Increase In Cell Surface Area Research Articles

Development of cellular hypertrophy by 8-hydroxyeicosatetraenoic acid in the human ventricular cardiomyocyte, RL-14 cell line, is implicated by MAPK and NF-κB.

Recent studies have established the role of mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) in the development of cardiovascular disease. Among these mid-chains, 8-HETE has been reported to have a proliferator and proinflammatory action. However, whether 8-HETE can induce cardiac hypertrophy has never been investigated before. Therefore, the overall objectives of the present study are to elucidate the potential hypertrophic effect of 8-HETE in the human ventricular cardiomyocytes, RL-14 cells, and to explore the mechanism(s) involved. Our results showed that 8-HETE induced cellular hypertrophy in RL-14 cells as evidenced by the induction of cardiac hypertrophy markers ANP, BNP, α-MHC, and β-MHC in a concentration- and time-dependent manner as well as the increase in cell surface area. Mechanistically, 8-HETE was able to induce the NF-κB activity as well as it significantly induced the phosphorylation of ERK1/2. The induction of cellular hypertrophy was associated with a proportional increase in the formation of dihydroxyeicosatrienoic acids (DHETs) parallel to the increase of soluble epoxide hydrolase (sEH) enzyme activity. Blocking the induction of NF-κB, ERK1/2, and sEH signaling pathways significantly inhibited 8-HETE-induced cellular hypertrophy. Our study provides the first evidence that 8-HETE induces cellular hypertrophy in RL-14 cells through MAPK- and NF-κB-dependent mechanism

Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca(2+). The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H2O2) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.

Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and α-Smooth Muscle Actin Expression of Retinal Müller Cells.

The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Müller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigidity of the extracellular matrix, our aim was to develop an in vitro model for studying Müller cell morphology and differentiation state in relation to matrix stiffness. A spontaneously immortalized human Müller cell line (MIO-M1) was cultured on type I collagen-coated polyacrylamide gels with Young's moduli ranging from 2 to 92 kPa. Cell surface area, focal adhesion, and the expression and morphology of α-smooth muscle actin induced by transforming growth factor β (TGF-β [10 ng/mL for 48 hours]) were analyzed by immunocytology. The images were documented by using fluorescence microscopy and confocal scanning laser microscopy. MIO-M1 cells cultured on stiff substrates exhibited a significant increase in cell surface area, stress fiber, and mature focal adhesion formation. Furthermore, Müller cells treated with TGF-β1 and TGF-β2 and cultured on stiff substrates showed an increased incorporation of α-smooth muscle actin into stress fibers when compared to those grown on soft surfaces. Compliance of the surrounding matrix seems to influence the morphology and contraction of retinal Müller cells in fibrotic conditions. Development of an in vitro model simulating both the normally compliant retinal tissue and the rigid retinal fibrotic tissue helps fill the gap between the results of petri-dish cell culture with rigid surfaces and in vivo findings.

The effect of TGF-beta-induced epithelial–mesenchymal transition on the expression of intracellular calcium-handling proteins in T47D and MCF-7 human breast cancer cells

The contribution of Ca2+ in TGF-β-induced EMT is poorly understood. We aimed to confirm the effect of TGF-β on the gene expression of intracellular calcium-handling proteins and to investigate the potential underlying mechanisms in TGF-β-induced EMT. T47D and MCF-7 cells were cultured in vitro and treated with TGF-β. The mRNA expression of EMT marker genes and intracellular calcium-handling proteins were quantified by qRT-PCR. qRT-PCR and Western blot analysis results verified the changes of EMT marker gene expression. Furthermore, we found that TGF-β induced cell morphological changes significantly with an increase of cell surface area and cell length. These results indicated that TGF-β induced EMT. The mRNA expression levels of SPCA1, SPCA2 and MCU were not influenced by TGF-β treatment, while NCX1 expression was decreased in T47D cells. In addition, the mRNA levels of SERCAs and IP3Rs were significantly changed due to TGF-β-induced EMT. The TGF-β-treated T47D cells exhibited markedly greater response to ATP than the control cells, and the descent velocity of cytosolic calcium concentration was faster in TGF-β-treated cells than in control cells. This is the first report to demonstrate that TGF-β-induced EMT in human breast cancer cells is associated with alterations in endoplasmic reticulum calcium homeostasis.

Abstract 17: Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca2+/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca2+. The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been explored. During developmental growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with an established lentiviral based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared to lentiviral transduction of CPCs with eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and increased length of cells compared to CPCe. CPCeδB are resistant to oxidative stress induced by H2O2 relative to CPCe, whereas a knockdown of CaMKIIδB using small hairpin RNA resulted in an up regulation of cell death compared to scrambled treated controls. Dexamethasone treatment to promote cardiac differentiation increased cardiomyogenic markers cardiac troponin T and α-smooth muscle actin measured by RT-PCR and immunoblot analyses in CPCeδB compared to control CPCe. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineage.

Differential ERK1/2 Signaling and Hypertrophic Response to Endothelin-1 in Cardiomyocytes from SHR and Wistar-Kyoto Rats: A Potential Target for Combination Therapy of Hypertension.

Extracellular signal regulated kinase½ (ERK1/2) signaling is critical to endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy. This study was to investigate ERK1/2 signaling and hypertrophic response to ET-1 stimulation in cardiomyocytes (CMs) from spontaneous hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Primary neonatal SHR and WKY CMs were exposed to ET-1 for up to 24 hrs. Minimal basal ERK1/2 phosphorylation was present in WKY CMs, while a significant baseline ERK1/2 phosphorylation was observed in SHR CMs. ET-1 induced a time- and dose-dependent increase in ERK1/2 phosphorylation in both SHR and WKY CMs. However, ET-1-induced ERK1/2 activation occurred much earlier with significantly higher peak phosphorylation level, and stayed elevated for longer duration in SHR CMs than that in WKY CMs. ET-1-induced hypertrophic response was more prominent in SHR CMs than that in WKY CMs as reflected by increased cell surface area, intracellular actin density, and protein synthesis. Pre-treatment with ERK1/2 phosphorylation inhibitor PD98059 completely prevented ET-1-induced ERK1/2 phosphorylation and increases in cell surface area and protein synthesis in SHR and WKY CMs. The specific PI3 kinase inhibitor LY294002 blocked ET-1-induced Akt and ERK1/2 phosphorylation, and protein synthesis in CMs. These data indicated that ERK1/2 signaling was differentially enhanced in CMs, and was associated with increased cardiac hypertrophic response to ET-1 in SHR. ET-1-induced ERK1/2 activation and cardiac hypertrophy appeared to be mediated via PI3 kinase/Akt signaling in SHR and WKY. The differential ERK1/2 activation in SHR CMs by ET-1 might represent a potential target for combination therapy of hypertension.

Role of MAPKs and NF‐κB in the Development of Cellular Hypertrophy by 8‐ Hydroxyeicosatetraenoic acids in Human Ventricular Cardiomyocyte, RL‐14 Cell Line

Recent studies have established the role of mid-chain hydroxyeicosatetraenoic acids (HETEs) in the development of cardiovascular disease. Among these mid-chains, 8-HETE has been reported to have proliferator and pro-inflammatory action. However, whether 8-HETE can induce cardiac hypertrophy has never been investigated before. Therefore, the overall objective of the present study is to elucidate the potential hypertrophic effect of 8-HETE in the human ventricular cardiomyocytes, RL-14 cells, and to explore the mechanism(s) involved. Our results showed that 8-HETE induced cellular hypertrophy in RL-14 cells as evidence by the induction of cardiac hypertrophy markers ANP, BNP, α-MHC, and β-MHC in a concentration- and time-dependent manner and the increase in cell surface area. Mechanistically, 8-HETE was able to induce the binding activity of NF-κB to its responsive element and significantly induced the phosphorylation of ERK1/2. The induction of cellular hypertrophy was associated with a proportional increase in the formation of dihydroxyeicosatrienoic acids (DHETs) parallel to the increase of soluble epoxide hydrolase enzyme activity. Blocking the induction of NF-κB, ERK1/2 and sEH signaling pathways significantly inhibited 8-HETE-induced cellular hypertrophy. Our study provides the first evidence that 8-HETEs induce cellular hypertrophy in RL-14 cells through MAPKs and NF-κB dependent mechanism.

Effects of ERK1/2/PPARα/SCAD signal pathways on cardiomyocyte hypertrophy induced by insulin-like growth factor 1 and phenylephrine

AimsShort-chain acyl-CoA dehydrogenase (SCAD) is a key enzyme in fatty acid oxidation. In the present study we aim to investigate the changes in SCAD between pathological and physiological cardiomyocyte hypertrophy. We also explore the different signaling pathways of pathological and physiological cardiomyocyte hypertrophy. Main methodsAfter neonatal rat cardiomyocytes were treated as setups, cell surface area, expression of SCAD, PPARα, phospho-ERK1/2, activity of SCAD, free fatty acid content and ATP content in the cardiomyocytes were measured. Key findingsNeonatal rat cardiomyocytes treated by PE showed an increased cell surface area and free fatty acid content, increased ERK1/2 phosphorylation, decreased expression of PPARα, decreased expression and activity of SCAD and decreased levels of ATP. Neonatal rat cardiomyocytes treated by IGF-1 showed the reverse effects except for the cell surface area. PPARα inhibitor GW6471 and PPARα activator Fenofibrate treatments abrogated the effects induced by IGF-1 and PE in cardiomyocytes respectively, as well as ERK1/2 activator EGF and ERK1/2 inhibitor PD98059. SignificanceSCAD has different changes between pathological and physiological cardiomyocyte hypertrophy. The ERK1/2/PPARα/SCAD signaling pathways play different roles in pathological and physiological cardiomyocyte hypertrophy. SCAD may be used as a new target to prevent the development of pathological cardiac hypertrophy.

Serotonin-promoted elevation of ROS levels may lead to cardiac pathologies in diabetic rat

Patients with diabetes mellitus (DM) develop tendencies toward heart disease. Hyperglycemia induces the release of serotonin from enterochromaffin cells (EC). Serotonin was observed to elevate reactive oxygen species (ROS) and downregulate antioxidant enzymes. As a result, elevated levels of serotonin could contribute to diabetic complications, including cardiac hypertrophy. In the present study, diabetes mellitus was induced in rats by alloxan administration; this was followed by the administration of serotonin to experimental animals. ROS, catalase (CAT), superoxide dismutase (SOD), B-type natriuretic peptide (BNP) expression, and histopathological assessments were performed. Elevated ROS concentrations and decreased antioxidant enzyme activities were detected. Further, we observed an increase in cell surface area and elevated BNP expression which suggests that events associated with cardiac hypertrophy were increased in serotonin-administered diabetic rats. We conclude that serotonin secretion in diabetes could contribute to diabetic complications, including cardiac hypertrophy, through enhanced ROS production.

Abstract 18420: Myozap-deficiency Inhibits SRF Signaling and Causes Severe Cardiomyopathy in Response to Pathological Biomechanical Stress in vivo

Background: We recently showed that the intercalated disc (ID) protein Myozap activates Rho-dependent SRF signaling both in vitro, and in vivo. Conversely, knockdown of its ortholog in Zebrafish led to severe contractile dysfunction and cardiomyopathy. Methods and Results: We generated a Myozap null mutant (MZP-ko) by global deletion of Myozap in mice. The absence of Myozap caused neither structural defects nor a baseline cardiac phenotype and led only to mild cardiac hypertrophy upon aging. Of note, induction of SRF target genes and the activation of SRF per se were markedly inhibited in MZP-ko mice. Nevertheless, biomechanical stress induced by transverse aortic constriction (TAC) triggered an excessive increase in cardiac hypertrophy (43% or 36% increased heart wt:body wt or LV wt:body wt ratios, p<0.001), an increased cell surface area, as well as accelerated fibrosis, followed by “super”- induction of the hypertrophic gene program (ANF/BNP). Moreover, MZP-ko mice revealed a severe reduction of fractional shortening (average %FS for MZP-ko 14.5% compared to 33.5% for wild-type littermates, p<0.001) and clinical signs of heart failure (54% increase in lung/body weights, p<0.001) which also caused a profound increase in mortality in response to TAC. Furthermore, expression of other ID proteins like N-Cadherin, Desmoplakin and Connexin 43 was found significantly altered upon pressure overload in MZP-ko mice. Finally, we observed a downregulation of Dysbindin, a novel interaction partner of Myozap and known inducer of ERK1/2 signaling in TAC operated MZP-ko mice. Consistently, activation of ERK1/2 was blunted in MZP-ko mice after TAC. Conclusions: We here show that myozap deficiency in vivo inhibits SRF- and ERK1/2-signaling leading to a maladaptative response to increased biomechanical stress, followed by cardiomyopathy, heart failure and cardiac death. Moreover, myozap deficiency severely altered the expression of its direct ID interaction partners such as Dysbindin and Desmoplakin. In a broader perspective, our data identify signaling at the level of the intercalated disc as a critical component of cardiac remodeling.

Polydatin prevents hypertrophy in phenylephrine induced neonatal mouse cardiomyocytes and pressure-overload mouse models

Recent evidence suggests that polydatin (PD), a resveratrol glucoside, may have beneficial actions on the cardiac hypertrophy. Therefore, the current study focused on the underlying mechanism of the PD anti-hypertrophic effect in cultured cardiomyocytes and in progression from cardiac hypertrophy to heart failure in vivo.Experiments were performed on cultured neonatal rat, ventricular myocytes as well as adult mice subjected to transverse aortic constriction (TAC). Treatment of cardiomyocytes with phenylephrine for three days produced a marked hypertrophic effect as evidenced by significantly increased cell surface area and atrial natriuretic peptide (ANP) protein expression. These effects were attenuated by PD in a concentration-dependent manner with a complete inhibition of hypertrophy at the concentration of 50µM. Phenylephrine increased ROCK activity, as well as intracellular reactive oxygen species production and lipid peroxidation. The oxidizing agent DTDP similarly increased Rho kinase (ROCK) activity and induced hypertrophic remodeling. PD treatment inhibited phenylephrine-induced oxidative stress and consequently suppressed ROCK activation in cardiomyocytes. Hypertrophic remodeling and heart failure were demonstrated in mice subjected to 13 weeks of TAC. Upregulation of ROCK signaling pathway was also evident in TAC mice. PD treatment significantly attenuated the increased ROCK activity, associated with a markedly reduced hypertrophic response and improved cardiac function.Our results demonstrated a robust anti-hypertrophic remodeling effect of polydatin, which is mediated by inhibition of reactive oxygen species dependent ROCK activation.

Astragalus polysaccharide inhibits isoprenaline-induced cardiac hypertrophy via suppressing Ca2+-mediated calcineurin/NFATc3 and CaMKII signaling cascades

Pathological cardiac hypertrophy induced by increased sympathetic drive can subsequently lead to congestive heart failure, which represents the major cause of morbidity and mortality worldwide. Astragalus polysaccharide (APS) is an active compound extracted from Chinese herb Astragalus membranaceus (AM), a frequently used “Qi-invigorating” herbal medicine in traditional medicine broadly used for the treatment of cardiovascular and other diseases. Currently, little is known about the effect of APS on cardiac hypertrophy. In the present study, we aimed to investigate its effect on cardiac hypertrophy and to clarify its possible mechanisms. In vitro cardiac hypertrophic model induced by isoprenaline (ISO) was employed to explore the anti-hypertrophic action of APS. We found that 10μM ISO treatment for 48h caused cultured cardiomyocytes to undergo significant increases in cell surface area, total protein content, protein synthesis as well as the expression of hypertrophic markers, including atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), which were effectively inhibited by APS in a dose dependent manner. Moreover, we found that APS pretreatment alleviated the augment of intracellular free calcium during cardiac hypertrophy induced by ISO. Our further study revealed that the upregulated expression of calcineurin, translocation of nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) into nucleus and activation of calmodulin kinase II (reflected by p-CaMKII) were dose dependently suppressed by the application of APS. According to this research, APS exerted its anti-hypertrophic action via inhibiting Ca2+-mediated calcineurin/NFATc3 and CaMKII signaling cascades, which provided new insights into the application of APS to the therapy of heart diseases.

208 The PKC Epsilon/AMPK ALPHA/ENOS Pathway is Implicated as a Mechanism by which Remote Ischaemic Conditioning Attenuates Endothelin-1 Mediated Cardiomyocyte Hypertrophy

IntroductionRemote ischaemic conditioning (rIC) has mainly been implicated in protection from ischemia/reperfusion injury. Using a model of endothelin-1 (ET-1) driven cardiomyocyte hypertrophy, we have previously shown that rIC attenuates ET-1...

P75NTR-dependent Rac1 activation requires receptor cleavage and activation of an NRAGE and NEDD9 signaling cascade.

The p75 neurotrophin receptor (p75NTR, also known as tumor necrosis factor receptor superfamily member 16) is implicated in diverse cellular events, but fundamental aspects of its signaling mechanisms remain unclear. To address this, we have established a novel bioassay to characterize signaling cascades activated by p75NTR. We show that in COS7 cells, p75NTR expression causes a large increase in cell surface area that relies on the activation of Rac1, and we demonstrate that the p75NTR-dependent COS7 phenotype is dependent on ADAM17- and c-secretase-dependent cleavage of p75NTR and generation of the p75NTR intracellular domain (p75NTRICD). We show that the p75NTR adaptor protein NRAGE (also known as MAGED1) acts downstream of the p75NTRICD in this cascade and, through a yeast two-hybrid screen, identify NEDD9, a Cas family adaptor protein, as a novel NRAGE-binding partner that mediates p75NTR-dependent Rac1 activation and cell spreading. Our results demonstrate a crucial role for p75NTR cleavage in small GTPase activation and define a novel Rac1 activation pathway involving the p75NTRICD, NRAGE andNEDD9.

23 The Role of AMPK and PKCε in the Attenuation of ET-1 Induced Cardiomyocyte Hypertrophy by RIC Human Serum

Rationale We have previously shown that serum from healthy volunteers who have undergone Remote Ischaemic Conditioning (RIC) reduces hypertrophy in rat cardiomyoblasts. Here we investigated the roles of AMPK and PKCe in this process. Methodology Blood was taken from healthy volunteers after 3 cycles of 5 min of upper arm cuff inflation/deflation and the serum applied to H9c2 cardiomyoblasts in culture. Cells were treated with endothelin-1 (ET-1) to stimulate hypertrophy, and cell area determined using immunofluorescence at 48 h. The role of AMPK-signalling in the anti-hypertrophic effect of RIC-serum was probed using the AMPK inhibitor compound-C and AMPK phosphorylation and PKCe translocation were analysed using Western blots. Results ET-1 increased cell surface area by >45% from 1.47 x 104 ± 0.08 μm 2 in control cells to 2.14 x 104 ± 0.09 μm 2 in ET-1 treated cells (n = 8, >1000 cells per treatment, p 2 (p 2 (p 2 ; ns). RIC-serum caused a transient translocation of PKCe after 30 min (76.3%), which dissipated after 48 h to be replaced by a significant increase in cytosolic PKCe (3.4 ± 0.5 fold, p Conclusion Our data suggests that PKCe and AMPK-signalling are involved in the anti-hypertrophic action of RIC-serum.

COX-2 is involved in ET-1-induced hypertrophy of neonatal rat cardiomyocytes: Role of NFATc3

Endothelin-1 (ET-1) is a critical molecule that involved in heart failure. It has been proved that ET-1 stimulation results in cardiac hypertrophy both in vitro and in vivo, but the mechanisms underlying remain largely unknown. In this study, we reported that cyclooxygenase-2 (COX-2) might be an important mediator of hypertrophic responses to ET-1 stimulation. In the cultured rat neonatal cardiomyocytes, ET-1 significantly upregulated the expression and activity of COX-2, which was accompanied by increase in cell surface area and BNP mRNA level. In contrast, ET-1-dependent cardiomyocyte hypertrophy was abolished by COX-2 selective inhibitors, NS-398 and celecoxib, or by COX-2 RNA interference, but the inhibitory effects could be diminished by pretreatment with PGE2. Furthermore, cyclosporin A (CsA) and knockdown of nuclear factor of activated T-cells c3 (NFATc3) inhibited the expression of COX-2 induced by ET-1, and NFATc3 could also bound to the -GGAAA- sequence in the promoter region of rat COX-2 gene, indicating that calcineurin/NFATc3 signaling participated in the transcriptional regulation of COX-2 following ET-1 treatment. These findings provided further insight into the roles of ET-1 in cardiac hypertrophy and suggested a potential therapeutic strategy against cardiac hypertrophy by inhibiting COX-2.

Adiponectin inhibits leptin‐induced cardiomyocyte hypertrophy by attenuation of calcineurin/NFAT activation

Leptin and adiponectin are adipokines which exert opposite effects on cardiac pathology. Leptin induces cardiomyocyte hypertrophy and hyperleptinemia has been shown to be a risk factor for developing heart failure. In contrast adiponectin is antihypertrophic and reduced plasma adiponectin levels constitute a cardiovascular risk factor. In the present study we determined whether adiponectin can modify leptin‐induced hypertrophy in cultured rat ventricular myocytes. Leptin (3.1 nM) treatment for 24 h produced a robust hypertrophy as evidenced by significantly increased cell surface area, α‐skeletal actin gene expression and rate of protein synthesis. These effects were associated with significantly increased calcineurin activity and import of the transcriptional factor NFAT3 into nuclei. The leptin–induced calcineurin activation was paralleled by increased intracellular Na+ and Ca2+ concentrations. The pro‐hypertrophic effect of leptin was completely prevented in myocytes co‐treated with adiponectin (0.5 μM). These effects were associated with attenuation of leptin‐induced elevations in intracellular Na+ and Ca2+ concentrations, calcineurin activation and NFAT3 nuclear translocation. Thus, adiponectin may represent an endogenous factor mitigating the pro‐hypertrophic effect of leptin by preventing calcineurin activation. Supported by the Canadian Institutes of Health Research.

Cytochrome P450 epoxygenase metabolite, 14,15-EET, protects against isoproterenol-induced cellular hypertrophy in H9c2 rat cell line

We have previously shown that isoproterenol-induced cardiac hypertrophy causes significant changes to cytochromes P450 (CYPs) and soluble epoxide hydrolase (sEH) gene expression. Therefore, in this study, we examined the effect of isoproterenol in H9c2 cells, and the protective effects of 14,15-EET against isoproterenol-induced cellular hypertrophy. Isoproterenol was incubated with H9c2 cells for 24 and 48h. To determine the protective effects of 14,15-EET, H9c2 cells were incubated with isoproterenol in the absence and presence of 14,15-EET. Thereafter, the expression of hypertrophic markers and different CYP genes were determined by real time-PCR. Our results demonstrated that isoproterenol significantly increased the expression of hypertrophic marker, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), parallel to a significant increase in cell surface area. Also, isoproterenol increased the mRNA expression of CYP1A1, CYP1B1, CYP2J3, CYP4F4 and CYP4F5, as well as the gene encoding sEH, EPHX2. On other hand, 14,15-EET significantly attenuated the isoproterenol-mediated induction of ANP, BNP, CYP1A1, CYP2J3, CYP4F4, CYP4F5 and EPHX2. Moreover 14,15-EET prevented the isoproterenol-mediated increase in cell surface area. Interestingly, 20-hydroxyeicosatetraenoic acid (20-HETE) treatment caused similar effects to that of isoproterenol treatment and induced cellular hypertrophy in H9c2 cells. In conclusion, isoproterenol induces cellular hypertrophy and modulates the expression of CYPs and EPHX2 in H9c2 cells. Furthermore, 14,15-EET exerts a protective effect against isoproterenol-induced cellular hypertrophy whereas, 20-HETE induced cellular hypertrophy in H9c2 cells.

Effects of Roscovitine on Schedule of Divisional Morphogenesis, Basal Bodies Proliferation and Cell Divisions in Tetrahymena thermophila

During cell cycle of a ciliate Tetrahymena thermophila the divisions of micro- and macronucleus, cortical morphogenesis and cytokinesis are temporarily coordinated. Cortical morphogenesis begins with proliferation of the new ciliary basal bodies (BBs) within meridional cortical rows of ciliary BBs, and with the local proliferation of BBs, which form the new oral apparatus (OA2), positioned subequatorialy and destined for prospective posterior daughter cell (opisthe). Prior to cytokinesis, two prospective daughter cells are of equal size and show metamery of their cortical patterns. We studied effects of 20 μM roscovitine (an inhibitor of several cyclin-dependent kinases) on the cell cycle progression of T. thermophila. We showed that roscovitine delayed cell division, delayed or arrested macronuclear division and induced increase of cell size and the number of BBs in the cortical rows. The increase in the number of BBs in cortical rows induced cell elongation which was proportional to the increase in cell surface area. There was uncoupling between this BBs proliferation which is continued during prolonged cell cycle and delayed cytokinesis, what resulted in topological alteration of the respective positions of the OA2 and of the contractile vacuole pores (CVPs). In roscovitine treated cells, the new OA2 was positioned subequatorialy, but the fission zone was shifted posterior to the equatorial plane of the cell and positioned across and in the extreme cases behind of the new OA2. This resulted in the formation of a large proter and small size opisthe. The roscovitine treatment induced a formation of a plethora of phenotypes of postdividing cells. We found that irrespective of changes in divisional morphogenesis induced by roscovitine treatment, all mature BBs were associated with the cdc14-like phosphatase. Taken together all these data indicate that during cell cycle of T. thermophila the normal morphology of the daughter cells depends on the proper division of micro- and macronucleus and on temporal control of BBs proliferation along the longitudinal rows, during OA2 stomatogenesis and during selection of BBs involved in differentiation of apical BBs (couplets) and cell division.

Tetrodotoxin attenuates isoproterenol-induced hypertrophy in H9c2 rat cardiac myocytes

Cardiac hypertrophy is often associated with an increased sympathetic drive, and both in vitro and in vivo studies have demonstrated the development of cardiomyocyte hypertrophy in response to either α- or β- adrenergic stimulation. The present study was carried out to determine whether the reversible sodium channel blocker tetrodotoxin (TTX) exerts a direct anti-hypertrophic effect on isoproterenol (ISO)-induced cell hypertrophy and find the underlying mechanism that regulate [Na(+)]( i ). The experiments were performed on cultured H9c2 cells exposed to ISO (10 μM) alone or combined with TTX (1 μM) for 48 h. Our results showed that ISO significantly increased cell surface area by 30 % and atrial natriuretic peptide gene expression by nearly twofold (p < 0.05 for both). These effects were associated with a significant reduction in the gene expression of Na(+)/K(+)-ATPase isoforms α2 and α3, whereas the α1 isoform was unaffected. Conversely, ISO increased Na(+)-H(+) exchanger 1 (NHE-1) gene expression by approximately 40 % and significantly increased [Na(+)]( i ) level by 50 % (p < 0.05 for both). ISO was also found to significantly increase aquaporin 4 gene expression by nearly ninefold (p < 0.05). All these effects were prevented when identical experiments were carried out in the presence of TTX, but the expression of NHE-1. The expression of sodium channel protein type 5 subunit alpha was unaffected by either ISO or TTX. When taken together, these studies show that TTX attenuates the hypertrophic effect of ISO and suggest a possible approach to limiting ISO-induced hypertrophy in clinical treatment.