT2R) and their modulation of intracellular calcium in vascular endothelial cells (VECs) in general and more particularly of human origin. Using western blots, our results showed that AT1Rs and AT2Rs are present in human VECs (hVECs). Using quantitative 3D confocal imaging, our results showed that AngII is present at the cytoplasmic and nucleoplasmic levels and its relative density is lower in the nucleoplasm. However, both AngII receptors AT1 and AT2 are present at both the plasma and the nuclear envelope membranes (NEMs). AngII (10-10 mol/L) induces a transient decrease of the relative density of cytosolic and nuclear AT1Rs. Blockade of AT1Rs with losartan or blocking protein synthesis with cycloheximide does not prevent internalization and nuclear translocation of AT1Rs but prevents de novo AT1R synthesis. In addition, AngII induces cytosolic and nuclear increases (EC50 near 5 × 10-14 mol/L) of calcium via the activation of AT1Rs. These results demonstrate that both AT1 and AT2 receptors are present in hVECs, and that only AT1Rs seem to undergo transcellular trafficking and modulate cytosolic and nuclear calcium homeostasis.

Neuropeptide Y and its receptors in ventricular endocardial endothelial cells.

Endocardial endothelial cells (EECs) constitute an important component of the heart. These cells form a monolayer that covers the cavities of the right (EECRs) and left (EECLs) ventricles. They play an important role in cardiac excitation-contraction coupling via their secretion of cardioactive factors such as neuropeptide Y (NPY). They also contribute to cardiac pathology such as arrhythmia, hypertrophy, and heart failure. Differences between EECRs and EECLs contribute to tuning of circulating factors at the entry and exit of the ventricles. NPY, via activation of its receptors, modulates the excitation-secretion coupling of EECs, thus, indirectly modulating cardiac function and remodeling.

Role of endothelin-1 and its receptors, ETA and ETB, in the survival of human vascular endothelial cells.

Our previous work showed the presence of endothelin-1 (ET-1) receptors, ETA and ETB, in human vascular endothelial cells (hVECs). In this study, we wanted to verify whether ET-1 plays a role in the survival of hVECs via the activation of its receptors ETA and (or) ETB (ETAR and ETBR, respectively). Our results showed that treatment of hVECs with ET-1 prevented apoptosis induced by genistein, an effect that was mimicked by treatment with ETBR-specific agonist IRL1620. Furthermore, blockade of ETBR with the selective ETBR antagonist A-192621 prevented the anti-apoptotic effect of ET-1 in hVECs. However, activation of ETA receptor alone did not seem to contribute to the anti-apoptotic effect of ET-1. In addition, the anti-apoptotic effect of ETBR was found to be associated with caspase 3 inhibition and does not depend on the density of this type of receptor. In conclusion, our results showed that ET-1 possesses an anti-apoptotic effect in hVECs and that this effect is mediated, to a great extent, via the activation of ETBR. This study revealed a new role for ETBR in the survival of hVECs.

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.

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).