Ablation of triadin causes loss of cardiac Ca2+ release units, impaired excitation-contraction coupling, and cardiac arrhythmias.
ABSTRACT Heart muscle excitation-contraction (E-C) coupling is governed by Ca(2+) release units (CRUs) whereby Ca(2+) influx via L-type Ca(2+) channels (Cav1.2) triggers Ca(2+) release from juxtaposed Ca(2+) release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn(-/-)). The structure and protein composition of the cardiac CRU is significantly altered in Trdn(-/-) hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn(-/-) hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn(-/-) myocytes. CRU function is impaired in Trdn(-/-) myocytes, with reduced SR Ca(2+) release and impaired negative feedback of SR Ca(2+) release on Cav1.2 Ca(2+) currents (I(Ca)). Uninhibited Ca(2+) influx via I(Ca) likely contributes to Ca(2+) overload and results in spontaneous SR Ca(2+) releases upon beta-adrenergic receptor stimulation with isoproterenol in Trdn(-/-) myocytes, and ventricular arrhythmias in Trdn(-/-) mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.
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ABSTRACT: The abrupt cessation of effective cardiac function due to an aberrant heart rhythm can cause sudden and unexpected death at any age, a syndrome called sudden cardiac death (SCD). Annually, more than 300,000 cases of SCD occur in the United States alone, making this a major public health concern. Our current understanding of the mechanisms responsible for SCD has emerged from decades of basic science investigation into the normal electrophysiology of the heart, the molecular physiology of cardiac ion channels, fundamental cellular and tissue events associated with cardiac arrhythmias, and the molecular genetics of monogenic disorders of heart rhythm. This knowledge has helped shape the current diagnosis and treatment of inherited arrhythmia susceptibility syndromes associated with SCD and has provided a pathophysiological framework for understanding more complex conditions predisposing to this tragic event. This Review presents an overview of the molecular basis of SCD, with a focus on monogenic arrhythmia syndromes.The Journal of clinical investigation 01/2013; 123(1):75-83. · 15.39 Impact Factor
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ABSTRACT: L-type Ca2 + channels and the Na+/Ca2 + exchanger are the main pathways for Ca2 + influx and efflux across the sarcolemma. The majority of Ca2 + channels are found in couplons adjacent to ryanodine receptors, but there are at least two smaller, physically and functionally distinct, extradyadic populations. NCX is more widely dispersed in the membrane although a subpopulation is closely associated with the alpha-2 isoform of the Na+/K+ ATPase and has a direct effect on ECC. In addition to Ca2 + channels and ryanodine receptors, couplons in adult animals contain a variety of other occupants that modulate their function. These modulators can vary from one couplon to another creating a variety of molecular architectures. In this review we examine our current understanding of the molecular composition, binding partners and determinants of the localization of these proteins. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.Journal of Molecular and Cellular Cardiology 12/2012; 58:22–31. · 5.15 Impact Factor
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ABSTRACT: Cardiac calsequestrin (CASQ2) contributes to intracellular Ca(2+) homeostasis by virtue of its low-affinity/high-capacity Ca(2+) binding properties, maintains sarcoplasmic reticulum (SR) architecture and regulates excitation-contraction coupling, especially or exclusively upon β-adrenergic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with cardiac arrest in children or young adults. Recessive CPVT variants are due to mutations in the CASQ2 gene. Molecular and ultra-structural properties were studied in hearts of CASQ2(R33Q/R33Q) and of CASQ2(-/-) mice from post-natal day 2 to week 8. The drastic reduction of CASQ2-R33Q is an early developmental event and is accompanied by down-regulation of triadin and junctin, and morphological changes of jSR and of SR-transverse-tubule junctions. Although endoplasmic reticulum stress is activated, no signs of either apoptosis or autophagy are detected. The other model of recessive CPVT, the CASQ2(-/-) mouse, does not display the same adaptive pattern. Expression of CASQ2-R33Q influences molecular and ultra-structural heart development; post-natal, adaptive changes appear capable of ensuring until adulthood a new pathophysiological equilibrium.Experimental Cell Research 12/2013; · 3.56 Impact Factor
Ablation of triadin causes loss of cardiac Ca2?
release units, impaired excitation–contraction
coupling, and cardiac arrhythmias
Nagesh Chopraa,1, Tao Yanga,1, Parisa Asgharib, Edwin D. Mooreb, Sabine Hukea, Brandy Akinc, Robert A. Cattolicad,
Claudio F. Pereze, Thinn Hlainga,2, Barbara E. C. Knollmann-Ritschelf, Larry R. Jonesc, Isaac N. Pessahd, Paul D. Allene,
Clara Franzini-Armstrongg,3, and Bjo ¨rn C. Knollmanna,3
aDivision of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, TN 37232;bDepartment of Cellular and
Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada V6T 1Z3;cDepartment of Medicine, Krannert Institute
of Cardiology, Indiana University, Indianapolis, IN 46202;dDepartment of Molecular Biosciences, School of Veterinary Medicine, University of California,
Davis, CA 95616;eDepartment of Anesthesia, Perioperative, and Pain Medicine, Brigham and Women’s Hospital, Boston, MA 02115;fDepartment
of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; andgDepartment of Cell and Developmental Biology,
University of Pennsylvania, Philadelphia, PA 19104
Contributed by Clara Franzini-Armstrong, March 18, 2009 (sent for review December 3, 2008)
Heart muscle excitation–contraction (E-C) coupling is governed by
Ca2?release units (CRUs) whereby Ca2?influx via L-type Ca2?
channels (Cav1.2) triggers Ca2?release from juxtaposed Ca2?
release channels (RyR2) located in junctional sarcoplasmic reticu-
lum (jSR). Although studies suggest that the jSR protein triadin
anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to
E-C coupling remains unclear. Here, we identify the role of triadin
using mice with ablation of the Trdn gene (Trdn?/?). The structure
and protein composition of the cardiac CRU is significantly altered
in Trdn?/?hearts. jSR proteins (RyR2, Casq2, junctin, and juncto-
philin 1 and 2) are significantly reduced in Trdn?/?hearts, whereas
Cav1.2 and SERCA2a remain unchanged. Electron microscopy
shows fragmentation and an overall 50% reduction in the contacts
between jSR and T-tubules. Immunolabeling experiments show
reduced colocalization of Cav1.2 with RyR2 and substantial Casq2
labeling outside of the jSR in Trdn?/?myocytes. CRU function is
impaired in Trdn?/?myocytes, with reduced SR Ca2?release and
impaired negative feedback of SR Ca2?release on Cav1.2 Ca2?
currents (ICa). Uninhibited Ca2?influx via ICalikely contributes to
Ca2?overload and results in spontaneous SR Ca2?releases upon
?-adrenergic receptor stimulation with isoproterenol in Trdn?/?
myocytes, and ventricular arrhythmias in Trdn?/?mice. We con-
clude that triadin is critically important for maintaining the struc-
tural and functional integrity of the cardiac CRU; triadin loss and
the resulting alterations in CRU structure and protein composition
impairs E-C coupling and renders hearts susceptible to ventricular
cardiac muscle ? sarcoplasmic reticulum ? calsequestrin ? Cav1.2 ? RyR2
nels (Cav1.2) juxtaposed to ryanodine receptor Ca2?release
channels (RyR2) (1). In cardiac excitation–contraction (E-C)
coupling, Ca2?current (ICa) via Cav1.2 triggers sarcoplasmic
reticulum (SR) Ca2?release through RyR2 release channels, a
process that is highly regulated by RyR2-associated proteins and
RyR2 phosphorylation (2). Among the CRU proteins, RyR2,
triadin, junctin, and cardiac calsequestrin (Casq2) form a protein
complex located in the junctional elements of the sarcoplasmic
reticulum (jSR) (3). Triadin is encoded by the Trdn gene (4),
which produces several isomeric forms that differ in size (5).
Among these, the triadin-1 isoform is predominant in cardiac
muscle (6). Triadin-1 has a membrane-spanning domain, a short
cytoplasmic N-terminal segment, and a long, positively charged
C-terminal domain extending into the lumen of the SR, which
also contains the putative binding domain for Casq2 (6).
he cardiac Ca2?release unit (CRU) is a multiprotein com-
plex whose principle components include L-type Ca2?chan-
Although the function of cardiac triadin-1 is not explicitly
known, attempts have been made to understand its physiologic
role in cardiac muscle using acute adenoviral (7) and transgenic
overexpression (8) and by reconstitution experiments in lipid
bilayers (9). Changes in expression of triadin-1 are frequently
accompanied by altered expression of its binding partners RyR2
(10), Casq2 (11), and junctin (10). The trimeric complex of
Casq2, triadin-1, and junctin is thought to regulate SR Ca2?
release (9, 12). Deletion (11) or even modest reductions (13) in
Casq2 protein increases diastolic SR Ca2?leak and causes
spontaneous SR Ca2?release (SCR) and catecholaminergic
cardiac arrhythmia. Deletion of junctin enhances SR Ca2?
cycling and contractility but is associated with delayed after-
depolarization–induced ventricular arrhythmias and premature
death under conditions of physiologic stress (14).
To better understand the physiologic role of triadin-1 in the
mammalian heart, we performed a comprehensive structural
and functional evaluation of the pan triadin null (Trdn?/?)
mouse model. We found that triadin-1 is critically important to
maintain the structural and functional integrity of the cardiac
CRU, which is pivotal for both E-C coupling and maintaining a
regular heart rhythm in mammalian hearts.
Triadin-1 Deletion Reduces Expression of jSR Proteins. The genera-
tion of Trdn?/?mice and characterization of their skeletal
muscle phenotype has been described previously (15). Here we
report the cardiac phenotype of Trdn?/?mice. As expected (6),
the immunoreactive 35/40-kDa doublet corresponding to the
unglycosylated and glycosylated triadin-1 is absent in Trdn?/?
microsomes (Fig. 1A). A 92-kDa immunoreactive protein, ten-
tatively identified as the putative triadin-3 (6), was also present
in Trdn?/?microsomes, further challenging the identity of this
immunoreactive band as a Trdn-related protein (ref. 16 and Fig.
1A). Thus, on the basis of our data the 92-kDa band represents
P.A., E.D.M., S.H., B.A., R.A.C., C.F.P., T.H., B.E.C.K.-R., L.R.J., C.F.-A., and B.C.K. performed
research; C.F.P. and P.D.A. contributed new reagents/analytic tools; N.C., T.Y., P.A., E.D.M.,
S.H., B.A., R.A.C., C.F.P., T.H., B.E.C.K.-R., L.R.J., C.F.-A., and B.C.K. analyzed data; and N.C.,
E.D.M., L.R.J., I.N.P., P.D.A., C.F.-A., and B.C.K. wrote the paper.
The authors declare no conflict of interest.
1N.C. and T.Y contributed equally to this article.
2Present address: Department of Medicine, University of Miami, Miami, FL 33136.
3To whom correspondence may be addressed. E-mail: email@example.com or
This article contains supporting information online at www.pnas.org/cgi/content/full/
May 5, 2009 ?
vol. 106 ?
nonspecific binding of the antibody, and triadin-1 is the only
triadin isoform that is detectable in murine myocardium.
SR proteins located in the jSR cisternae (RyR2, Casq2,
junctin, and junctophilin 1 and 2) were all significantly decreased
in Trdn?/?hearts (Fig. 1 B and C). Expression levels of the SR
Ca2?uptake pump, SERCA2a (Fig. 1 B and C) and phospho-
lamban (data not shown) located in the free SR domain were not
changed. Consistent with the reduction of RyR2 protein (Fig.
1B), Bmax for high affinity [3H]ryanodine binding sites was
significantly reduced (Trdn?/?: 570 ? 30 fmol/mg, n ? 6;
Trdn?/?: 430 ? 25 fmol/mg, n ? 4; P ? 0.0013) in Trdn?/?
preparations compared with Trdn?/?[supporting information
(SI) Fig. S1]. When [3H]ryanodine binding studies were repeated
in a buffer containing high salt (1 M KCl) to optimize RyR2
binding, the Bmax measured in Trdn?/?cardiac muscle was
reduced by 42% compared with Trdn?/?(data not shown). In
contrast, Bmaxof [3H]PN200–110 binding, which binds with high
affinity to Cav1.2, was not different between the 2 groups (Fig.
S1). The KDof neither radioligand was altered due to triadin-1
RyR2 and associated jSR proteins without significant change in
Cav1.2 expression in the plasma membrane.
Loss of Triadin-1 Alters the Architecture of Cardiac CRUs. In mam-
malian myocardium, RyR2, Casq2, triadin, junctin, and juncto-
philin are all located in terminal SR cisternae, where the jSR
comes in close contact with either the plasma membrane (pe-
ripheral coupling) or the T-tubules (dyads), at the level of
Z-lines. Together with juxtaposed Cav1.2 channels in the plas-
malemma, jSR cisternae form the cardiac CRU.
Analysis of electron micrographs demonstrates that jSR cis-
ternae were less frequent and had shorter RyR2-bearing junc-
tional contacts with T-tubules (Fig. 1 D and E and Table 1).
Overall, the reduced number and shorter contacts resulted in
?56% reduction in total extent of RyR2-bearing jSR cisternae
(Table 1), in good agreement with the 50% reduction in RyR2
protein measured by Western blot (Fig. 1C). At the same time,
the remaining jSR cisternae were more variable in width and
significantly wider in Trdn?/?hearts (Fig. 1E and Table 1).
Casq2 was visible in the form of small condensed nodules in
Trdn?/?(Fig. 1D) but was barely noticeable, showing only as a
microsomal preparations from Trdn?/?and Trdn?/?hearts. The 35/40-kDa double band represents triadin-1 and its glycosylated form, the only triadin isoform
significantly expressed in adult mouse heart. The band at 92 kDa is a nonspecific cross-reacting band unrelated to triadin. (B and C) Representative examples of
immunoblots of whole-heart homogenates (B) and summarized data (C) demonstrate reduced expression of jSR proteins in Trdn?/?(?/?) mice. n ? 5 hearts per
group.*, P ? 0.05. (D and E) Electron micrographs from thin sections of age-matched Trdn?/?(D) and Trdn?/?(E) myocardium from the left ventricle showing
details of dyads. For ease of identification, a transparent yellow overlay covers the lumen of T-tubules (T) and a green overlay that of the jSR domains. Structural
details are visible under the overlay. A narrow cleft containing profiles of ‘‘feet’’ representing the cytoplasmic domains of RyR2 occupy the narrow junctional
gap. The images were selected to illustrate 2 major differences between ?/? and ?/? myocytes: The junctional SR domains of ?/? myocytes are less extensive
areas results in a decrease of approximately 50% in areas occupied by RyR2 (see Table 1).
Table 1. Quantitative electron microscopic morphometry of ventricular cardiac myocytes
Genotype 1. jSR lumen width (nm) 2. jSR contact length (nm) 3. jSR contacts per IMS4. jSR extent (arbitrary units)
21.7 ? 6.3 (179)
37.1 ? 15.0* (192)
227 ? 142 (108)
139 ? 62* (82)
0.42 ? 0.20 (85)
0.30 ? 0.22* (89)
95 ? 28
42 ? 13*
jSR lumen width was significantly larger and more variable in Trdn?/?cells (see also Fig. 1 C and D). jSR extent (where feet are located) was obtained by
multiplying the jSR contact length (column 2) by the frequency of dyads (? jSR contacts per intermyofibrillar space (IMS), column 3). jSR extent was reduced by
56% in the Trdn?/?myocardium. Data are mean ? SD. All data are from 3 mice. In parenthesis are the number of dyads (columns 1 and 2) and number of images
(column 3).*, P ? 0.001, Student’s t test.
Chopra et al. PNAS ?
May 5, 2009 ?
vol. 106 ?
no. 18 ?
diffuse density, in the Trdn?/?jSR (Fig. 1E). There was no
significant widening of the free SR elements and no change in
total SR volume (data not shown).
We next examined whether loss of triadin-1 also changes the
relative positioning of 3 key CRU proteins. Immunolabeling of
isolated ventricular myocytes from Trdn?/?mice with anti-RyR2
antibody (red) and anti-Cav1.2 antibody (green) shows well-
defined and colocalized (white) foci located at the Z-lines
corresponding to dyads (Fig. 2A). In Trdn?/?myocytes, the
degree of colocalization of Cav1.2 with RyR2 was significantly
reduced (Fig. 2 B and E). The non-colocalized Cav1.2 are part
of either CRUs with reduced RyR2 content or nonfunctional
CRUs lacking RyR2. Immunolabeling with anti-RyR2 and anti-
Casq2 antibodies shows the dyadic colocalization of both pro-
of RyR2 with Casq2 was not significantly different in Trdn?/?
myocytes, but colocalization of Casq2 with RyR2 was significantly
reduced (Fig. 2 D and E). These data demonstrate that although
some Casq2 protein is retained in jSR cisternae, a significant
fraction of the Casq2 protein moves into longitudinal SR.
Triadin-1 Deletion Impairs Cardiac E-C Coupling. For cardiac E-C
coupling, ICathrough Cav1.2 is required to trigger Ca2?release
(17). As would be predicted by our ligand-binding studies (Fig.
S1), ICa amplitude was not significantly different in Trdn?/?
compared with Trdn?/?myocytes under control conditions (Fig.
3A). However, ICa inactivation was significantly slower in
Trdn?/?myocytes (Fig. 3B). ICainactivation is both voltage and
Ca2?dependent (18). Because of the close proximity of RyR2
and Cav1.2 in the dyad, Ca2?released from the SR mediates a
large fraction of Ca2?-dependent ICa inactivation (19). Given
that there are fewer RyR2 channels in Trdn?/?hearts (Fig. 1B
and Fig. S1), a substantial fraction of Cav1.2 channels are not
associated with RyR2 (Fig. 2E), and the differences in Ica
inactivation could be the result of impaired negative feedback on
Icafrom SR Ca2?release. Consistent with this idea, preventing
SR Ca2?release by blocking RyR2 channels with ryanodine
abolished the differences in Icainactivation (Fig. 3B). Incubating
myocytes for 30 min with 2 ?M thapsigargin, which empties SR
Ca2?stores by blocking SR Ca2?uptake, also abolished the
differences in ICainactivation time constants [tau at 0 mV (ms):
Trdn?/?: 62 ? 2.36, n ? 9 vs. Trdn?/?: 65.21 ? 2.57, n ? 12;
P ? 0.36).
In ventricular myocytes, ?-adrenergic receptor stimulation
with isoproterenol (ISO) significantly increases ICa amplitude
and accelerates ICa inactivation, in part by increasing the SR
Ca2?content and release (ref. 19 and Fig. 3C, Left). Ryanodine
pretreatment further enhances ICa peak current amplitude in
ISO-stimulated Trdn?/?myocytes, because large SR Ca2?re-
leases can curtail peak ICa(ref. 20 and Fig. 3C, Left). In Trdn?/?
myocytes, ISO caused a greater percentage increase in ICa
amplitude (Fig. 3C) than in Trdn?/?myocytes, which was
accompanied by very slow ICa inactivation (Fig. 3 C and D).
Casq2 in subcellular areas outside RyR2-containing dyads. (A–D) Isolated
ventricular myocytes from ?/? (A and C) and ?/? (B and D) were colabeled
(green; C and D). White pixels indicate colocalization. (Scale bar, 5 ?m.) (E)
Colocalization of Cav1.2 and Casq2 with RyR2 is significantly reduced in ?/?
myocytes, demonstrating that a significant number of Cav1.2 and Casq2 are
located outside the dyads. Data are mean ? SEM.***, P ? 0.001;**, P ? 0.01;
?/? n ? 5 myocytes; ?/? n ? 6 myocytes.
(ICa) recorded from Trdn?/?(?/?) and Trdn?/?(?/?) myocytes in control
conditions (CON) and in presence of 10 ?M ryanodine (RY). (Bottom) Average
not significantly different (?/? 152 ? 4.3 pF; ?/? 154 ? 3.7 pF; n ? 20 each;
of RyR2 channels with ryanodine abolished the differences in ICainactivation.
(C) ICarecordings in the presence of 1 ?M ISO. ?/? myocytes exhibit signifi-
cantly larger ICaamplitudes. Note that ryanodine abolished the differences
Although ryanodine reduced the differences between the 2 groups, ICainactiva-
Trdn?/?myocytes exhibit impaired Ca2?-dependent inactivation of
www.pnas.org?cgi?doi?10.1073?pnas.0902919106 Chopra et al.
Ryanodine pretreatment abolished the differences in ICaampli-
tude between the 2 groups (Fig. 3C), but both activation and
inactivation of ICa remained significantly slower in Trdn?/?
myocytes (Fig. 3D). Similar results were obtained in thapsigar-
gin-treated myocytes (data not shown). Thus, although the
difference in ISO-stimulated ICaamplitude can be explained by
the reduced negative feedback from SR Ca2?release, a com-
ponent of ICainactivation is independent of Ca2?release. To test
whether channel gating is altered, we used Ba2?as a charge
carrier, which does not trigger SR Ca2?release and does not
cause Ca2?-dependent inactivation (19). Although not different
in control conditions (data not shown), in the presence of ISO,
both activation and inactivation of IBahad significantly slower
kinetics in Trdn?/?compared with Trdn?/?myocytes (Fig. S2).
We next examined the consequences of triadin deletion and
CRU remodeling on SR Ca2?release and storage. The ampli-
tude and rate of SR Ca2?release was significantly reduced in
Trdn?/?compared with Trdn?/?myocytes (Fig. 4 A–C). ISO
abolished the differences in Ca2?transient amplitude (Fig. 4 A
and B), but the rate of SR Ca2?release remained significantly
slower in Trdn?/?compared with Trdn?/?myocytes (Fig. 4C).
Diastolic Ca2?(Fig. 4D) and SR Ca2?content (Fig. 4 A and E)
were significantly increased in Trdn?/?compared with Trdn?/?
and RyR2 channels (Fig. 2E), the fraction of SR Ca2?content
released in response to a steady-state field stimulus (? fractional
release) was significantly reduced in Trdn?/?myocytes (Fig. 4F).
Together with the finding that ICaof Trdn?/?myocytes was not
different in control conditions (Fig. 3A) and that it increased in
coupling efficiency is impaired in Trdn?/?myocytes.
To investigate what caused the increased SR Ca2?content of
Trdn?/?myocytes, we estimated SR Ca2?uptake by fitting the
decay of Ca2?transients to a monoexponential function. Com-
pared with Trdn?/?myocytes, the average decay time constant
of Trdn?/?myocytes was significantly slower in control condi-
tions [tau (s): Trdn?/?: 0.175 ? 0.005, n ? 37 vs. Trdn?/?: 0.347 ?
0.037, n ? 38; P ? 0.001] and not significantly different during
ISO challenge [tau (s): Trdn?/?: 0.081 ? 0.16, n ? 31 vs. Trdn?/?:
0.080 ? 0.34, n ? 23; P ? 0.89]. NaCa exchanger function,
estimated by the time constant of cytosolic Ca2?decay during
caffeine application was also not significantly different between
the 2 groups [tau (s): Trdn?/?: 2.02 ? 0.14, n ? 37 vs. Trdn?/?:
1.82 ? 0.31, n ? 38; P ? 0.56]. On the other hand, measuring SR
Ca2?content in unpaced, quiescent myocytes (Fratio: Trdn?/?
1.35 ? 0.10, n ? 33 vs. Trdn?/?1.34 ? 0.11, n ? 30; P ? 0.95),
or blocking ICawith 20 ?M nifedipine (Fratio: Trdn?/?1.13 ?
0.11, n ? 17 vs. Trdn?/?1.15 ? 0.11, n ? 12; P ? 0.90) abolished
the differences between the 2 groups. Taken together, these data
suggest that the impaired inactivation of ICacauses excess Ca2?
influx, which was responsible for the increased SR Ca2?content
ISO Challenge Causes SCRs in Trdn?/?Myocytes and Ventricular
Arrhythmia in Trdn?/?Mice. Myocyte Ca2?overload can cause
SCR, Ca2?waves, and triggered beats (22). On the other hand,
because triadin-1 reportedly sensitizes the CRU to luminal Ca2?
(7, 9), triadin-1 deletion may prevent SCR even under conditions
of high SR Ca2?load observed in Trdn?/?myocytes. The net
effect of Ca2?overload in myocytes lacking triadin was an
increased incidence of SCRs during ISO exposure (Fig. 5 A and
B). Conducting the ISO challenge in quiescent myocytes without
field stimulation (SCR/min: Trdn?/?13 ? 2.8, n ? 41 vs. Trdn?/?
11.8 ? 2.9, n ? 32; P ? 0.64) or pretreatment with nifedipine (20
?M) abolished the differences in SCR incidence between the 2
groups (Fig. 5B).
We next studied global cardiac function in vivo (Table S1).
Trdn?/?mice had increased cardiac contractility measured by
echocardiography. Heart/body weight ratio of Trdn?/?mice was
SR Ca2?content. (A) Representative examples of rapid caffeine (10 mM)
application to Trdn?/?(?/?) and Trdn?/?(?/?) myocytes that were field
stimulated at 1 Hz to maintain consistent SR Ca2?load. The 2 last paced Ca2?
transients (CaT) are also shown. The amplitude of the caffeine transient was
used as a measure of total SR Ca2?content. Experiments were carried out in
control conditions (CON) and in the presence of 1 ?M ISO. (B–F) Comparisons
of average CaT amplitudes (B), CaT rise time (0 to 90% of peak) (C), end-
diastolic [Ca2?] (D), SR Ca2?content (E), and SR Ca2?release fraction (F) in the
2 groups. Trdn?/?(?/?): n ? 37 (CON) and 31 (ISO); Trdn?/?(?/?): n ? 38
(CON) and 23 (ISO);*, P ? 0.05;***, P ? 0.001. CaT time to peak 90%, time to
reach 90% of peak CaT height.
Trdn?/?myocytes display impaired SR Ca2?release despite increased
of premature SCR (*) in a Trdn?/?myocyte during exposure to 1 ?M ISO.
Myocytes were loaded with Fura2 AM and paced at 1 Hz (vertical lines). (B)
Average rate of SCRs during a 20-s recording period. Note that Ca2?channel
block with nifedipine (NIF, 20 ?M) abolished the differences between the
groups. Trdn?/?(?/?): n ? 39 (CON), 37 (ISO), and 61 (ISO ? NIF); Trdn?/?
(?/?): n ? 46 (CON), 33 (ISO), and 67 (ISO ? NIF);*, P ? 0.001, Mann-Whitney
test. (C) ECG records showing representative examples of ventricular extra-
systoles (VES, #) and an episode of nonsustained ventricular tachycardia (VT)
in conscious Trdn?/?mice after i.p. injection of ISO (1.5 mg/kg). (D) Average
rate of VES and VT during a 1.5-h period after ISO challenge.*, P ? 0.05,
Mann-Whitney test. n ? 8 mice per group.
Catecholamine challenge with ISO caused premature SCR in Trdn?/?
Chopra et al. PNAS ?
May 5, 2009 ?
vol. 106 ?
no. 18 ?
increased by 26% (P ? 0.001). Histologic analysis of ventricular
sections did not reveal any evidence for fibrosis, inflammation,
myofibrillar disarray, or myocyte hypertrophy in Trdn?/?hearts
(data not shown). Electrocardiogram (ECG) evaluation dem-
onstrated significant sinus bradycardia, increased P-wave ampli-
tude, and widened QRS complex, but normal repolarization
parameters (Table S1). Upon ISO challenge, Trdn?/?mice
displayed a significantly higher rate of ventricular ectopy and
nonsustained ventricular tachycardia than Trdn?/?mice (Fig. 5
C and D).
Taken together, these results suggest that the structural re-
modeling of the dyad coupled with myocyte Ca2?overload as a
result of impaired Ca2?-dependent inactivation of Cav1.2 was
sufficient to cause stress-induced ventricular tachycardia in
Our results indicate that cardiac triadin-1 is critically important
for maintaining the structural integrity of the cardiac CRU. The
absence of triadin-1 leads to a loss of dyads and to a reduction
in the size of T-tubule–jSR contacts. The structural abnormal-
ities are accompanied by reduction of jSR proteins located in the
dyad. The CRU remodeling significantly impaired Ca2?release-
dependent inactivation of Cav1.2, resulting in myocyte Ca2?
overload and SCR events, and ventricular arrhythmias in vivo.
Although it is difficult to know which functional changes are
caused directly by the absence of Trdn and which are conse-
quences of secondary changes such as the CRU remodeling, our
results clearly suggest that TRDN should be considered as a
candidate gene for arrhythmia susceptibility in humans.
The mechanism that underlies the Trdn-linked arrhythmia
phenotype is surprisingly different from that of genes associated
with catecholaminergic ventricular tachycardia (11, 13). Unlike
ventricular tachycardia caused by Casq2 or RyR2 mutations,
which cause SR Ca2?leak and triggered beats at normal or
decreased SR Ca2?load (11), deletion of triadin-1 increases the
frequency of SCRs and extra beats by increasing SR Ca2?
content. We speculate that the dramatically reduced feedback
influx into the cell. Consistent with this hypothesis, ICainhibition
with nifedipine prevented excessive Ca2?loading of the SR and
the resultant SCRs. Furthermore, although not tested here, it is
possible that slow inactivation of ICa contributed to the high
incidence of ventricular ectopy via generation of early after-
Our data suggest that 2 mechanisms exist whereby absence of
triadin-1 impairs ICa inactivation, resulting in myocyte Ca2?
overload. First, the dyadic membranes within Trdn?/?cardio-
myocytes exhibit an altered architecture, with a reduced colo-
calization of Cav1.2 with RyR2 channels according to our
electron microscopy, immunolabeling, and ligand-binding re-
sults. We hypothesize that these structural changes reduce the
negative feedback that SR Ca2?release normally exerts on Ica
(19), given that most differences in ICacan be prevented by block
of SR Ca2?release with ryanodine or depletion of Ca2?stores
with thapsigargin. A second component of ICa inactivation
even when Ba2?was used as charge carrier, suggesting that
Cav1.2 channel gating properties are altered independently of
RyR2 activity and the filling state of SR. Interestingly, altered
channel gating is only evident in ISO-stimulated Trdn?/?myo-
cytes. It is possible that Cav1.2 channels located outside of dyads
nonconducting unless activated by ISO. Whether disruption of a
form of bidirectional signaling between Cav1.2 and RyR2 could
have contributed to the differences in channel gating remains to
be explored. Weak conformational coupling between these
proteins has been proposed (24) but remains elusive in cardiac
muscle. Other possibilities include enhanced ICamode 2 gating
(25), altered subunit composition favoring ICafacilitation (26),
or CaMKII-mediated ICa facilitation (27), although intact SR
Ca2?release is reportedly required for the latter (27).
It has been previously shown that overexpression of triadin-1
sensitizes RyR2 channels, increases E-C coupling gain, and
causes SCR (7). According to these studies, loss of triadin-1
should desensitize the SR-release complex and decrease E-C
coupling efficiency. Although Trdn?/?myocytes exhibited im-
in the extent of junctional contacts between SR and T-tubules)
is likely to be a major contributor. The increased rate of SCR
observed here is not consistent with a primary effect of RyR2
desensitization (22) but more likely the consequence of the
increased SR Ca2?content observed in Trdn?/?myocytes.
However, our experiments do not rule out that loss of triadin-1
desensitized the RyR2 complex (9). The concomitant reduction
in Casq2 that occurs in Trdn?/?myocytes may offset any
modulation of RyR2 activity imparted by the lack of triadin-1 (9,
13). The net effect on intrinsic RyR2 activity will have to be
addressed by direct measurements of RyR2 function.
Triadin and junctin reportedly anchor Casq2 to RyR2 (3, 6,
28). In the presence of the 2 proteins Casq2 is restricted to the
jSR cisternae and arranged in small clusters (29). The less dense
configuration of Casq2 in the jSR lumen and the partial move-
ment of Casq2 into the free SR are consistent with the drastic
reduction in junctin and complete loss of triadin-1 in Trdn?/?
myocytes. Overall, the structural analysis indicates a much looser
association of Casq2 with the RyR2 complex. Although the
functional significance of these changes remains to be explored,
it may decrease the rate of Ca2?diffusion within the SR lumen
and thereby contribute to the slow rate of Ca2?release observed
in Trdn?/?myocytes. The presence of Casq2 in the free SR
lumen is not easily detected in electron micrographs, probably
form a gel of the type seen when Casq2 is overexpressed (30).
The second effect of Trdn deletion, the fragmentation of the jSR
cisternae and the resulting decrease in jSR–T-tubule contacts
and decreased Cav1.2-RyR2 colocalization, may be the result of
a decrease in junctophilin 1 and 2 rather than a direct effect of
triadin absence. By partially uncoupling Cav1.2 from RyR2, this
configuration change may be the one with the strongest func-
tional effect, as discussed above. The underlying mechanism of
how loss of triadin causes a reduction in the jSR proteins will
have to be determined in future studies.
A recent report showed a dramatic downregulation of junctin
(below level of detection) and triadin (22%) in human failing
hearts (31). Downregulation of triadin-1 in human heart failure
may contribute to the decreased E-C coupling efficiency ob-
served in models of heart failure (32). Surprisingly, despite
decreased Ca2?transients and contractility of individual myo-
cytes, fractional left ventricular shortening was increased in
anesthetized Trdn?/?mice. Several factors likely contributed to
enhanced contractility in vivo: absence of heart fibrosis and
myocyte hypertrophy suggests that myocyte hyperplasia may
increase the muscle mass of Trdn?/?hearts, which would offset
hypocontractility at the individual myocyte level. Circulating
catecholamines may further enhance contractility in vivo, con-
sidering that in individual ISO-stimulated Trdn?/?myocytes,
Ca2?transients and contractility were not different from wild-
type myocytes. Finally, the slower heart rate of Trdn?/?mice
allows increased left ventricular filling during diastole, which
would increase apparent left ventricular fractional shortening
regardless of intrinsic myocardial contractility (33).
We conclude that ablation of the jSR protein triadin causes
susceptibility to ventricular arrhythmias in mice. The underlying
remodeling of the cardiac Ca2?release unit, which results in
www.pnas.org?cgi?doi?10.1073?pnas.0902919106 Chopra et al.