doi: 10.1161/01.RES.0000162162.97722.38
© 2005 American Heart Association, Inc.
Editorials |
Triadin
The New Player on Excitation-Contraction Coupling Block
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
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Introduction |
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 Top Introduction Voltage-Dependence of CICRAdenovirus-Mediated Versus...Arrhythmia and TRDReferences |
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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.
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Voltage-Dependence of CICR |
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TopIntroductionVoltage-Dependence of CICRAdenovirus-Mediated Versus...Arrhythmia and TRDReferences |
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In the present context of regulation of ICa-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 ICa 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 ICa-gated Ca2+ release, such that the small ICa 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 ICa 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 ICa-triggered Ca2+ release significantly, even though producing sharply opposite results on the efficacy of ICa to release SR Ca2+. In myocytes overexpressing CSQ (adenoviral approach), there was a large enhancement of both caffeine- and ICa-triggered Ca2+ transients.9 In transgenic CSQ overexpressing mice, ICa-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 ICa-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 ICa, the ability of ICa to trigger Ca2+ release is strongly enhanced only when ICa 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 ICa (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 ICa-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 ICa-induced Ca2+ release to behave more like the depolarization-induced Ca2+ release of skeletal muscle. Quantifying the gain of ICa-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.
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Adenovirus-Mediated Versus Transgenic TRD Overexpression |
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TopIntroductionVoltage-Dependence of CICRAdenovirus-Mediated Versus...Arrhythmia and TRDReferences |
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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 ICa 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.
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Arrhythmia and TRD |
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TopIntroductionVoltage-Dependence of CICRAdenovirus-Mediated Versus...Arrhythmia and TRDReferences |
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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 data15,16 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 previously13,14 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.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionVoltage-Dependence of CICRAdenovirus-Mediated Versus...Arrhythmia and TRDReferences |
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1. Terentyev D, Cala SE, Houle TD, Viatchenko-Karpinski S, Gyorke I, Terentyeva R, Williams SC, Gyorke S. Triadin overexpression stimulates excitation-contraction coupling and increases predisposition to cellular arrhythmia in cardiac myocytes. Circ Res. 2005; 96: 651–658.
2. Zhang L, Kelley J, Schmeisser G, Kobayashi YM, Jones LR. Complex formation between junctin, triadin, calsequestrin, and the ryanodine receptor. Proteins of the cardiac junctional sarcoplasmic reticulum membrane. J Biol Chem. 1997; 272: 23389–23397.
3. Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol. 2004; 37: 417–429.[CrossRef][Medline] [Order article via Infotrieve]
4. Nabauer M, Callewaert G, Cleemann L, Morad M. Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes. Science. 1989; 244: 800–803.
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6. Woo SH, Soldatov NM, Morad M. Modulation of Ca2+ signalling in rat atrial myocytes: possible role of the alpha1C carboxyl terminal. J Physiol. 2003; 552: 437–447.
7. Sham JSK, Cleemann L, Morad M. Epinephrine stimulates Ca release in cardiomyocytes by enhancing Ca loading of sarcoplasmic reticulum. Biophys J. 1992; 61: 22.(Abstract.)
8. Terentyev D, Viatchenko-Karpinski S, Valdivia HH, Escobar AL, Gyorke S. Luminal Ca2+ controls termination and refractory behavior of Ca2+-induced Ca2+ release in cardiac myocytes. Circ Res. 2002; 91: 414–420.
9. Terentyev D, Viatchenko-Karpinski S, Gyorke I, Volpe P, Williams SC, Gyorke S. Calsequestrin determines the functional size and stability of cardiac intracellular calcium stores: mechanism for hereditary arrhythmia. Proc Natl Acad Sci U S A. 2003; 100: 11759–11764.
10. Jones LR, Suzuki YJ, Wang W, Kobayashi YM, Ramesh V, Franzini-Armstrong C, Cleemann L, Morad M. Regulation of Ca2+ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin. J Clin Invest. 1998; 101: 1385–1393.[Medline] [Order article via Infotrieve]
11. Wang W, Cleemann L, Jones LR, Morad M. Modulation of focal and global Ca2+ release in calsequestrin-overexpressing mouse cardiomyocytes. J Physiol. 2000; 524 pt 2: 399–414.
12. Gyorke I, Hester N, Jones LR, Gyorke S. The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium. Biophys J. 2004; 86: 2121–2128.[Medline] [Order article via Infotrieve]
13. Kirchhefer U, Jones LR, Begrow F, Boknik P, Hein L, Lohse MJ, Riemann B, Schmitz W, Stypmann J, Neumann J. Transgenic triadin 1 overexpression alters SR Ca2+ handling and leads to a blunted contractile response to beta-adrenergic agonists. Cardiovasc Res. 2004; 62: 122–134.
14. Kirchhefer U, Neumann J, Baba HA, Begrow F, Kobayashi YM, Reinke U, Schmitz W, Jones LR. Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1. J Biol Chem. 2001; 276: 4142–4149.
15. Priori SG, Napolitano C, Memmi M, Colombi B, Drago F, Gasparini M, DeSimone L, Coltorti F, Bloise R, Keegan R, Cruz Filho FE, Vignati G, Benatar A, DeLogu A. Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia. Circulation. 2002; 106: 69–74.
16. Postma AV, Denjoy I, Hoorntje TM, Lupoglazoff JM, Da Costa A, Sebillon P, Mannens MM, Wilde AA, Guicheney P. Absence of calsequestrin 2 causes severe forms of catecholaminergic polymorphic ventricular tachycardia. Circ Res. 2002; 91: e21–6.[CrossRef][Medline] [Order article via Infotrieve]
17. Viatchenko-Karpinski S, Terentyev D, Gyorke I, Terentyeva R, Volpe P, Priori SG, Napolitano C, Nori A, Williams SC, Gyorke S. Abnormal calcium signaling and sudden cardiac death associated with mutation of calsequestrin. Circ Res. 2004; 94: 471–477.
18. George CH, Higgs GV, Lai FA. Ryanodine receptor mutations associated with stress-induced ventricular tachycardia mediate increased calcium release in stimulated cardiomyocytes. Circ Res. 2003; 93: 531–540.
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Circ. Res. 2005 96: 651-658.
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