Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes

2001 ◽  
Vol 281 (5) ◽  
pp. H2079-H2088 ◽  
Author(s):  
Xue-Qian Zhang ◽  
Jianliang Song ◽  
Lawrence I. Rothblum ◽  
Mingyue Lun ◽  
Xujun Wang ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Contractile Function ◽  
Electrical Field Stimulation ◽  
Western Blots ◽  
Adult Rat ◽  
Gfp Fluorescence ◽  
Intracellular Ca ◽  
Functional Consequences ◽  
Rat Myocytes ◽  
Green Fluorescent

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant ( P ≤ 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.

Overexpression of phospholemman alters contractility and [Ca2+]i transients in adult rat myocytes

2002 ◽  
Vol 283 (2) ◽  
pp. H576-H583 ◽  
Author(s):  
Jianliang Song ◽  
Xue-Qian Zhang ◽  
Lois L. Carl ◽  
Anwer Qureshi ◽  
Lawrence I. Rothblum ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Contractile Function ◽  
Maximal Contraction ◽  
Western Blots ◽  
Adult Rat ◽  
Rat Myocytes ◽  
Green Fluorescent ◽  
Rat Cardiac Myocytes

Previous studies showed increased phospholemman (PLM) mRNA after myocardial infarction (MI) in rats (Sehl PD, Tai JTN, Hillan KJ, Brown LA, Goddard A, Yang R, Jin H, and Lowe DG. Circulation 101: 1990–1999, 2000). We tested the hypothesis that, in normal adult rat cardiac myocytes, PLM overexpression alters contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis in a manner similar to that observed in post-MI myocytes. Compared with myocytes infected by control adenovirus expressing green fluorescent protein (GFP) alone, Western blots indicated a 41% increase in PLM expression after 72 h ( P < 0.001) but no changes in Na+/Ca2+ exchanger, SERCA2, and calsequestrin levels in myocytes infected by adenovirus expressing GFP and PLM. At 5 mM extracellular [Ca2+] ([Ca2+]o), maximal contraction amplitudes in PLM-overexpressed myocytes were 24% ( P < 0.005) and [Ca2+]i transient amplitudes were 18% ( P < 0.05) lower than control myocytes. At 0.6 mM [Ca2+]o, however, contraction and [Ca2+]i transient amplitudes were significantly ( P < 0.05) higher in PLM-overexpressed than control myocytes (18% and 42%, respectively); at 1.8 mM [Ca2+]o, the differences in contraction and [Ca2+]i transient amplitudes were narrowed. This pattern of contractile and [Ca2+]itransient abnormalities in PLM-overexpressed myocytes mimics that observed in post-MI rat myocytes. We suggest that PLM overexpression observed in post-MI myocytes may partly account for contractile abnormalities by perturbing Ca2+ fluxes during excitation-contraction.

scholarly journals Effects of sprint training on contractility and [Ca2+]i transients in adult rat myocytes

2002 ◽  
Vol 93 (4) ◽  
pp. 1310-1317 ◽  
Author(s):  
Xue-Qian Zhang ◽  
Jianliang Song ◽  
Lois L. Carl ◽  
Weixing Shi ◽  
Anwer Qureshi ◽  
...  
Keyword(s):  
Contractile Function ◽  
Sprint Training ◽  
Cell Width ◽  
Western Blots ◽  
Adult Rat ◽  
Protein Levels ◽  
Intracellular Ca ◽  
Uptake Activity ◽  
Rat Myocytes ◽  
Myocyte Contractility

The effects of 6–8 wk of high-intensity sprint training (HIST) on rat myocyte contractility and intracellular Ca2+ concentration ([Ca2+]i) transients were investigated. Compared with sedentary (Sed) myocytes, HIST induced a modest (5%) but significant ( P < 0.0005) increase in cell length with no changes in cell width. In addition, the percentage of myosin heavy chain α-isoenzyme increased significantly ( P < 0.02) from 0.566 ± 0.077% in Sed rats to 0.871 ± 0.006% in HIST rats. At all three (0.6, 1.8, and 5 mM) extracellular Ca2+concentrations ([Ca2+]o) examined, maximal shortening amplitudes and maximal shortening velocities were significantly ( P < 0.0001) lower and half-times of relaxation were significantly ( P < 0.005) longer in HIST myocytes. HIST myocytes had significantly ( P < 0.0001) higher diastolic [Ca2+]i levels. Compared with Sed myocytes, systolic [Ca2+]ilevels in HIST myocytes were higher at 0.6 mM [Ca2+]o, similar at 1.8 mM [Ca2+]o, and lower at 5 mM [Ca2+]o. The amplitudes of [Ca2+]i transients were significantly ( P < 0.0001) lower in HIST myocytes. Half-times of [Ca2+]i transient decline, an estimate of sarcoplasmic reticulum (SR) Ca2+ uptake activity, were not different between Sed and HIST myocytes. Compared with Sed hearts, Western blots demonstrated a significant ( P < 0.03) threefold decrease in Na+/Ca2+ exchanger, but SR Ca2+-ATPase and calsequestrin protein levels were unchanged in HIST hearts. We conclude that HIST effected diminished myocyte contractile function and [Ca2+]itransient amplitudes under the conditions studied. We speculate that downregulation of Na+/Ca2+ exchanger may partly account for the decreased contractility in HIST myocytes.

Phospholemman modulates Na+/Ca2+exchange in adult rat cardiac myocytes

2003 ◽  
Vol 284 (1) ◽  
pp. H225-H233 ◽  
Author(s):  
Xue-Qian Zhang ◽  
Anwer Qureshi ◽  
Jianliang Song ◽  
Lois L. Carl ◽  
Qiang Tian ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Contractile Function ◽  
Resting Membrane Potential ◽  
Adult Rat ◽  
Protein Levels ◽  
Rat Myocytes ◽  
T Tubules ◽  
Green Fluorescent ◽  
Myocyte Contractility ◽  
Rat Cardiac Myocytes

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca2+ homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na+/Ca2+exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold ( P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na+ out:1 Ca2+ in) was significantly ( P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca2+] ([Ca2+]o), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca2+]o, the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na+/Ca2+ exchange.

Rescue of contractile abnormalities by Na+/Ca2+ exchanger overexpression in postinfarction rat myocytes

2002 ◽  
Vol 93 (6) ◽  
pp. 1925-1931 ◽  
Author(s):  
Xue-Qian Zhang ◽  
Jianliang Song ◽  
Anwer Qureshi ◽  
Lawrence I. Rothblum ◽  
Lois L. Carl ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Electrical Field Stimulation ◽  
Green Fluorescent Protein Fluorescence ◽  
Protein Fluorescence ◽  
Electrical Field ◽  
Rat Hearts ◽  
Intracellular Ca ◽  
Rat Myocytes ◽  
Green Fluorescent

Previous studies on myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated increased cell length, reduced Na+/Ca2+ exchange (NCX1) activity, altered contractility, and intracellular Ca2+ concentration ([Ca2+]i) transients. In the present study, we investigated whether NCX1 overexpression in MI myocytes would restore contraction and [Ca2+]i transients to normal. When myocytes were placed in culture under continued electrical-field stimulation conditions, differences in contraction amplitudes and cell lengths between sham and MI myocytes were preserved for at least 48 h. Infection of both sham and MI myocytes by adenovirus expressing green fluorescent protein resulted in >95% infection, as evidenced by green fluorescent protein fluorescence, but contraction amplitudes at 6-, 24-, and 48-h postinfection were not affected. NCX1 overexpression in MI myocytes resulted in lower diastolic [Ca2+]i levels at all extracellular Ca2+ concentrations ([Ca2+]o) examined, suggesting enhanced forward NCX1 activity. At 5 mM [Ca2+]o, subnormal contraction and [Ca2+]i transient amplitudes in MI myocytes (compared with sham myocytes) were restored toward normal levels by overexpressing NCX1. At 0.6 mM [Ca2+]o, supranormal contraction and [Ca2+]i transient amplitudes in MI myocytes (compared with sham myocytes) were lowered by NCX1 overexpression. We conclude that overexpression of NCX1 in MI myocytes was effective in improving contractile dysfunction, most likely because of enhancement of both Ca2+ efflux and influx during a cardiac cycle. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in postinfarction myocytes.

Effects of phospholemman downregulation on contractility and [Ca2+]i transients in adult rat cardiac myocytes

2004 ◽  
Vol 286 (4) ◽  
pp. H1322-H1330 ◽  
Author(s):  
M. Ayoub Mirza ◽  
Xue-Qian Zhang ◽  
Belinda A. Ahlers ◽  
Anwer Qureshi ◽  
Lois L. Carl ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Contractile Function ◽  
Transient Behavior ◽  
Normal Adult ◽  
Adult Rat ◽  
Rat Hearts ◽  
Rat Myocytes ◽  
Green Fluorescent ◽  
Rat Cardiac Myocytes

Phospholemman (PLM) expression was increased in rat hearts after myocardial infarction (MI). Overexpression of PLM in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis in a manner similar to that observed in post-MI myocytes. In this study, we tested whether PLM downregulation in normal adult rat myocytes resulted in contractility and [Ca2+]i transient changes opposite to those observed in post-MI myocytes. Compared with control myocytes infected with adenovirus (Adv) expressing green fluorescent protein (GFP) alone, myocytes infected with Adv expressing both GFP and rat antisense PLM (rASPLM) had 23% less PLM protein ( P < 0.012) at 3 days, but no differences were found in sarcoplasmic reticulum (SR) Ca2+-ATPase, Na+/Ca2+ exchanger (NCX1), Na+-K+-ATPase, and calsequestrin levels. SR Ca2+ uptake and whole cell capacitance were not affected by rASPLM treatment. Relaxation from caffeine-induced contracture was faster, and NCX1 current amplitudes were higher in rASPLM myocytes, indicating that PLM downregulation enhanced NCX1 activity. In native rat cardiac myocytes, coimmunoprecipitation experiments indicated an association of PLM with NCX1. At 0.6 mM [Ca2+]o, rASPLM myocytes had significantly ( P < 0.003) lower contraction and [Ca2+]i transient amplitudes than control GFP myocytes. At 5 mM [Ca2+]o, both contraction and [Ca2+]i transient amplitudes were higher in rASPLM myocytes. This pattern of contractile and [Ca2+]i transient behavior in rASPLM myocytes was opposite to that observed in post-MI rat myocytes. We conclude that downregulation of PLM in normal rat cardiac myocytes enhanced NCX1 function and affected [Ca2+]i transient and contraction amplitudes. We suggest that PLM downregulation offers a potential therapeutic strategy for ameliorating contractile abnormalities in MI myocytes.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R

2007 ◽  
Vol 292 (5) ◽  
pp. F1303-F1313 ◽  
Author(s):  
Xianhua Yi ◽  
Richard Bouley ◽  
Herbert Y. Lin ◽  
Shaliha Bechoua ◽  
Tian-xiao Sun ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Human Kidney ◽  
Lysosomal Degradation ◽  
Interacting Protein ◽  
Parathyroid Hormone Receptor ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
G Protein Coupled ◽  
Regulatory Event

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK1 epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

scholarly journals Could Cells from Your Nose Fix Your Heart? Transplantation of Olfactory Stem Cells in a Rat Model of Cardiac Infarction

2010 ◽  
Vol 10 ◽  
pp. 422-433 ◽  
Author(s):  
Cameron McDonald ◽  
Alan Mackay-Sim ◽  
Denis Crane ◽  
Wayne Murrell
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Fluorescent Protein ◽  
Genetically Engineered ◽  
Olfactory Mucosa ◽  
Cell Transplant ◽  
Adult Rat ◽  
Cardiac Ventricle ◽  
Green Fluorescent

This study examines the hypothesis that multipotent olfactory mucosal stem cells could provide a basis for the development of autologous cell transplant therapy for the treatment of heart attack. In humans, these cells are easily obtained by simple biopsy. Neural stem cells from the olfactory mucosa are multipotent, with the capacity to differentiate into developmental fates other than neurons and glia, with evidence of cardiomyocyte differentiationin vitroand after transplantation into the chick embryo. Olfactory stem cells were grown from rat olfactory mucosa. These cells are propagated as neurosphere cultures, similar to other neural stem cells. Olfactory neurospheres were grownin vitro, dissociated into single cell suspensions, and transplanted into the infarcted hearts of congeneic rats. Transplanted cells were genetically engineered to express green fluorescent protein (GFP) in order to allow them to be identified after transplantation. Functional assessment was attempted using echocardiography in three groups of rats: control, unoperated; infarct only; infarcted and transplanted. Transplantation of neurosphere-derived cells from adult rat olfactory mucosa appeared to restore heart rate with other trends towards improvement in other measures of ventricular function indicated. Importantly, donor-derived cells engrafted in the transplanted cardiac ventricle and expressed cardiac contractile proteins.

Taste Receptor Cell Responses to the Bitter Stimulus Denatonium Involve Ca2+ Influx Via Store-Operated Channels

2002 ◽  
Vol 87 (6) ◽  
pp. 3152-3155 ◽  
Author(s):  
Tatsuya Ogura ◽  
Robert F. Margolskee ◽  
Sue C. Kinnamon
Keyword(s):  
Prolonged Exposure ◽  
Fluorescent Protein ◽  
Bitter Taste ◽  
Taste Receptor ◽  
Taste Receptor Cell ◽  
Cell Responses ◽  
Necturus Maculosus ◽  
Taste Cells ◽  
Intracellular Ca ◽  
Green Fluorescent

Previous studies in rat and mouse have shown that brief exposure to the bitter stimulus denatonium induces an increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores, rather than Ca2+influx. We report here that prolonged exposure to denatonium induces sustained increases in [Ca2+]i that are dependent on Ca2+ influx. Similar results were obtained from taste cells of the mudpuppy, Necturus maculosus, as well as green fluorescent protein (GFP) tagged gustducin-expressing taste cells of transgenic mice. In a subset of mudpuppy taste cells, prolonged exposure to denatonium induced oscillatory Ca2+responses. Depletion of Ca2+ stores by thapsigargin also induced Ca2+ influx, suggesting that Ca2+store-operated channels (SOCs) are present in both mudpuppy taste cells and gustducin-expressing taste cells of mouse. Further, treatment with thapsigargin prevented subsequent responses to denatonium, suggesting that the SOCs were the source of the Ca2+ influx. These data suggest that SOCs may contribute to bitter taste transduction and to regulation of Ca2+ homeostasis in taste cells.

scholarly journals Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic β-cells

2005 ◽  
Vol 185 (1) ◽  
pp. 57-67 ◽  
Author(s):  
L B Hays ◽  
B Wicksteed ◽  
Y Wang ◽  
J F McCuaig ◽  
L H Philipson ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Fluorescent Protein ◽  
Zymogen Granule ◽  
Β Cells ◽  
Rat Islets ◽  
Intracellular Ca ◽  
Specific Localization ◽  
Glucagon Like Peptide 1 ◽  
Green Fluorescent ◽  
Insulin Exocytosis

Several proteins play a role in the mechanism of insulin exocytosis. However, these ‘exocytotic proteins’ have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet β-cells as experimental tools. Syncollin is not normally expressed in β-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of β-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5μM). Consistent with specific localization of syncollin to β-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in β-cells has highlighted the importance of intralumenal β-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a ‘fluorescent tag’ to track β-granule transport and exocytosis in real time.

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