Triadin Gene Research Articles

Cardiomyocyte-Specific Long Noncoding RNA Regulates Alternative Splicing of the Triadin Gene in the Heart.

Abnormalities in Ca2+ homeostasis are associated with cardiac arrhythmias and heart failure. Triadin plays an important role in Ca2+ homeostasis in cardiomyocytes. Alternative splicing of a single triadin gene produces multiple triadin isoforms. The cardiac-predominant isoform, mouse MT-1 or human Trisk32, is encoded by triadin exons 1 to 8. In humans, mutations in the triadin gene that lead to a reduction in Trisk32 levels in the heart can cause cardiac dysfunction and arrhythmias. Decreased levels of Trisk32 in the heart are also common in patients with heart failure. However, mechanisms that maintain triadin isoform composition in the heart remain elusive. We analyzed triadin expression in heart explants from patients with heart failure and cardiac arrhythmias and in hearts from mice carrying a knockout allele for Trdn-as, a cardiomyocyte-specific long noncoding RNA encoded by the antisense strand of the triadin gene, between exons 9 and 11. Catecholamine challenge with isoproterenol was performed on Trdn-as knockout mice to assess the role of Trdn-as in cardiac arrhythmogenesis, as assessed by ECG. Ca2+ transients in adult mouse cardiomyocytes were measured with the IonOptix platform or the GCaMP system. Biochemistry assays, single-molecule fluorescence in situ hybridization, subcellular localization imaging, RNA sequencing, and molecular rescue assays were used to investigate the mechanisms by which Trdn-as regulates cardiac function and triadin levels in the heart. We report that Trdn-as maintains cardiac function, at least in part, by regulating alternative splicing of the triadin gene. Knockout of Trdn-as in mice downregulates cardiac triadin, impairs Ca2+ handling, and causes premature death. Trdn-as knockout mice are susceptible to cardiac arrhythmias in response to catecholamine challenge. Normalization of cardiac triadin levels in Trdn-as knockout cardiomyocytes is sufficient to restore Ca2+ handling. Last, Trdn-as colocalizes and interacts with serine/arginine splicing factors in cardiomyocyte nuclei and is essential for efficient recruitment of splicing factors to triadin precursor mRNA. These findings reveal regulation of alternative splicing as a novel mechanism by which a long noncoding RNA controls cardiac function. This study indicates potential therapeutics for heart disease by targeting the long noncoding RNA or pathways regulating alternative splicing.

A novel homozygous mutation in the TRDN gene causes a severe form of pediatric malignant ventricular arrhythmia

Triadin is a protein expressed in cardiac and skeletal muscle that has an essential role in the structure and functional regulation of calcium release units and excitation-contraction coupling. Mutations in the triadin gene (TRDN) have been described in different forms of human arrhythmia syndromes with early onset and severe arrhythmogenic phenotype, including triadin knockout syndrome. The purpose of this study was to characterize the pathogenetic mechanism underlying a case of severe pediatric malignant arrhythmia associated with a defect in the TRDN gene. We used a trio whole exome sequencing approach to identify the genetic defect in a 2-year-old boy who had been resuscitated from sudden cardiac arrest and had frequent episodes of ventricular fibrillation and a family history positive for sudden death. We then performed invitro functional analysis to investigate possible pathogenic mechanisms underlying this severe phenotype. We identified a novel homozygous missense variant (p.L56P) in the TRDN gene in the proband that was inherited from the heterozygous unaffected parents. Expression of a green fluorescent protein (GFP)-tagged mutant human cardiac triadin isoform (TRISK32-L56P-GFP) in heterologous systems revealed that the mutation alters protein dynamics. Furthermore, when co-expressed with the type 2 ryanodine receptor, caffeine-induced calcium release from TRISK32-L56P-GFP was relatively lower compared to that observed with the wild-type construct. The results of this study allowed us to hypothesize a pathogenic mechanism underlying this rare arrhythmogenic recessive form, suggesting that the mutant protein potentially can trigger arrhythmias by altering calcium homeostasis.

The Microtubule-Associated Protein CLIMP-63 is a New Member of the Calcium Release Complex

Muscle contraction is achieved when an efficient excitation signal at the plasma membrane triggers intracellular calcium release. This process called “excitation-contraction (E-C) coupling” relies on a macromolecular protein complex, spanning the plasma membrane and the sarcoplasmic reticulum (SR), containing the calcium channel of the SR, the ryanodine receptor (RyR). This calcium release complex is present exclusively in highly organized membrane structures called triads. A triad is composed of two SR terminal cisternae surrounding a plasma membrane transverse-tubule. This architecture is essential to sustain the activity of the calcium channel RyR1, which is located in the membrane of SR terminal cisternae. However, little is known about the molecular mechanisms allowing the formation and maintenance of SR terminal cisternae. Triadin is a member of this complex, present in the SR membrane and interacting with RyR1. Deletion of the triadin gene leads to partial disorganisation of SR membranes in skeletal muscles, with abnormal orientation of part of the triads. Triadin could play a role in the structure of sarcoplasmic reticulum to allow efficient E-C coupling. We have shown that triadin could indirectly interact with the microtubules, and therefore anchor the sarcoplasmic reticulum to the microtubule network (Fourest-Lieuvin, J.Cell.Science, 2012). Using mass spectrometry analysis of the proteins co-immunoprecipitated with triadin, we have identified a new partner of triadin, CLIMP-63 which could be involved in this function. CLIMP-63 is a shaping protein able to mediate the anchoring of the reticulum to microtubules. CLIMP-63 has been involved in the shaping of endoplasmic reticulum in neurons (Cui-Wang, Cell, 2012). We have dissected the interacting domains between CLIMP-63 and triadin, and studied the consequences of this association for muscle function, and triad formation or maintenance.

New description of a family with an autosomal recessive cathecholergic ventricular tachycardia due to Triadin gene

We describe a family with suspicion of genetic arrhythmia that has benefited from a wide genetic exploration. The eldest of the siblings presented syncope at age 5.5 years and cardiac explorations were normal. A few months later, her elder sister presented a sudden death at age 4.5 years, while she was playing in the garden. The cardiac explorations showed a heart of normal structure but presence of polymorphic premature ventricular complexes. Isoprenaline test was positive. Treatment with beta-blockers (nadolol 50 mg/m 2 ) was introduced. There was no family history of sudden death or other cardiac defects. Because of these two serious rhythmic events occurring in two young children, a genetic study was initiated by next generation sequencing of 42 genes involved in cardiac arrhythmias (long QT, Brugada, cathecholaminergic ventricular tachycardia). Two heterozygous mutations (c.613C > T/p.Gln205* and c.22 + 29 A > G) were identified in the Triadin gene, coding for a protein of the calcium release complex, recently involved in cathecholaminergic ventricular tachycardia in two families (Roux-Buisson et al., 2012). The parents of our two cases were each carriers of a heterozygous mutation and had no cardiac symptoms. Their cardiac assessment did not show any abnormality (ECG Holter, exercise test, Isoprenaline test). The nonsense p.Gln205* mutation was present in one of the published families; however the splicing mutation in intron 1 had never been identified. Minigene experiments helped to confirm its pathogenicity. Presymptomatic testing was then proposed to the third child of the family (age 3), finding the two pathogenic mutations. She was therefore put under the same treatment as her sisters. This is the second report of an autosomal recessive cathecholaminergic ventricular tachycardia due to the Triadin gene. This case illustrates the interest of Next Generation Sequencing exploring simultaneously several candidate genes, in cases of sudden death of unknown origin.

Triadin Function in Sarcoplasmic Reticulum Structure

Muscle contraction is achieved when an efficient excitation signal at the plasma membrane triggers intracellular calcium release. This process called “excitation-contraction (E-C) coupling” relies on a multimolecular protein complex, spanning the plasma membrane and the sarcoplasmic reticulum (SR), containing the calcium channel of the SR, the ryanodine receptor (RyR). Triadin is a member of this complex, present in the SR membrane and interacting with RyR in skeletal muscle. We have shown that overexpression of triadin in cultured myotubes abolishes E-C coupling, although RyR is still functional. Moreover in knock-out mice, deletion of the triadin gene leads to disorganisation of SR membranes in skeletal muscles. In the present work, we have expressed triadin in COS-7 cells to dissect its intrinsic properties on membrane organisation. We show that triadin expression leads to important modification of the endoplasmic reticulum (ER) morphology, already observed with the expression of proteins regulating ER morphology, and known as “rope-like structures”. These modifications of ER morphology are correlated to alteration of the microtubule network. Indeed, in cells expressing triadin, microtubules are bundled, often running parallel to the plasma membrane, and more stable than in untransfected cells. Surprisingly, suppression of the cytosolic N-terminal part of triadin did not reverse this phenotype. Using splice versions of the triadin protein and C-terminal deletion mutants, we show that ER/microtubules modifications depend on an intra-lumenal sequence. Altogether, our work lead to the hypothesis that modifications of ER morphology and microtubule dynamics observed in cells expressing triadin are mediated by an intermediate protein currently under investigation. Expression of triadin in COS-7 cells can modify endoplasmic reticulum morphology. It thus suggests that in skeletal muscle, triadin could play a role in the structure of sarcoplasmic reticulum to allow efficient E-C coupling.

Katecholaminerge polymorphe ventrikuläre Tachykardien

Catecholaminergic polymorphic ventricular tachycardia (PCVT) is a rare, congenital ventricular tachyarrhythmia which occurs in the setting of adrenergic activation. It potentially leads to syncope and/or sudden cardiac death (SCD). PCVT represents one of the most dangerous congenital ion channel diseases. Mutations of the ryanodine receptor gene (RYR2), the calsequestrin gene (CASQ2), and the triadin gene (TRDN) have been identified as an underlying correlate. β-Blockers are employed as therapy and are sometimes combined with class IC antiarrhythmic drugs, or calcium antagonists of the verapamil type. ICD implantation is recommended in case of persisting syncope in the presence of β-blocker therapy or survived SCD. Left thoracic sympathectomy represents a subsidiary interventional therapy for individual cases. In addition, modifications of the patient's lifestyle including avoidance of physical stress and heart rates> 120/min are recommended.

Identification of the First Mutations in the Human Triadin Gene, Associated to Catecholaminergic Tachycardia, a Pathology of the Cardiac Calcium Release Complex

Cardiac contraction is triggered when a membrane depolarisation induces a massive increase in intracellular calcium concentration. This process called “excitation-contraction (E-C) coupling” relies on a multimolecular protein complex, the calcium release complex (CRC) organized around the sarcoplasmic reticulum calcium channel, the ryanodine receptor (RyR2). Among the proteins involved in the efficient function of the CRC, calsequestrin, triadin and junctin are sarcoplasmic reticulum proteins able to interact with RyR2 and regulate calcium release.Mutations in RyR2 and calsequestrin are associated to a rare but fatal cardiac arrhythmia: catecholaminergic polymorphic ventricular tachycardia (CPVT). Nevertheless, variations in these two genes (RYR2 and CASQ2) account so far for only 50 to 70% of the cases, suggesting that other genes are most probably involved. To reveal new genes involved in CPVT, we have based a candidate gene approach on the hypothesis that the pathology could be considered as a disease of the calcium release complex. We therefore searched for variations in the genes encoding proteins of the CRC in a large French cohort of CPVT patients with no detected mutations in RYR2 or CASQ2. We have identified for the first time mutations in the human triadin gene TRDN, and studied the functional consequences of a missense mutation both in a cell model and in vivo after expression in triadin KO mice. Our results confirmed the hypothesis that CPVT can be more generally considered as a defect in the CRC.

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca2+ going local?

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca²⁺ going local?

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

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. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca(2+).[(3)H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca(2+) activation or Ca(2+) and Mg(2+) inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca(2+) homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.

Human skeletal muscle triadin: gene organization and cloning of the major isoform, Trisk 51

We obtained the gene organization of human triadin gene by aligning the DNA coding sequence of human 95-kDa triadin (Trisk 95) with human genomic database. We identified a novel human triadin isoform, a potential human homologue of rat Trisk 51. We show that both isoforms of triadin, Trisk 51 and Trisk 95, are alternative splice variants of the same gene. We demonstrated experimentally the existence of this Trisk 51 transcript in human skeletal muscle and cloned its full length cDNA. We further demonstrated that the protein encoded by this transcript is expressed in the human skeletal muscle. In addition, unlike other species, Trisk 51 is the major triadin isoform expressed in human skeletal muscle, whereas Trisk 95 is below the detection level in the two types of muscles tested.