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During mammalian cardiomyocyte excitation-contraction coupling, Ca2+ influx through voltage-gated Ca2+ channels triggers Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptor channels. This Ca2+-induced Ca2+ release mechanism controls cardiomyocyte contraction and is exquisitely regulated by SR Ca2+ levels. The histidine-rich calcium-binding protein (HRC) and its aspartic acid-rich paralogue aspolin are high-capacity, low-affinity Ca2+-binding proteins. Aspolin also acts as a trimethylamine N-oxide demethylase. At low intraluminal Ca2+ concentrations, HRC binds to the SR Ca2+-ATPase 2, inhibiting its Ca2+-pumping activity. At high intraluminal Ca2+ levels, HRC interacts with triadin to reduce Ca2+ release through ryanodine receptor channels. This Review analyses the evolution of these Ca2+-regulatory proteins, to gain insights into their roles. It reveals that HRC homologues are present in chordates, annelid worms, molluscs, corals and sea anemones. In contrast, triadin appears to be a chordate innovation. Furthermore, HRC is evolving more rapidly than other cardiac excitation-contraction coupling proteins. This positive selection (or relaxed negative selection) occurs along most of the mammalian HRC protein sequence, with the exception being the C-terminal cysteine-rich region, which is undergoing negative selection. The histidine-rich region of HRC might be involved in pH sensing, as an adaptation to air-breathing, endothermic and terrestrial life. In addition, a cysteine-rich pattern within HRC and aspolin is also found in a wide range of iron-sulfur cluster proteins, suggesting roles in redox reactions and metal binding. The polyaspartic regions of aspolins are likely to underlie their trimethylamine N-oxide demethylase activity, which might be mimicked by the acidic regions of HRCs. These potential roles of HRCs and aspolins await experimental verification., Competing Interests: Competing interests The author declares no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Catecholaminergic polymorphic ventricular tachycardia (CPVT), a rare autosomal dominant or recessive disease, usually results in syncope or sudden cardiac death. Most CPVT patients do not show abnormal cardiac structure and electrocardiogram features and symptoms, usually onset during adrenergically mediated physiological conditions. CPVT tends to occur at a younger age and is not easy to be diagnosed and managed. The main cause of CPVT is associated with mishandling Ca2+ in cardiomyocytes. Intracellular Ca2+ is strictly controlled by a protein located in the sarcoplasm reticulum (SR), such as ryanodine receptor, histidine-rich Ca2+-binding protein, triadin, and junctin. Mutation in these proteins results in misfolding or malfunction of these proteins, thereby affecting their Ca2+-binding affinity, and subsequently disturbs Ca2+ homeostasis during excitation–contraction coupling (E-C coupling). Furthermore, transient disturbance of Ca2+ homeostasis increases membrane potential and causes Ca2+ store overload-induced Ca2+ release, which in turn leads to delayed after depolarization and arrhythmia. Previous studies have focused on the interaction between ryanodine receptors and protein kinase or phosphatase in the cytosol. However, recent studies showed the regulation signaling for ryanodine receptor not only from the cytosol but also within the SR. The changing of Ca2+ concentration is critical for protein interaction inside the SR which changes protein conformation to regulate the open probability of ryanodine receptors. Thus, it influences the threshold of Ca2+ released from the SR, making it easier to release Ca2+ during E-C coupling. In this review, we briefly discuss how Ca2+ handling protein variations affect the Ca2+ handling in CPVT.

TRDN mutations cause catecholaminergic polymorphic ventricular tachycardia (CPVT) but may present with abnormal electrocardiogram (ECG) findings provoking a diagnosis of long QT syndrome (LQTS). We report two novel cases of sudden cardiac death in children due to mutations of TRDN, providing further insight into this rare and aggressive inherited arrhythmia syndrome. Whole exome sequencing (WES) was performed in two unrelated children who experienced cardiac arrest during exercise and were negative for targeted testing of LQTS. WES identified a novel homozygous splice‐site mutation in both patients, denoted c.22+1G>T, absent from gnomAD and suggesting a founder variant in the Iranian population. We now summarize the genetic architecture of all reported TRDN‐related patients, including 27 patients from 21 families. The average age‐onset was 30 months (range 1–10) for all cases. Adrenergic‐mediated cardiac events were common, occurring in 23 of 27 cases (85%). LQTS was diagnosed in 10 cases (37%), CPVT in 10 (37%) cases, and in 7 cases. No phenotypic diagnosis was provided. Five cases (15%) had evidence for associated skeletal myopathy. Four missense TRDN variants (24%) were observed in diseased cases, while the remaining variants reflect putative loss‐of‐function (LOF) mutations. No disease phenotype was reported in 26 heterozygous carriers. In conclusion, TRDN mutations cause a rare autosomal recessive arrhythmia syndrome presenting with adrenergic‐mediated arrhythmic events, but with ECG abnormalities leading to a diagnosis of LQTS in a proportion of cases. Heterozygous carriers are free of disease manifestations. [ABSTRACT FROM AUTHOR]

Catecholaminergic polymorphic ventricular tachycardia (CPVT), a rare autosomal dominant or recessive disease, usually results in syncope or sudden cardiac death. Most CPVT patients do not show abnormal cardiac structure and electrocardiogram features and symptoms, usually onset during adrenergically mediated physiological conditions. CPVT tends to occur at a younger age and is not easy to be diagnosed and managed. The main cause of CPVT is associated with mishandling Ca2+ in cardiomyocytes. Intracellular Ca2+ is strictly controlled by a protein located in the sarcoplasm reticulum (SR), such as ryanodine receptor, histidine-rich Ca2+-binding protein, triadin, and junctin. Mutation in these proteins results in misfolding or malfunction of these proteins, thereby affecting their Ca2+-binding affinity, and subsequently disturbs Ca2+ homeostasis during excitation–contraction coupling (E-C coupling). Furthermore, transient disturbance of Ca2+ homeostasis increases membrane potential and causes Ca2+ store overload-induced Ca2+ release, which in turn leads to delayed after depolarization and arrhythmia. Previous studies have focused on the interaction between ryanodine receptors and protein kinase or phosphatase in the cytosol. However, recent studies showed the regulation signaling for ryanodine receptor not only from the cytosol but also within the SR. The changing of Ca2+ concentration is critical for protein interaction inside the SR which changes protein conformation to regulate the open probability of ryanodine receptors. Thus, it influences the threshold of Ca2+ released from the SR, making it easier to release Ca2+ during E-C coupling. In this review, we briefly discuss how Ca2+ handling protein variations affect the Ca2+ handling in CPVT. [ABSTRACT FROM AUTHOR]

The review covers studies evaluating the role of genes and proteins of phospholamban (PLN), triadin (TRDN), and junctin (ASPH) in the development of myocardial contractile dysfunction. The results reflecting the effect of changes in the structure and expression of these genes on the functioning of the Ca2+ transporting system of the sarcoplasmic reticulum of cardiomyocytes are presented. [ABSTRACT FROM AUTHOR]

Aim: To perform a comprehensive phenotype-genotype correlation of all rare variants in Triadin leading to malignant arrhythmias in pediatrics.Methods: Triadin knockout syndrome is a rare entity reported in pediatric population. This syndrome is caused by rare variants in the TRDN gene. Malignant ventricular arrhythmias and sudden cardiac death can be a primary manifestation of disease. Although pharmacological measures are effective, some patients require an implantable defibrillator due to high risk of arrhythmogenic episodes.Main Results: Fourteen rare genetic alterations in TRDN have been reported to date. All of these potentially pathogenic alterations are located in a specific area of TRDN, highlighting this hot spot as an arrhythmogenic gene region.Conclusions: Early recognition and comprehensive interpretation of alterations in Triadin are crucial to adopt preventive measures and avoid malignant arrhythmogenic episodes in pediatric population.

Background: Triadin knockout syndrome (TKOS) is a rare arrhythmia syndrome caused by recessive null variants in TRDN-encoded cardiac triadin 1. TKOS has presented frequently with cardiac arrest in childhood.Objective: The purpose of this study was to elucidate the underlying genetic mechanism of disease in a genetically elusive patient displaying a characteristic TKOS phenotype.Methods: Genome sequencing and a TRDN gene-specific trio analysis were completed on the patient. RNA and protein isolated from patient-specific human-induced pluripotent stem cell-derived cardiomyocytes were used to determine the effects of the identified variants using reverse transcription polymerase chain reaction (RT-PCR) and Western blot.Results: Genome sequencing revealed compound heterozygous putative splice-error variants (maternal c.22+29A>G and paternal c.484+1189G>A). The novel paternally derived c.484+1189G>A variant is located within 24 base pairs of a predicted alternative exon 6 (exon 6a), which resides within the intron between canonical exons 5 and 6. We determined that this previously unrecognized exon 6a produces a short TRDN transcript and potentially a novel protein isoform in the normal human heart. The c.484+1189G>A variant not only results in abnormal splicing of the exon 6a-containing transcript leading to a frameshift mutation but also results in the abolishment of the 8-exon cardiac triadin 1 transcript.Conclusion: Here, we present evidence for a novel alternative exon 6a-containing TRDN transcript in the normal heart. The novel deep intronic TRDN variant identified in a patient with TKOS leads to splicing error of a newly recognized exon 6a and loss of triadin. Considering that both TRDN variants in this patient were missed after commercial testing, these results highlight the importance of using genome sequencing when identifying patients with TKOS. [ABSTRACT FROM AUTHOR]

Background: 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.Objective: 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.Methods: 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 in vitro functional analysis to investigate possible pathogenic mechanisms underlying this severe phenotype.Results: 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.Conclusion: 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. [ABSTRACT FROM AUTHOR]

Non-coding RNAs play major roles in cardiac pathophysiology. Recent studies reported that long non-coding RNAs (lncRNAs) are dysregulated in the failing heart, but how they contribute to heart failure development is unclear. In this study, we aimed to identify heart-enriched lncRNAs and investigate their regulation and function in the failing heart. Results Analysis of a RNA-seq dataset of 15 Caucasian tissues allowed the identification of 415 heart-enriched lncRNAs. Fifty-three lncRNAs were located on the genome in close vicinity to protein-coding genes associated with cardiac function and disease. Analysis of a second RNA-seq dataset of 16 failing human hearts highlighted one lncRNA which we arbitrarily named TRDN-AS due to its localisation in the antisense position of the gene encoding triadin (TRDN). Expression of TRDN-AS and cardiac TRDN was up-regulated in biopsies from failing human hearts compared to control hearts. In failing hearts, TRDN-AS was positively correlated with a cardiac isoform of TRDN and negatively correlated with a skeletal muscle isoform of TRDN. A murine homolog of human TRDN-AS was identified and found to be enriched in the heart and localised in the nuclear compartment of cardiomyocytes. Trdn-AS expression as well as the ratio between cardiac and skeletal muscle isoforms were down-regulated after experimental myocardial infarction. In murine cardiomyocytes, activation of Trdn-AS transcription with the CRISPR/dCas9-VPR system enhanced the ratio between cardiac and skeletal isoforms of Trdn. Conclusion The lncRNA TRDN-AS regulates the balance between cardiac and skeletal isoforms of triadin. This finding may have implications for the treatment of heart failure. [ABSTRACT FROM AUTHOR]

The core skeletal muscle ryanodine receptor (RyR1) calcium release complex extends through three compartments of the muscle fibre, linking the extracellular environment through the cytoplasmic junctional gap to the lumen of the internal sarcoplasmic reticulum ( SR) calcium store. The protein complex is essential for skeletal excitation-contraction ( EC)-coupling and skeletal muscle function. Its importance is highlighted by perinatal death if any one of the EC-coupling components are missing and by myopathies associated with mutation of any of the proteins. The proteins essential for EC-coupling include the DHPR α1S subunit in the transverse tubule membrane, the DHPR β1a subunit in the cytosol and the RyR1 ion channel in the SR membrane. The other core proteins are triadin and junctin and calsequestrin, associated mainly with SR. These SR proteins are not essential for survival but exert structural and functional influences that modify the gain of EC-coupling and maintain normal muscle function. This review summarises our current knowledge of the individual protein/protein interactions within the core complex and their overall contribution to EC-coupling. We highlight significant areas that provide a continuing challenge for the field. Additional important components of the Ca2+ release complex, such as FKBP12, calmodulin, S100A1 and Stac3 are identified and reviewed elsewhere. [ABSTRACT FROM AUTHOR]

Triadin knockout syndrome (TKOS) is a malignant arrhythmia disorder caused by recessive null variants in TRDN-encoded cardiac triadin. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from two unrelated TKOS patients and an unrelated control. CRISPR-Cas9 gene editing was used to insert homozygous TRDN-p.D18fs ∗ 13 into a control line to generate a TKOS model (TRDN -/- ). Western blot confirmed total knockout of triadin in patient-specific and TRDN -/- iPSC-CMs. iPSC-CMs from both patients revealed a prolonged action potential duration (APD) at 90% repolarization, and this was normalized by protein replacement of triadin. APD prolongation was confirmed in TRDN -/- iPSC-CMs. TRDN -/- iPSC-CMs revealed that loss of triadin underlies decreased expression and co-localization of key calcium handling proteins, slow and decreased calcium release from the sarcoplasmic reticulum, and slow inactivation of the L-type calcium channel leading to frequent cellular arrhythmias, including early and delayed afterdepolarizations and APD alternans., Competing Interests: Conflict of interests M.J.A. is a consultant for Abbott, ARMGO Pharma, Boston Scientific, Daiichi Sankyo, Invitae, LQT Therapeutics, Medtronic, and UpToDate. M.J.A. and Mayo Clinic are involved in equity/royalty relationships with AliveCor and Anumana. These relationships are all modest, and none of these entities have contributed to this study in any manner., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Triadin isoforms, splice variants of one gene, maintain healthy Ca homeostasis in skeletal muscle by subserving several functions including an influence on Ca release through the ligand-gated ryanodine receptor (RyR1) ion channels. The predominant triadin isoform in skeletal muscle, Trisk 95, activates RyR1 in vitro via binding to previously unidentified amino acids between residues 200 and 232. Here, we identify three amino acids that influence Trisk 95 binding to RyR1 and ion channel activation, using peptides encompassing residues 200-232. Selective alanine substitutions show that K, K, and K together facilitate normal Trisk 95 binding to RyR1 and channel activation. Neither RyR1 binding nor activation are altered by alanine substitution of K alone or of K and K. Therefore K, K, and K contribute to a robust binding and activation site that is disrupted only when the charge on all three residues is neutralized. We suggest that charged pair interactions between acidic RyR1 residues D, D, and E and Trisk 95 residues K, K, and K facilitate Trisk 95 binding to RyR1 and channel activation. Since K, K, and K are also required for CSQ binding to RyRs (Kobayashi et al. 17, J Biol Chem 275, 17639-17646), the results suggest that Trisk 95 may not simultaneously bind to RyR1 and CSQ, contrary to the widely held belief that triadin monomers form a quaternary complex with junctin, CSQ and RyR1. Therefore, the in vivo role of triadin monomers in modulating RyR1 activity is likely unrelated to CSQ. [ABSTRACT FROM AUTHOR]

In skeletal muscle, the triad is a structure comprising a transverse (T)- tubule and sarcoplasmic reticulum (SR) cisternae. Triads constitute the basis of excitation-contraction coupling as the cradle of the Ca2+ release complex. We have shown previously that triadin, a member of this complex, has shaping properties on reticulum membrane and is indirectly involved in a link between triads and microtubules. We have identified here that CLIMP-63 (also known as CKAP4), as the partner of triadin, is responsible for this association of triads and microtubules. Triadin and CLIMP-63 interact through their respective luminal domains and the shaping properties of triadin depend on the capacity of CLIMP-63 to bind microtubules with its cytosolic portion. In skeletal muscle, CLIMP-63 is localized in the SR, including triads, and is associated with the Ca2+ release complex through its interaction with triadin. Knockout of triadin in muscles results in the delocalization of CLIMP-63 from triads, its dissociation from the Ca2+ release complex and a disorganization of the microtubule network. Our results suggest that the association of triadin and CLIMP-63 could be involved in the shaping of SR terminal cisternae and in the guidance of microtubules close to the triads. [ABSTRACT FROM AUTHOR]

We present the case of two siblings who both presented with an out-of-hospital cardiac arrest at 2 years of age. Both siblings underwent internal cardiac defibrillator implantation and both had recurrent episodes of ventricular fibrillation (VF). A compound heterozygous mutation in the triadin gene was discovered; one of these mutations has been described previously in the homozygous state, and the other one is unreported. The combination of these mutations has resulted in a particularly arrhythmogenic phenotype, with cardiac arrest occurring at a very young age and recurrent episodes of VF despite β-blockade. Flecainide seems to have been very effective in preventing clinical arrhythmias for this particular mutation. [ABSTRACT FROM AUTHOR]

Sudden Death Linked to the Triadin Gene We describe a new family with cathecholaminergic polymorphic ventricular tachycardia (CPVT) linked to the Triadin gene. This is the second report of such a CPVT of autosomal recessive inheritance. Using an NGS panel including 42 genes involved in cardiac sudden death, 2 heterozygous pathogenic mutations (c.613C> T/p.Gln205* and c.22 + 29 A>G) were identified in the Triadin gene in 2 sibs who experienced early severe arrhythmias without evidence of CPVT diagnosis at first cardiac evaluation. However, significant arrhythmias occurred after catecholaminergic stimulation. Each of the TRDN mutations was inherited from a healthy parent. In this family, genetic studies permit confirmation of the CPVT diagnosis in the 2 affected sibs and permit the early diagnosis of the third asymptomatic child. It also helped guide the therapeutic strategy in this family. [ABSTRACT FROM AUTHOR]

The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca 2 + release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species–specific antibodies. Newly synthesized JCT dog and TRD dog appeared by 12–24 h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24–48 h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRD dog and JCT dog , demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQ-DsRed interactions occurred when a form of TRD dog was used in which CSQ2-binding sites are removed ( del TRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRD dog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRD dog and del TRD were therefore able to elucidate areas of ER–SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER–SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic. [ABSTRACT FROM AUTHOR]

Ca2+ release, which is necessary for muscle contraction, occurs at the j-SR (junctional domain of the sarcoplasmic reticulum). It requires the assembly of a large multiprotein complex containing the RyR (ryanodine receptor) and additional proteins, including triadin and calsequestrin. The signals which drive these proteins to the j-SR and howthey assemble to form this multiprotein complex are poorly understood. To address aspects of these questions we studied the localization, dynamic properties and molecular interactions of triadin. We identified three regions, named TR1 (targeting region 1), TR2 and TR3, that contribute to the localization of triadin at the j-SR. FRAP experiments showed that triadin is stably associated with the j-SR and that this association is mediated by TR3. Protein pull-down experiments indicated that TR3 contains binding sites for calsequestrin-1 and that triadin clustering can be enhanced by binding to calsequestrin-1. These findings were confirmed by FRET experiments. Interestingly, the stable association of triadin to the j-SRwas significantly decreased in myotubes from calsequestrin-1 knockout mice. Taken together, these results identify three regions in triadin thatmediate targeting to the j-SR and reveal a role for calsequestrin-1 in promoting the stable association of triadin to the multiprotein complex associated with RyR. [ABSTRACT FROM AUTHOR]

Since the discovery of triadin >20 years ago as one of the major proteins located in the junctional sarcoplasmic reticulum, the field has come a long way in understanding the pivotal role of triadin in orchestrating sarcoplasmic reticulum Ca2+-release and hence excitation–contraction (EC) coupling. Building on the information gathered from earlier lipid bilayer and myocyte overexpression studies, the gene-targeted ablation of Trdn demonstrated triadin's indispensable role for maintaining the structural integrity of the couplon. More recently, the discovery of inherited and acquired diseases displaying altered expression and function of triadin has further emphasized the role of triadin in health and disease. Novel therapeutic approaches could be aimed at correcting the loss of triadin in diseased hearts, and thereby correcting the sub-cellular EC coupling defect. This review summarizes current concepts of the impact of triadin on cardiac EC coupling with a focus towards triadin's role for ventricular arrhythmia. [ABSTRACT FROM PUBLISHER]

The terminal cisternae represent one of the functional domains of the skeletal muscle sarcoplasmic reticulum (SR). They are closely apposed to plasma membrane invaginations, the T-tubules, with which they form structures called triads. In triads, the physical interaction between the T-tubule-anchored voltage-sensing channel DHPR and the SR calcium channel RyR1 is essential because it allows the depolarization-induced calcium release that triggers muscle contraction. This interaction between DHPR and RyR1 is based on the peculiar membrane structures of both T-tubules and SR terminal cisternae. However, little is known about the molecular mechanisms governing the formation of SR terminal cisternae. We have previously shown that ablation of triadins, a family of SR transmembrane proteins that interact with RyR1, induced skeletal muscle weakness in knockout mice as well as a modification of the shape of triads. Here we explore the intrinsic molecular properties of the longest triadin isoform Trisk 95. We show that when ectopically expressed, Trisk 95 can modulate reticulum membrane morphology. The membrane deformations induced by Trisk 95 are accompanied by modifications of the microtubule network organization. We show that multimerization of Trisk 95 by disulfide bridges, together with interaction with microtubules, are responsible for the ability of Trisk 95 to structure reticulum membrane. When domains responsible for these molecular properties are deleted, anchoring of Trisk 95 to the triads in muscle cells is strongly decreased, suggesting that oligomers of Trisk 95 and microtubules contribute to the organization of the SR terminal cisternae in a triad. [ABSTRACT FROM AUTHOR]

Aims Junctin and triadin are calsequestrin-binding proteins that regulate sarcoplasmic reticulum (SR) Ca2+ release by interacting with the ryanodine receptor. The levels of these proteins are significantly down-regulated in failing human hearts. However, the significance of such decreases is currently unknown. Here, we addressed the functional role of these accessory proteins in the heart's responses to ischaemia/reperfusion (I/R) injury. Methods and results Isolated mouse hearts were subjected to global I/R, and contractile parameters were assessed in wild-type (WT), junctin-knockout (JKO), and triadin-knockout (TKO) hearts. Both JKO and TKO were associated with significantly depressed post-I/R contractile recovery. However, ablation of triadin resulted in the most severe post-I/R phenotype. The additional contractile impairment of TKO hearts was not related to a mitochondrial death pathway, but attributed to endoplasmic reticulum (ER) stress-mediated apoptosis. Activation of the X-box-binding protein-1 and transcriptional up-regulation of C/EBP-homologous protein (CHOP) provided a molecular mechanism of caspase-12-dependent apoptosis in myocytes. In addition, elevation of cytosolic Ca2+ during reperfusion was associated with the activation of calpain proteases and troponin I breakdown. Accordingly, treatment with the calpain inhibitor MDL-28170 significantly ameliorated post-I/R impairment of contractile recovery in intact hearts. Conclusion These findings indicate that deficiency of either junctin or triadin impairs the contractile recovery in post-ischaemic hearts, which appears to be primarily attributed to increased ER stress and activation of calpain. [ABSTRACT FROM PUBLISHER]

The contractile function of the heart requires the release of Ca2+ from intracellular Ca2+ stores in the sarcoplasmic reticulum ( SR) of cardiac muscle cells. The efficacy of Ca2+ release depends on the amount of Ca2+ loaded into the Ca2+ store and the way in which this 'Ca2+ load' influences the activity of the cardiac ryanodine receptor Ca2+ release channel ( Ry R2)., The effects of the Ca2+ load on Ca2+ release through Ry R2 are facilitated by: (i) the sensitivity of RyR2 itself to luminal Ca2+ concentrations; and (ii) interactions between the cardiac Ca2+-binding protein calsequestrin ( CSQ) 2 and Ry R2, transmitted through the 'anchoring' proteins junctin and/or triadin., Mutations in Ry R2 are linked to catecholaminergic polymorphic ventricular tachycardia ( CPVT) and sudden cardiac death. The tachycardia is associated with changes in the sensitivity of Ry R2 to luminal Ca2+. Triadin-, junctin- or CSQ-null animals survive, but their longevity and ability to tolerate stress is compromised. These studies reveal the importance of the proteins in normal muscle function, but do not reveal the molecular nature of their functional interactions, which must be defined before changes in the proteins leading to CPVT and heart disease can be understood., Herein, we discuss known interactions between the Ry R, triadin, junctin and CSQ with emphasis on the cardiac isoforms of the proteins. Where there is little known about the cardiac isoforms, we discuss evidence from skeletal isoforms. [ABSTRACT FROM AUTHOR]

Abstract: In the neonatal mammalian heart, the role of ryanodine receptor (=Ca2+ release channel)-mediated sarcoplasmic reticulum (SR) Ca2+ release for excitation–contraction coupling is still a matter of debate. Using an adenoviral system, we overexpressed separately the junctional SR proteins triadin, junctin, and calsequestrin, which are probably involved in regulation of ryanodine receptor function. Infection of neonatal rat cardiac myocytes with triadin, junctin, or calsequestrin viruses, controlled by green fluorescent protein expression, resulted in an increased protein level of the corresponding transgenes. Measurement of Ca2+ transients of infected cardiac myocytes revealed unchanged peak amplitudes under basal conditions but with overexpression of calsequestrin and triadin caffeine-releasable SR Ca2+ content was increased. Our results demonstrate that an increased expression of triadin or calsequestrin is associated with an increased SR Ca2+ storage but unchanged Ca2+ signaling in neonatal rat cardiac myocytes. This is consistent with an ancillary role of the sarcoplasmic reticulum in excitation–contraction coupling in the developing mammalian heart. [Copyright &y& Elsevier]

This review focuses on molecular interactions between calsequestrin, triadin, junctin and the ryanodine receptor in the lumen of the sarcoplasmic reticulum. These interactions modulate changes in Ca2+ release in response to changes in the Ca2+ load within the sarcoplasmic reticulum store in striated muscle and are of fundamental importance to Ca2+ homeostasis, since massive adaptive changes occur when expression of the proteins is manipulated, while mutations in calsequestrin lead to functional changes which can be fatal. We find that calsequestrin plays a different role in the heart and skeletal muscle, enhancing Ca2+ release in the heart, but depressing Ca2+ release in skeletal muscle. We also find that triadin and junctin exert independent influences on the ryanodine receptor in skeletal muscle where triadin alone modifies excitation–contraction coupling, while junctin alone supports functional interactions between calsequestrin and the ryanodine receptor. [ABSTRACT FROM AUTHOR]

Abstract: Normal Ca2+ signalling in skeletal muscle depends on the membrane associated proteins triadin and junctin and their ability to mediate functional interactions between the Ca2+ binding protein calsequestrin and the type 1 ryanodine receptor in the lumen of the sarcoplasmic reticulum. This important mechanism conserves intracellular Ca2+ stores, but is poorly understood. Triadin and junctin share similar structures and are lumped together in models of interactions between skeletal muscle calsequestrin and ryanodine receptors, however their individual roles have not been examined at a molecular level. We show here that purified skeletal ryanodine receptors are similarly activated by purified triadin or purified junctin added to their luminal side, although a lack of competition indicated that the proteins act at independent sites. Surprisingly, triadin and junctin differed markedly in their ability to transmit information between skeletal calsequestrin and ryanodine receptors. Purified calsequestrin inhibited junctin/triadin-associated, or junctin-associated, ryanodine receptors and the calsequestrin re-associated channel complexes were further inhibited when luminal Ca2+ fell from 1mM to ≤100μM, as seen with native channels (containing endogenous calsequestrin/triadin/junctin). In contrast, skeletal calsequestrin had no effect on the triadin/ryanodine receptor complex and the channel activity of this complex increased when luminal Ca2+ fell, as seen with purified channels prior to triadin/calsequestrin re-association. Therefore in this cell free system, junctin alone mediates signals between luminal Ca2+, skeletal calsequestrin and skeletal ryanodine receptors and may curtail resting Ca2+ leak from the sarcoplasmic reticulum. We suggest that triadin serves a different function which may dominate during excitation–contraction coupling. [Copyright &y& Elsevier]

The junctional domain of sarcoplasmic reticulum (jSR) is specialized for receiving signals from the plasmalemma-transverse tubules and for releasing Ca2+ during muscle activation. The junctional face of the jSR, facing the transverse tubules, is occupied by a molecular complex composed of the transmembrane Ca2+ release channels (ryanodine receptors); the luminal protein calsequestrin (CSQ); the 2 membrane proteins, junctin (Jct), and triadin (Tr), which mediate CSQ-ryanodine receptor interactions; and several other components. Under the conditions prevailing within the sarcoplasmic reticulum lumen (physiological ionic strength, mostly due to K+ and Ca2+ ions), CSQ forms long linear polymers and the fixed protein gel is clearly visible in the electron microscope. The luminal domains of Jct and Tr are detectable but, overall, the 2 molecules are not clearly delineated. Cardiac muscles either overexpressing or bearing null mutations for 3 proteins of the junctional complex (CSQ, Jct, and Tr) reveal the contribution of these 3 components to the general architecture of the jSR. [ABSTRACT FROM AUTHOR]

Summary: Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20–25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400μM 4-chloro-m-cresol, 10mM caffeine, 400μM UTP, or 1μM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins’ mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes. [Copyright &y& Elsevier]

Summary: Calcium signaling, intrinsic to skeletal and cardiac muscle function, is critically dependent on the amount of calcium stored within the sarcoplasmic reticulum. Calsequestrin, the main calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for release through the ryanodine receptor and acts as a luminal calcium sensor for the channel via its interactions with triadin and junctin. We examined the influence of phosphorylation of calsequestrin on its ability to store calcium, to polymerise and to regulate ryanodine receptors by binding to triadin and junctin. Our hypothesis was that these parameters might be altered by phosphorylation of threonine 353, which is located near the calcium and triadin/junctin binding sites. Although phosphorylation increased the calcium binding capacity of calsequestrin nearly 2-fold, it did not alter calsequestrin polymerisation, its binding to triadin or junctin or inhibition of ryanodine receptor activity at 1mM luminal calcium. Phosphorylation was required for calsequestrin binding to junctin when calcium concentration was low (100nM), and ryanodine receptors were activated by dephosphorylated calsequestrin when it bound to triadin alone. These novel data shows that phosphorylated calsequestrin is required for high capacity calcium buffering and suggest that ryanodine receptor inhibition by calsequestrin is mediated by junctin. [Copyright &y& Elsevier]

Abstract: In the heart, Ca2+ released from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), is the principal determinant of cardiac contractility. SR Ca2+ release is controlled by dedicated molecular machinery, composed of the cardiac ryanodine receptor (RyR2) and a number of accessory proteins, including FKBP12.6, calsequestrin (CASQ2), triadin (TRD) and junctin (JN). Acquired and genetic defects in the components of the release channel complex result in a spectrum of abnormal Ca2+ release phenotypes ranging from arrhythmogenic spontaneous Ca2+ releases and Ca2+ alternans to the uniformly diminished systolic Ca2+ release characteristic of heart failure. In this article, we will present an overview of the structure and molecular components of the SR and Ca2+ release machinery and its modulation by different intracellular factors, such as Ca2+ levels inside the SR as well as phosphorylation and redox modification of RyR2s. We will also discuss the relationships between abnormal SR Ca2+ release and various cardiac disease phenotypes, including, arrhythmias and heart failure, and consider SR Ca2+ release as a potential therapeutic target. [Copyright &y& Elsevier]

Abstract: The single genomic locus, AβH-J-J, encodes three functionally distinct proteins aspartyl beta-hydroxylase, junctin and junctate by alternative splicing. Among these three proteins, junctin and junctate could play important roles in the regulation of intracellular Ca2+ by regulating either Ca2+ release from intracellular Ca2+ stores or Ca2+ influx in various biological processes. Here we review recent findings concerning the expressional regulations and the proposed functions of junctin and junctate. [Copyright &y& Elsevier]

Depressed cardiac Ca cycling by the sarcoplasmic reticulum (SR) has been associated with attenuated contractility, which can progress to heart failure. The histidine-rich Ca-binding protein (HRC) is an SR component that binds to triadin and may affect Ca release through the ryanodine receptor. HRC overexpression in transgenic mouse hearts was associated with decreased rates of SR Ca uptake and delayed relaxation, which progressed to hypertrophy with aging. The present study shows that HRC may mediate part of its regulatory effects by binding directly to sarco(endo)plasmic reticulum Ca-ATPase type 2 (SERCA2) in cardiac muscle, which is confirmed by coimmunostaining observed under confocal microscopy. This interaction involves the histidine- and glutamic acid-rich domain of HRC (320-460 aa) and the part of the NH2-terminal cation transporter domain of SERCA2 (74-90 aa) that projects into the SR lumen. The SERCA2-binding domain is upstream from the triadin-binding region in human HRC (609-699 aa). Specific binding between HRC and SERCA was verified by coimmunoprecipitation and pull-down assays using human and mouse cardiac homogenates and by blot overlays using glutathione S-transferase and maltose-binding protein recombinant proteins. Importantly, increases in Ca concentration were associated with a significant reduction of HRC binding to SERCA2, whereas they had opposite effects on the HRC-triadin interaction in cardiac homogenates. Collectively, our data suggest that HRC may play a key role in the regulation of SR Ca cycling through its direct interactions with SERCA2 and triadin, mediating a fine cross talk between SR Ca uptake and release in the heart. [ABSTRACT FROM AUTHOR]

Abstract: Reduced function of the cardiac ryanodine receptor or calsequestrin causes catecholaminergic ventricular tachycardia (VT). These proteins regulate sarcoplasmic Ca2+ release in close conjunction with two accessory proteins, triadin and junctin. Based on data from cardiomyocytes, we hypothesized that enhanced triadin expression could cause VT. We assessed arrhythmias and electrophysiological changes in vivo and in the beating heart in mice expressing junctin, triadin, or both proteins (TRD×JCN), and measured calcium transients in isolated ventricular cardiomyocytes. TRD×JCN mice were studied to compensate the down-regulation of junctin expression in triadin-expressing mice. Exercise or stress provoked repetitive VT in freely roaming TRD×JCN mice whenever heart rate increased above approximately 600 bpm (p <0.05 vs. the three other genotypes). TRD×JCN mice expressed total triadin 2.9-fold (p <0.05) and total junctin not different to wildtype (p =ns). Left ventricular systolic function was not different between lineages. β-adrenoreceptor stimulation (orciprenaline 1.7 μM) provoked early-coupled ventricular ectopy and repetitive VT in isolated, Langendorff-perfused TRD×JCN hearts (p <0.05). Under conditions associated with VT (high pacing rate, catecholamine stimulation), action potential duration was shorter in TRD×JCN with VT than in the other genotypes and shorter than in TRD×JCN hearts without VT (p <0.05). Ca2+ transient duration was prolonged in Indo1-loaded TRD×JCN cardiomyocytes under VT-provoking conditions. Action potential prolongation by mexiletine (2 μM or 4 μM) or clarithromycine (150 μM) suppressed VT. Expression of triadin provokes stress- and tachycardia-related ventricular arrhythmias in mice. An imbalance between prolonged intracellular calcium release and shortening of the ventricular action potential may contribute to genesis of arrhythmias in this model. [Copyright &y& Elsevier]

Abstract: The 95kDa triadin (or T95), the main skeletal muscle triadin isoform, negatively regulates the mechanism of excitation–contraction coupling. T95 is a ryanodine receptor (RyR)-interacting protein but it also possesses a calsequestrin-interacting domain. RyR and calsequestrin are involved in Ca2+ signalling and, for instance, influence the activity of store-dependent Ca2+ channels (SOC). This work was undertaken to determine whether T95 was able to modulate the entry of Ca2+ through SOC. The experiments were carried out on differentiated rat myotubes over-expressing T95 or DsRed (control cells) by means of an adenovirus infection. Intracellular Ca2+ signals were analyzed using the Ca2+ indicator Fluo-4. The sarco–endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ stores. When applied in the presence of a Ca2+-free medium, thapsigargin elicited transient but long-lasting Fluo-4 responses by elevating the cytoplasmic concentration of Ca2+ ([Ca2+]i). The over-expression of T95 reduced the thapsigargin-dependent [Ca2+]i increase, with respect to control myotubes. Addition of extracellular Ca2+ after the depletion of this Ca2+ pool was accompanied by a [Ca2+]i increase that was sensitive to the SOC blockers 2-APB, SKF-96365 and La3+. The over-expression of T95 reduced this Ca2+ influx, without changing its pharmacological properties, showing that T95 over-expression did not alter the properties of the SOC. In conclusion, the RyR-interacting molecule T95, recently shown to inhibit the excitation–contraction coupling, has also the ability to interfere with the skeletal muscle Ca2+ signalling by depressing thapsigargin-dependent Ca2+ release and influx. [Copyright &y& Elsevier]