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(Circulation Research. 2005;96:607.)
© 2005 American Heart Association, Inc.

Editorials

Triadin

The New Player on Excitation-Contraction Coupling Block

Martin Morad, Lars Cleemann, Björn C. Knollmann

From the Department of Pharmacology, Georgetown University, Washington, DC.

Correspondence to Martin Morad, Georgetown University, Dept. of Pharmacology, 3900 Reservoir Road, NW, Washington DC, 20057. E-mail moradm{at}georgetown.edu

See related article, pages 651–658

Key Words: triadin overexpression • cardiac Ca²⁺ signaling • cardiac EC coupling • ryanodine receptors • calsequestrin • arrythmia

	Introduction

Top Introduction Voltage-Dependence of CICR Adenovirus-Mediated Versus... Arrhythmia and TRD References

 
The Ca²⁺ movements that control many cellular functions, including contraction of striated and smooth muscle cells and vesicular secretion from endocrine cells and nerve terminals, are increasingly recognized to involve macromolecular Ca²⁺-signaling complexes that, in addition to the key Ca²⁺-transporting proteins, include large numbers of associated proteins that provide a variety of regulatory, structural and Ca²⁺-sensing/buffering functions. The importance of working out the subtle interactions within these macromolecular complexes is underscored by the genetic diseases that have been associated with mutations of their constituent proteins.

In this issue, Terentyev et al¹ use a spectrum of molecular and electrophysiological techniques to demonstrate that triadin (TRD) plays an unexpectedly important role in regulating the ryanodine receptors (RyRs), found primarily in dyadic junctions of cardiac sarcoplasmic reticulum (SR; Figure). It has been previously suggested that TRD and junctin are integral membrane proteins of the junctional SR, and serve as linker proteins from the SR Ca release channel (RyR) to calsequestrin (CSQ) complexes, the major Ca²⁺-buffer in the lumen of the SR (Figure).² The large (4500 aa) cytoplasmic domain of RyR appears to have multiple binding sites for an ever-growing list of proteins that includes: calmodulin, PKA, FKBP 12.6.³ The major findings of the present communication are that overexpression of TRD leads to 3-fold increase in open probability of RyRs in bilayers, a 60% increase in spontaneous spark frequency with only minor decreases in spark amplitude (10%) and SR Ca²⁺ content (30%), as well as a marked alteration in the voltage dependence of Ca²⁺ release. The authors propose the activity of RyRs to be directly modulated by the level of expression of TRD, most likely mediated by amino acid residues 200 to 224 of TRD, associating with RyR in a manner similar to that of CSQ. They provide fairly clear evidence that these residues are critical for the described excitation-contraction (EC) coupling phenotype, as transfection of myocytes without this domain failed to alter the control EC coupling phenotype. Because a decrease of the Ca²⁺-content of the SR by 30% would have a direct inhibitory effect on the frequency of occurrence of spontaneous sparks and the open probability of RyRs as previously proposed, the observed increases in frequency of sparks and open probability of single RyRs is even more impressive, suggesting that TRD may be a more critical regulator of RyR activity, perhaps even more than luminal SR Ca²⁺ concentrations.

View larger version (37K): [in this window] [in a new window]   Macromolecular Ca2+ signaling complex at junctions between t-tubular and SR membranes.

	Voltage-Dependence of CICR

Top Introduction Voltage-Dependence of CICR Adenovirus-Mediated Versus... Arrhythmia and TRD References

 
In the present context of regulation of I_Ca-gated Ca²⁺ release (CICR) via interaction of TRD with RyRs, and the resultant significant change in the bell-shaped voltage-dependence of Ca²⁺ release (Figure 2 of Terentyev et al),¹ it may be appropriate to consider the present understanding of cardiac Ca²⁺ signaling cascade, including the possible steric interactions between RyRs and the cytoplasmic tail of the _1c subunit of the Ca²⁺ channel.

The dominant Ca²⁺-signaling pathway that underlies cardiac EC coupling involves activation of _1c subunit of Ca²⁺ channel and mandatory influx of Ca²⁺ through the channel leading to release of Ca²⁺ from the RyR,⁴ which in turn inactivate the Ca²⁺ channel helping to terminate the release process.⁵ Deviations from a strict Ca²⁺-dependent process, however, were recognized early when quantifying the gain of CICR. It was surprising to find that the gain of CICR was voltage-dependent, showing 10x higher gain at negative voltages of –30 to –40 mV where I_Ca was minimally activated. Recent studies introducing small segments of the carboxylic tail (ie, LA peptide)⁶ of the Ca²⁺ channel _1c subunit into atrial myocytes suggest that only the apocalmodulin binding domain of the LA peptide was the critical domain required to enhance the spontaneous spark frequency and the gain factor of the Ca²⁺ channel (dihydropyridine receptors [DHPR]) uncoupled central release sites. The specific interaction of the LA peptide with the RyR at –30 mV and –40 mV, but not at +10 mV, could provide for the voltage-dependence of CICR, as well as the 4x higher spontaneous frequency of peripheral Ca²⁺ sparks (where DHPR and RyRs are coexpressed), as compared with the DHPR-uncoupled central sites of atrial myocytes.⁶ These findings suggest that CICR mechanism maybe regulated also by molecular processes that could involve direct protein–protein interaction.

In this issue, using adenoviral transfection of adult rat myocytes, Terentyev et al¹ provide compelling evidence that overexpression of TRD leads to altered voltage-dependence of I_Ca-gated Ca²⁺ release, such that the small I_Ca activated at –30 mV and –20 mV produce the same amount of Ca²⁺ release as that at 0 mV to +60 mV, making the voltage-dependence of Ca²⁺ release less bell-shaped. This more sigmoid voltage-dependence of peak Ca²⁺ release approximates those recorded in skeletal muscle. Such "squaring off" of the voltage-dependence of Ca²⁺ release has been previously reported when intracellular Ca²⁺ pools were increased by incubating the myocytes in 10 mmol/L Ca²⁺, even when I_Ca was triggered in solutions with normal Ca²⁺ concentrations⁷ or by introducing large concentrations of low-affinity Ca²⁺ buffers (eg, citrate) into the SR,⁸ even though in the former case the voltage-dependence of the rate of Ca²⁺ release continued to remain bell-shaped.

In contrast, large increases in Ca²⁺ content of the SR by overexpression of the endogenous Ca²⁺ buffering protein, cardiac CSQ via adenoviral approach,⁹ or via the transgenic mice approach¹⁰ failed to change the voltage-dependence of I_Ca-triggered Ca²⁺ release significantly, even though producing sharply opposite results on the efficacy of I_Ca to release SR Ca²⁺. In myocytes overexpressing CSQ (adenoviral approach), there was a large enhancement of both caffeine- and I_Ca-triggered Ca²⁺ transients.⁹ In transgenic CSQ overexpressing mice, I_Ca-triggered Ca²⁺ release was markedly suppressed, and coordinated activation of Ca²⁺ sparks failed to occur, leading to smaller and slower Ca²⁺ transients, even though caffeine triggered stores were 3 to 5x larger than in wild mice.¹⁰ In this model, interestingly, increasing the Ca²⁺ sensitivity of RyRs with 0.2 to 0.5 mmol/L of caffeine restored coordinated sarcomeric Ca²⁺ striping.¹¹ Irrespective of the differences in the 2 sets of data, the voltage-dependence of I_Ca-triggered Ca²⁺ release was not significantly different between the 2 models, suggesting that CSQ serves a dual role in regulating CICR, ie, CSQ enhances Ca²⁺ release by increasing SR Ca²⁺ content and, at the same time, CSQ inhibits Ca²⁺ release by its Ca²⁺-dependent binding to the RyR, possibly via its interaction with TRD. Consistent with this idea are in vitro data suggesting that CSQ binds to TRD (Figure), and that both proteins together may represent the sarcoplasmic Ca²⁺ sensor that regulates the intraluminal Ca²⁺ sensitivity of the RyR.¹²

When comparing the voltage-dependence of the Ca²⁺ current of control and TRD overexpressing myocytes it becomes quite clear that even though there is no effect on I_Ca, the ability of I_Ca to trigger Ca²⁺ release is strongly enhanced only when I_Ca is very small, suggesting either an increased sensitivity of RyRs to Ca²⁺ (easily testable from bilayer single RyRs studies), or that the gating of RyRs is fundamentally altered in TRD overexpressing myocytes. It is suprising to note that the Ca²⁺ release actually may precede I_Ca (Figure 2 of Terentyev et al), suggesting that the depolarization signal may directly regulate Ca²⁺ release process similar to the mechanism of skeletal muscle. It is intriguing to consider whether the level of expression of TRD in part determines the "purity" of the CICR mechanism. If that were the case, does the higher expression of TRD drive the reaction toward a less Ca²⁺-dependent phenotype as found in skeletal muscle? In this respect it would be critical to determine the stoichiometry of TRD and RyR in control, and TRD-overexpressing myocytes, as well as in skeletal muscle. A cursory quantification of the gain of I_Ca-gated Ca²⁺ release, based on the data of their Figure 2, suggests orders of magnitude increase in the amplification factor at –30 mV, –20 mV and +60 mV, allowing the I_Ca-induced Ca²⁺ release to behave more like the depolarization-induced Ca²⁺ release of skeletal muscle. Quantifying the gain of I_Ca-gated Ca²⁺ release corrected for the Ca²⁺ content of SR as a ratio of the extent of TRD overexpression may provide critical insight in determining how TRD amplifies Ca²⁺ release.

	Adenovirus-Mediated Versus Transgenic TRD Overexpression

Top Introduction Voltage-Dependence of CICR Adenovirus-Mediated Versus... Arrhythmia and TRD References

 
It should be noted that chronic overexpression of TRD in cardiac tissue via a transgenic approach resulted in a quite different EC coupling phenotype. Unlike the results from rat myocytes acutely overexpressing TRD presented by Terentyev et al in this current issue,¹ chronic overexpression of TRD in mouse myocytes increased spark amplitude, and SR Ca²⁺ load, but did not change spark frequency.¹³ Inactivation of I_Ca was slowed, consistent with impaired CICR.¹³ There are many possible reasons for the discrepancies between acute and chronic overexpression of TRD. For example, chronic overexpression of TRD causes down-regulation of the RyR and junctin,¹⁴ which may in turn contribute to the observed differences in the TRD transgenic Ca²⁺ signaling phenotype. On the other hand, keeping adult cardiomyocytes in culture for over 48 hours (necessary for the adenovirus transfection experiments) significantly changes myocyte structure and protein expression (ie, loss of t-tubules, down-regulation of K-channels). Furthermore, in neither model system the exact subcellular location of the overexpressed TRD is known. Depending on the degree of association of the overexpressed TRD molecules with the native RyRs and their stoichiometry, the resultant phenotype could be quite different. For example, rapid production of TRD protein driven by a highly active adenovirus promoter may lead to TRD that is not coupled to the native RyRs. The excess TRD would still bind to the more ubiquitous CSQ and possibly disrupt the Ca²⁺ binding of CSQ, resulting in increased free luminal Ca²⁺ and decreased SR Ca²⁺ buffering capacity.

	Arrhythmia and TRD

Top Introduction Voltage-Dependence of CICR Adenovirus-Mediated Versus... Arrhythmia and TRD References

 
If indeed TRD is an important regulator of the SR Ca²⁺ release channel (RyR, Figure), as the data of Terentyev et al¹ suggest, it may not be surprising that TRD overexpression also would increase the proclivity for arrhythmogenesis in a manner similar the abnormalities of expression of other SR associated Ca²⁺-signaling proteins. Specifically, recent clinical data^15,16 strongly suggest that mutations that render cardiac CSQ or RyR dysfunctional can cause a syndrome of catecholamine-induced polymorphic ventricular tachycardia (CPVT). The finding of catecholamine-induced Ca²⁺ waves that trigger delayed after-depolarizations in TRD overexpressing myocytes reported here¹ resembles results obtained in myocytes that have decreased levels of functional CSQ¹⁷ or harbor CPVT-linked RyR mutations.¹⁸ Together, this raises the exciting possibility that mutations in or increased expression of TRD may represent a novel mechanism that could be responsible for the CPVT syndrome in humans. However, the data from transgenic mice with cardiac-targeted overexpression of TRD appear to suggest otherwise. It has been reported that chronic overexpression of TRD in mouse myocytes was not accompanied by catecholamine-induced arrhythmia, and Ca²⁺ release was less sensitive to catecholamines.¹³ Finally, TRD transgenic mice appeared to develop cardiac hypertrophy,¹⁴ not a usual feature of the clinical CPVT syndrome.

What can we conclude? The data given by Terentyev et al¹ demonstrate that TRD plays a critical role in the regulation of EC coupling, clearly a major step forward. On the other hand, the large discrepancies of the experimental results between the adenovirus-based increased TRD expression levels reported here and TRD transgenic mice reported previously^13,14 suggests that the function of TRD in regulation of CICR and cardiac pathophysiology maybe more complex than what might be predicted from the available data.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

Top Introduction Voltage-Dependence of CICR Adenovirus-Mediated Versus... Arrhythmia and TRD References

 

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Related Article:

Triadin Overexpression Stimulates Excitation-Contraction Coupling and Increases Predisposition to Cellular Arrhythmia in Cardiac Myocytes

Dmitry Terentyev, Steven E. Cala, Timothy D. Houle, Serge Viatchenko-Karpinski, Inna Gyorke, Radmila Terentyeva, Simon C. Williams, and Sandor Gyorke
Circ. Res. 2005 96: 651-658. [Abstract] [Full Text] [PDF]

This Article Extract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Morad, M. Articles by Knollmann, B. C. Search for Related Content PubMed PubMed Citation Articles by Morad, M. Articles by Knollmann, B. C. Related Collections Related Article