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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.37 no.4 Santiago  2004

http://dx.doi.org/10.4067/S0716-97602004000400014 

 

Biol Res 37: 603-607, 2004

ARTICLE

Modulation of sarcoplasmic reticulum calcium release by calsequestrin in cardiac myocytes

SANDOR GYÖRKE, INNA GYÖRKE, DMITRY TERENTYEV, SERGE VIATCHENKO-KARPINSKI and SIMON C. WILLIAMS

Texas Tech University Health Sciences Center, Lubbock, Texas USA

Dirección para Correspondencia


ABSTRACT

Calsequestrin (CASQ2) is a high capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR). Mutations in the cardiac calsequestrin gene (CASQ2) have been linked to arrhythmias and sudden death induced by exercise and emotional stress. We have studied the function of CASQ2 and the consequences of arrhythmogenic CASQ2 mutations on intracellular Ca signalling using a combination of approaches of reverse genetics and cellular physiology in adult cardiac myocytes. We have found that CASQ2 is an essential determinant of the ability of the SR to store and release Ca2+ in cardiac muscle. CASQ2 serves as a reservoir for Ca2+ that is readily accessible for Ca2+-induced Ca2+ release (CICR) and also as an active Ca2+ buffer that modulates the local luminal Ca-dependent closure of the SR Ca2+ release channels. At the same time, CASQ2 stabilizes the CICR process by slowing the functional recharging of SR Ca2+ stores. Abnormal restitution of the Ca2+ release channels from a luminal Ca-dependent refractory state could account for ventricular arrhythmias associated with mutations in the CASQ2 gene.

Key words: Excitation-contraction coupling, calcium-induced calcium release, ryanodine receptor, calsequestrin; arrhythmia.


INTRODUCTION

In cardiac muscle the process of excitation contraction (EC) coupling relies on Ca2+ influx through voltage-dependent Ca2+ channels activating the SR Ca2+ release channels (ryanodine receptors, RyRs) in the sarcoplasmic reticulum (SR), a mechanism known as Ca2+-induced Ca2+ release (CICR) (1,2) (See also Franzini-Armstrong, this issue (2a)). In addition to cytosolic Ca2+, SR Ca2+ release is controlled by Ca2+ levels at the luminal side of the RyR channels (3). Deactivation of Ca2+ release channels upon a decline in intra-SR Ca levels appears to account for termination of CICR following its activation (4). Calsequestrin (CASQ2) is a high capacity Ca2+ binding protein targeted to the junctional SR in the vicinity of the RyR (5). Recently, mutations in the cardiac calsequestrin gene have been linked to a certain form of cardiac arrhythmia and sudden death inducible by catecholamine infusion and exercise (a condition called catecholaminergic polymorphic ventricular tachycardia, CPVT) (6,7). However, the specific role of this protein in heart physiology and the mechanism by which mutations in the CASQ2 gene lead to arrhythmias is only beginning to emerge. Here we summarize the results of our recent studies aimed at defining the function of this protein in cardiac EC coupling and the pathogenesis of arrhythmia associated with mutations of CASQ2.

MODULATION OF GLOBAL AND FOCAL Ca2+ RELEASE BY CASQ2

To explore CASQ2 function we utilized recombinant adenoviruses to either increase or decrease CASQ2 protein levels in rat ventricular myocytes (8). To overexpress CASQ2, we constructed an adenovirus that contained the full-length coding region of CASQ2 (Ad-CASQ2). To knock down CASQ2 levels, we constructed a virus that contained the same sequence in the antisense orientation (Ad-CASQ2as). In addition, a third adenovirus containing a short CASQ2 sequence with a stop codon was used as a control for viral infection (Ad-control). We studied the functional consequences of changes in CASQ2 levels using Ca2+ imaging and patch clamp methods. As revealed by Western blot analysis (Fig. 1A), CASQ2 levels were elevated approximately 4-fold in cells infected with Ad-CASQ2 and reduced to approximately 30% of control levels in cells infected with the Ad-CASQ2as virus. CASQ2 protein levels were unchanged in cells infected with the control virus. Importantly, the levels of other SR proteins such as SERCA2a and phospholamban were similar in all samples (8). In addition, CASQ2 targeting to the cisternae of SR was unchanged by overexpression of CASQ2, as revealed by immunofluorescence staining (8).

We used caffeine application to assess changes in the total Ca2+ storage capacity of the SR caused by increased or decreased CASQ2 expression (Fig. 1B). As judged from the changes in caffeine-induced Ca2+ transients, the total SR Ca2+ content was increased in Ad-CASQ2 myocytes about 2.5-fold and decreased in Ad-CASQ2as myocytes to about 40% of control levels. Therefore CASQ2 is an important determinant of the Ca2+ storage capacity of the SR.

Figure 1. Effects of increased and decreased CASQ2 levels on SR Ca2+ content, ICa-induced Ca2+ transients and spontaneous Ca2+ sparks in rat ventricular myocytes. A: Representative immunoblots detecting CASQ2 in non-infected (control) myocytes and myocytes infected with different Ad vectors (as indicated). B: Traces of intracellular Ca2+ transients induced by 10 mM caffeine. C: Recordings of Ca2+ transients and ICa currents during a depolarizing step from -50 to 0 mV. D: Surface plots of averaged spontaneous Ca2+ sparks. Experiments were performed at ~48 hrs after infection.

 

The effects of altered CASQ2 levels on active SR Ca2+ release during EC coupling was studied in myocytes undergoing voltage clamp stimulation (Fig. 1C). The amplitude of depolarization induced Ca2+ transients was dramatically increased in Ad-CASQ2 myocytes and dramatically reduced in Ad-CASQ2as myocytes. In addition, the rising phase of Ca transients was prolonged in CASQ2 overexpressing cells and shortened in CASQ2 underexpressing cells compared to control. The altered rise times suggest that the duration of active Ca2+ release underlying these signals was prolonged in Ad-CASQ2 and shortened in Ad-CASQ2as myocytes. Importantly, there was no significant change in the amplitude of ICa, the Ca2+ trigger for SR Ca2+ release. Taken together, these results show that CASQ2 is an essential determinant of the Ca2+ releasing function of the SR. CASQ2 seems to control the amount of Ca2+ released to the cystosol by influencing the duration of the release process based on effects on rise-time of Ca2+ transients.

Measurements of elementary Ca2+ release events, Ca2+ sparks, were performed to gain further insight into the role of CASQ2 in cardiac EC coupling (Fig. 1D). The overall size and the spatiotemporal spread of sparks were increased dramatically in Ad-CASQ2 cells and dramatically reduced in Ad-CASQ2as cells. The magnitude of the spark was larger in CASQ2 overexpressing cells because the sparks continued to rise longer, apparently due to slowed termination of the underlying Ca2+ release. The opposite was true for CASQ2-underexpressing cells where Ca2+ sparks were smaller due to faster release termination. Therefore, the changes in properties of global Ca2+ release caused by altered CASQ2 levels could be attributed, at least in part, to altered termination of elementary Ca2+ release events. As will be shown below, CASQ2 levels not only modulate termination of Ca2+ release but also its refractoriness. Furthermore, alterations in the repriming behavior of release sites may contribute to the pathogenic causes of arrhythmias caused by mutations of CASQ2.

CELLULAR MECHANISMS OF CPVT

To date four mutations in the cardiac CASQ2 gene have been linked to CPVT. Three of these mutations (a nonsense R33X, a splicing 532+1 G>1, and a 1 base-pare deletion, 62delA) are thought to induce premature stop codons, thus, potentially compromising normal expression of CASQ2 and resulting in lowered CASQ2 levels (7). The fourth mutation converts a negatively charged aspartic acid into a histidine in a highly conserved region of CASQ2 that is proposed to be involved in Ca2+ binding (D307H, ref. 6). We used myocytes infected with the CASQ2-antisense virus as a model system to study the cellular mechanism of arrhythmia caused by reduced CASQ2 levels. In control cells, exposure to isoproterenol (ISO) caused an increase in the amplitude of Ca2+ transients without any apparent disturbances in periodic Ca2+ cycling (Fig. 2). In cells with reduced CASQ2 levels, introduction of ISO caused profound disturbances in Ca2+ cycling. These disturbances were manifested by extrasystolic, spontaneous Ca2+ transients. Importantly, the membrane potential (MP) traces showed clear signs of arrythmogenic delayed after depolarizations (DADs) at the time points of spontaneous Ca2+ transients. These oscillations in membrane potential are the underlying causes of arrhythmia (9,10). These results indicate that lowered CASQ2 abundance destabilized the Ca2+ release mechanism leading to spontaneous, premature discharges of SR Ca2+ stores. The dependency of the observed effects on ISO suggest that our results are relevant to clinical episodes of CPVT that are also associated with increased levels of circulating catecholamines. More recently, we showed that expression of the arrythmogenic CASQ2 mutant D307H in cardiac myocytes resulted in similar alterations in Ca2+ handling to those observed in Ad-CASQ2as myocytes (11). Thus both reduced CASQ2 levels and altered CASQ2 protein activity induce arrhythmogenic behavior in cardiac myocytes.

Figure 2. Pro-arrhythmic behavior induced by isoproterenol in myocytes underexpressing CASQ2. Recordings of membrane potential along with line-scan images and time-dependent profiles of [Ca2+]i in a myocyte with a knocked down CASQ2 level before and after application of 1 µM ISO; the myocyte was stimulated at 2 Hz.

 

What are the specific subcellular or molecular mechanisms underlying the pro-arrhythmic behavior of myocytes underexpressing CASQ2? One possibility that we considered is that the premature, spontaneous Ca2+ transients are due to abnormal restitution of the Ca2+ release mechanism. Normally, a certain time must pass following CICR before Ca2+ release can be triggered again. This refractoriness of Ca2+ release sites is important for stability of CICR as it prevents the SR Ca store from premature reactivation. We therefore investigated the effects of altered CASQ2 protein levels on the restitution behavior of the Ca release mechanism. To study restitution behavior at the level of individual release sites we used the high affinity RyR activator Imperatoxin A (ImpTxA). Our protocol takes advantage of the ability of ImpTxA to induce high frequency flickering between the closed and open states of the cardiac RyR2 at resting cytosolic [Ca2+] (~100 nM) (12) so that in myocytes, toxin-activated RyR2s provide Ca2+ stimuli for igniting repetitive Ca2+ sparks from the same individual release sites (4,8). Because the repetitive firing of sparks in the presence of the toxin is determined by the ability of RyR2s that compose a release unit to follow the activity of the toxin-modified channel, inter-spark intervals (or frequency of repetitive sparks) provide a measure of release site refractoriness (4,8). The frequency of repetitive Ca2+ sparks was dramatically reduced in Ad-CASQ2 and increased in Ad-CASQ2as cells. The frequency of repetitive sparks in Ad-CASQ2 cells was further enhanced after activation of endogenous protein kinase A (PKA) by cAMP. These results suggest that CASQ2 is an important determinant of the refractory behavior of the release sites. When CASQ2 levels were reduced the functional recovery of release sites was accelerated making them prone to premature or spontaneous reactivation.

SUMMARY

In summary, CASQ2 is an essential determinant of the ability of the SR to store and release Ca2+ in cardiac muscle. CASQ2 appears to function as a Ca2+ reservoir that is readily accessible for CICR and also as an active Ca2+ buffer that modulates local luminal Ca2+ dependent closure of RyRs. CASQ2 stabilizes the CICR mechanism by slowing the functional recharging of SR Ca2+ stores. Abnormal repriming behavior of Ca2+ release sites leads to premature spontaneous Ca2+ transients triggering arrythmogenic DADs, thereby providing the pathogenesis of arrhythmia associated with mutations in CASQ2.

 

ACKNOWLEDGEMENTS

This work was supported by the by the American Heart Association Grant 0245088N (SCW) and the NIH Grants HL-74045 and HL-63043 (SG).

REFERENCES

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Corresponding author: Sandor Györke, Department of Physiology, Texas Tech University HSC, 3601 4th Street, Lubbock, TX 79430. Phone: (806) 743-2520, Fax: (806) 743-1512, E-mail: sandor.gyorke@ttuhsc.edu

Received: March 31, 2004. Accepted: May 4, 2004.

 

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