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.

Hyperthyroid adult rat cardiomyocytes. I. Nucleotide content, beta- and alpha-adrenoreceptors, and cAMP production

1989 ◽  
Vol 257 (5) ◽  
pp. C948-C956 ◽  
Author(s):  
C. M. Hohl ◽  
S. Wetzel ◽  
R. H. Fertel ◽  
D. K. Wimsatt ◽  
G. P. Brierley ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Adenine Nucleotide ◽  
Phosphodiesterase Inhibitor ◽  
Cardiac Cells ◽  
Beta Agonist ◽  
Camp Production ◽  
Adult Rats ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Half Maximal Effective Concentration

Ventricular myocytes isolated from the hypertrophied hearts of thyrotoxic adult rats have an increase in mean protein content per myocyte (6.3 +/- 0.2 vs. 4.4 +/- 0.2 ng) compared with euthyroid cells. Viability and adenine nucleotide profiles are similar in both populations, but NAD content of the hyperthyroid myocytes is depressed (4.9 +/- 0.2 vs. 5.5 +/- 0.2 nmol/mg for controls) and UTP is higher (1.2 +/- 0.09 vs. 0.9 +/- 0.04 nmol/mg). Binding of (-)-[125I]iodocyanopindolol to intact hyperthyroid myocytes is increased by 42% compared with controls, with no change in the dissociation constant (Kd). This elevation in beta-receptor number is correlated to enhanced beta-agonist-induced adenosine 3',5'-cyclic monophosphate (cAMP) production. The half-maximal effective concentration (EC50) for the euthyroid isoproterenol dose-response curve is 2.14 x 10(-7) M but is decreased to 2.51 x 10(-8) M in hyperthyroid cardiac cells. Basal adenylate cyclase activity is apparently not affected by thyroid hormones, since basal cAMP levels for both groups are identical (5 pmol/mg) and both rise roughly twofold in the presence of a phosphodiesterase inhibitor. Forskolin-induced cAMP production and cAMP-specific phosphodiesterase activity are similar as well. In contrast to beta-adrenergic response, there are no significant differences in alpha 1-antagonist [3H]prazosin binding parameters between hyperthyroid and euthyroid cardiomyocytes.

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.

Evidence for functional sodium and calcium ion channels in the membrane of cultured cardiomyocytes of the adult rat

1983 ◽  
Vol 61 (11) ◽  
pp. 1312-1316 ◽  
Author(s):  
S. L. Jacobson ◽  
C. B. Kennedy ◽  
G. A. R. Mealing
Keyword(s):  
Action Potentials ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Culture Cells ◽  
Calcium Ion Channels ◽  
Spontaneous Action ◽  
Fetal Bovine ◽  
Spontaneous Action Potentials

Characteristics are reported for electrical activity of adult rat cardiomyocytes in long-term primary culture. Cells in vitro for 12 to 28 days have mean membrane potential of −53 mV, are electrically excitable, and some are spontaneously contractile. The action potential of these cells has a slow rate of depolarization and is abolished by methoxyverapamil (D-600) but not by tetrodotoxin (TTX). When cells are hyperpolarized by passage of an inward current, spontaneous action potentials cease and action potentials evoked by depolarizing pulses are then TTX sensitive. Fetal bovine serum is a constituent of the culture medium. Its temporary removal causes spontaneous contractility to cease but the cells remain electrically excitable.

Effect of angiotensin II on cytosolic free calcium in neonatal rat cardiomyocytes

1991 ◽  
Vol 261 (1) ◽  
pp. C77-C85 ◽  
Author(s):  
D. C. Kem ◽  
E. I. Johnson ◽  
A. M. Capponi ◽  
D. Chardonnens ◽  
U. Lang ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Acute Effect ◽  
Neonatal Rat ◽  
Free Calcium ◽  
Receptor Subtype ◽  
Ang Ii ◽  
Rat Cardiomyocytes ◽  
Neonatal Rat Ventricular Myocytes

The effect of angiotensin II (ANG II) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured neonatal rat ventricular myocytes. [Ca2+]i was estimated in groups of one to three cells by dual-wavelength microfluorometry or in cell populations using conventional fluorometry. ANG II (10(-8) M) produced an acute short-lived increase over the control basal diastolic [Ca2+]i and increased the frequency of the [Ca2+]i transients. The amplitude of the [Ca2+]i transients was decreased to 64.4% of basal values. The effect of ANG II on [Ca2+]i was blocked by the selective AT1 receptor subtype antagonist Du Pont 753 but not by the AT2 antagonist PD 123319. Removal of extracellular Ca2+ or blockade of voltage-gated Ca2+ channels in cells cultured for 5-7 days abolished the [Ca2+]i transients, but only partially diminished the effect of ANG II on [Ca2+]i. Thapsigargin, an inhibitor of sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase, reduced or abolished the [Ca2+]i response to ANG II. Phorbol 12-myristate 13-acetate (PMA), 10(-6) and 10(-7) M, also decreased the amplitude of the Ca2+ transients similar to ANG II. Pretreatment with 10(-6) M PMA or 10(-6) M 1-oleoyl-2-acetyl-glycerol (OAG) inhibited the initial rise in [Ca2+]i and the Ca2+ transients. Thus ANG II produces an acute rise in [Ca2+]i which is derived predominantly from sarcoplasmic reticulum intracellular stores. This acute effect is followed by a significant reduction in the amplitude for the Ca2+ transient and may be mediated by activation of protein kinase C.

Development of instrumentation to measure rapid calcium transits in neonatal rat ventricular myocytes

1988 ◽  
Vol 46 ◽  
pp. 44-45
Author(s):  
H. K. Hagler ◽  
A. C. Morris ◽  
L. M. Buja
Keyword(s):  
Intracellular Calcium ◽  
Cell Injury ◽  
Ventricular Myocytes ◽  
Calcium Transients ◽  
Neonatal Rat ◽  
Free Calcium ◽  
Total Calcium ◽  
X Ray ◽  
Calcium Indicators ◽  
Electron Microscopes

Changes in the intracellular calcium levels in cardiac myocytes are important in the regulation of normal cardiac function and have been implicated in contributing to irreversible cell injury with ischemia or hypoxia. Intracellular measurement of total calcium changes with subcellular resolution have become routine using rapid cryofixation, cryosectioning, cryotransfer and energy dispersive x-ray microanalysis in analytical electron microscopes. The x-ray microanalysis technique measures total calcium changes within subcellular compartments, but does not distinguish between the bound and free calcium. With the successful development of fluorescent calcium indicators which may be introduced into cells without significantly buffering the intracellular calcium levels, it has become possible to measure rapid calcium transients during contraction. The primary requirements in the development of a system to utilize the fluorescent calcium indicators were to resolve calcium transients in individual cells (since the response to perturbations such as hypoxia is heterogeneous) and develop a system which would be flexible enough to accommodate new indicators as they become available.

The effects of sodium, hydrogen and magnesium ions on mitochondrial calcium sequestration in adult rat ventricular myocytes

1984 ◽  
Vol 223 (1231) ◽  
pp. 239-254 ◽  
Keyword(s):  
Ventricular Myocytes ◽  
Calcium Handling ◽  
Magnesium Ions ◽  
Cardiac Mitochondria ◽  
Inotropic Effects ◽  
Adult Rat ◽  
Calcium Sequestration ◽  
Sodium Hydrogen ◽  
Calcium Exchange ◽  
Semi Empirical

The effect of Na + , H + and Mg 2+ ions on net calcium exchange induced in digitonin-treated myocytes has been investigated. Raising the [Na] from 1.4 to 31.4 mm revealed a sodium-sensitive fraction of net calcium exchange with a K 1/2 2 for Na + ions of 12 mm, alongside the respiration-dependent accumulation of calcium. An acidosis, but not an alkalosis, was found to depress both of these processes. Mg 2+ ions exerted an effect solely on the respiration-dependent calcium sequestration. A simple semi-empirical model based on the experimental data was formulated to assess the effects that altering sarcoplasmic [Na + ] and [H + ] would have on the calcium-handling properties of cardiac mitochondria. It is concluded that part of the inotropic effects of these ions could be mediated via this organelle.

Thapsigargin inhibits Ca2+ uptake, and Ca2+ depletes sarcoplasmic reticulum in intact cardiac myocytes

1993 ◽  
Vol 265 (2) ◽  
pp. H517-H522 ◽  
Author(s):  
A. M. Janczewski ◽  
E. G. Lakatta
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Rat Myocardium ◽  
Half Time ◽  
Adult Rat ◽  
Time To Peak ◽  
Rapid Pacing ◽  
Ethanesulfonic Acid

We examined the effects of thapsigargin on Ca2+ accumulation by the sarcoplasmic reticulum (SR) and on electrically stimulated beats in single adult rat ventricular myocytes loaded with indo 1 and bathed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer containing 1 mM Ca2+ at 23 degrees C. The SR Ca2+ content was assessed from the magnitude of intracellular Ca2+ (Ca2+i) transients and contractions elicited by rapid, brief applications of caffeine. After 20-30 min of exposure to 200 nM thapsigargin, the caffeine-dependent Ca2+i transients were abolished or markedly diminished (by 89 +/- 4%). The postrest potentiation of the Ca2+i transient and contraction, typical for rat myocardium, was abolished. Thapsigargin did not significantly change resting Ca2+i but diminished the amplitude of the steady-state Ca2+i transients by 73%, prolonged the time to peak by 24%, and prolonged the half-time (t1/2) of the Ca2+i transient decline by 42%. Progressive SR Ca2+ depletion by thapsigargin was strongly related (r = -0.78) to the prolongation of the t1/2 of relaxation of the steady-state Ca2+i transients, suggesting that the thapsigargin-dependent SR Ca2+ depletion results from an inhibition of the SR Ca2+ uptake. This interpretation was corroborated by comparison of the effects of thapsigargin with those of ryanodine (100 nM), which depletes SR of Ca2+ by accelerating the SR Ca2+ efflux but does not inhibit the SR Ca2+ pump. During rapid pacing (5 Hz), which raises Ca2+i and thus Ca2+ available for SR uptake, the caffeine-dependent SR Ca2+ release was restored in ryanodine-treated cells but not in the presence of thapsigargin.(ABSTRACT TRUNCATED AT 250 WORDS)

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