Molecular cloning and characterization of mouse cardiac triadin isoforms

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca 2؉ imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca 2؉ transients. release mediated by these two channels (for review see Refs. 1-4). These proteins, including calsequestrin (Csq), calmodulin, triadin, junctin, Junctophilins 1 and 2, MG-29, FKBP12, and others yet to be discovered, along with the RyR and DHPR make up the so-called calcium release units (CRUs) (5).Triadins, a multimember family of proteins that are the product of alternative splicing from a single gene and expressed almost exclusively in striated muscle (3, 6), have generated significant attention in recent years for their involvement in a variety of cellular events in muscle cells, but their precise role in muscle function is mostly unknown. Triadin was first identified in skeletal muscle as a 94-to 95-kDa transmembrane protein (7,8) that is abundantly expressed on the junctional sarcoplasmic reticulum (jSR), were it colocalizes with RyR1 and DHPR (9).Early studies of binding assays of solubilized SR proteins showed that triadin could not only be coimmunoprecipitated with other triadic proteins (10) but also could associate into macromolecular complexes with both the DHPR and RyR1 (7,11,12). Based in this association triadin was proposed as the key molecular linker mediating the DHPR/RyR1 communication during muscle contraction. Although functional interactions between triadin and the DHPR in skeletal muscle have proven difficult to confirm, functional and structural interactions between triadin and RyR1 have been documented by several investigators. In vitro studies have shown that the SR luminal domain of triadin not only interacts with RyR1 but appears to anchor Csq to it, mediating the functional coupling between these two proteins via specific domains (13-17).Several studies have suggested a major role for triadin 95 in modulating RyR channel properties. Both an anti-triadin anti-* This work was supported by American Heart Association Grant 0530250N (to C. F. P.) and National Institute of Health Grant PO1AR47605 (to P. D. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Together these studies suggest a negative regulatory role for triadin on RyR...

Section: Resultsmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Triadins Modulate Intracellular Ca2+ Homeostasis but Are Not Essential for Excitation-Contraction Coupling in Skeletal Muscle

Shen

Franzini‐Armstrong

López

et al. 2007

“…One is the rat homolog of the 95-kDa triadin identified in rabbit skeletal muscle, named Trisk 95, and the second one, Trisk 51, is a truncated form of Trisk 95 (461 amino acids instead of 687 amino acids) almost identical for the major part of the protein but with a unique C-terminal end of 6 amino acids. In cardiac muscle, multiple isoforms of triadin have also been identified (11,12), but the major if not only isoform expressed is CT1, a 32-kDa triadin isoform (13).…”

mentioning

confidence: 99%

Triadin (Trisk 95) Overexpression Blocks Excitation-Contraction Coupling in Rat Skeletal Myotubes

Rezgui

Vassilopoulos

Brocard

et al. 2005

To identify the function of triadin in skeletal muscle, adenovirusmediated overexpression of Trisk 95 or Trisk 51, the two major skeletal muscle isoforms, was induced in rat skeletal muscle primary cultures, and the physiological behavior of the modified cells was analyzed. Overexpression did not modify the expression level of their protein partners ryanodine receptor, dihydropyridine receptor, and the other triadin. Caffeine-induced calcium release was also unaffected by triadin overexpression. Nevertheless, in the absence of extracellular calcium, depolarization-induced calcium release was almost abolished in Trisk 95 overexpressing myotubes (T95 myotubes), and not modified in Trisk 51 overexpressing myotubes (T51 myotubes). This was not because of a modification of dihydropyridine receptors, as depolarization in presence of external calcium still induced a calcium release, and the activation curve of dihydropyridine receptor was unchanged, in both T95 and T51 myotubes. The calcium release complex was also maintained in T95 myotubes as Trisk 95, ryanodine receptor, dihydropyridine receptor, and Trisk 51 were still co-localized. The effect of Trisk 95 overexpression on depolarization-induced calcium release was reversed by a simultaneous infection with an antisense Trisk 95 adenovirus, indicating the specificity of this effect. Thus, the level of Trisk 95 and not Trisk 51 is important on regulating the calcium release complex, and an excess of this protein can lead to an inhibition of the physiological function of the complex. In skeletal muscle cells, excitation-contraction (E-C)2 coupling is the process by which depolarization of the plasma membrane produces a large transient release of Ca 2ϩ from the sarcoplasmic reticulum, which in turn triggers contraction. Dihydropyridine receptors (DHPRs), the L-type Ca 2ϩ channels localized in the T-tubule membrane, serve as the voltage sensors for E-C coupling (1, 2) and activate ryanodine receptors (RyRs), the intracellular Ca 2ϩ release channels of the sarcoplasmic reticulum membrane (3, 4). In skeletal muscle, entry of Ca 2ϩ through DHPR is not required for E-C coupling (5). Instead, a voltage-dependent conformational change in the II-III loop of the skeletal muscle DHPR ␣1 subunit (Cav1.1) activates the skeletal muscle ryanodine receptor, RyR1. Activation of RyR1 results in a release of Ca 2ϩ from the sarcoplasmic reticulum intracellular stores into the cytosol, which in turn activates the cellular contractile apparatus to initiate muscle contraction. In cardiac muscle, the entry of external calcium through DHPR is required to induce activation of RyR and release of internal Ca 2ϩ via RyR, a mechanism known as calcium-induced calcium release. Calcium-induced calcium release is not the initial mechanism responsible of Ca 2ϩ release in skeletal muscle, but it can contribute to the amplification of the signal, and both calcium-induced calcium release and conformational coupling are involved in skeletal muscle contraction, to different extents (6).Triadin is a transmem...

“…Of these, CT1 (35 kDa) is the major triadin isoform expressed in canine heart muscle, whereas CT2 (40 kDa) is not detectable as a protein, and CT3 (92 kDa) is expressed at very low levels in this species (19). More recently, three triadin isoforms have been cloned from mouse heart muscle with molecular masses of 35, 35.5, and 40 kDa (20). Whereas the 35-and 40-kDa isoforms presumably correspond to CT1 and CT2 isoforms of rabbit heart muscle, the 35.5-kDa protein presumably represents a new isoform.…”

mentioning

confidence: 99%

Triadins Are Not Triad-specific Proteins

Vassilopoulos

Thevenon

Rezgui

et al. 2005

We have cloned two new triadin isoforms from rat skeletal muscle, Trisk 49 and Trisk 32, which were named according to their theoretical molecular masses (49 and 32 kDa, respectively). Specific antibodies directed against each protein were produced to characterize both new triadins. Both are expressed in adult rat skeletal muscle, and their expression in slow twitch muscle is lower than that in fast twitch muscle. Using double immunofluorescent labeling, the localization of these two triadins was studied in comparison to wellcharacterized proteins such as ryanodine receptor, calsequestrin, desmin, Ca 2؉ -ATPase, and titin. None of these two triadins are localized within the rat skeletal muscle triad. Both are instead found in different parts of the longitudinal sarcoplasmic reticulum. We attempted to identify partners for each isoform: neither is associated with ryanodine receptor; Trisk 49 could be associated with titin or another sarcomeric protein; and Trisk 32 could be associated with IP 3 receptor. These results open further fields of research concerning the functions of these two proteins; in particular, they could be involved in the set up and maintenance of a precise sarcoplasmic reticulum structure.Skeletal muscle cells have highly organized structures. Sarcomeres, the contractile units of striated muscles, are assembled from thousands of proteins to produce the largest and most regular macromolecular complex known. In addition, this organized structure is designed to undergo strong mechanical stress during muscle contraction. Muscle contraction is activated by Ca 2ϩ release from the sarcoplasmic reticulum in response to plasma membrane depolarization. This process is referred to as excitation-contraction coupling and takes place at the skeletal muscle triad junction, where T-tubules and the sarcoplasmic reticulum terminal cisternae are in close contact (1). To perform its function, the sarcoplasmic reticulum is built as a sleeve-like structure around the myofibrils, and it is compartmentalized in highly specialized structures (terminal cisternae and longitudinal reticulum) with specialized functions (calcium release and calcium uptake, respectively) (2).Calcium release occurs via a macromolecular complex, the calcium release complex, specifically localized in the skeletal muscle triad (3). Major components of this calcium release complex are the two calcium channels, the ryanodine receptor (RyR) 1 and the dihydropyridine receptor (4). Triadin is an integral membrane protein of the sarcoplasmic reticulum, first identified in rabbit skeletal muscle in 1990 (5, 6) as a 95-kDa glycoprotein specifically located in the triads. Because of its co-localization with RyR in the triads, involvement of triadin in excitation-contraction coupling has been presumed (7, 8). Protein interaction studies have shown that molecular partners of triadin are RyR (9 -11); calsequestrin (CSQ), the protein that traps calcium inside the sarcoplasmic reticulum (12-14); junctin (15); and histidine-rich Ca 2ϩ -binding protein (16). F...