Energy depletion-repletion and calcium transients in single cardiomyocytes

1989 ◽  
Vol 257 (3) ◽  
pp. C427-C434 ◽  
Author(s):  
Q. Li ◽  
C. M. Hohl ◽  
R. A. Altschuld ◽  
B. T. Stokes
Keyword(s):  
Electrical Stimulation ◽  
Cellular Response ◽  
Contractile Activity ◽  
Calcium Transients ◽  
Free Calcium ◽  
Variable Rate ◽  
Time Resolved ◽  
Adult Rat ◽  
Carbonyl Cyanide ◽  
Rat Heart Myocytes

Rapid fluctuations of intracellular free calcium in single adult rat heart myocytes were monitored by time-resolved fura-2 fluorescence microscopy. Under controlled aerobic conditions (35 degrees C, pH 7.3), electrical stimulation at 0.5 Hz produced a concave negative staircase of calcium transients. When the myocytes were challenged with 3 mM amobarbital (Amytal) and 2 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to deplete ATP, the cells became unresponsive to electrical stimulation within 1 min but responded to 10 mM caffeine with a large increase in free calcium. After the development of rigor contracture, the cellular response to caffeine was blunted. Free calcium increased at a variable rate in individual cells, reaching values of 300-1,000 nM after 15 min. When the inhibitors were removed, calcium declined toward control values, and spontaneous contractile activity and calcium transients were invariably observed. During subsequent electrical stimulation, there was a decrease in the half-widths of the calcium transients and an attenuation of the negative staircase. Parallel experiments with cells in suspension indicated that Amytal and CCCP caused ATP to fall from 27.6 +/- 1.6 to 0.7 +/- 0.2 nmol/mg protein, and the percent rod-shaped cells to fall from 70 to 0% in 5 min. Removal of the inhibitors after 15 min caused a rebound in ATP to 5.3 +/- 1.5 nmol/mg within 2 min and 6.6 +/- 1.3 nmol/mg after 10 min.

Hyperthyroid adult rat cardiomyocytes. II. Single cell electrophysiology and free calcium transients

1989 ◽  
Vol 257 (5) ◽  
pp. C957-C963 ◽  
Author(s):  
Q. Li ◽  
Z. Guan ◽  
B. A. Biagi ◽  
B. T. Stokes ◽  
R. A. Altschuld
Keyword(s):  
Single Cell ◽  
Action Potentials ◽  
Mechanical Performance ◽  
Ventricular Myocytes ◽  
Calcium Transients ◽  
Calcium Handling ◽  
Free Calcium ◽  
Half Time ◽  
Rat Cardiomyocytes ◽  
Adult Rat

The effects of hyperthyroidism on electrophysiological properties and intracellular free calcium transients in single adult rat cardiomyocytes were studied using conventional microelectrodes and time-resolved single cell fura-2 fluorescence microscopy. Under control conditions, resting membrane potentials and triggered action potentials were not different in euthyroid and hyperthyroid myocytes. Calcium transients produced by electrical stimulation, however, were markedly abbreviated in hyperthyroid myocytes. During a train of stimuli, the duration of the calcium transients at half peak amplitude (half time) was 124 +/- 14 ms at the fifth beat in hyperthyroid cells vs. 287 +/- 35 ms in euthyroid cells. Isoproterenol (1 microM) prolonged time to 50% repolarization (APD50) of the action potentials and increased the peak calcium transients in both euthyroid and hyperthyroid myocytes. It also shortened the half time of the calcium transients in euthyroid myocytes but had little effect on the half time in hyperthyroid cells. These data are consistent with the electrophysiology and mechanical performance in intact euthyroid and hyperthyroid cardiac tissues, and the intrinsic changes in hyperthyroid tissues can therefore be illustrated in single ventricular myocytes. Furthermore, the results suggest that alterations in intracellular calcium handling by sarcoplasmic reticulum may account for contractile changes of the heart induced by hyperthyroidism.

Myocyte deenergization and intracellular free calcium dynamics

1988 ◽  
Vol 255 (2) ◽  
pp. C162-C168 ◽  
Author(s):  
Q. A. Li ◽  
R. A. Altschuld ◽  
B. T. Stokes
Keyword(s):  
Ventricular Myocytes ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Calcium Dynamics ◽  
Adult Rat ◽  
Quiescent Cells ◽  
A Value ◽  
Rate Of Increase ◽  
Carbonyl Cyanide ◽  
High Concentrations

Intracellular free calcium in adult rat heart ventricular myocytes was monitored by single cell fura-2 fluorescence microscopy. The average resting free calcium in rod-shaped quiescent cells was 125 nM (range 70-200 nM). When cells were deenergized with an inhibitor (amytal) and an uncoupler (carbonyl-cyanide m-chlorophenylhydrazone) of oxidative phosphorylation, there was a small but significant increase (125-380 nM) in intracellular free calcium during the transition to a highly contracted (square) rigor form. After the onset of contracture, which occurred 5-15 min after addition of the above compounds, the increase in free calcium was slow for the first 20 min, reaching a value of only 750 nM. Thereafter, the rate of increase accelerated and 50 min after contracture, free calcium was approximately 3 microM. The increase in free calcium was absolutely dependent on extracellular calcium but was not inhibited by high concentrations of verapamil (2-7 microM), suggesting influx via the Na+-Ca2+ exchange transporter as the cause of calcium increase. However, in calcium repletion protocols the rate of increase in sodium-loaded myocytes was greatly accelerated if cells were not depleted of ATP, confirming suggestions that ATP loss partially inhibits Na+-Ca2+ exchange.

Increase vs. decrease of calcium uptake by isolated heart cells induced by H2O2 vs. HOCl

1989 ◽  
Vol 256 (3) ◽  
pp. C598-C607 ◽  
Author(s):  
T. Kaminishi ◽  
T. Matsuoka ◽  
T. Yanagishita ◽  
K. J. Kako
Keyword(s):  
Calcium Uptake ◽  
Isolated Heart ◽  
Atp Depletion ◽  
Heart Cell ◽  
Heart Cells ◽  
Adult Rat ◽  
Isolated Heart Cells ◽  
Highly Sensitive ◽  
Carbonyl Cyanide ◽  
Rat Heart Myocytes

Adult rat heart myocytes were labeled rapidly with exogenous [45Ca2+]. Addition of 2.5 mM H2O2 to the heart cell suspension raised the content of rapidly exchangeable intracellular Ca2+ twofold, whereas addition of 1-30 mM HOCl decreased the Ca2+ content. The H2O2-induced increase in Ca2+ content was dependent on the medium Na+, pH, and temperature but was not significantly affected by addition of verapamil, diltiazem, amiloride, or 3-aminobenzamide. The [3H]ouabain binding to myocytes was suppressed by H2O2, whereas the Ca2+ efflux from myocytes was not influenced. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, reduced Ca2+ content, implying that the H2O2-induced change in Ca2+ content was not directly related to ATP depletion. On the other hand, the H2O2-induced Ca2+ accumulation in myocytes was prevented by deferoxamine or o-phenanthroline. These results suggest that H2O2 inhibited Na+-K+-ATPase, resulting in an increase in intracellular Na+ concentration and stimulation of sarcolemmal Na+-Ca2+ exchange activity, which caused a transient net Ca2+ influx into myocytes. By contrast, HOCl decreased the Ca2+ content of the rapidly exchangeable pool below control levels and this action of HOCl was antagonized by 1,4-dithiothreitol. HOCl accelerated Ca2+ efflux from myocytes. Ca2+ uptake and Ca2+-ATPase of the isolated sarcoplasmic reticular (SR) fraction were highly sensitive to the action of HOCl. Ca2+ uptake by intracellular sites, studied with myocytes permeabilized with digitonin, was inhibited by both H2O2 and HOCl. Thus these results suggest that HOCl inhibits the SR Ca2+ pump, resulting in the observed acceleration of Ca2+ efflux from and decline in Ca2+ content of myocytes.

Myocyte contractile activity modulates norepinephrine cytotoxicity and survival effects of neuregulin-1β

2004 ◽  
Vol 286 (2) ◽  
pp. C222-C229 ◽  
Author(s):  
Yukio Kuramochi ◽  
Chee Chew Lim ◽  
Xinxin Guo ◽  
Wilson S. Colucci ◽  
Ronglih Liao ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Cell Survival ◽  
Ventricular Myocytes ◽  
Contractile Activity ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Adult Rat ◽  
Quiescent Cells ◽  
Stress Kinase ◽  
Induced Apoptosis ◽  
Myocyte Contractility

The purpose of this study is to test the hypothesis that mechanical and electrical activity in adult rat ventricular myocytes (ARVM) alters responses to proapoptotic and prosurvival ligands. The effects of electrical stimulation on myocyte survival, stress signaling, response to β-adrenergic receptor (β-AR)-stimulated apoptosis, and neuregulin-1β (NRG) were examined. Electrical stimulation (6.6 V/cm; 0, 2, and 5 Hz; 2-ms duration; alternating polarity) of ARVM resulted in more than 70% capture. Although ARVM paced for 48 h showed higher mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( P < 0.05, 0 vs. 2 and 5 Hz), electrical stimulation had little effect on cell survival assessed by trypan blue uptake, CPK release, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Electrical stimulation for 24 h did not induce stress response (heat shock protein 70, 90) nor stress kinase (Erk, JNK, p38) activation. NRG stimulation of Erk and Akt was similar between paced and quiescent cells. Pacing sensitized myocytes to β-AR-stimulated JNK phosphorylation and cell death with 0.1 μM norepinephrine (NE) in paced myocytes causing equivalent cytotoxicity to 10 μM NE in quiescent cells. NRG suppressed β-AR-induced apoptosis through a phosphatidylinositol-3-kinase-dependent pathway in both paced and quiescent cells, although it is overwhelmed by high-NE concentration in paced cells. Thus myocyte contractility modulates both NE cytotoxicity as well as the cytoprotective effect of NRG. These results demonstrate the feasibility and importance of using electrically paced cardiomyocytes in primary culture when examining the signaling pathways of cell survival.

Interactions between sustained contractile activity and beta-adrenergic receptors in regulation of gene expression in skeletal muscles

1989 ◽  
Vol 256 (3) ◽  
pp. C506-C514 ◽  
Author(s):  
W. E. Kraus ◽  
T. S. Bernard ◽  
R. S. Williams
Keyword(s):  
Electrical Stimulation ◽  
Steady State ◽  
Oxidative Metabolism ◽  
Adrenergic Receptors ◽  
Contractile Activity ◽  
Mitochondrial Enzymes ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Induced Changes ◽  
Aldolase A

Continuous electrical stimulation for 10-21 days of the motor nerve innervating the anterior compartment muscles of adult rabbits increased both the density of beta-adrenergic receptors (beta-AR) and tissue concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) by two to threefold. Changes in cAMP and in beta-AR occurred in parallel with stimulation-induced adaptations in the specific activity of mitochondrial enzymes (2- to 6-fold increases) and with changes in steady-state concentrations of mitochondrial RNA, beta-F1ATPase mRNA, and myoglobin mRNA (2- to 11-fold increases). These increases in muscle cAMP, in beta-AR, and in expression of protein and mRNA products of genes encoding proteins of oxidative metabolism occurred even in animals receiving high doses of propranolol during the period of electrical stimulation. In contrast to genes that encode proteins of oxidative metabolism, the direction and the time course of activity-induced changes in expression of the glycolytic enzyme aldolase A appeared to be unrelated to changes in muscle cAMP; suppression of steady-state concentrations of aldolase A mRNA was maximal (20-25% of control) at early time points preceding the maximal rise in cAMP. In addition, administration of propranolol attenuated the suppressive effect of continuous contractile activity on expression of aldolase A, even in the absence of an effect of this drug on cAMP in stimulated muscles. We conclude that activity-induced changes in cAMP, in beta-AR, and in expression of genes that encode proteins important for oxidative metabolism occur as a direct consequence of contractile activity and do not require concomitant stimulation of beta-AR.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular free Ca2+ and the hypercontracture of adult rat heart myocytes

1986 ◽  
Vol 245 (2) ◽  
pp. 426-435 ◽  
Author(s):  
Mindy R. Lambert ◽  
J.David Johnson ◽  
Karla G. Lamka ◽  
Gerald P. Brierley ◽  
Ruth A. Altschuld
Keyword(s):  
Rat Heart ◽  
Adult Rat ◽  
Rat Heart Myocytes

scholarly journals Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos

2000 ◽  
Vol 113 (19) ◽  
pp. 3519-3529 ◽  
Author(s):  
C. Leclerc ◽  
S.E. Webb ◽  
C. Daguzan ◽  
M. Moreau ◽  
A.L. Miller
Keyword(s):  
Xenopus Laevis ◽  
Calcium Signalling ◽  
Neural Induction ◽  
Calcium Transients ◽  
Free Calcium ◽  
Treated Animal ◽  
Cell Stage ◽  
Subsequent Formation ◽  
Xenopus Laevis Embryos

Through the injection of f-aequorin (a calcium-sensitive bioluminescent reporter) into the dorsal micromeres of 8-cell stage Xenopus laevis embryos, and the use of a Photon Imaging Microscope, distinct patterns of calcium signalling were visualised during the gastrulation period. We present results to show that localised domains of elevated calcium were observed exclusively in the anterior dorsal part of the ectoderm, and that these transients increased in number and amplitude between stages 9 to 11, just prior to the onset of neural induction. During this time, however, no increase in cytosolic free calcium was observed in the ventral ectoderm, mesoderm or endoderm. The origin and role of these dorsal calcium-signalling patterns were also investigated. Calcium transients require the presence of functional L-type voltage-sensitive calcium channels. Inhibition of channel activation from stages 8 to 14 with the specific antagonist R(+)BayK 8644 led to a complete inhibition of the calcium transients during gastrulation and resulted in severe defects in the subsequent formation of the anterior nervous system. BayK treatment also led to a reduction in the expression of Zic3 and geminin in whole embryos, and of NCAM in noggin-treated animal caps. The possible role of calcium transients in regulating developmental gene expression is discussed.

Protein kinase Cε and the antiadrenergic action of adenosine in rat ventricular myocytes

2004 ◽  
Vol 287 (4) ◽  
pp. H1721-H1729 ◽  
Author(s):  
Koji Miyazaki ◽  
Satoshi Komatsu ◽  
Mitsuo Ikebe ◽  
Richard A. Fenton ◽  
James G. Dobson
Keyword(s):  
Electrical Stimulation ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Adrenergic Agonist ◽  
Inhibitory Peptide ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Tubular System ◽  
Stimulation Of

Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine A1 receptors (A1R). The role of PKCε in the antiadrenergic action of adenosine was explored with adult rat ventricular myocytes in which PKCε was overexpressed. Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKCε construct and compared with normal myocytes. The extent of myocyte shortening elicited by electrical stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKCε was found localized primarily in transverse tubules. The A1R agonist chlorocyclopentyladenosine (CCPA) at 1 μM rendered an enhanced localization of PKCε in the t-tubular system. The β-adrenergic agonist isoproterenol (Iso; 0.4 μM) elicited a 29–36% increase in myocyte shortening in all three groups. Although CCPA significantly reduced the Iso-produced increase in shortening in all three groups, the reduction caused by CCPA was greatest with PKCε overexpression. The CCPA reduction of the Iso-elicited shortening was eliminated in the presence of a PKCε inhibitory peptide. These results suggest that the translocation of PKCε to the t-tubular system plays an important role in A1R-mediated antiadrenergic actions in the heart.

scholarly journals Release of enzymes from adult rat heart myocytes.

1982 ◽  
Vol 51 (5) ◽  
pp. 560-568 ◽  
Author(s):  
M P Murphy ◽  
C Hohl ◽  
G P Brierley ◽  
R A Altschuld
Keyword(s):  
Rat Heart ◽  
Adult Rat ◽  
Rat Heart Myocytes
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