Calsequestrin Mutations and Sudden Death
A Case of Too Little Sarcoplasmic Reticulum Calcium Buffering?
See related article, pages 298–306
In cardiac muscle, calcium plays a crucial role in excitation–contraction coupling, but it is also implicated in arrhythmogenesis. Calcium is released from the sarcoplasmic reticulum (SR), resulting in the systolic Ca transient. This release occurs through a specialized channel, the ryanodine receptor (RyR). The RyR is formed by the assembly of 4 identical subunits and binds several accessory proteins that are involved in the control of its function. Channel opening is influenced by Ca levels on both the luminal and cytosolic side and the amount of Ca released depends steeply1 on SR Ca content.
Diastolic Calcium Release and Arrhythmias
In various conditions, the SR can release Ca independently from an action potential. This diastolic release propagates through the cell as a wave of calcium-induced calcium release. Some of the calcium is pumped out of the cell by the electrogenic Na–Ca exchange, resulting in delayed afterdepolarizations (DADs) and triggered arrhythmias (reviewed elsewhere2). Diastolic Ca release occurs when the SR Ca concentration reaches a critical, threshold level.3 Recent studies have suggested that DADs and arrhythmias can be produced not only as a consequence of elevated SR Ca content but also if the properties of Ca release from the SR are altered.
Cathecholiminergic Polymorphic Ventricular Tachycardia
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder characterized by the onset of ventricular tachycardia (VT) during stress. Two forms have been described, an autosomal dominant (CPVT-1) resulting from mutations of RyR4,5 and an autosomal recessive (CPVT-2) resulting from mutations of calsequestrin (CSQ).6 Both animal7 and human8 studies have demonstrated that CPVT-1 arrhythmias are attributable to DADs. Studies on isolated cells expressing mutant RyR have demonstrated that these mutations increase the incidence of diastolic Ca release and DADs.7 When these mutant RyRs were expressed in HEK cells, there was a decrease in threshold for diastolic Ca release compared to control cells.9 One important point is that the mutations in isolation are not sufficient to cause diastolic Ca release and DADs. Increasing RyR open probability with low concentrations of caffeine produces diastolic Ca release only transiently. This results in a decrease of SR Ca, so that no diastolic Ca release is seen in the steady state.10 However, if a β-adrenergic agonist is added potentiation of the RyR results in Ca waves in the steady state, an effect that is attributed to increased SR Ca content. This result explains why patients with mutations in the RyR or CSQ only develop arrhythmias during stress when presumably adrenergic stimulation increases SR Ca content.
Normal function of CSQ
CSQ is a Ca binding protein that provides a store of Ca for release during systole. It also allows the RyR to sense luminal Ca. Work on single RyRs reconstituted into lipid bilayers has shown that, in the presence of CSQ, an increase of luminal Ca increases RyR open probability. Gyorke et al have proposed a model to explain the action of CSQ on RyR.11 CSQ binds to RyR via triadin or junctin when intra-SR free Ca is low, and this reduces RyR open probability. When free intra-SR Ca increases, CSQ dissociates from RyR and RyR open probability increases. Central to the action of CSQ is the fact that its binding to triadin and RyR is influenced by free intra-SR Ca.
CSQ Mutations and Generation of DADs
Several CSQ mutations that cause CPVT have been identified, and their effects on CSQ function and Ca handling have been studied either by using overexpression studies or transgenic mice. In a homozygous knock-in mouse model, the D307H mutation decreased CSQ levels and increased calreticulin, another SR Ca-buffering protein.12 The decreased CSQ increased the incidence of diastolic Ca release, DADs and VT after β-adrenergic stimulation. This was ascribed to a reduced inhibitory action of CSQ on RyR. A study on CSQ knockout mice showed that total deletion of CSQ resulted in dilatation of the SR terminal cisternae and loss of the condensed CSQ and increase in SR volume.13 The removal of CSQ is associated with reduction in the levels of both triadin and junctin. Exposure to catecholamines causes diastolic Ca release, DADs, and VT. Terentyev et al14 studied the CSQ mutation R33Q in both bilayers and in rat myocytes (by overexpression) and found that the mutant CSQ is unable to reduce RyR activity at low Ca concentrations. In this issue of Circulation Research, Rizzi et al report an elegant study on a knock-in mouse model for the same mutation (R33Q).15 Using a broad spectrum of techniques they obtain several key findings. Arrhythmias can be produced very easily in these animals by simple environmental stress (in contrast to other RyR and CSQ mutants, in which catecholamine injection is often required to produce arrhythmias). The R33Q mutation decreases total CSQ levels. This is mainly attributable to the fact that the mutant protein is more prone to degradation and not because of low mRNA transcription rates. Electron microscopic analysis found dilatation of the SR terminal cisternae without increase in total SR area (in contrast to the CSQ KO mice, in which an increase of SR volume was observed13). Levels of triadin and junctin are decreased, but other Ca-handling proteins levels are unchanged including Na–Ca exchange, SERCA, phospholamban, and calreticulin. The mice develop bidirectional or polymorphic VT after exposure to environmental stress. Cellular studies confirm the increased incidence of DADS after exposure to catecholamines. Measurement of SR Ca content show that this is significantly decreased. The authors conclude that the arrhythmogenic effects of these mutations are attributable to a combination of decreased intra-SR buffering and abnormal interaction among CSQ, triadin, and RyR that fails to decrease RyR activity at low levels of free luminal Ca. This is certainly the most likely explanation. It will be important to study the relative contribution to the phenotype of (1) decreased SR Ca buffering and (2) abnormal interactions between RyR and triadin/junction. One way to do this would be to measure free SR Ca and see whether at a given SR Ca, the Ca transient in the R33Q mouse differs from that in control. As mentioned above, viral overexpression of R33Q results in a decrease of the free SR Ca required for diastolic Ca release.14 It will be interesting to discover whether a similar effect is seen in the R33Q knock-in mice or, alternatively, whether the increased diastolic Ca release is simply attributable to the fact that at a given SR total Ca there is a higher free Ca. The last interesting finding is that the heterozygous mouse has normal levels of CSQ and an almost normal phenotype. What is unclear, however, is how much of this CSQ in the heterozygote is wild-type as opposed to R33Q. It is possible that an increased susceptibility of R33Q to degradation will mean that most of the CSQ in a heterozygote is wild-type. The fact that the animals are free from arrhythmias supports this. If the 2 forms of CSQ were expressed in equal amounts, then on the basis of the conclusions of the previous overexpression studies, some arrhythmias should be observed because that percentage of mutant CSQ would impair CSQ ability to inhibit RyR.
The clear picture that emerges from all of these studies is that decreased levels of CSQ are central to pathogenesis of arrhythmias in patients with CSQ mutations and that different compensatory changes induced by different mutations (ie, changes in other SR Ca-handling proteins) determine the severity of the phenotype. In the light of this conclusion, one can also hypothesize that some individuals heterozygous for CSQ mutations develop arrhythmias because they are not able to achieve normal levels of CSQ in their SR.
It has become increasingly clear that catecholamines cause arrhythmias in CPVT mainly because they increase SR Ca content up to the threshold for diastolic Ca release. An area that still needs to be clarified is what are the effects of catecholamines on mutant RyR and CSQ? Recent studies have suggested that catecholamines directly increase the activity of RyR.16 It is still unclear whether mutant RyRs respond differently to catecholamines. In the case of CSQ, it is not known whether catecholamines have any effects on its affinity for Ca or its capacity to bind triadin and inhibit RyR. Understanding of these factors would help us to find new treatments for CPVT. A recent study has clearly demonstrated that most VT is CPVT is mainly generated in the His-Purkinje system.17 It would be extremely interesting and useful to establish whether the alteration in Ca handling produced by RyR and CSQ mutations ventricular myocytes are also present in His bundle cells.
Finally, with the development of agents that stabilize RyR and increase threshold for diastolic Ca release,18,19 it will be very interesting to see whether these agents are effective in reducing arrhythmias in mice with CSQ mutations.
Sources of Funding
British Heart Foundation.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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