Triadin/Junctin double null mouse reveals a differential role for Triadin and Junctin in anchoring CASQ to the jSR and regulating Ca(2+) homeostasis

Abstract

Triadin (Tdn) and Junctin (Jct) are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ) and ryanodine receptor (RyRs) at the junctional sarcoplasmic reticulum (jSR). However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c) coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null), Jct (Jct-null) or both (Tdn/Jct-null), we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca(2+) imaging and Ca(2+) selective microelectrodes we found that changes in e-c coupling, SR Ca(2+)content and resting [Ca(2+)] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca(2+) regulation than Jct/CASQ association.

Figure 1. Relative levels of CRU proteins in crude homogenates from skeletal muscle.

Identical amounts (25 µg/lane) of microsomal fraction of skeletal muscles from WT, Tdn-null, Jct-null and Tdn-/Jct null mice were loaded and immunoblotted with several antibodies. Membranes were tested for expression of triadin (Tdn), junctin (Jct), ryanodine receptor (RyR1), Dihydropyridine receptors (Cav1.1), Calsequestrin (CASQ), SERCA-1 pump (SERCA), FK506 binding protein (FKBP12), Junctophilin-1 (JP-1) and Histidin-rich Ca²⁺ binding protein (HRC) as described in Material and Methods. Band intensity for each protein was normalized to GAPDH expression to correct for loading and plotted as fraction of its WT counterpart (dotted line). Data presented as mean ± SD of 3–7 independent blots. *p<0.05, **p<0.01, ***p<0.001 (ANOVA, One-way analysis of variance, Tukey's multiple comparison test). Representative blots for each series, including three anti-GAPDH blots (lower panel), are presented.

Figure 2. Images from thin sections at right angle to the T-tubule long axis (A) and parallel to it (B, C and inset) from WT EDL muscles.

A) In WT muscles the two jSR cisternae (jSR) facing the central T-tubule (TT, white space) are relatively large, they contain electron dense polymer of CASQ and are joined to T-tubules by two feet. B, C and inset). A row of periodically disposed feet (RyRs, blue dots in C) fills the jSR-T tubule junctional gap and the profiles of small anchors (green in C) project into the jSR lumen (yellow in C) in a position alternate to that of feet (arrowheads in C, better seen in the inset). The distal ends of anchor connect to a thin linear density, particularly prominent in C, presumably a long CASQ polymer. Bars: A–C, 0.1 µm; inset, 0.05 µm.

Figure 3. Sections from sternomastoid muscle in mutated mice.

A and B) In the absence of Jct the overall structure is not visibly altered. A polymer of CASQ fills the jSR and is anchored to the feet-bearing jSR membrane (arrowheads). The size of the transversely cut jSR profiles is slightly smaller than wild type in this image (see detail in Fig. 4C and D). In the absence of Tdn anchors are missing and the visible jSR content quite reduced although still visible. The jSR cisternae are considerably smaller. E and F) In the double mutant, the jSR profiles are very narrow and they seem to be basically empty. In all cases, the jSR-T tubule junctional gap and the rows of feet are unaltered. Colors: white: T-Tubule; yellow: jSR lumen; blue RyRs; Green: anchors. Bar: A–F, 0.1 µm.

Figure 4. Sections at right angle to triads in sternomastoid (top row) and soleus (bottom row) muscles illustrating changes in dimensions of the jSR cisternae relative to WT.

Compare with Table 1. C and D) In Jct-null muscles the triads are slightly smaller than WT in sternomastoid (A), but somewhat larger in soleus (compare B and D; E and F) in Tdn-null fibers the jSR cisternae are smaller in all muscles; G and H) in the double null the dimensions are further reduced. Bar: A–H, 0.1 µm.

Figure 5. Additional structural alterations:

a shift of triads orientation from transverse to longitudinal in fast twitch fibers of EDL and sternomastoid (A, larger arrow) resulting in the development of large jSR plaques carrying multiple rows of feet (D and E). This occurs in fast twitch fibers of Tdn-null fibers, as previously reported, and in the double null mutants. B) The double null mutant fibers show a small number of quite large sacs always located in correspondence of the Z-line (not shown) and filled with a finely granular material similar to the CASQ content of the jSR (star) and some flat SR cisternae (A, B, white arrows, C and detail in F). The flat SR cisternae are separated by small densities that are clearly different from feet (compare D and F, at the same magnification). Bars: A, 0.5 µm; B, 0.5 µm; C, 0.1 µm; D–F, 0.1 µm.

Figure 6. Effect of Tdn and Jct on e-c coupling and ECCE.

A) Representative traces of K⁺ -dose responses of primary cultured myotubes from wild type (WT), triadin-null (Tdn), junctin-null (Jct) and triadin/juctin double-null (Tdn/Jct) mouse muscles. B) Average peak fluorescent amplitude of depolarization-induced Ca²⁺ release response of WT (black, n = 114 cells), Jct-null (green, n = 83 cells), Tdn-null (blue, n = 57 cells) and Tdn-/Jct null (red, n = 68 cells) myotubes. Myotubes were loaded with 5 µM Fura-4F and exposed to increased concentrations of K⁺ for 5 s. C) Average rate of decrease in Fura-2 signal by Mn²⁺ quench during depolarization with 80 mM KCl. Numbers in the bars indicate the number of cells analyzed per condition. The data are from 3–4 experiments and are presented as mean ± SEM (***p<0.001 (ANOVA, One-way analysis of variance, Tukey's multiple comparison test)).

Figure 7. Effect of Tdn and Jct ablation on SR Ca²⁺ content of cultured myotubes.

A) Average peak fluorescent amplitude of caffeine-induced Ca²⁺ transients of Fura-4F loaded myotubes from WT (black, n = 59 cells), Tdn-null (blue, n = 52 cells), Jct-null (green, n = 60 cells) and Tdn-/Jct null (red, n = 59 cells) mice. B) Representative traces of CPA-induced Ca²⁺ transients of WT (black), Jct-null (green), Tdn-null (blue) and Tdn/Jct-null (red) myotubes loaded with Fura-2 used to estimate SR Ca²⁺ content. C) Comparison of average peak Ca²⁺ transient amplitude induced by 10 µM CPA. Numbers in the bars indicate the number of cells analyzed per condition. Data presented as mean ± SEM. **p<0.01, ***p<0.001 (ANOVA, One-way analysis of variance, Tukey's multiple comparison test).

Figure 8. A model proposing the contribution of Tdn's to the jSR anchors and a possible positioning of Jct.

In the model the triad is seen in a view parallel to the T-tubule axis (as in Figs. 2 B–C and 3 B, D and F) and the RyR array is modeled as seen in this orientation. The proportion between RyR heights and their spacing is appropriate, as suggested to us by Dr M. Samso . Clusters (polymers) of six Tdn molecules, modeled roughly according to the topology proposed by Knudson et al., (1993) and Marty et al., (1995) are located between the RyRs as initially suggested by Fan et al. (1995). Their aggregated mass is responsible for the visible anchors, but the exact ratio of Tdns to anchors is not known. Individual triadin molecules are connected to RyRs and to a long linear CASQ polymer on the luminal side of the SR. The latter corresponds to the fine line visible in the EM images at the tips of anchors (see Fig. 2 C and inset). The jSR lumen is filled by long CASQ polymers that randomly intersect each other . Junctin is depicted as monomers associated with RyRs , . Although we cannot visualize them directly, it is likely that individual Jct molecules are positioned as indicated along the jSR face and also possibly at the sites where CASQ is linked to the lateral sides of the SR.