Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes

1993 ◽  
Vol 264 (3) ◽  
pp. C677-C686 ◽  
Author(s):  
L. Hove-Madsen ◽  
D. M. Bers
Keyword(s):  
Ventricular Myocytes ◽  
Ruthenium Red ◽  
Cell Protein ◽  
Uptake Curve ◽  
Spatial Gradients ◽  
Ca Uptake ◽  
Intracellular Ca ◽  
Rabbit Ventricular Myocytes ◽  
The Difference ◽  
Binding Curve

Passive Ca binding was measured with a Ca-selective minielectrode in suspensions of permeabilized rabbit ventricular myocytes equilibrated with 5 microM thapsigargin and 30 microM ruthenium red to prevent sarcoplasmic reticulum (SR) or mitochondrial Ca uptake. Passive Ca binding was obtained by titration of the myocytes with Ca and subtraction of Ca binding in a blank titration without myocytes. Passive Ca binding could be described by a Michaelis binding curve with two sites: K1 = 0.42 microM n1 = 1.27 nmol/mg cell protein and K2 = 79 microM, n2 = 4.13 nmol/mg cell protein. The passive Ca buffering over the physiological Ca concentration was approximately twice the value expected from the values compiled by Fabiato [A. Fabiato. Am. J. Physiol. 245 (Cell Physiol. 14): C1-C14, 1983]. The maximal SR Ca uptake in the presence of 30 microM ruthenium red was fit by an uptake curve with a maximal uptake of 5.16 nmol/mg cell protein and a K 1/2 of 1.0 microM. In the presence of 5 microM thapsigargin and no ruthenium red, a significant Ca uptake attributed to mitochondria was measured between 10 and 100 microM free Ca. Rapid changes in free Ca concentration ([Ca]) measured with a Ca electrode were slower than simultaneous measurements of free [Ca] with indo-1 in permeabilized myocytes. However, oxalate, which buffers Ca and maximizes SR Ca uptake, increased the uptake rate and eliminated the difference in free [Ca] measured with Ca electrode and indo-1. This suggests that spatial gradients of [Ca] exist in permeabilized myocytes without Ca buffering. The new estimates of the buffering of intracellular Ca in cardiac myocytes should be valuable in developing quantitative insights into cardiac Ca regulation.

Sarcoplasmic reticulum and Na+/Ca2+exchanger function during early and late relaxation in ventricular myocytes

1997 ◽  
Vol 273 (6) ◽  
pp. H2765-H2773 ◽  
Author(s):  
Atsushi Yao ◽  
Hiroshi Matsui ◽  
Kenneth W. Spitzer ◽  
John H. B. Bridge ◽  
William H. Barry
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Initial Rate ◽  
Terminal Phase ◽  
Adult Rabbit ◽  
High K ◽  
Intracellular Ca ◽  
Rabbit Ventricular Myocytes ◽  
The Difference ◽  
Rate Of Decline

The relative importance of the Na+/Ca2+exchanger in the initial and terminal phases of relaxation and the decline in the [Ca2+]itransient was investigated in adult rabbit ventricular myocytes loaded with the Ca2+ indicator fluo 3. For electrically stimulated contractions, the peak intracellular Ca2+ concentration ([Ca2+]i) was 700 ± 87 nM and end-diastolic [Ca2+]iwas 239 ± 30 nM (0.25 Hz, 37°C, 1.08 mM extracellular Ca2+ concentration; n = 14). Abrupt inhibition of Na+/Ca2+exchange was produced by removal of extracellular Na+ (KCl substitution) and Ca2+ [2 mM Ca2+-free ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid] by means of a rapid switcher device (SW). Abrupt exposure to high K+ induced an action potential, although sufficient Ca2+ remained adjacent to the sarcolemma to induce a contraction (SW beat) and [Ca2+]itransient that were identical in amplitude to those induced by electrical stimulation (ES beat). The initial relaxation and decline in the [Ca2+]itransient was not significantly prolonged by abrupt elimination of the Na+/Ca2+exchanger, but the rate and extent of the terminal phase of the decline in the [Ca2+]itransient were significantly reduced. The first derivative of [Ca2+]iwith respect to time versus [Ca2+]iduring the decline of the [Ca2+]itransient attributable to sarcoplasmic reticulum (SR) function was estimated from the average SW transients, and that attributable to Na+/Ca2+exchange was estimated from the difference between SW and ES transients. By this analysis, the Na+/Ca2+exchanger produces 13% of the first half of the decline in [Ca2+]iand 45% of the second half of the decline. We conclude that abrupt inhibition of forward Na+/Ca2+exchange does not significantly affect the amplitude or the initial rate of decline of the [Ca2+]itransient and relaxation. However, its contribution to the reduction of [Ca2+]ibecomes apparent late during the [Ca2+]itransient, when cytosolic [Ca2+]ihas been reduced.

scholarly journals Mechano-arrhythmogenicity is enhanced during late repolarisation in ischemia and driven by a TRPA1-, calcium-, and reactive oxygen species-dependent mechanism

2021 ◽  
Author(s):  
Breanne Ashleigh Cameron ◽  
T Alexander Quinn
Keyword(s):  
Reactive Oxygen Species ◽  
Ventricular Myocytes ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Cellular Mechanisms ◽  
Sustained Activity ◽  
Intracellular Ca ◽  
Rabbit Ventricular Myocytes ◽  
Induced Changes ◽  
Dependent Mechanism

Background: Cardiac dyskinesis in regional ischemia results in arrhythmias through mechanically-induced changes in electrophysiology ('mechano-arrhythmogenicity') that involve ischemic alterations in voltage-calcium (Ca2+) dynamics, creating a vulnerable period (VP) in late repolarisation. Objective: To determine cellular mechanisms of mechano-arrhythmogenicity in ischemia and define the importance of the VP. Methods and Results: Voltage-Ca2+ dynamics were simultaneously monitored in rabbit ventricular myocytes by dual-fluorescence imaging to assess the VP in control and simulated ischemia (SI). The VP was longer in SI than in control (146±7 vs 54±8 ms; p<0.0001) and was reduced by blocking KATP channels with glibenclamide (109±6 ms; p<0.0001). Cells were rapidly stretched (10-18% increase in sarcomere length over 110-170 ms) with carbon fibres during diastole or the VP. Mechano-arrhythmogenicity, associated with stretch and release in the VP, was greater in SI than control (7 vs 1% of stretches induced arrhythmias; p<0.005) but was similar in diastole. Arrhythmias during the VP were more complex than in diastole (100 vs 69% had sustained activity; p<0.05). In the VP, incidence was reduced with glibenclamide (2%; p<0.05), by chelating intracellular Ca2+ (BAPTA; 2%; p<0.05), blocking mechano-sensitive TRPA1 (HC-030031; 1%; p<0.005), or by scavenging (NAC; 1%; p<0.005) or blocking reactive oxygen species (ROS) production (DPI; 2%; p<0.05). Ratiometric Ca2+ imaging revealed that SI increased diastolic Ca2+ (+9±1%, p<0.0001), which was not prevented by HC-030031 or NAC. Conclusion: In ischemia, mechano-arrhythmogenicity is enhanced specifically during the VP and is mediated by ROS, TRPA1, and Ca2+.

Twitch-dependent SR Ca accumulation and release in rabbit ventricular myocytes

1993 ◽  
Vol 265 (2) ◽  
pp. C533-C540 ◽  
Author(s):  
J. W. Bassani ◽  
R. A. Bassani ◽  
D. M. Bers
Keyword(s):  
Steady State ◽  
Ventricular Myocytes ◽  
State Level ◽  
Exposure Period ◽  
Steady State Level ◽  
Fluorescent Indicator ◽  
Intracellular Ca ◽  
Rabbit Ventricular Myocytes ◽  
Time Required ◽  
Ca Depletion

Using caffeine-induced contractures (Ccaf) and thapsigargin (TG), we estimated the fraction of sarcoplasmic reticulum (SR) Ca released at one twitch and also the number of twitches required to reload a Ca-depleted SR. Similar results were obtained for twitches or intracellular Ca (Cai) transient with the fluorescent indicator, indo 1. Sustained exposure to 10 mM caffeine completely depletes the SR of Ca in < 5 s (as assessed by a second Ccaf). After such Ca depletion, four to five twitches are necessary to reload the SR to the steady-state level (with a twitch constant, tau = 1.6 twitches). We also determined the time required for complete inhibition of the SR Ca-adenosinetriphosphatase (ATPase) by TG. After SR Ca depletion, 5 microM TG was applied for different periods of time before a train of "reloading" twitches. A TG exposure period of 90 s was sufficient to completely prevent Ccaf after these reloading twitches. When SR is Ca depleted, the twitch is larger in the presence of TG, indicating that the SR Ca-ATPase can limit the ability of Ca influx to activate contraction. To assess SR Ca released at one twitch in cells with normally Ca-loaded SR, 5 microM TG was applied for 90 s to prevent SR Ca reuptake. Then one or several twitches were activated (causing SR Ca release, but with reuptake completely blocked). After the twitch (or train), a Ccaf was used to assess remaining SR Ca.(ABSTRACT TRUNCATED AT 250 WORDS)

CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias

1999 ◽  
Vol 276 (6) ◽  
pp. H2168-H2178 ◽  
Author(s):  
Yuejin Wu ◽  
Leigh B. MacMillan ◽  
R. Blair McNeill ◽  
Roger J. Colbran ◽  
Mark E. Anderson
Keyword(s):  
Ventricular Myocytes ◽  
Ryanodine Receptors ◽  
Cam Kinase ◽  
Early Afterdepolarizations ◽  
Ultrastructural Studies ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
T Tubules ◽  
Rabbit Ventricular Myocytes

Early afterdepolarizations (EAD) caused by L-type Ca2+ current ( I Ca,L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in I Ca,L during repolarization. I Ca,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. I Ca,Laugmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase.

Abstract 14409: TRPA1 Channels Are a Source of Calcium-driven Cellular Mechano-arrhythmogenicity

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Breanne A Cameron ◽  
Matthew R Stoyek ◽  
T Alexander Quinn
Keyword(s):  
Molecular Mechanisms ◽  
Sarcomere Length ◽  
Fluorescent Dyes ◽  
Ventricular Myocytes ◽  
Cardiac Mechanics ◽  
Cell Stiffness ◽  
Intracellular Ca ◽  
Rabbit Ventricular Myocytes ◽  
Induced Changes ◽  
Stretch Activated Channels

Introduction: Pathologic changes in myocardial mechanics and hemodynamic load result in arrhythmias via mechanically-induced changes in electrophysiology or intracellular Ca 2+ (‘mechano-arrhythmogenicity’). While molecular mechanisms driving mechano-arrhythmogenicity are poorly defined, they are associated with disease-related alterations in voltage-Ca 2+ dynamics. Objective: Define mechanisms of mechano-arrhythmogenicity during alterations in voltage-Ca 2+ dynamics in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100ms) during diastole or late repolarisation in control or during K ATP channel activation (pinacidil). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), non-selectively block stretch-activated channels (streptomycin), or specifically block (HC-030031) or activate (AITC) mechano-sensitive TRPA1 channels. Voltage-Ca 2+ dynamics were simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system and diastolic Ca 2+ was measured using Fura Red. Results: Pinacidil caused greater shortening of the AP than Ca 2+ transient (-144±17 vs -74±11ms; n =24 cells, N =7 rabbits; p <0.001) with no change in cell stiffness or contractility. Stretch during pinacidil application caused arrhythmias in both diastole and late repolarisation (8 and 10% of stretches; n =46, N =5), which voltage-Ca 2+ imaging revealed were Ca 2+ -driven. Arrhythmias were reduced with BAPTA (3 and 0% of stretches; p <0.05), streptomycin (4 and 2%; p <0.05), and HC-030031(2 and 1%; p <0.01), while dantrolene had no effect ( n =40, N =5 for each). Stretch in diastole during AITC application also caused arrhythmias (15%; p <0.001), which were blocked by HC-030031 (4%; p <0.001) or BAPTA (3%; p <0.001; n =40, N =5 each). Both AITC and pinacidil caused an increase in diastolic Ca 2+ (112±29 and 78±29% of control; p<0.05), which was reduced by HC-030031 with AITC (26±24%; p <0.05), but not with pinacidil ( n =25, N =5 each). Conclusions: TRPA1 activation increases mechano-arrhythmogenicity via a Ca 2+ -driven mechanism and may represent a novel anti-arrhythmic target in pathologies involving altered cardiac mechanics.

scholarly journals Revisiting the ionic mechanisms of early afterdepolarizations in cardiomyocytes: predominant by Ca waves or Ca currents?

2012 ◽  
Vol 302 (8) ◽  
pp. H1636-H1644 ◽  
Author(s):  
Zhenghang Zhao ◽  
Hairuo Wen ◽  
Nadezhda Fefelova ◽  
Charelle Allen ◽  
Akemichi Baba ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Ryanodine Receptors ◽  
Convincing Evidence ◽  
Rate Dependency ◽  
Early Afterdepolarizations ◽  
Relative Contribution ◽  
Intracellular Ca ◽  
Ionic Mechanisms ◽  
Rabbit Ventricular Myocytes ◽  
Bayk 8644

Early afterdepolarizations (EADs) have been implicated in severe cardiac arrhythmias and sudden cardiac deaths. However, the mechanism(s) for EAD genesis, especially regarding the relative contribution of Ca2+ wave (CaW) vs. L-type Ca current ( ICa,L), still remains controversial. In the present study, we simultaneously recorded action potentials (APs) and intracellular Ca2+ images in isolated rabbit ventricular myocytes and systematically compared the properties of EADs in the following two pharmacological models: 1) hydrogen peroxide (H2O2; 200 μM); and 2) isoproterenol (100 nM) and BayK 8644 (50 nM) (Iso + BayK). We assessed the rate dependency of EADs, the temporal relationship between EADs and corresponding CaWs, the distribution of EADs over voltage, and the effects of blockers of ICa,L, Na/Ca exchangers, and ryanodine receptors. The most convincing evidence came from the AP-clamp experiment, in which the cell membrane clamp was switched from current clamp to voltage clamp using a normal AP waveform without EAD; CaWs disappeared in the H2O2 model, but persisted in the Iso + BayK model. We postulate that, although CaWs and reactivation of ICa,L may act synergistically in either case, reactivation of ICa,L plays a predominant role in EAD genesis under oxidative stress (H2O2 model), while spontaneous CaWs are a predominant cause for EADs under Ca2+ overload condition (Iso + BayK model).

Diastolic SR Ca efflux in atrial pacemaker cells and Ca-overloaded myocytes

1997 ◽  
Vol 273 (2) ◽  
pp. H886-H892 ◽  
Author(s):  
R. A. Bassani ◽  
J. W. Bassani ◽  
S. L. Lipsius ◽  
D. M. Bers
Keyword(s):  
Spontaneous Activity ◽  
Ventricular Myocytes ◽  
Cardiac Cells ◽  
Transport Systems ◽  
Pacemaker Activity ◽  
Pacemaker Cells ◽  
Sa Node ◽  
Ca Uptake ◽  
Rabbit Ventricular Myocytes ◽  
Ca Depletion

Evidence has shown that the sarcoplasmic reticulum (SR) of cardiac cells releases Ca not only during excitation-contraction coupling but also during diastole, albeit at a much lower rate. This diastolic SR Ca release (leak) has also been implicated in the generation of spontaneous depolarization in latent atrial pacemaker cells of the cat right atrium. In the present work, we sought to measure Ca transients in pacemaker and nonpacemaker cells of the cat using the fluorescent Ca indicator indo 1. Atrial latent pacemaker cells develop a slow Ca transient when rested in the presence of both Na- and Ca-free solution and thapsigargin [used to inhibit Na/Ca exchange and SR Ca adenosinetriphosphatase (Ca-ATPase), respectively]. This increase in cytosolic Ca concentration ([Ca]i) is probably caused by the rate of SR Ca leak exceeding the capacity of the remaining Ca transport systems (e.g., sarcolemmal Ca-ATPase and mitochondrial Ca uptake). However, neither cat sinoatrial (SA) node cells nor myocytes from cat atrium or ventricle exhibited a similar increase in [Ca]i during the same protocol. This indicates that SR Ca leak in these cells occurred at a rate low enough to be within the capacity of the slow Ca transporters, as observed previously in rabbit ventricular myocytes. When atrial and ventricular myocytes were stimulated at higher frequencies, sufficient to markedly increase diastolic and systolic [Ca]i and approach Ca overload (and spontaneous activity), they responded to inhibition of SR Ca-ATPase and Na/Ca exchange with a slow Ca transient similar to that normally observed in atrial latent pacemaker cells. Furthermore, the SR Ca depletion by thapsigargin did not affect spontaneous activity of SA node cells, but it prevented or slowed pacemaker activity in the atrial latent pacemaker cells. These findings suggest that enhanced diastolic SR Ca efflux contributes significantly to the generation of spontaneous activity in atrial subsidiary pacemakers under normal conditions and in Ca-overloaded myocytes but not in SA node cells.

scholarly journals Cardiac small-conductance calcium-activated potassium channels in health and disease

2021 ◽  
Vol 473 (3) ◽  
pp. 477-489 ◽  
Author(s):  
Xiao-Dong Zhang ◽  
Phung N. Thai ◽  
Deborah K. Lieu ◽  
Nipavan Chiamvimonvat
Keyword(s):  
Ventricular Myocytes ◽  
Pulmonary Veins ◽  
Differential Sensitivity ◽  
Sk Channels ◽  
Sk Channel ◽  
Ventricular Cells ◽  
Intracellular Ca ◽  
Life Threatening ◽  
Health And Disease ◽  
Small Conductance

AbstractSmall-conductance Ca2+-activated K+ (SK, KCa2) channels are encoded by KCNN genes, including KCNN1, 2, and 3. The channels play critical roles in the regulation of cardiac excitability and are gated solely by beat-to-beat changes in intracellular Ca2+. The family of SK channels consists of three members with differential sensitivity to apamin. All three isoforms are expressed in human hearts. Studies over the past two decades have provided evidence to substantiate the pivotal roles of SK channels, not only in healthy heart but also with diseases including atrial fibrillation (AF), ventricular arrhythmia, and heart failure (HF). SK channels are prominently expressed in atrial myocytes and pacemaking cells, compared to ventricular cells. However, the channels are significantly upregulated in ventricular myocytes in HF and pulmonary veins in AF models. Interests in cardiac SK channels are further fueled by recent studies suggesting the possible roles of SK channels in human AF. Therefore, SK channel may represent a novel therapeutic target for atrial arrhythmias. Furthermore, SK channel function is significantly altered by human calmodulin (CaM) mutations, linked to life-threatening arrhythmia syndromes. The current review will summarize recent progress in our understanding of cardiac SK channels and the roles of SK channels in the heart in health and disease.

scholarly journals Effects of acidosis on resting cytosolic and mitochondrial Ca2+ in mammalian myocardium.

1993 ◽  
Vol 102 (3) ◽  
pp. 575-597 ◽  
Author(s):  
G Gambassi ◽  
R G Hansford ◽  
S J Sollott ◽  
B A Hogue ◽  
E G Lakatta ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Ventricular Myocytes ◽  
Free Acid ◽  
Left Ventricular ◽  
Ruthenium Red ◽  
Acetoxymethyl Ester ◽  
Cytosolic Ph ◽  
Bicarbonate Buffer ◽  
Adult Rat ◽  
Mammalian Myocardium

Acidosis increases resting cytosolic [Ca2+], (Cai) of myocardial preparations; however, neither the Ca2+ sources for the increase in Cai nor the effect of acidosis on mitochondrial free [Ca2+], (Cam) have been characterized. In this study cytosolic pH (pHi) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl ester (AM form) of SNARF-1. A stable decrease in the pHi of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a bicarbonate buffer equilibrated with 5% CO2 to a buffer equilibrated with 20% CO2. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pHi in 5% CO2, nor did it affect the magnitude of pHi decrease in response to hypercarbic acidosis. Cai was measured in myocytes loaded with indo-1/free acid and Cam was monitored in cells loaded with indo-1/AM after quenching cytosolic indo-1 fluorescence with MnCl2. In quiescent intact myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Cai from 130 +/- 5 to 221 +/- 13 nM. However, when acidosis was effected in electrically stimulated myocytes, diastolic Cai increased more than resting Cai in quiescent myocytes, and during pacing at 1.5 Hz diastolic Cai was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P &lt; 0.05). The magnitude of Cai increase in quiescent myocytes was not affected either by sarcoplasmic reticulum (SR) Ca2+ depletion with ryanodine or by SR Ca2+ depletion and concomitant superfusion with a Ca(2+)-free buffer. In unstimulated intact myocytes hypercarbia increased Cam from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by ryanodine and a Ca(2+)-free buffer or by 50 microM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspensions loaded either with BCECF/AM or indo-1/AM, acidosis produced by lactic acid addition decreased both intra- and extramitochondrial pH and increased Cam. Studies of mitochondrial suspensions bathed in indo-1/free acid-containing solution showed an increase in extramitochondrial Ca2+ after the addition of lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial buffers, rather than transsarcolemmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Cai and Cam, respectively; additionally, Ca2+ efflux from the mitochondria may contribute to the raise in Cai. In contrast, in response to acidosis, diastolic Cai in electrically stimulated myocytes increases more than resting Cai in quiescent cells; this suggests that during pacing, net cell Ca2+ gain contributes to enhance diastolic Cai.

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