Adult rat cardiomyocytes exhibit capacitative calcium entry

2004 ◽  
Vol 286 (3) ◽  
pp. H1124-H1132 ◽  
Author(s):  
Dacia L. Hunton ◽  
LuYun Zou ◽  
Yi Pang ◽  
Richard B. Marchase
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Physiological Responses ◽  
Atpase Inhibitor ◽  
Capacitative Calcium Entry ◽  
Rat Cardiomyocytes ◽  
Inward Current ◽  
Adult Rat ◽  
Adult Cardiomyocytes ◽  
Inositol 1,4,5 Trisphosphate

Capacitative Ca2+ entry (CCE) refers to the influx of Ca2+ through plasma membrane channels activated on depletion of endoplasmic-sarcoplasmic reticulum Ca2+ stores. We utilized two Ca2+-sensitive dyes (one monitoring cytoplasmic free Ca2+ and the other free Ca2+ within the sarcoplasmic reticulum) to determine whether adult rat ventricular myocytes exhibit CCE. Treatments with inhibitors of the sarcoplasmic endoplasmic reticulum Ca2+-ATPases were not efficient in releasing Ca2+ from stores. However, when these inhibitors were coupled with either Ca2+ ionophores or angiotensin II (an agonist generating inositol 1,4,5 trisphosphate), depletion of stores was observed. This depletion was accompanied by a significant influx of extracellular Ca2+ characteristic of CCE. CCE was also observed when stores were depleted with caffeine. This influx of Ca2+ was sensitive to four inhibitors of CCE (glucosamine, lanthanum, gadolinium, and SKF-96365) but not to inhibitors of L-type channels or the Na+/Ca2+ exchanger. In the whole cell configuration, an inward current of ∼0.7 pA/pF at –90 mV was activated when a Ca2+ chelator or inositol (1,4,5)-trisphosphate was included in the pipette or when Ca2+ stores were depleted with a Ca2+-ATPase inhibitor and ionophore. The current was maximal at hyperpolarizing voltages and inwardly rectified. The channel was relatively permeant to Ca2+ and Ba2+ but only poorly to Mg2+ or Mn2+. Taken together, these data support the existence of CCE in adult cardiomyocytes, a finding with likely implications to physiological responses to phospholipase C-generating agonists.

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.

scholarly journals Forced expression of the cyclin B1–CDC2 complex induces proliferation in adult rat cardiomyocytes

2004 ◽  
Vol 382 (2) ◽  
pp. 411-416 ◽  
Author(s):  
Katrina A. BICKNELL ◽  
Carmen H. COXON ◽  
Gavin BROOKS
Keyword(s):  
Cell Division ◽  
Cyclin B1 ◽  
Cell Division Cycle ◽  
Myocardial Regeneration ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Adult Cardiomyocytes ◽  
Mammalian Myocardium

Repair of the mature mammalian myocardium following injury is impaired by the inability of the majority of cardiomyocytes to undergo cell division. We show that overexpression of the cyclin B1–CDC2 (cell division cycle 2 kinase) complex re-initiates cell division in adult cardiomyocytes. Thus strategies targeting the cyclin B1–CDC2 complex might re-initiate cell division in mature cardiomyocytes in vivo and facilitate myocardial regeneration following injury.

Free radicals enhance Na+/Ca2+ exchange in ventricular myocytes

1996 ◽  
Vol 271 (3) ◽  
pp. H823-H833 ◽  
Author(s):  
J. I. Goldhaber
Keyword(s):  
Free Radicals ◽  
Free Radical ◽  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Similar Response ◽  
Inward Current ◽  
Oxygen Derived Free Radicals ◽  
Triggered Arrhythmias ◽  
Rabbit Ventricular Myocytes ◽  
Generating System

Oxygen-derived free radicals (OFR) have been implicated in the pathogenesis of intracellular Ca2+ overload and the arrhythmias that characterize cardiac reperfusion. These arrhythmias may in large part be due to activation of the pathological transient inward current (ITI). However, the identity of the ITI generated by OFR is uncertain. We previously found that H2O2, an OFR-generating compound, markedly stimulated the ITI elicited by brief caffeine pulses in patch-clamped guinea pig ventricular myocytes. In the present study, using patch-clamped rabbit ventricular myocytes loaded with the Ca(2+)-sensitive indicator fura 2, we have further characterized this ITI and have identified its major component to be Na+/Ca2+ exchange based on its dependence on extracellular Na+ and sarcoplasmic reticulum Ca2+ release, its sensitivity to Ni2+, and the effects of its inhibition on relaxation. The effect on ITI was not unique to H2O2, because another free radical-generating system, xanthine + xanthine oxidase, produced a similar response. We hypothesize that enhancement of Na+/Ca2+ exchange by OFR during reperfusion, when intracellular Na+ is elevated, may promote intracellular Ca2+ overload and triggered arrhythmias.

scholarly journals Inositol 1,4,5-Trisphosphate Directs Ca2+Flow between Mitochondria and the Endoplasmic/Sarcoplasmic Reticulum: A Role in Regulating Cardiac Autonomic Ca2+ Spiking

2000 ◽  
Vol 11 (5) ◽  
pp. 1845-1858 ◽  
Author(s):  
Marisa Jaconi ◽  
Claire Bony ◽  
Stephen M. Richards ◽  
André Terzic ◽  
Serge Arnaudeau ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Excitable Cells ◽  
Rat Cardiomyocytes ◽  
Cell Functions ◽  
Inositol 1,4,5 Trisphosphate ◽  
Cellular Ca ◽  
Cardiac Autonomic Activity

The signaling role of the Ca2+ releaser inositol 1,4,5-trisphosphate (IP3) has been associated with diverse cell functions. Yet, the physiological significance of IP3 in tissues that feature a ryanodine-sensitive sarcoplasmic reticulum has remained elusive. IP3 generated by photolysis of caged IP3 or by purinergic activation of phospholipase Cγ slowed down or abolished autonomic Ca2+ spiking in neonatal rat cardiomyocytes. Microinjection of heparin, blocking dominant-negative fusion protein, or anti-phospholipase Cγ antibody prevented the IP3-mediated purinergic effect. IP3 triggered a ryanodine- and caffeine-insensitive Ca2+ release restricted to the perinuclear region. In cells loaded with Rhod2 or expressing a mitochondria-targeted cameleon and TMRM to monitor mitochondrial Ca2+ and potential, IP3 induced transient Ca2+ loading and depolarization of the organelles. These mitochondrial changes were associated with Ca2+ depletion of the sarcoplasmic reticulum and preceded the arrest of cellular Ca2+ spiking. Thus, IP3 acting within a restricted cellular region regulates the dynamic of calcium flow between mitochondria and the endoplasmic/sarcoplasmic reticulum. We have thus uncovered a novel role for IP3 in excitable cells, the regulation of cardiac autonomic activity.

Reduced expression of the Na+/Ca2+ exchanger in adult cardiomyocytes via adenovirally delivered shRNA results in resistance to simulated ischemic injury

2010 ◽  
Vol 298 (2) ◽  
pp. H360-H366 ◽  
Author(s):  
Thane G. Maddaford ◽  
Elena Dibrov ◽  
Cecilia Hurtado ◽  
Grant N. Pierce
Keyword(s):  
Gene Expression ◽  
Nucleotide Sequence ◽  
Contractile Activity ◽  
Ischemic Injury ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Adult Cardiomyocytes ◽  
Adenoviral Delivery ◽  
Significant Depression ◽  
Short Time

The Na+/Ca2+ exchanger (NCX) is proposed to be an important protein in the regulation of Ca2+ movements in the heart. This Ca2+ regulatory action is thought to modulate contractile activity in the heart under normal physiological conditions and may contribute to the Ca2+ overload that occurs during ischemic reperfusion challenge. To evaluate these hypotheses, adult rat cardiomyocytes were exposed to an adenovirus that codes for short hairpin RNA (shRNA) targeting NCX gene expression through RNA interference. An adenovirus transcribing a short RNA with a scrambled nucleotide sequence was compared with the NCX-shRNA nucleotide sequence and used as a control. Freshly isolated rat cardiomyocytes were infected with virus for 48 h before examination. Cardiomyocytes maintained their characteristic morphological appearance during this short time period after isolation. NCX expression was inhibited by up to ∼60% by the shRNA treatment as determined by Western blot analysis. The depletion in NCX protein was accompanied by a significant depression of NCX activity in shRNA-treated cells. Ca2+ homeostasis was unaltered in the shRNA-treated cells upon electrical stimulation compared with control cells. However, when cardiomyocytes were exposed to a simulated ischemic solution, NCX-depleted cells were significantly protected from the rise in cytoplasmic Ca2+ and damage that was detected in control cells during ischemia and reperfusion. Our data support the role for NCX in ischemic injury to the heart and demonstrate the usefulness of altering gene expression with an adenoviral-delivery system of shRNA in adult cardiomyocytes.

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.

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.

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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