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 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 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 Ca²⁺ release and impaired negative feedback of SR Ca²⁺ release on Cav1.2 Ca²⁺ currents (I_Ca). Uninhibited Ca²⁺ influx via I_Ca likely contributes to Ca²⁺ overload and results in spontaneous SR Ca²⁺ releases upon β-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.

The cardiac Ca²⁺ release unit (CRU) is a multiprotein complex whose principle components include L-type Ca²⁺ channels (Cav1.2) juxtaposed to ryanodine receptor Ca²⁺ release channels (RyR2) (1). In cardiac excitation–contraction (E-C) coupling, Ca²⁺ current (I_Ca) 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).

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.

Results

Triadin-1 Deletion Reduces Expression of jSR Proteins.

The generation 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, tentatively 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 nonspecific binding of the antibody, and triadin-1 is the only triadin isoform that is detectable in murine myocardium.

Fig. 1.

Gene-targeted ablation of triadin reduces expression of jSR proteins and the extent of T-tubule jSR interfaces of cardiac CRUs. (A) Immunoblot of pooled 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 and have an increased width. Not shown is the result that jSR profiles are also less frequent in −/− myocytes. The combination of less-frequent and smaller contact areas results in a decrease of approximately 50% in areas occupied by RyR2 (see Table 1).

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 phospholamban (data not shown) located in the free SR domain were not changed. Consistent with the reduction of RyR2 protein (Fig. 1B), B_max 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 B_max measured in Trdn^−/− cardiac muscle was reduced by 42% compared with Trdn^+/+ (data not shown). In contrast, B_max of [³H]PN200–110 binding, which binds with high affinity to Cav1.2, was not different between the 2 groups (Fig. S1). The K_D of neither radioligand was altered due to triadin-1 deletion. Thus, triadin-1 deletion caused significant reductions in 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 mammalian myocardium, RyR2, Casq2, triadin, junctin, and junctophilin are all located in terminal SR cisternae, where the jSR comes in close contact with either the plasma membrane (peripheral coupling) or the T-tubules (dyads), at the level of Z-lines. Together with juxtaposed Cav1.2 channels in the plasmalemma, jSR cisternae form the cardiac CRU.

Analysis of electron micrographs demonstrates that jSR cisternae were less frequent and had shorter RyR2-bearing junctional 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 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).

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 IMS	4. jSR extent (arbitrary units)
Trdn+/+	21.7 ± 6.3 (179)	227 ± 142 (108)	0.42 ± 0.20 (85)	95 ± 28
Trdn−/−	37.1 ± 15.0* (192)	139 ± 62* (82)	0.30 ± 0.22* (89)	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.

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 proteins in Trdn^+/+ myocytes (Fig. 2C). The degree of colocalization 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.

Fig. 2.

Gene-targeted ablation of triadin increases localization of Cav1.2 and Casq2 in subcellular areas outside RyR2-containing dyads. (A–D) Isolated ventricular myocytes from +/+ (A and C) and −/− (B and D) were colabeled with antibodies against RyR2 (red) and either Cav1.2 (green; A and B) or Casq2 (green; C and D). White pixels indicate colocalization. (Scale bar, 5 μm.) (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.

Triadin-1 Deletion Impairs Cardiac E-C Coupling.

For cardiac E-C coupling, I_Ca through Cav1.2 is required to trigger Ca²⁺ release (17). As would be predicted by our ligand-binding studies (Fig. S1), I_Ca amplitude was not significantly different in Trdn^−/− compared with Trdn^+/+ myocytes under control conditions (Fig. 3A). However, I_Ca inactivation was significantly slower in Trdn^−/− myocytes (Fig. 3B). I_Ca inactivation 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 I_Ca 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 I_ca inactivation could be the result of impaired negative feedback on I_ca from SR Ca²⁺ release. Consistent with this idea, preventing SR Ca²⁺ release by blocking RyR2 channels with ryanodine abolished the differences in I_ca inactivation (Fig. 3B). Incubating myocytes for 30 min with 2 μM thapsigargin, which empties SR Ca²⁺ stores by blocking SR Ca²⁺ uptake, also abolished the differences in I_Ca inactivation time constants [tau at 0 mV (ms): Trdn^+/+: 62 ± 2.36, n = 9 vs. Trdn^−/−: 65.21 ± 2.57, n = 12; P = 0.36).

Fig. 3.

Trdn^−/− myocytes exhibit impaired Ca²⁺-dependent inactivation of L-type Ca²⁺ current. (A) (Top) Representative examples of L-type Ca²⁺ currents (I_Ca) recorded from Trdn^+/+ (+/+) and Trdn^−/− (−/−) myocytes in control conditions (CON) and in presence of 10 μM ryanodine (RY). (Bottom) Average current–voltage relationships. Myocyte size estimated by cell capacitance was not significantly different (+/+ 152 ± 4.3 pF; −/− 154 ± 3.7 pF; n = 20 each; P = 0.73). (B) Representative examples of superimposed normalized I_Ca records at 0 mV (Left) and averaged data (Right) in control conditions. Note that block of RyR2 channels with ryanodine abolished the differences in I_Ca inactivation. (C) I_Ca recordings in the presence of 1 μM ISO. −/− myocytes exhibit significantly larger I_Ca amplitudes. Note that ryanodine abolished the differences between +/+ and −/− myocytes. (D) I_Ca inactivation in ISO-stimulated myocytes. Although ryanodine reduced the differences between the 2 groups, I_Ca inactivation remained significantly slower in Trdn-/- myocytes even in the presence of RY. n = 7–10 myocytes from 3–4 different mice per genotype; ***, P < 0.001 vs. +/+.

In ventricular myocytes, β-adrenergic receptor stimulation with isoproterenol (ISO) significantly increases I_Ca amplitude and accelerates I_Ca inactivation, in part by increasing the SR Ca²⁺ content and release (ref. 19 and Fig. 3C, Left). Ryanodine pretreatment further enhances I_Ca peak current amplitude in ISO-stimulated Trdn^+/+ myocytes, because large SR Ca²⁺ releases can curtail peak I_Ca (ref. 20 and Fig. 3C, Left). In Trdn^−/− myocytes, ISO caused a greater percentage increase in I_Ca amplitude (Fig. 3C) than in Trdn^+/+ myocytes, which was accompanied by very slow I_Ca inactivation (Fig. 3 C and D). Ryanodine pretreatment abolished the differences in I_Ca amplitude between the 2 groups (Fig. 3C), but both activation and inactivation of I_Ca remained significantly slower in Trdn^−/− myocytes (Fig. 3D). Similar results were obtained in thapsigargin-treated myocytes (data not shown). Thus, although the difference in ISO-stimulated I_Ca amplitude can be explained by the reduced negative feedback from SR Ca²⁺ release, a component of I_Ca inactivation 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 I_Ba had 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 amplitude 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^+/+ myocytes. Consistent with the decreased colocalization of Cav1.2 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 I_Ca of Trdn^−/− myocytes was not different in control conditions (Fig. 3A) and that it increased in the presence of ISO (Fig. 3C), these results demonstrate that E-C coupling efficiency is impaired in Trdn^−/− myocytes.

Fig. 4.

Trdn^−/− myocytes display impaired SR Ca²⁺ release despite increased 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 μM 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.

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. Compared with Trdn^+/+ myocytes, the average decay time constant of Trdn^−/− myocytes was significantly slower in control conditions [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 (F_ratio: Trdn^+/+ 1.35 ± 0.10, n = 33 vs. Trdn^−/− 1.34 ± 0.11, n = 30; P = 0.95), or blocking I_Ca with 20 μM nifedipine (F_ratio: 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 I_Ca causes excess Ca²⁺ influx, which was responsible for the increased SR Ca²⁺ content of Trdn^−/− myocytes.

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 μM) abolished the differences in SCR incidence between the 2 groups (Fig. 5B).

Fig. 5.

Catecholamine challenge with ISO caused premature SCR in Trdn^−/− myocytes and ventricular ectopy in Trdn^−/− mice. (A) Representative examples of premature SCR (*) in a Trdn^−/− myocyte during exposure to 1 μM 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 μM) 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 extrasystoles (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.

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 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 demonstrated significant sinus bradycardia, increased P-wave amplitude, 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 remodeling 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 Trdn^−/− mice.

Discussion

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 abnormalities 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 consequences 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 inhibition of I_Ca by SR Ca²⁺ release caused a net increase of Ca²⁺ influx into the cell. Consistent with this hypothesis, I_Ca inhibition 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 I_Ca contributed to the high incidence of ventricular ectopy via generation of early after-depolarizations (23).

Our data suggest that 2 mechanisms exist whereby absence of triadin-1 impairs I_Ca inactivation, resulting in myocyte Ca²⁺ overload. First, the dyadic membranes within Trdn^−/− cardiomyocytes exhibit an altered architecture, with a reduced colocalization of Cav1.2 with RyR2 channels according to our electron microscopy, immunolabeling, and ligand-binding results. We hypothesize that these structural changes reduce the negative feedback that SR Ca²⁺ release normally exerts on I_ca (19), given that most differences in I_Ca can be prevented by block of SR Ca²⁺ release with ryanodine or depletion of Ca²⁺ stores with thapsigargin. A second component of I_Ca inactivation remains insensitive to SR Ca²⁺ release inhibition and is observed 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^−/− myocytes. It is possible that Cav1.2 channels located outside of dyads exhibit different gating properties, and/or that these channels are 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 I_Ca mode 2 gating (25), altered subunit composition favoring I_Ca facilitation (26), or CaMKII-mediated I_Ca 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 impaired SR Ca²⁺ release, the altered jSR anatomy (50% reduction 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 movement 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 because Casq2 concentration is relatively low and thus it does not 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 functional 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 observed in models of heart failure (32). Surprisingly, despite decreased Ca²⁺ transients and contractility of individual myocytes, 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, considering 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 mechanism for the arrhythmia phenotype seems to be the structural remodeling of the cardiac Ca²⁺ release unit, which results in impaired E-C coupling and impaired contractility at the level of the myocyte. Although catecholamines can normalize contractile function by increasing I_Ca and SR Ca²⁺ content, it comes at the price of an increased risk for spontaneous Ca²⁺ releases in myocytes and triggered ventricular arrhythmias in vivo.

Methods

Animal Model.

The use of animals in this study was in accordance to National Institutes of Health guidelines and approved by the Vanderbilt University Laboratory Animal Care and Use Committee. Sex-matched Trdn^+/+ and Trdn^−/− mice, C57/black6 strain, of 4–7 months of age were used for all of the experiments.

Protein Analysis.

Mouse ventricular homogenates and microsomes were prepared, immunoblotted, and quantified as previously described (11); see also SI Methods.

Electron Microscopy.

Hearts from 6–7-month-old Trdn^+/+ and Trdn^−/− mice were harvested, the aorta cannulated, and hearts fixed by retrograde reperfusion with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Hearts were processed, imaged by electron microscopy, and estimates of relative surface areas and volumes of the total obtained as previously described (11), with a factor of 2 correction. Lengths and widths of the jSR cisternae profiles were measured with Photoshop (Adobe Systems). All quantitative data were obtained from 3 hearts each for Trdn^+/+ and Trdn^−/− mice.

Heart Histology.

Hearts were harvested from 6 age- and sex-matched mice per genotype, fixed, sectioned, stained, and the amount of inflammation, cell hypertrophy, myofiber disarray, and fibrosis quantified by an experienced pathologist blinded to the genotype; see also SI Methods.

Immunolabeling and Colocalization Experiments.

Isolated ventricular myocytes were used for fixation, permeabilization, and immunolabeling, as well as processing, deconvolving, and analyzing images as previously described (34); see also SI Methods.

Myocyte Isolation and Ca²⁺ Fluorescence Measurements.

Single ventricular myocytes were isolated, loaded with the membrane-permeable fluorescent Ca²⁺ indicator Fura-2 AM, and [Ca²⁺] measured as previously described (11); see also SI Methods.

Analysis of SCRs.

An SCR was defined as any spontaneous increase of 0.1 ratiometric units (3 times the average background noise) or more from the diastolic F_ratio other than when triggered by field stimulation or caffeine (11). For each myocyte, SCRs were counted over a 20-s period. SCRs and SR Ca²⁺ content were also analyzed in myocytes after 20-min incubation with the Cav1.2 channel blocker nifedipine 20 μM.

Voltage-Clamp Studies.

Cav1.2 Ca²⁺ currents were measured as previously described (11); see also SI Methods.

ECG Recordings and Echocardiography.

For the surface ECG and echocardiography, recordings were done as previously described (11). ISO challenge was performed on unrestrained telemetry-implanted mice as described previously (11).

Statistical Analysis.

All experiments were done in random sequence with respect to the genotype, and measurements were taken by a single observer who was blinded to the genotype. Differences between groups were assessed using a one-way analysis of variance (for normally distributed parameters) or by Kruskal-Wallis test (for parameters that are not normally distributed). If statistically significant differences were found, individual groups were compared with Student's t test or by nonparametric tests, as indicated in the text. Results were considered statistically significant if the P value was <0.05. Unless otherwise indicated, results are expressed as arithmetic means ± SEM.