Altered expression of triadin 95 causes parallel changes in localized Ca²⁺ release events and global Ca²⁺ signals in skeletal muscle cells in culture

Abstract

The 95 kDa triadin (Trisk 95), an integral protein of the sarcoplasmic reticular membrane in skeletal muscle, interacts with both the ryanodine receptor (RyR) and calsequestrin. While its role in the regulation of calcium homeostasis has been extensively studied, data are not available on whether the overexpression or the interference with the expression of Trisk 95 would affect calcium sparks the localized events of calcium release (LCRE). In the present study LCRE and calcium transients were studied using laser scanning confocal microscopy on C2C12 cells and on primary cultures of skeletal muscle. Liposome-or adenovirus-mediated Trisk 95 overexpression and shRNA interference with triadin translation were used to modify the level of the protein. Stable overexpression in C2C12 cells significantly decreased the amplitude and frequency of calcium sparks, and the frequency of embers. In line with these observations, depolarization-evoked calcium transients were also suppressed. Similarly, adenoviral transfection of Trisk 95 into cultured mouse skeletal muscle cells significantly decreased both the frequency and amplitude of spontaneous global calcium transients. Inhibition of endogenous triadin expression by RNA interference caused opposite effects. Primary cultures of rat skeletal muscle cells expressing endogenous Trisk 95 readily generated spontaneous calcium transients but rarely produced calcium sparks. Their transfection with specific shRNA sequence significantly reduced the triadin-specific immunoreactivity. Functional experiments on these cells revealed that while caffeine-evoked calcium transients were reduced, LCRE appeared with higher frequency. These results suggest that Trisk 95 negatively regulates RyR function by suppressing localized calcium release events and global calcium signals in cultured muscle cells.

Trisk 32 regulates IP3 receptors in rat skeletal myoblasts

“…From these experiments and from those presented below (see Fig. 4), as well as from our earlier results [9] we concluded that the transfection with the empty vector does not affect the IP 3 pathway. To examine the effect of the inhibition of IP 3 Fig.…”

“…By measuring the amplitude and the integral of the thapsigargin-evoked Ca 2+ transients and finding no significant difference in either when comparing control and transfected cells, suggest that the overexpression of Trisk 32 does not affect the Ca 2+ content of the ER Ca 2+ store. The 95 kDa isoform of triadin is known to regulate Ca 2+ release via RyRs [9,24]. To exclude the possibility that such an interaction from Trisk 32 or an enhanced CICR was responsible for the increased Ca 2+ transients evoked by bradykinin or AVP, functional [Ca 2+ ] i measurements were performed.…”

“…RyRs have been shown to interact with a number of different proteins present in the triadic junction including, among others, calsequestrin, junctate, junctin, JP45, mitsugumins, and triadin (for review see [31]). From this group of SR membrane proteins triadin was first to be identified in rabbit skeletal muscle in 1990 [5,16] as a 95 kDa glycoprotein specifically located in the triads, and was later proved to decrease the extent of Ca 2+ release via RyRs [9,24]. Since then several isoforms have been identified both in skeletal and cardiac muscle [13,14,19,33].…”

Ablation of Skeletal Muscle Triadin Impairs FKBP12/RyR1 Channel Interactions Essential for Maintaining Resting Cytoplasmic Ca2+

“…Although originally thought to be the link between RyR1 and DHPR (6 -8) and a key modulator of EC coupling (9 -11), it appears evident now that triadin acts primarily as a negative regulator of RyR1 activity (12)(13)(14)(15). Although the mechanism by which triadin regulate RyRs remains unclear, there is support for the hypothesis that triadin is involved in facilitating the cross-communication be-tween calsequestrin (CSQ) and the RyRs (16 -18).…”

Triadin Deletion Induces Impaired Skeletal Muscle Function

“…Overexpression studies using adenoviruses have shown that Trisk 95 is able to block the depolarization-induced calcium release, but in the same conditions, no effect was observed with Trisk 51 overexpression, and therefore no function could be attributed to this isoform (21). Triadin knockdown has also been realized using small interference RNA in cultured cells (22,23), and resulted in reduction of the depolarization-induced calcium release. In addition to various functional effects resulting from expression modification, the triadin isoforms have different localizations and partners, and whereas Trisk 95 and Trisk 51 are both located within the…”