cADP ribose and [Ca2+]iregulation in rat cardiac myocytes

cADP ribose (cADPR)-induced intracellular Ca2+ concentration ([Ca2+]i) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1–10 μM) induced sustained, concentration-dependent [Ca2+]i responses ranging from 50 to 500 nM, which were completely inhibited by 20 μM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca2+]i waves, increasing concentrations of cADPR increased wave frequency up to ∼250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca2+]i transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca2+]i responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca2+]i responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca2+]i response. These results indicate that in cardiac myocytes, cADPR induces Ca2+ release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.

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Effects of Saponin Monomer 13 of Dwarf Lilyturf Tuber on L-Type Calcium Currents in Adult Rat Ventricular Myocytes

The American Journal of Chinese Medicine ◽

10.1142/s0192415x05003417 ◽

2005 ◽

Vol 33 (05) ◽

pp. 797-806 ◽

Cited By ~ 12

Author(s):

Jin Tao ◽

Hongyi Wang ◽

Jiandong Chen ◽

Huae Xu ◽

Shengnan Li

Keyword(s):

Cardiac Myocytes ◽

Ventricular Myocytes ◽

Calcium Currents ◽

Inhibitory Effects ◽

Patch Clamp Recording ◽

Inactivation Curve ◽

Control Value ◽

Adult Rat ◽

Cardioprotective Effects ◽

Rat Cardiac Myocytes

The saponin monomer 13 of dwarf lilyturf tuber (DT-13), one of the saponin monomers of dwarf lilyturf tuber, has been found to have potent cardioprotective effects. In order to investigate the effect of DT-13 on L-type calcium currents ( I Ca,L ), exploring the mechanisms of DT-13's cardioprotective effects, we directly measured the I Ca,L in the adult rat cardiac myocytes exposed to DT-13 using standard whole-cell patch-clamp recording technique. Our results showed that DT-13 exerted inhibitory effects on the I Ca,L of the single adult rat cardiac myocytes. The current density was reduced by about 38% after exposure of the cells to DT-13 (0.1 μM) for 10 minutes, from the control value of 7.46 ± 1.31 pA/pF to 4.25 ± 0.35 pA/pF ( n = 6, p < 0.05). This I Ca,L -inhibiting action of DT-13 was concentration-dependent. DT-13 up-shifted the current-voltage (I-V) curve, but did not significantly affect the half activation potential (V0.5). V0.5 was from -11.8 ± 0.9 mV in the control to -12.6 ± 1.9 mV in the presence of DT-13 at 0.1 μmol/L. DT-13 at 0.1 μM did not markedly affect the activation of I Ca,L , but shifted the inactivation curve of I Ca,L to the left. In combination with previous reports, these results suggest that there might be a close relationship between the cardioprotective effects of dwarf lilyturf tuber and the inhibitory effects of DT-13 on L-type calcium currents.

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Abstract 5302: Control of Reversible Phosphorylation of Ryanodine Receptor by MiR-1 : Mechanism of Cardiac Arrhythmia

Circulation ◽

10.1161/circ.118.suppl_18.s_523 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Dmitry Terentyev ◽

Andriy Belevych ◽

Radmila Terentyeva ◽

Donald E Kuhn ◽

Geraldine E Malana ◽

...

Keyword(s):

Ryanodine Receptor ◽

Ventricular Myocytes ◽

Heart Association ◽

Regulatory Subunit ◽

Resting Cells ◽

Cellular Mechanisms ◽

Adult Rat ◽

Disease States ◽

Intracellular Ca ◽

Regulate Protein

MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1 , have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation-contraction coupling and Ca cycling in adult rat ventricular myocytes by using methods of electrophysiology, confocal Ca imaging and quantitative immunoblotting. Adenoviral-mediated overexpressions of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca current and flatten cytosolic Ca transients voltage dependency. The frequency of spontaneous Ca sparks recorded in intact resting cells was enhanced, while the sarcoplasmic reticulum Ca content was reduced in miR-1 - overexpressing myocytes as compared with controls. In the presence of beta-adrenergic receptor agonist isoproterenol, rhythmically paced miR-1 -overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca, events that occurred only rarely in control myocytes. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Immunological analysis with phospho-specific antibodies showed that while phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor at Ser-2814 (CaMKII) but not at Ser-2808 (PKA). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56alpha involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 through translational inhibition of this mRNA target, causes CaMKII-dependent hyperphosphorylation of RyR2 via disrupting localization of PP2A activity to this channel, enhances RyR2 activity, and promotes arrhythmogenic SR Ca release. This mechanism could contribute to induction of arrhythmia in disease states accompanied by elevated miR-1 . This research has received full or partial funding support from the American Heart Association, AHA National Center.

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A real-time three-dimensional confocal imaging system captures Ca2+ wave propagation in adult rat cardiac myocytes

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2006.08.103 ◽

2006 ◽

Vol 41 (6) ◽

pp. 1071-1071

Author(s):

Y EGUCHI ◽

N HIGASHIJIMA ◽

T TANAAMI ◽

Y HIROTA ◽

H ISHIDA

Keyword(s):

Wave Propagation ◽

Real Time ◽

Cardiac Myocytes ◽

Imaging System ◽

Three Dimensional ◽

Confocal Imaging ◽

Adult Rat ◽

Rat Cardiac Myocytes

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.265.2.h517 ◽

1993 ◽

Vol 265 (2) ◽

pp. H517-H522 ◽

Cited By ~ 12

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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Effects of β-adrenoceptor stimulation on contractility, [Ca2+]i, and Ca2+ current in diabetic rat cardiomyocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.274.6.h1849 ◽

1998 ◽

Vol 274 (6) ◽

pp. H1849-H1857 ◽

Cited By ~ 5

Author(s):

Atsushi Tamada ◽

Yuichi Hattori ◽

Hideki Houzen ◽

Yoichi Yamada ◽

Ichiro Sakuma ◽

...

Keyword(s):

Sarcoplasmic Reticulum ◽

Cardiac Myocytes ◽

Ventricular Myocytes ◽

Diabetic Rat ◽

Inotropic Response ◽

Dibutyryl Camp ◽

Rat Cardiomyocytes ◽

Adrenergic Response ◽

Significant Difference ◽

Intracellular Ca

The mechanism of the diminished inotropic response to β-adrenoceptor stimulation in diabetic hearts was studied in enzymatically isolated diabetic rat ventricular myocytes in comparison with age-matched controls. The increases in contractions and intracellular Ca2+ concentration ([Ca2+]i) transients produced by isoproterenol were markedly diminished in diabetic myocytes. The inotropic and [Ca2+]iresponses to forskolin and dibutyryl cAMP (DBcAMP) were also reduced. No significant difference was found in the stimulating effects of isoproterenol, forskolin, and DBcAMP on the L-type Ca2+ current ( I Ca) between control and diabetic myocytes. The rise of [Ca2+]iin response to rapid caffeine application, an index of sarcoplasmic reticulum (SR) Ca2+ content, was significantly decreased in diabetic myocytes. Isoproterenol, forskolin, and DBcAMP enhanced this [Ca2+]iresponse to caffeine in control myocytes more markedly than in diabetic myocytes. The changes in the isoproterenol responses observed in diabetic myocytes were prevented by insulin therapy. We conclude that 1) diabetes causes an impairment of the contractile and [Ca2+]iresponses of cardiac myocytes when stimulated at both β-adrenoceptors and the postreceptor level without affecting the I Ca response and 2) altered SR functions of uptake and/or release of Ca2+ may primarily contribute to the diminished β-adrenergic response.

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Response of isolated adult canine cardiac myocytes to prolonged hypoxia and reoxygenation

AJP Cell Physiology ◽

10.1152/ajpcell.1991.260.3.c383 ◽

1991 ◽

Vol 260 (3) ◽

pp. C383-C391 ◽

Cited By ~ 10

Author(s):

C. M. Hohl ◽

R. A. Altschuld

Keyword(s):

Cardiac Myocytes ◽

Species Differences ◽

Ventricular Myocytes ◽

Lactate Production ◽

Atp Production ◽

Adult Rat ◽

Respiratory Inhibitor ◽

Constant Rate ◽

Initial Content ◽

Rat Cardiac Myocytes

Isolated adult canine ventricular myocytes incubated in the absence of glucose with the respiratory inhibitor rotenone retained 67% of ATP (control, 26.0 +/- 0.9 nmol/mg protein) during 3-h incubation, yet phosphocreatine fell to 23% of initial content. Lactate production proceeded at a constant rate of 5 nmol.mg-1.min-1 in rotenone-treated glucose-free myocytes. A 36% decline in rod-shaped cells and an increase in percent 22Na permeation from 37% in aerobic cells (approximately 13 mM intracellular sodium) to 68% in rotenone-treated glucose-free myocytes paralleled the loss of ATP. Total exchangeable calcium was maintained at control aerobic levels. Exposure of canine cells to 3-h hypoxia in the absence of glucose followed by 5-min reoxygenation resulted in a 73% decrease in ATP, a rise in calcium from 3.3 +/- 0.2 to 6.6 +/- 1.6 nmol/mg, and an increase in 22Na permeation to 111%. Under these conditions the number of rod-shaped myocytes declined by 77%, with corresponding increases in viable contracted and hypercontracted myocytes. The response of canine myocytes to severe hypoxia and reaeration contrasts greatly to earlier studies using adult rat cardiac myocytes [see Hohl et al. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H1022-H1030, 1982]. Species differences with respect to basal metabolism, rates of ATP production and degradation, and regulation of cation movements are most likely responsible for the observed differences.

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Caffeine-induced Ca2+ sparks in mouse ventricular myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.2.h666 ◽

2000 ◽

Vol 278 (2) ◽

pp. H666-H669 ◽

Cited By ~ 6

Author(s):

Michael Ritter ◽

Zhi Su ◽

Kenneth W. Spitzer ◽

Hideyuki Ishida ◽

William H. Barry

Keyword(s):

Sarcoplasmic Reticulum ◽

Cell Surface ◽

Ryanodine Receptor ◽

Ventricular Myocytes ◽

Surface Membrane ◽

Release Channel ◽

Whole Cell ◽

Cell Surface Membrane ◽

Naturally Occurring ◽

Intracellular Ca

Ca2+ sparks are spatially localized intracellular Ca2+ release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor, RyR) opening induced by Ca2+ influx via L-type Ca2+ channels or by spontaneous RyR openings and have been thought to reflect Ca2+ release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca2+ concentration ([Ca2+]i) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca2+]i transient consists of the summation of sparks and that Ca2+ sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca2+ channel-RyR relation determine spark properties.

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ATP receptor-induced Ca2+ transients in cardiac myocytes: sources of mobilized Ca2+

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.4.c750 ◽

1989 ◽

Vol 257 (4) ◽

pp. C750-C758 ◽

Cited By ~ 52

Author(s):

M. B. De Young ◽

A. Scarpa

Keyword(s):

Sarcoplasmic Reticulum ◽

Ethylene Glycol ◽

Cardiac Myocytes ◽

Ventricular Myocytes ◽

Channel Blockers ◽

Tetraacetic Acid ◽

Free Medium ◽

Fura 2 ◽

Adult Rat ◽

Ca2 Channels

Addition of micromolar concentrations of extracellular ATP to adult rat cardiac ventricular myocytes increases cytosolic Ca2+ concentration ([Ca2+]). Experiments were performed on fura-2-loaded myocytes to determine whether the [Ca2+] rise was due to Ca2+ influx, release of Ca2+ from the sarcoplasmic reticulum (SR), or a combination of both. BAY K 8644 and nifedipine affected ATP-induced [Ca2+] transients, indicating involvement of voltage-sensitive Ca2+ channels. Addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or Ca2+ channel blockers significantly reduced cytosolic [Ca2+] changes due to addition of ATP or KCl without depleting Ca2+ stores (shown by ionomycin treatment in a Ca2+-free medium), demonstrating that these responses require Ca2+ influx. Depletion of intracellular Ca2+ stores by caffeine or ryanodine also diminished cytosolic [Ca2+] responses, indicating that a portion of the increased cytosolic [Ca2+] is due to Ca2+ release from SR. Norepinephrine potentiates the ATP-Ca2+ response, and this effect was not inhibited by depletion of intracellular stores. Although the data show that there are two Ca2+ sources in the cytosolic Ca2+ response to ATP, the pattern is also consistent with the hypothesis of Ca2+-induced Ca2+ release from cardiac SR.

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