[Show abstract][Hide abstract] ABSTRACT: The E2F/Rb pathway regulates cardiac growth and development and holds great potential as a therapeutic target. The E2F6 repressor is a unique E2F member that acts independently of pocket proteins. Forced expression of E2F6 in mouse myocardium induced heart failure and mortality, with severity of symptoms correlating to E2F6 levels. Echocardiography demonstrated a 37% increase (P<0.05) in left ventricular end-diastolic diameter and reduced ejection fraction (<40%, P<0.05) in young transgenic (Tg) mice. Microarray and qPCR analysis revealed a paradoxical increase in E2F-responsive genes, which regulate the cell cycle, without changes in cardiomyocyte cell number or size in Tg mice. Young adult Tg mice displayed a 75% (P<0.01) decrease in gap junction protein connexin-43, resulting in abnormal electrocardiogram including a 24% (P<0.05) increase in PR interval. Further, mir-206, which targets connexin-43, was up-regulated 10-fold (P<0.05) in Tg myocardium. The mitogen-activated protein kinase pathway, which regulates the levels of miR-206 and connexin-43, was activated in Tg hearts. Thus, deregulated E2F6 levels evoked abnormal gene expression at transcriptional and post-transcriptional levels, leading to cardiac remodeling and dilated cardiomyopathy. The data highlight an unprecedented role for the strict regulation of the E2F pathway in normal postnatal cardiac function.
The FASEB Journal 03/2012; 26(6):2569-79. DOI:10.1096/fj.11-203174 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sarcolemmal membrane-associated proteins (SLMAPs) are components of cardiac membranes involved in excitation-contraction (E-C) coupling. Here, we assessed the role of SLMAP in cardiac structure and function. We generated transgenic (Tg) mice with cardiac-restricted overexpression of SLMAP1 bearing the transmembrane domain 2 (TM2) to potentially interfere with endogenous SLMAP through homodimerization and subcellular targeting. Histological examination revealed vacuolated myocardium; the severity of which correlated with the expression level of SLMAP1-TM2. High resolution microscopy showed dilation of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) and confocal imaging combined with biochemical analysis indicated targeting of SLMAP1-TM2 to the SR/ER membranes and inappropriate homodimerization. Older (28 wk of age) Tg mice exhibited reduced contractility with impaired relaxation as assessed by left ventricle pressure monitoring. The ventricular dysfunction was associated with electrophysiological abnormalities (elongated QT interval). Younger (5 wk of age) Tg mice also exhibited an elongated QT interval with minimal functional disturbances associated with the activation of the fetal gene program. They were less responsive to isoproterenol challenge (ΔdP/dt(max)) and developed electrical and left ventricular pressure alternans. The altered electrophysiological and functional disturbances in Tg mice were associated with diminished expression level of calcium cycling proteins of the sarcoplasmic reticulum such as the ryanodine receptor, Ca(2+)-ATPase, calsequestrin, and triadin (but not phospholamban), as well as significantly reduced calcium uptake in microsomal fractions. These data demonstrate that SLMAP is a regulator of E-C coupling at the level of the SR and its perturbation results in progressive deterioration of cardiac electrophysiology and function.
[Show abstract][Hide abstract] ABSTRACT: Using immunofluorescence and 3-dimensional confocal microscopy techniques, the present study was designed to verify if NHE-1 is present at the level of the nuclear membrane in cells that are known to express this type of exchanger. Nuclei were isolated from aortic tissues of adult human, rabbit, and rats, as well as from liver tissues of human fetus, and adult rabbit and rat. In addition, cultured ventricular cardiomyocytes were isolated from 2-week-old rat. Our results showed the presence of NHE-1 in isolated nuclei of aortic vascular smooth muscle and liver of human, rabbit, and rat. NHE-1 seems to be distributed throughout the isolated nucleus and more particularly at the level of the nuclear membranes. The relative fluorescence density of NHE-1 was significantly higher (p < 0.05) in isolated liver nuclei of human, when compared with those of rabbit and rat. However, in isolated nuclei of aortic vascular smooth muscle, the relative fluorescence density of NHE-1 was significantly (p < 0.001) higher in the rabbit when compared with human and rat. In cultured rat ventricular cardiomyocytes, NHE-1 fluorescent labeling could be easily seen throughout the cell, including the nucleus, and more particularly at both the sarcolemma and the nuclear membranes. In rat cardiomyocytes, the relative fluorescence density of NHE-1 of the sarcolemma membrane, including the cytosol, was significantly lower than that of the whole nucleus (including the nuclear envelope membranes). In conclusion, our results showed that NHE-1 is present at the nuclear membranes and in the nucleoplasm and its distribution and density may depend on cell type and species used. These results suggest that nuclear membranes' NHE-1 may play a role in the modulation of intranuclear pH.
Canadian Journal of Physiology and Pharmacology 02/2011; 82(8-9):805-11. DOI:10.1139/y04-119 · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Using immunofluorescence and real 3-D confocal microscopy, our results showed the presence of ET-1, ETA, and ETB receptors in isolated human aortic vascular endothelial cells (hVECs). The level of the peptide and its receptors was significantly higher in the nucleus (including the nuclear envelope membranes) than in the cytosol (including the cell membrane). Furthermore, using the Western blot technique we demonstrated the presence of both ETA and ETB receptors. Using intact and isolated human hVECs and the Fura-2 calcium (Ca2+) measurement technique, we showed that ET-1 induced a dose-dependent increase of total intracellular free Ca2+, with an EC50 of 1.3 x 10-10 mol/L. The specific ETA receptor antagonist ABT-627 (10-7 mol/L), but not the ETB receptor antagonist A-192621 (10-7 mol/L), prevented the ET-1 (10-9 mol/L) induced increase of total intracellular Ca2+. In conclusion, these results clearly show that similar to ETB receptors, ETA receptors are also present in human aortic vascular endothelial cells and their levels are higher than ETB in the nucleus when compared with the cytosol. Furthermore, we suggest that ETA, but not ETB, receptors mediate the effect of ET-1 on total intracellular Ca2+ of human aortic vascular endothelial cells.
Canadian Journal of Physiology and Pharmacology 08/2010; 88(8):817-29. DOI:10.1139/Y10-057 · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Canadian Journal of Physiology and Pharmacology 09/2008; 86(8):546-56. DOI:10.1139/Y08-048 · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuropeptide Y (NPY), a sympathetic cotransmitter, acts via G protein-coupled receptors to stimulate constriction and vascular smooth muscle cell (VSMC) proliferation through interactions with its Y1 receptors. However, VSMC proliferation appears bimodal, with high- and low-affinity peaks differentially blocked by antagonists of both Y1 and Y5 receptors. Here, we sought to determine the signaling mechanisms of NPY-mediated bimodal mitogenesis. In rat aortic VSMCs, NPY's mitogenic effect at all concentrations was blocked by pertussis toxin and was associated with decreased forskolin-stimulated cAMP levels. NPY also increased intracellular calcium levels; in contrast to mitogenesis, this effect was dose dependent. The rise in intracellular Ca2+ depended on extracellular Ca2+ and was mediated via activation of Y1 receptors, but not Y5 receptors. Despite differences in calcium, the signaling pathways activated at low and high NPY concentrations were similar. The mitogenic effect of the peptide at all doses was completely blocked by inhibitors of calcium/calmodulin-dependent kinase II (CaMKII), protein kinase C (PKC), and mitogen-activated protein kinase kinase, MEK1/2. Thus, in VSMCs, NPY-mediated mitogenesis signals primarily via Y1 receptors activating 2 Ca2+-dependent, growth-promoting pathways -- PKC and CaMKII. At the high-affinity peak, these 2 pathways are amplified by Y5 receptor-mediated, calcium-independent inhibition of the adenylyl cyclase - protein kinase A (PKA) pathway. All 3 mechanisms converge to the extracellular signal-regulated kinases (ERK1/2) signaling cascade and lead to VSMC proliferation.
Canadian Journal of Physiology and Pharmacology 07/2008; 86(7):438-48. DOI:10.1139/y08-054 · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS3Cn) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS3C, by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS3C,. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS3C6-C9 is a major factor in their overall photodynamic activity.
Photochemistry and Photobiology 11/2006; 82(6):1712-20. DOI:10.1562/2005-12-13-RA-752 · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prostaglandins (PG) are important mediators of bone metabolism with direct and indirect effects on bone cells. They may have important effects on osteoclasts, but it is not known if these cells can synthesize PG. We used 2 experimental models in order (1) to determine the presence and functionality of cyclooxygenase (COX) and phospholipase A2 (PLA2) enzymes in human osteoclasts and (2) to study their role in cell metabolism.
Experiments were undertaken on authentic human osteoclasts extracted from human fetuses (fhOC) and on human osteoclast-like (hOCL) cells differentiated from peripheral blood mononuclear cells. The presence of COX proteins was determined by immunohistochemistry. COX and PLA2 enzymatic activity was evaluated at the single-cell level by fluorescence microscopy. An enriched population of hOCL cells was used to evaluate total PG production and the influence of COX activity on bone resorption.
COX-1 was expressed in the cytoplasm and COX-2 was distributed mainly near the nuclear membrane of osteoclasts. These cells showed a high basal level of COX activity that could be inhibited by pretreatment with COX inhibitors. Cytosolic PLA2 was present in both models. Human osteoclasts actively produced PG, and the COX-1 pathway was implicated in the control of bone resorption.
These results indicate that PG may be important autacoids for the control of osteoclast biology and that the COX-1 pathway is implicated in the inhibition of bone resorption.
The Journal of Rheumatology 08/2006; 33(7):1320-8. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA1-receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA1-receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor L-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca2+ transients, activation of p42 MAPK, NF-kappaB binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.
[Show abstract][Hide abstract] ABSTRACT: 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.
Canadian Journal of Physiology and Pharmacology 06/2006; 84(3-4):431-41. DOI:10.1139/y06-002 · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of angiotensin II (Ang II) on the T- and L-type calcium currents (I(Ca)) in single ventricular heart cells of 18-week-old fetal human and 10-day-old chick embryos was studied using the whole-cell voltage clamp technique. Our results showed that in both, human and chick cardiomyocytes, Ang II (10(-7)M) increased the T-type calcium current and decreased the L-type I(Ca). The effect of Ang II on both types of currents was blocked by the AT1 peptidic antagonist, [Sar1, Ala8] Ang II (2 x 10(-7)M). Protein kinase C activator, phorbol 12,13-dibutyrate, mimicked the effect of Ang II on the T- and L-type calcium currents. These results demonstrate that in fetal human and chick embryo cardiomyocytes Ang II affects the T- and L-type Ca2+ currents differently, and this effect seems to be mediated by the PKC pathway.
[Show abstract][Hide abstract] ABSTRACT: The aim of this work is to verify if Angiotensin II (Ang II) affects the frequency of spontaneous cytosolic and nuclear Ca2+ waves in chick embryonic cardiomyocytes and if this effect is mediated via the activation of AT1 and/or AT2 receptors. Using the rapid scan technique of confocal microscopy, we observed that Ang II (10(-8)M) increases the frequency of cytosolic and nuclear Ca2+ waves. This effect was accompanied by a decrease in the amplitude of nuclear Ca2+ waves and an absence of effect on the amplitude of cytosolic Ca2+ waves. The effect of the octapeptide on both frequency and amplitude of the nuclear waves was prevented by the AT1 receptor antagonist L158809. However, blockade of the AT2 receptor using the antagonist PD123319 (10(-7)M) only prevented the effect of Ang II on the frequency of Ca2+ waves. Furthermore, the effect was prevented by both a PKC inhibitor (bisindolylmaleimide) and a PKC activator (phorbol 12,13-dibutyrate). In addition, the Ang II effect was not prevented by the blocker of the pacemaker current If. These results demonstrate that Ang II, via the activation of its receptors AT1 and AT2, affects the frequency of spontaneous Ca2+ waves and this effect seems to be mediated by the PKC pathway.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Molecular and Cellular Cardiology 05/2005; 38(4):571-82. DOI:10.1016/j.yjmcc.2005.01.003 · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of an ET-1 fluorescent probe in human heart and vascular smooth muscle cells showed that ET-1 receptors are present at both the sarcolemma and nuclear envelope membranes. The use of immunofluorescence studies showed that the ETA receptor was mainly present at the sarcolemma and cytosolic levels. However, the ETB receptor was present at the sarcolemma and the cytosol, as well as the nuclear envelope membranes and the nucleoplasm. In addition, ET-1 immunoreactivity was seen in the cytosol and the nucleus. Using Ca2+ fluorescent probes such as Fluo-3, Indo 1, and yellow cameleon, as well as confocal microscopy three-dimensional image measurement technique, stimulation of ET-1 receptors at the sarcolemma membranes induced an increase of cytosolic and nuclear free Ca2+ levels. This effect of extracellular ET-1 was blocked by removal of extracellular calcium. Direct stimulation of ET-1 receptors at the nuclear envelope membranes also induced an increase of intranuclear free Ca2+ level. Our results suggest that the stimulation of sarcolemmal Ca2+ influx by ET-1 seems to be due to the activation of ETA and ETB receptors. However, the increase of nucleoplasmic Ca2+ levels by cytosolic ET-1 seems to be mediated via the activation of ETB receptors. Activation of nuclear membranes ETB receptors seems to prevent nuclear Ca2+ overload and may protect the cell from apoptosis.
Canadian Journal of Physiology and Pharmacology 07/2003; 81(6):654-62. DOI:10.1139/y03-020 · 1.77 Impact Factor