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.

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.

scholarly journals Phospholamban overexpression in rabbit ventricular myocytes does not alter sarcoplasmic reticulum Ca transport

2009 ◽  
Vol 296 (3) ◽  
pp. H698-H703 ◽  
Author(s):  
Jason R. Waggoner ◽  
Kenneth S. Ginsburg ◽  
Bryan Mitton ◽  
Kobra Haghighi ◽  
Jeffrey Robbins ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Mammalian Species ◽  
Cardiac Contractility ◽  
Adult Rabbit ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Important Species ◽  
Plasmic Reticulum ◽  
Rabbit Ventricular Myocytes ◽  
Serca 2A

Phospholamban has been suggested to be a key regulator of cardiac sarcoplasmic reticulum (SR) Ca cycling and contractility and a potential therapeutic target in restoring the depressed Ca cycling in failing hearts. Our understanding of the function of phospholamban stems primarily from studies in genetically altered mouse models. To evaluate the significance of this protein in larger mammalian species, which exhibit Ca cycling properties similar to humans, we overexpressed phospholamban in adult rabbit cardiomyocytes. Adenoviral-mediated gene transfer, at high multiplicities of infection, resulted in an insignificant 1.22-fold overexpression of phospholamban. There were no effects on twitch Ca-transient amplitude or decay under basal or isoproterenol-stimulated conditions. Furthermore, the SR Ca load and Na/Ca exchanger function were not altered. These apparent differences between phospholamban overexpression in rabbit compared with previous findings in the mouse may be due to a significantly higher (1.5-fold) endogenous phospholamban-to-sarco(endo)plasmic reticulum Ca-ATPase (SERCA) 2a ratio and potential functional saturation of SERCA2a by phospholamban in rabbit cardiomyocytes. The findings suggest that important species-dependent differences in phospholamban regulation of SERCA2a occur. In larger mammals, a higher fraction of SERCA2a pumps are regulated by phospholamban, and this may influence therapeutic strategies to enhance cardiac contractility and functional cardiac reserve.

Steady-state twitch Ca2+ fluxes and cytosolic Ca2+ buffering in rabbit ventricular myocytes

1996 ◽  
Vol 270 (1) ◽  
pp. C192-C199 ◽  
Author(s):  
L. M. Delbridge ◽  
J. W. Bassani ◽  
D. M. Bers
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Whole Cell ◽  
Rabbit Ventricular Myocytes ◽  
The Mean ◽  
State Contraction ◽  
Caffeine Contractures

Intracellular Ca2+ ([Ca2+]i) transients and transsarcolemmal Ca2+ currents were measured in indo 1-loaded isolated rabbit ventricular myocytes during whole cell voltage clamp to quantitate the components of cytosolic Ca2+ influx and to describe the dynamic aspects of cytosolic Ca2+ buffering during steady-state contraction (0.5 Hz, 22 degrees C). Sarcolemmal Ca2+ influx was directly measured from the integrated Ca2+ current (Ica) recorded during the clamp (158 +/- 10 attomoles; amol). Sarcoplasmic reticulum (SR) Ca2+ content was determined from the integrated electrogenic Na+/Ca2+ exchange current (Ix) induced during rapid application and sustained exposure of cells to caffeine to elicit the release of the SR Ca2+ load (1,208 +/- 170 amol). The mean steady-state SR Ca2+ load was calculated to be 87 +/- 13 microM (mumol/l nonmitochondrial cytosolic volume). Ca2+ influx via Ica represented approximately 14% of the stored SR Ca2+ and 23% of the total cytosolic Ca2+ flux during a twitch (47 +/- 6 microM). Comparison of electrophysiologically measured Ca2+ fluxes with Ca2+ transients yields apparent buffering values of 60 for caffeine contractures and 110 for twitches (delta Ca2+ total/delta Ca2+ free). This is consistent with the occurrence of "active" buffering of cytosolic Ca2+ by SR Ca2+ uptake during the twitch.

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.

Effect of 2,3-butanedione monoxime on myocyte resting force during prolonged metabolic inhibition

1994 ◽  
Vol 267 (2) ◽  
pp. H419-H430 ◽  
Author(s):  
H. Ikenouchi ◽  
L. Zhao ◽  
W. H. Barry
Keyword(s):  
Ventricular Myocytes ◽  
Metabolic Inhibition ◽  
Atp Depletion ◽  
Luciferase Assay ◽  
Initial Increase ◽  
Adult Rabbit ◽  
Force Development ◽  
Butanedione Monoxime ◽  
Rabbit Ventricular Myocytes ◽  
Transient Relaxation

The chemical phosphatase 2,3-butanedione monoxime (BDM) has been reported to inhibit both Ca(2+)-induced myofilament force development and rigor due to ATP depletion. However, during prolonged hypoxia in cultured ventricular myocytes BDM delays but does not prevent a marked increase in resting force. To investigate the mechanisms involved we measured the effects of BDM on intracellular Ca2+ concentration ([Ca2+]i; indo 1), force development (video motion detector), and ATP contents (luciferase assay) in cultured embryonic chick ventricular myocytes and adult rabbit ventricular myocytes subjected to prolonged metabolic inhibition with 1 mM NaCN and 20 mM 2-deoxyglucose. In the absence of metabolic inhibition, 20 mM BDM depressed force development even when [Ca2+]i was markedly elevated by exposure to zero-Na solution or 10 mM caffeine in chick cells, and 30 mM BDM completely inhibited Ca(2+)-induced force development in rabbit myocytes. During metabolic inhibition, 20 mM BDM delayed the onset of an increase in resting force (from 5.44 +/- 0.87 to 13.67 +/- 1.34 min in chick myocytes; from 19.13 +/- 2.23 to 32.43 +/- 3.30 min in rabbit myocytes, means +/- SE, n = 8-9). However, the rates of ATP depletion and rise in [Ca2+]i after metabolic inhibition were not altered by BDM. In the presence of BDM, during prolonged metabolic inhibition in both chick and rabbit myocytes, abrupt spontaneous or evoked alterations in [Ca2+]i were associated with corresponding changes in force. During the initial increase in resting force induced by metabolic inhibition, exposure to BDM caused a partial transient relaxation. We conclude that the delayed increase in resting force during metabolic inhibition in the presence of BDM is due to redevelopment of Ca2+ sensitivity of the myofilaments in the presence of an increased [Ca2+]i as a consequence of severe ATP depletion, whereas in the absence of BDM the more rapidly developing increase in resting force during metabolic inhibition is initially due to a rise in [Ca2+]i followed by development of rigor.

Shortening and [Ca2+] dynamics of left ventricular myocytes isolated from exercise-trained rats

1998 ◽  
Vol 85 (6) ◽  
pp. 2159-2168 ◽  
Author(s):  
Bradley M. Palmer ◽  
Anne M. Thayer ◽  
Steven M. Snyder ◽  
Russell L. Moore
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Left Ventricular ◽  
Trained Group ◽  
Effective Coupling ◽  
Temporal Characteristics ◽  
Fura 2 ◽  
Intracellular Ca ◽  
Maximal Extent

The effects of run endurance training and fura 2 loading on the contractile function and Ca2+ regulation of rat left ventricular myocytes were examined. In myocytes not loaded with fura 2, the maximal extent of myocyte shortening was reduced with training under our pacing conditions [0.5 Hz at 2.0 and 0.75 mM external Ca2+ concentration ([Ca2+]o)], although training had no effect on the temporal characteristics. The “light” loading of myocytes with fura 2 markedly suppressed (∼50%) maximal shortening in the sedentary and trained groups, although the temporal characteristics of myocyte shortening were significantly prolonged in the trained group. No discernible differences in the dynamic characteristics of the intracellular Ca2+ concentration ([Ca2+]) transient were detected at 2.0 mM [Ca2+]o, although peak [Ca2+] and rate of [Ca2+] rise during caffeine contracture were greater in the trained state at 0.75 mM [Ca2+]o. We conclude that training induced a diminished myocyte contractile function under the conditions studied here and a more effective coupling of inward Ca2+ current to sarcoplasmic reticulum Ca2+ release at low [Ca2+]o, and that fura 2 and its loading vehicle DMSO significantly alter the intrinsic characteristics of myocyte contractile function and Ca2+ regulation.

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