Skip Navigation

Cardiovascular Research 2003 58(1):99-108; doi:10.1016/S0008-6363(02)00854-4
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Baartscheer, A.
Right arrow Articles by Fiolet, J. W.T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baartscheer, A.
Right arrow Articles by Fiolet, J. W.T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

SR calcium handling and calcium after-transients in a rabbit model of heart failure

Antonius Baartscheer*, Cees A. Schumacher, Charly N.W. Belterman, Ruben Coronel and Jan W.T. Fiolet

Experimental and Molecular Cardiology Group, Department of Experimental Cardiology, Room M-0-052, Academic Medical Center, University of Amsterdam, P.O. Box 22700, Meibergdreef 9, 1100 DE Amsterdam, The Netherlands

* Corresponding author. Tel.: +31-20-566-3265; fax: +31-20-697-5458. a.baartscheer{at}amc.uva.nl

Received 19 July 2002; accepted 6 December 2002


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: After-depolarization associated arrhythmias are frequently observed in heart failure and associated with spontaneous calcium release from sarcoplasmic reticulum (SR), calcium after-transients. We hypothesize that disturbed SR calcium handling underlies calcium after-transients in heart failure (HF). Methods: We measured the stimulation rate dependence (0.2–3 Hz) of diastolic calcium, calcium transient amplitude and SR calcium content in left ventricular myocytes isolated from hearts of rabbits with pressure and volume overload-induced HF and age-matched control animals. Cytosolic calcium was measured with indo-1. In some experiments, delayed after-depolarizations (DADs) were monitored with the voltage sensitive dye di-4-Annepps. SR calcium content was estimated from the response to rapid cooling (RC). After-transients were elicited in the presence of norepinephrine (100 nmol/l) after cessation of burst pacing. Results: With increasing stimulation rate (0.2–3.0 Hz): (1) steady state diastolic [Ca]i increased from 102 to 174 nmol/l in HF and from 44 to 103 nmol/l in control, (2) calcium transient amplitudes decreased from 310 to 254 nmol/l in HF and increased from 186 to 429 nmol/l in control, (3) SR calcium content decreased from 1.25 to 1.09 mmol/l in HF and increased from 1.51 to 2.48 mmol/l in control, (4) in HF and control, the end diastolic SR membrane calcium gradient decreased by about 30%; at any stimulation rate, the magnitude of gradient in HF was one-third of control, (5) systolic depletion of SR was 85% in HF and 60% in control. In HF, noradrenaline (100 nmol/l) increased SR calcium content and SR membrane gradient by 40% versus about 7% in control. Calcium after-transients were observed in 14 out of 18 HF rabbits, and none in eight control animals and were associated with DADs. Calcium after-transients were associated with a 35% decrease in SR calcium content. The frequency of occurrence of calcium after-transients was related to diastolic calcium. Conclusions: in HF, diastolic calcium is increased and both SR calcium content and SR membrane calcium gradient are decreased in a stimulation rate-dependent manner. In HF, β-adrenergic stimulation can partly restore the SR calcium content and SR membrane gradient at higher stimulation rates in a meta-stable condition; upon transition to low stimulation rates, the SR membrane can no longer maintain this high unbalanced SR calcium load at increased diastolic calcium, the magnitude of which is causally related to the occurrence of calcium after-transients.

KEYWORDS Arrhythmia (mechanisms); Calcium (cellular); Heart failure; Myocytes; SR function


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Patients with heart failure have a poor prognosis and a high risk of sudden death most likely related to ventricular tachycardias [1–3]. Two characteristic events may precede the fatal arrhythmia leading to sudden death: increase in heart rate and sinus pause [4,5]. In a rabbit model of volume and pressure overload induced heart failure, a high incidence of delayed after-depolarizations (DADs) and automaticity has been observed after burst pacing in the presence of norepinephrine [6]. In hypertrophy and heart failure, disturbed intracellular calcium handling is directly involved in contractile dysfunction, and contributes to DAD-related arrhythmias [7–9]. Inward current carried by the electrogenic Na+/Ca2+-exchanger (NCX) in conditions of increased cytoplasmic calcium ([Ca2+]i) [10,11] may be responsible for generation of DADs. NCX is up-regulated in heart failure [12] and relatively large depolarizing currents may occur when intracellular calcium becomes suddenly elevated. Spontaneous calcium release from sarcoplasmic reticulum (SR) may set the stage for such a condition. Calcium after-transients and DADs can be elicited in normal myocytes with interventions that increase the SR calcium content [13]. Although increased SR calcium loading alone does not suffice to generate spontaneous calcium release, it is conceivable that a highly loaded SR becomes more susceptible to spontaneous calcium release when calcium is raised at the cytosolic side of the SR membrane [14]. Although in heart failure diastolic calcium is indeed increased, there is no evidence showing SR calcium overload. On the contrary, down-regulated expression of SR Ca2+-ATPase [15–17] as well as increased open probability of ryanodine receptors [18,19] would rather favor a decrease in the SR calcium content.

We hypothesize that in HF, spontaneous SR calcium release (including DADs) occurs following SR calcium over-loading at increased cytoplasmic diastolic calcium. To that purpose we studied cytoplasmic calcium and SR calcium content in left ventricular myocytes of a rabbit model of volume and pressure overload induced heart failure having ventricular arrhythmias and sudden death [6,20].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
This investigation was approved by the local ethical committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 The rabbit model of pressure and volume overload induced heart failure and isolation of left ventricular myocytes
Volume and pressure overload HF was induced in 18 rabbits (New Zealand White, SPF, 3–3.5 kg as described previously [6,21]); volume overload by rupture of the aortic valve until pulse pressure increased by 100%, pressure overload 3 weeks later after the first operation by a suprarenal stenosis of approximately 50% of the abdominal aorta. Twelve weeks after the second operation, left ventricular myocytes were isolated as described previously [22]. Heart failure was indexed (0.0–1.0) based on relative heart weight, relative lung weight, gallop rhythm, LVEDP and ascites as described previously [6,20]. Myocytes were stored at room temperature in separate vials, each containing about 105 myocytes in 5 ml Hepes buffered solution (pH 7.3) containing in mmol/l [Na+] 156, [K+] 4.7, [Ca2+] 1.3, [Mg2+] 2.0, [Cl–] 150.6, [HCO3–] 4.3, [HPO42–] 1.4, [Hepes] 17, [Glucose] 11 supplied with 1% fatty acid free albumin. Cell dimensions of 100 randomly chosen rod-shaped myocytes were determined. Age-matched untreated animals (n = 8) served as controls.

2.2 Experimental design and determination of free cytosolic [Ca2+]i and SR calcium content
Before each individual experiment, cells were loaded during 30 min with 5 µmol/l indo-1/AM and 0.01% pluronic, washed twice with fresh Hepes solution (without albumin), and kept for another 15 min to ensure complete de-esterification of indo-1/AM. Myocytes were attached to a poly-D-lysine (0.1 g/l) treated cover slip placed on a microscope stage of an inverted fluorescence microscope (Nikon Diaphot). A perfusion chamber (height 0.4 mm, diameter 10 mm, volume 30 µl), having two needles at opposite sides for superfusion purposes and two thin parallel platinum electrodes for field stimulation (distance of 8 mm, pulses of 40 V/cm and 0.5 ms duration), was tightly positioned onto the cover slip. Microscope stage and perfusion chamber were temperature controlled at 37°C. The contents of the chamber could be replaced within 100 ms. One quiescent rod-shaped myocyte was selected and the measuring area was adjusted to the cellular surface with a rectangular diaphragm. Indo-1 fluorescence was excited at 340 nm with a 100 W xenon lamp. Dual wavelength emission was measured at 410 and 516 nm, respectively. Signals were digitized at 1 kHz and corrected for background signals recorded from indo-1 free myocytes. In order to obtain ‘true’ cytosolic free [Ca2+]i fluorescent signals were corrected for 35% mitochondrially compartmentalized indo-1 measured in control and HF myocytes with the Mn2+ technique as described previously [24,25] and stimulation rate dependency of mitochondrial calcium [25]. Free cellular calcium ([Ca2+]i) and total cytoplasmic buffered calcium was calculated as described previously [23,26]. In some experiments, myocytes were superfused with the voltage-sensitive dye di-4-Annepps (1 µmol/l) to monitor DADs. Annepps fluorescence was excited at 516 nm and measured at 583 nm.

Stimulation frequency dependence (0.2–3 Hz) of diastolic calcium ([Ca2+]i), calcium transient amplitude and SR calcium content were measured after 2 min of stimulation at each frequency. Each experiment started with a conditioning 2-min episode of stimulation at 2 Hz. Calcium after-transients (at least three myocytes per animal) were evoked by cessation of the stimulation following 10 s of burst pacing (3 Hz) in the presence of 100 nmol/l noradrenaline.

Rapid cooling (RC) was used to estimate SR calcium content; RC causes complete depletion of calcium from SR and calcium released remains confined to the cytoplasm [27,28]. RC was carried out by rapid superfusion with ice-cold Tyrode's solution of the same composition; low temperature (0–1°C) was reached within 200 ms. SR calcium content was measured as the response of [Ca2+]i to rapid cooling (within 200 ms) with ice-cold Hepes solution of the same composition and was calculated from the change in total cytosolic calcium and a fractional SR volume of 10%. To study the change in SR calcium content before and after the occurrence of a calcium after-transient, RC was applied either during the last paced beat or 5 s after cessation of stimulation (separate experiments).

2.3 Statistical analysis
Data obtained from cells were pooled per heart. Pooled data were averaged and expressed as mean±S.E.M. Comparisons were carried out using Student's t-test with P<0.05 indicating statistical significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Two rabbits that developed only moderate HF after 3 months (failure index less than 0.6 [6]) were excluded from this study. Table 1 summarizes parameters relevant to heart failure in 18 HF and eight age-matched control animals. All parameters, except bodyweight, were significantly higher in HF animals. The left ventricles of all HF animals were hypertrophied and dilated and cell dimensions were increased. Ascites or gallop rhythm was observed in about 25% of HF rabbits. The average failure index was 0.76±0.04.


View this table:
[in this window]
[in a new window]

  		Table 1 Animal characteristics

 
Fig. 1 shows representative examples of measured, ‘true’ cytoplasmic and total cytosolic calcium transients in myocytes (2 Hz) from a control and a HF heart; transients in the middle and lower panels are calculated from the data in the upper panel (see Methods and Ref. [23]). Measured, ‘true’ and total diastolic calcium are increased in HF compared to control. Measured and ‘true’ amplitudes of calcium transients in HF and control are almost similar. However, note that the amplitude of total calcium transient of the HF myocyte is substantially smaller than that of control. This is a direct consequence of the calcium buffering characteristics and the difference in diastolic calcium.


Figure 1
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Representative example of calcium transients in a control (left panel) and HF myocyte (right panel). Upper panel: measured calcium (nmol/l). Middle panel: ‘true’ cytosolic calcium (nmol/l, see Methods). Lower panel: total cytosolic calcium (µmol/l, see Methods).

 
Fig. 2 summarizes the stimulation rate dependence of diastolic calcium (panel A), the amplitude of the calcium transient expressed as ‘true’ free [Ca2+]i (panel B) and the associated values expressed as total cytoplasmic calcium (panel C) in control (closed symbols) and the HF group (open symbols). At all stimulation rates (from 0 to 3 Hz), diastolic [Ca2+]i was significantly higher in the HF group compared to control. The transient amplitude, and total calcium, increased with stimulation rate in control, but decreased in HF. At lower frequencies, the amplitudes expressed as free [Ca2+]i were higher in HF than in control, but lower at the higher frequencies. When expressed as total calcium, the crossover point is shifted to a lower stimulation rate; the difference between amplitudes substantially decreased at the lower frequencies and increased at the higher stimulation rates.


Figure 2
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Stimulation rate dependence of calcium transients in eight control rabbits (closed symbols, 24 myocytes) and 18 HF rabbits (open symbols, 54 myocytes). Stimulation rates were 0.2, 0.5, 1.0, 2.0 and 3.0 Hz. Data for diastolic calcium of quiescent myocytes are included. Panel A shows ‘true’ diastolic calcium (nmol/l, see Methods). Panel B ‘true’ calcium transient amplitude (nmol/l, see Methods). Panel C total calcium transient amplitude (µmol/l, see Methods). Data for three myocytes of each animal were pooled. Error bars indicate S.E.M. of the pooled data per animal.

 
Fig. 3A shows the stimulation rate dependence (0.2–3 Hz) of the SR calcium content. SR calcium content increased with frequency in control and decreased slightly in HF. At all stimulation rates, SR calcium content in HF was less than control, the difference increasing with frequency. Fig. 3B shows the ratio of this estimate of SR calcium content (Fig. 3A) and diastolic calcium (Fig. 2A). This is a measure of the calcium gradient across the SR membrane during diastole. Both in the control and HF, the calcium gradient across the SR membrane decreased with stimulation rate. At all frequencies the gradient in HF was about threefold less than in control. Fig. 3C shows the stimulation rate dependence of the degree to which SR becomes depleted during systole (ratio of the calcium transient amplitude from Fig. 2C corrected for SR volume) and SR calcium content Fig. 3A). Relative SR depletion was significantly larger in HF than in control and was rather independent of frequency in both groups.


Figure 3
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Stimulation rate dependence of SR calcium content, SR membrane calcium gradient and systolic SR depletion. Number of animals, myocytes and conditions as in Fig. 2. Panel A: SR calcium content is expressed as the change in cytosolic total calcium (µmol/l) in response to rapid cooling (RC). Panel B: ratio of SR calcium content (panel A) and diastolic calcium (Fig. 2A). Panel C: the stimulation rate dependence of the degree to which SR becomes depleted during systole (ratio of the calcium transient amplitude from Fig. 2C corrected for SR volume) and SR calcium content (A).

 
Table 2 summarizes the effects of β-adrenergic stimulation (100 nmol/l noradrenaline) on diastolic [Ca2+]i, calcium transient amplitude and SR calcium content in control and HF (stimulation rate 2 Hz). The calcium transient amplitude increased both in control and HF. SR calcium content substantially increased in HF (P<0.05), but was hardly affected in control. No significant change in diastolic calcium occurred in both groups. Consequently, in HF in contrast to control, noradrenaline caused an increase in the calcium gradient across the SR membrane although control values were not reached. Also relative systolic SR calcium depletion decreased with noradrenaline, but values as low as in control were not reached.


View this table:
[in this window]
[in a new window]

  		Table 2 Effects of β-adrenergic stimulation on calcium handling in control and HF animals

 
Fig. 4 shows representative examples of after-transients and DADs subsequently elicited in the same HF myocytes following cessation of burst pacing in the presence of noradrenaline (100 nmol/l). With noradrenaline, calcium after-transients were never observed in control animals. Calcium after-transients were absent in four HF animals. In three animals, two out of three myocytes and in 11 animals all three myocytes exhibited after-transients. In 30 HF myocytes, one single after-transient occurred (right panel). The left panel shows a train of calcium after-transients found in six myocytes. In three myocytes, repetitive full-blown calcium transients occurred over a period of 5 s (not shown). In 10 HF myocytes, DADs (four rabbits) were measured, subsequent to measurement of the calcium after-transient. Calcium after-transients were always associated with DADs; the initial increase in cytosolic calcium always preceded membrane depolarization (right panel) or full-blown action potentials (left panel). In order to substantiate the temporal relation between the after-transient and DAD, the order of subsequent measurements was reversed in six HF myocytes from two rabbits; measurement of the calcium after-transient was sandwiched between two measurements of DADs. In these myocytes, calcium after-transients also started earlier than the DADs. Without noradrenaline, calcium after-transients and DADs were never observed, either in HF or in control myocytes.


Figure 4
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Representative examples of calcium after-transients (lower panels) and DADs (upper panels) subsequently (within 60 s) measured in the same HF myocytes. After-transients and DADs were evoked by cessation of 10 s of burst stimulation (3 Hz) in the presence of 100 nmol/l noradrenaline. Arrows mark the last two stimulated beats.

 
Fig. 5 shows the frequency of occurrence of calcium after-transients in all HF myocytes as a function of diastolic calcium. The data suggest a critical region of diastolic calcium from about 100 to 150 nmol/l for enhanced susceptibility to development of calcium after-transients.


Figure 5
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Probability to observe calcium after-transients in HF rabbits as a function of diastolic calcium. Probability was calculated in 10 nmol/l classes of diastolic calcium using all myocytes (n = 54) from all 18 HF rabbits.

 
Fig. 6 shows that the calcium after-transients are of SR origin. RC was applied either before the last stimulated beat or 5 s after cessation of burst pacing. In control myocytes in the presence of noradrenaline there was no difference in SR calcium content during (open bars) and after (hatched bars) burst pacing. In HF myocytes, SR calcium content during burst pacing was less than control, however substantially higher than without adrenaline (compare Fig. 3A). In HF myocytes having no after-transients, SR calcium content remained unchanged at 5 s after cessation of burst pacing. However, in HF myocytes exhibiting after-transients, SR became substantially depleted following the calcium after-transient. These data strongly suggest SR to be the source of calcium associated with the after-transients and that this calcium is subsequently removed from the cytosol presumably by action of NCX.


Figure 6
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 The origin of calcium associated with calcium after-transients. SR calcium content was measured either just before the last stimulated beat (open bars) in 12 control myocytes, 18 HF myocytes or 5 s after the last beat (dotted bars) in 12 control myocytes, 6 HF myocytes without after-transients and 12 myocytes with after-transients. SR calcium content is expressed as the change in total cytosolic calcium (µmol/l) (see Methods) in response to RC (±S.E.M.).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The data presented in this study provide evidence for a direct and possibly causal relationship between increased diastolic [Ca2+]i and disturbed SR calcium handling in HF which leads to generation of calcium after-transients, which may underlie the genesis of after-depolarization related arrhythmias in heart failure.

For this study, we used a rabbit model of combined volume and pressure overload, which induces heart failure over a period of 2–3 months [6,20,21], during which a progressive increase is observed of ventricular premature beats and episodes of ventricular tachycardia [20,29]. Development of these types of arrhythmias is particularly apparent in rabbits with a high (>0.6) index of failure as defined previously [6,20]. For this reason only rabbits with failure index >0.6 were included in this study and age-matched animals (Table 1) served as control.

Abnormal calcium handling of myocytes has been implicated in arrhythmogenesis in heart failure [7–9]. In our model of heart failure, cytosolic and SR calcium handling is disturbed as documented by increased diastolic [Ca2+]i, decreased calcium transient amplitude and rate of decay, decreased SR calcium content, decreased SR membrane calcium gradient and a negative calcium amplitude frequency relationship (Figs. 1 to 3GoGo). These results corroborate previously reported data on the negative force frequency relation in HF [30–32], decreased SR calcium uptake due to down-regulation of SERCA [15–17] and decreased driving force of the Na+/Ca2+-exchanger [33]. However, Pogwidz et al., using the same model of heart failure, reported unaltered diastolic calcium content and SR calcium [34]. It may be of relevance to note that in their model, pulse pressure was increased by aortic valve rupture by only 50% rather than 100% in our model, which might have resulted in less severe heart failure of lower fail index.

In isolated ventricular trabeculae from both failing rabbits and hearts from patients undergoing cardiac transplantation, triggered activity based on DADs occurred in about 50% of the preparations only in the presence of noradrenaline using the same stimulation protocol [6]. This corresponds with our findings that in the absence of noradrenaline, calcium after-transients could never be elicited either in healthy or in HF myocytes, but in the presence of noradrenaline only in 77% of the HF myocytes.

It has been inferred from experiments with induced SR calcium overload or with selective SR calcium channel (RyR) inhibition that calcium after-transients result from release of calcium from SR [6,29,34]. We provide direct experimental evidence for this contention demonstrating that calcium associated with after-transients originates from SR (Fig. 6). The occurrence of after-transients in HF myocytes was, in contrast to myocytes not exhibiting after-transients, invariably associated with a decrease in SR calcium content by about 35%, which was not restored at least until 5 s following the after-transient. This indicates that after-transient associated calcium is effectively removed from the cell by electrogenic sarcolemmal mechanisms giving rise to transient depolarization (DAD), the most likely candidate being NCX. It has been demonstrated that in calcium overloaded ‘normal’ rabbit myocytes, NCX and calcium-activated Cl–-channel inward currents were responsible for generation of DADs [35]. In similar experiments with human myocytes, only NCX was found operative [36]. It seems plausible that the same mechanisms are operative in the generation of DADs in HF myocytes. NCX is usually found up-regulated in HF [12], which makes its role potentially even more prominent.

We not only demonstrate that DADs were always associated with calcium after-transients in HF (Fig. 4), but also that the onset of depolarization always occurred later than the onset of the after-transient, irrespective of the order in which after-transients and DADs were measured subsequently in the same cell. This strongly suggests that the SR calcium released during the after-transient is instrumental in the onset of the DAD. Although the evidence for this is compelling, it should be realized that final proof of this temporal relationship requires simultaneous measurement of after-transients and membrane potential.

Calcium release from SR is mediated by opening of SR calcium release channels (RyR). Our data indicate that in HF, SR has a higher propensity to spontaneous calcium release than in control myocytes. This suggests increased (persistent) RyR-channel open probability, which would be expected to decrease and prolong calcium transients. This indeed is commonly observed in HF and is also demonstrated in our data (Figs. 1 and 2Go). Our demonstration that the fractional systolic SR calcium release was significantly higher in HF than in control (Fig. 3C) may provide support for a mechanism involving increased open probability of RyR. It should be realized, however, that in HF increased systolic NCX reversed mode activity is responsible for this [33].

Spontaneous SR calcium release is non-zero even in normal cardiac tissue, which may be inferred from the finite occurrence of calcium sparks [37] and from slow depletion of SR in quiescent myocytes [38]; apparently, however, the rate of spark generation is insufficient to induce calcium after-transients. Therefore, a substantial increase in RyR-channel open probability seems to be required to account for genesis of calcium after-transients and DADs. Calcium concentrations both at the cytosolic and luminal side of the SR membrane calcium are important mediators of the open probability of the RyR channels [14,39,40]. Evidence for luminal calcium effects has been obtained in normal myocytes where increase in SR calcium content was shown to enhance the incidence of calcium waves, DADs and calcium after-transients [13]. However, in HF myocytes we demonstrate that, if anything, SR calcium content is reduced compared to control (Fig. 3). This would imply that calcium overload of SR is at least not the only cause for the occurrence of calcium after-transients in HF. After-transients in HF could be elicited only with β-adrenergic stimulation. On the one hand, noradrenaline indeed caused an increase in SR calcium content, which however never reached control values (Table 2). On the other hand, it partly restored the calcium gradient across the SR membrane. It may be speculated that this condition in combination with increased RyR-channel open probability constitutes an unbalanced overload condition, which might also prevail in HF in vivo at elevated catecholamine plasma levels [41]. Apparently, noradrenaline did not cause such an unbalanced condition in control myocytes, where it only slightly elevated SR calcium and did not give rise to calcium after-transients.

Open probability of RyR is not only affected by SR calcium content but also by cytosolic calcium [14]. Therefore, increased diastolic calcium in HF (Fig. 2) might provide an alternative mechanism underlying increased open probability of RyR and increased propensity for genesis of calcium after-transients and DADs. Indeed, it has been demonstrated in normal tissue that the open probability of RyR increases with increasing cytosolic [Ca2+]i. The incidence of calcium sparks increased with elevation of cytoplasmic [Ca2+]i from 100 to 150 nmol/l [42], which notably well coincides with the critical range of diastolic [Ca2+]i around 100–150 nmol/l we measured for the genesis of after-transients (Fig. 6). Fig. 6 also shows that there is not an all-or-nothing relationship between diastolic [Ca2+]i and the occurrence of after-transients. Although this heterogeneity may reflect the stochastic nature of RyR-channel opening events of the entire cellular ensemble of RyR-channels in HF, it might also result from the spatial origin of the myocytes. To minimize such heterogeneity we only used midmural left ventricular myocytes. Heterogeneity might also originate from intrinsic variation in severity of heart failure between rabbits. In this respect, it is of relevance to mention that the variation of diastolic calcium in myocytes per animal was always smaller than that in the total myocyte population.

Obviously, calcium is not the only factor influencing open probability of RyR. The recently reported modulation of open probability of RyR by binding to FKBP is of particular interest in this respect [18,19]. Dissociation of FKBP from RyR by rapamycin or FK506 resulted in an increase in the frequency of Ca2+ sparks in myocytes [43]. Hyperphosphorylation by PKA resulted in dissociation of FKBP and increased sensitivity to Ca2+-dependent activation of RyR in HF [18]. This might underlie the beneficial effects of beta-blockade treatment in patients with heart failure [44], although this could equally well be attributed to reduction of SR calcium content. Dissociation of FKBP from RyR-channels also attenuated coupled gating, resulting in more stochastic openings [45].

In summary, in HF myocytes, the SR content as well as the SR trans-membrane calcium gradient are decreased but systolic SR depletion is increased. All of these parameters are partly restored by β-adrenergic stimulation. However, the SR of β-adrenergically stimulated HF myocytes have a reduced capability to maintain the increased SR trans-membrane gradient due to increased open probability of the SR calcium release channels secondary to increased diastolic calcium. This leads to a higher incidence of spontaneous calcium release from SR, calcium after-transients and DADs by transient inward current carried by NCX and calcium-activated Cl–-channels. This may underlie triggered activity and ventricular tachycardia.

Time for primary review 38 days.


   Acknowledgements
 
This work was supported by the Netherland Heart Association (96039).


   Notes
 
* For this manuscript Dr. A. Bril acted as Guest Editor.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Dargie HJ, Cleland JGF, Leckie BJ et al. The relation of arrhythmias and electrolyte abnormalities to survival in patients with severe chronic heart failure. Circulation 1987;75:IV-98–IV-107.
  2. Hofmann T., Meinertz T., Kasper W., et al. Mode of cardiac death in idiopathic dilated cardiomyopathy: a multivariate analysis of prognostic determinates. Am Heart J (1988) 116:1455–1463.[CrossRef][Web of Science][Medline]
  3. Wilson J.R., Schwartz J.S., St John Sutton M. Prognosis in severe heart failure: relation to hemodynamic measurements and ventricular ectopic activity. J Am Coll Cardiol (1983) 2:403–410.[Web of Science][Medline]
  4. Bayés de Luna A., Coumel P., Leclercq J.F. Ambulatory sudden death: mechanism of production of fatal arrhythmias on the basis of data from 157 cases. Am Heart J (1989) 117:151–159.[CrossRef][Web of Science][Medline]
  5. Coumel P., Leenhardt A., Houven X. Atrial and ventricular fibrillation: mechanisms and device therapy. Allesie M.A., Fromer M., eds. (1997) Armonk, NY: Futura Publishing. 37–51.
  6. Vermeulen J.T., McGuire M.A., Opthof T., et al. Triggered activity and automaticity in ventricular trabeculae of failing and rabbits hearts. Cardiovasc Res (1994) 28:1547–1554.[Abstract/Free Full Text]
  7. Pye M.P., Cobbe S.M. Mechanisms of ventricular arrhythmias in cardiac failure and hypertrophy. Cardiovasc Res (1992) 26:740–750.[Free Full Text]
  8. Aronson RS, Ming Z. Cellular mechanisms of arrhythmia's in hypertrophied and failing myocardium. Circulation 1993;83:VII76–VII83.
  9. Lakatta E.G., Talo A., Capogrossi M.C., Spurgeon H.A., Stern M.D. Spontaneous sarcoplasmic reticulum Ca2+ release leads to heterogeneity of contractile and electrical properties of the heart. Basic Res Cardiol (1992) 87:93–104.[Web of Science][Medline]
  10. Witt A.L., Rosen M.R. The heart and cardiovascular system. Fozard H.A., Haber H., Jennings R.B., Katz A.M., Morgan H.E., eds. (1986) New York: New York Press. 1449–1490.
  11. Cranefield P.E., Aronson R.S. Cardiac arrhythmias. The role of triggered activity and other mechanisms. (1988) Mount Kisco, NY: Futura Publishing. pp. 91–292.
  12. Studer R., Reinecke H., Bilger J., et al. Gene expression of the cardiac Na+/Ca2+-exchanger in end-stage human heart failure. Circ Res (1994) 75:443–453.[Abstract/Free Full Text]
  13. Tweedie D., Harding S.E., MacLeod K.T. Sarcoplasmic reticulum Ca content sarcolemmal Ca influx and the genesis of arrhythmias in isolated guinea-pig cardio myocytes. J Mol Cell Cardiol (2000) 32:261–272.[CrossRef][Web of Science][Medline]
  14. Zahradníková A., Zahradní I. Analysis of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles using models derived from single-channel data. Biochim Biophys Acta (1999) 1418:268–284.[Medline]
  15. Mercadier J.-J., Lompre A.-M., Duc P., et al. Altered sarcoplasmic reticulum Ca2+-ATPase gene expression in the human ventricle during end-stage heart failure. J Clin Invest (1990) 85:305–309.[Web of Science][Medline]
  16. Arai M., Alpert N.R., MacLennan D.H., Barton P., Periasamy M. Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties for the failing myocardium. Circ Res (1993) 72:463–469. Abstract.[Abstract/Free Full Text]
  17. Arai M., Matsui H., Periasamy M. Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ Res (1994) 74:555–564.[Free Full Text]
  18. Marx S.O., Reiken S., Hisamatsu Y., et al. PKA phosphorylation dissociates FKBP 12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell (2000) 101:365–376.[CrossRef][Web of Science][Medline]
  19. Yano M., Ono K., Ohkusa T., et al. Altered stoichiometry of FKBP 12.6 versus ryanodine receptor in heart failure. Circulation (2000) 102:2131–2136.[Abstract/Free Full Text]
  20. Opthof T., Coronel R., Rademaker H.M.E., et al. Changes in sinus function in a rabbit model of heart failure with ventricular arrhythmias and sudden death. Circulation (2000) 101:2975–2980.[Abstract/Free Full Text]
  21. Bril A., Forest M., Gout B. Ischemia and reperfusion-induced arrhythmias in rabbits with chronic heart failure. Am J Physiol (1991) 261:H301–H307.[Web of Science][Medline]
  22. ter Welle H.F., Baartscheer A., Fiolet J.W.T., Schumacher C.A. The cytoplasmic free energy of ATP hydrolysis in isolated rod-shaped rat ventricular myocytes. J Mol Cell Cardiol (1988) 20:435–441.[CrossRef][Web of Science][Medline]
  23. Baartscheer A., Schumacher C.A., Opthof T., Fiolet J.W.T. The origin of increased cytoplasmic calcium upon reversal of the Na+/Ca2+-exchanger in isolated rat ventricular myocytes. J Mol Cell Cardiol (1996) 28:1963–1996.[CrossRef][Web of Science][Medline]
  24. Baartscheer A., Schumacher C.A., Fiolet J.W.T. SR calcium depletion following reversal of the Na+/Ca2+-exchanger in rat ventricular myocytes. J Mol Cell Cardiol (2000) 32:1025–1037.[CrossRef][Web of Science][Medline]
  25. Miyata H., Silverman H.S., Sollot S.J., et al. Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes. Am J Physiol (1991) 261:H1123–H1134.[Web of Science][Medline]
  26. Hove-Madsen L., Bers D.M. Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes. Am J Physiol (1993) 33:C677–C686.
  27. Bers D.M. Ryanodine and the calcium content of cardiac SR assessed by caffeine and rapid cooling contracture. Am J Physiol (1987) 253:C408–C411.[Web of Science][Medline]
  28. Bers D.M., Bridge J.H.B., Spitzer K.W. Intracellular calcium transients during rapid cooling contractures in guinea-pig ventricular myocytes. J Physiol (1989) 417:537–553.[Abstract/Free Full Text]
  29. Pogwizd S.M., Schlotthauer K., Weilong Y., Bers D.M. Arrhythmogenesis and contractile dysfunction in heart failure. Circ Res (2001) 88:1159–1167.[Abstract/Free Full Text]
  30. Sipido K.R., Stankovicova T., Flameng W., Vanhaecke J., Verdonck F. Frequency dependence of Ca2+ release from sarcoplasmic reticulum in human ventricular myocytes from end-stage heart failure. Cardiovasc Res (1998) 37:478–479.[Abstract/Free Full Text]
  31. Gwatmey J.K., Copelas L., Mac Kinnon R., et al. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res (1987) 61:70–76.[Abstract/Free Full Text]
  32. Pieske B., Krethmann B., Meyer M., et al. Alteration in intracellular calcium handling associated with the inverse force–frequency relation in human dilated cardiomyopathy. Circulation (1995) 92:1169–1178.[Abstract/Free Full Text]
  33. Baartscheer A., Schumacher C.A., Belterman C.N.W., Fiolet J.W.T. The driving force of the Na/Ca-exchanger and the increase of diastolic calcium in a rabbit model of pressure and volume overload induced heart failure. Eur J Heart Failure (2001) 3:S61–S62.
  34. Pogwizd S.M., Qi M., Yuan W., Samarel A.M., Bers D.M. Upregulation of Na+/Ca2+-exchanger expression and function in an arrhythmogenic rabbit model of heart failure. Circ Res (1999) 85:1009–1019.[Abstract/Free Full Text]
  35. Verkerk A.O., Veldkamp M.W., Bouman L.N., van Ginneken A.C.G. Calcium-activated Cl– current contributes to delayed afterdepolarizations in single purkinje and ventricular myocytes. Circulation (2000) 101:2639–2644.[Abstract/Free Full Text]
  36. Verkerk A.O., Veldkamp M.W., Baartscheer A., et al. Ionic mechanism of delayed afterdepolarizations in ventricular cells isolated from human end-stage failing hearts. Circulation (2001) 104:2728–2733.[Abstract/Free Full Text]
  37. Chen W., Steenbergen C., Levy L.A., et al. Measurement of free Ca2+ in sarcoplasmic reticulum in perfused rabbit heart loaded with 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N'N'-tetraacetic by 19F NMR. J Biol Chem (1996) 271:7398–7403.[Abstract/Free Full Text]
  38. Bers D.M. Excitation–contraction coupling and cardiac contractile force. (1991) Dordrecht: Kluwer Academic. 93–118.
  39. Diaz M.E., Trafford A.W., O'Neill S.C., Eisner D.A. Measurement of sarcoplasmic Ca2+ content and sarcolemmal Ca2+ fluxes in isolated rat ventricular myocytes during spontaneous Ca2+ release. J Physiol (1997) 501:3–16.[Abstract/Free Full Text]
  40. Capogrossi M.C., Lakatta E.G. Frequency modulation and synchronization of spontaneous oscillations in cardiac cells. Am J Physiol (1985) 248:H412–H418.[Web of Science][Medline]
  41. Cohn J.N., Levine T.B., Olivari M.T., et al. Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med (1984) 311:819–823.[Abstract]
  42. Jafri M.S., Sobie E.A., Lederer W. Modeling the effects of sarcoplasmic reticulum lumenal and subspace calcium on spontaneous spark rate in cardiac myocytes. Biophys J (2002) 82(1):281.
  43. McCall E., Li L., Satoh H., et al. Effects of FK-506 on contraction and Ca2+ transients in rat cardiac myocytes. Circ Res (1996) 79:1110–1121.[Abstract/Free Full Text]
  44. Doughty R.N., Rodgers A., Sharpe N., MacMahon S. Effects of beta-blocker therapy on mortality in patients with heart failure: A systematic overview of randomized trials. Eur Heart J (1997) 8:560–565.
  45. Marx S.O., Ondrias K., Marks A.R. Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors). Science (1998) 281:818–821.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G. S. Hoeker, R. P. Katra, L. D. Wilson, B. N. Plummer, and K. R. Laurita
Spontaneous calcium release in tissue from the failing canine heart
Am J Physiol Heart Circ Physiol, October 1, 2009; 297(4): H1235 - H1242.
[Abstract] [Full Text] [PDF]

Home page
 Circ Heart FailHome page
K. D. Varian, A. Kijtawornrat, S. C. Gupta, C. A.A. Torres, M. M. Monasky, N. Hiranandani, D. A. Delfin, J. A. Rafael-Fortney, M. Periasamy, R. L. Hamlin, et al.
Impairment of Diastolic Function by Lack of Frequency-Dependent Myofilament Desensitizationin Rabbit Right Ventricular Hypertrophy
Circ Heart Fail, September 1, 2009; 2(5): 472 - 481.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Wang, R. W. Joyner, M. B. Wagner, J. Cheng, D. Lai, and B. H. Crawford
Stretch-activated channel activation promotes early afterdepolarizations in rat ventricular myocytes under oxidative stress
Am J Physiol Heart Circ Physiol, May 1, 2009; 296(5): H1227 - H1235.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart JHome page
I. A. Brouwer, M. H. Raitt, C. Dullemeijer, D. F. Kraemer, P. L. Zock, C. Morris, M. B. Katan, W. E. Connor, J. A. Camm, E. G. Schouten, et al.
Effect of fish oil on ventricular tachyarrhythmia in three studies in patients with implantable cardioverter defibrillators
Eur. Heart J., April 1, 2009; 30(7): 820 - 826.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. Wang, J.-F. Du, F. Wu, and H.-C. Wang
Apelin decreases the SR Ca2+ content but enhances the amplitude of [Ca2+]i transient and contractions during twitches in isolated rat cardiac myocytes
Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2540 - H2546.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
H. M. Den Ruijter, G. Berecki, A. O. Verkerk, D. Bakker, A. Baartscheer, C. A. Schumacher, C. N.W. Belterman, N. de Jonge, J. W.T. Fiolet, I. A. Brouwer, et al.
Acute Administration of Fish Oil Inhibits Triggered Activity in Isolated Myocytes From Rabbits and Patients With Heart Failure
Circulation, January 29, 2008; 117(4): 536 - 544.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. D. Varian and P. M. L. Janssen
Frequency-dependent acceleration of relaxation involves decreased myofilament calcium sensitivity
Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2212 - H2219.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
H. M. Den Ruijter, G. Berecki, T. Opthof, A. O. Verkerk, P. L. Zock, and R. Coronel
Pro- and antiarrhythmic properties of a diet rich in fish oil
Cardiovasc Res, January 15, 2007; 73(2): 316 - 325.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
A. Proven, H. L. Roderick, S. J. Conway, M. J. Berridge, J. K. Horton, S. J. Capper, and M. D. Bootman
Inositol 1,4,5-trisphosphate supports the arrhythmogenic action of endothelin-1 on ventricular cardiac myocytes
J. Cell Sci., August 15, 2006; 119(16): 3363 - 3375.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K. R. Sipido and D. Eisner
Something old, something new: Changing views on the cellular mechanisms of heart failure
Cardiovasc Res, November 1, 2005; 68(2): 167 - 174.
[Full Text] [PDF]

Home page
 Cardiovasc ResHome page
J. Fauconnier, A. Lacampagne, J.-M. Rauzier, G. Vassort, and S. Richard
Ca2+-dependent reduction of IK1 in rat ventricular cells: A novel paradigm for arrhythmia in heart failure?
Cardiovasc Res, November 1, 2005; 68(2): 204 - 212.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Saba, A. M. Janczewski, L. C. Baker, V. Shusterman, E. C. Gursoy, A. M. Feldman, G. Salama, C. F. McTiernan, and B. London
Atrial contractile dysfunction, fibrosis, and arrhythmias in a mouse model of cardiomyopathy secondary to cardiac-specific overexpression of tumor necrosis factor-{alpha}
Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1456 - H1467.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
R. P. Katra and K. R. Laurita
Cellular Mechanism of Calcium-Mediated Triggered Activity in the Heart
Circ. Res., March 18, 2005; 96(5): 535 - 542.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. Baartscheer, C. A. Schumacher, M. M.G.J. van Borren, C. N.W. Belterman, R. Coronel, T. Opthof, and J. W.T. Fiolet
Chronic inhibition of Na+/H+-exchanger attenuates cardiac hypertrophy and prevents cellular remodeling in heart failure
Cardiovasc Res, January 1, 2005; 65(1): 83 - 92.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
L. Yin, H. Bien, and E. Entcheva
Scaffold topography alters intracellular calcium dynamics in cultured cardiomyocyte networks
Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1276 - H1285.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. Jiang, B. Xiao, D. Yang, R. Wang, P. Choi, L. Zhang, H. Cheng, and S. R. W. Chen
RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR)
PNAS, August 31, 2004; 101(35): 13062 - 13067.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. J Janse
Electrophysiological changes in heart failure and their relationship to arrhythmogenesis
Cardiovasc Res, February 1, 2004; 61(2): 208 - 217.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Baartscheer, A.
Right arrow Articles by Fiolet, J. W.T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baartscheer, A.
Right arrow Articles by Fiolet, J. W.T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?