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.

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).

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.

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. +/+.

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.

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.