Stable and reusable calcium-responsive biopolymer for affinity precipitation of therapeutic antibodies.

Affinity precipitation is an attractive method for protein purification due to its many advantages, including the rapid capture of target proteins, simple processing, high specificity, and ease of scale-up. We previously reported a robust antibody purification method using Ca2+-dependent precipitation of ZZ-hCSQ2, a fusion protein of human calsequestrin 2, and the antibody-binding protein ZZ. However, the stability of this fusion protein was not sufficiently high for industrial use because the antibody recovery yield decreased to 60% after being reused 10 times. To identify a more stable calsequestrin (CSQ), we calculated Rosetta energy values for the folding stabilities of various CSQ homologs and selected human CSQ1 (hCSQ1) with lowest energy value (-992.6) as the new CSQ platform. We also identified that the linker sequence between ZZ and CSQ was vulnerable to proteases and alkaline pH by N-terminal protein sequencing. Therefore, we changed the linker to four asparagine (4N) sequences, which were shorter and less flexible than the previous glycine-rich linker. The new version of ZZ-CSQ, ZZ-4N-hCSQ1, was stable in a protease-containing conditioned medium obtained from the cultured Chinese hamster ovary cell or high pH condition (0.1M sodium hydroxide) for more than 5 days and could be reused at least 25 times for antibody purification without loss of recovery yield. The antibodies purified by ZZ-4N-hCSQ1 precipitation also showed greater purity (~33.6-fold lower host cell DNA and ~6.4-fold lower host cell protein) than those purified by protein A chromatography. These data suggest that ZZ-4N-hCSQ1 precipitation is more efficient and can achieve cost-effectiveness of up to 12.5-fold cheaper than previous antibody purification methods and can lower the production costs of therapeutic antibodies.

Investigation of a Large Kindred Reveals Cardiac Calsequestrin (CASQ2) as a Cause of Brugada Syndrome.

BACKGROUND: Brugada syndrome (BrS) is an inherited primary channelopathy syndrome associated with the risk of ventricular fibrillation (VF) and sudden cardiac death in a structurally normal heart. AIM OF THE STUDY: The aim of this study was to clinically and genetically evaluate a large family with severe autosomal dominant Brugada syndrome. METHODS: Clinical and genetic studies were performed. Genetic analysis was conducted with NGS technologies (WES) using the Illumina instrument. According to the standard procedure, variants found by WES were confirmed in all available families by Sanger sequencing. The effect of the variants was studied by using in silico prediction of pathogenicity. RESULTS: The proband was a 52-year-old man who was admitted to the emergency department for syncope at rest. WES of the index case identified a heterozygous VUS CASQ2, c.532T>C, p.(Tyr178His). We studied the segregation of the variation in all pedigree members. All the patients were heterozygous for the variation CASQ2 p.(Tyr178His), whereas the remaining healthy individuals in the family were homozygous for the normal allele. Structural analysis of CASQ2 p.(Tyr178His) was performed and revealed an important effect of the missense variation on monomer stability. The CASQ2 Tyr180 residue is located inside the sarcoplasmic reticulum (SR) junctional face membrane interaction domain and is predicted to disrupt filamentation. CONCLUSIONS: Our data suggest that the p.Tyr178His substitution is associated with BrS in the family investigated, affecting the stability of the protein, disrupting filamentation at the interdimer interface, and affecting the subsequent formation of tetramers and polymers that contain calcium-binding sites.

[Identification of a novel variant in a patient with Calsequestrin 1 related myopathy].

OBJECTIVE: To explore the genetic basis of a myopathic patient with pathological characteristics including tubular aggregates and vacuoles. METHODS: Next generation sequencing was carried out for the patient, and candidate variant was verified by Sanger sequencing. RESULTS: Genetic testing revealed that the patient has harbored a heterozygous c.730G>C (p.D244H) variant of Calsequestrin 1 (CASQ1) gene. The same variant was not found in his unaffected parents. Based on guidelines from the American College of Medical Genetics and Genomics, the variant was rated as pathogenic (PS1+PM2+PP3). CONCLUSION: The novel c.730G>C (p.D244H) variant of the CASQ1 gene probably underlay the myopathy in this patient. Above finding has enriched the mutational spectrum of the CASQ1 gene.

Catecholaminergic polymorphic ventricular tachycardia (and seizure) caused by a novel homozygous likely pathogenic variant in CASQ2 gene.

BACKGROUND: Although structural heart disease is frequently present among patients who experience sudden cardiac death (SCD), inherited arrhythmia syndromes can also play an important role in the occurrence of SCD. CPVT2, which is the second-most prevalent form of CPVT, arises from an abnormality in the CASQ2 gene. OBJECTIVE: We represent a novel CASQ2 variant that causes CPVT2 and conduct a comprehensive review on this topic. METHODS: The proband underwent Whole-exome sequencing (WES) in order to ascertain the etiology of CPVT. Subsequently, the process of segregating the available family members was carried out through the utilization of PCR and Sanger Sequencing. We searched the google scholar and PubMed/Medline for studies reporting CASQ2 variants, published up to May 10,2023. We used the following mesh term "Calsequestrin" and using free-text method with terms including "CASQ2","CASQ2 variants", and "CASQ2 mutation". RESULTS: The CASQ2 gene was found to contain an autosomal recessive nonsense variant c.268_269insTA:p.Gly90ValfsTer4, which was identified by WES. This variant was determined to be the most probable cause of CPVT in the pedigree under investigation. CONCLUSION: CASQ2 variants play an important role in pathogenesis of CPVT2. Notabely, based on results of our study and other findings in the literature the variant in this gene may cause an neurological signs in the patients with CPVT2. Further studies are needed for more details about the role of this gene in CPVT evaluation, diagnosis, and gene therapy.

The Structural-Functional Crosstalk of the Calsequestrin System: Insights and Pathological Implications.

Calsequestrin (CASQ) is a key intra-sarcoplasmic reticulum Ca2+-handling protein that plays a pivotal role in the contraction of cardiac and skeletal muscles. Its Ca2+-dependent polymerization dynamics shape the translation of electric excitation signals to the Ca2+-induced contraction of the actin-myosin architecture. Mutations in CASQ are linked to life-threatening pathological conditions, including tubular aggregate myopathy, malignant hyperthermia, and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). The variability in the penetrance of these phenotypes and the lack of a clear understanding of the disease mechanisms associated with CASQ mutations pose a major challenge to the development of effective therapeutic strategies. In vitro studies have mainly focused on the polymerization and Ca2+-buffering properties of CASQ but have provided little insight into the complex interplay of structural and functional changes that underlie disease. In this review, the biochemical and structural natures of CASQ are explored in-depth, while emphasizing their direct and indirect consequences for muscle Ca2+ physiology. We propose a novel functional classification of CASQ pathological missense mutations based on the structural stability of the monomer, dimer, or linear polymer conformation. We also highlight emerging similarities between polymeric CASQ and polyelectrolyte systems, emphasizing the potential for the use of this paradigm to guide further research.

Constitutive assembly of Ca2+ entry units in soleus muscle from calsequestrin knockout mice.

Calcium (Ca2+) entry units (CEUs) are junctions within the I band of the sarcomere between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of the transverse (T)-tubule. CEUs contain STIM1 and Orai1 proteins, the molecular machinery of store-operated Ca2+ entry (SOCE). In extensor digitorum longus (EDL) fibers of wild-type (WT) mice, CEUs transiently assemble during acute exercise and disassemble several hours thereafter. By contrast, calsequestrin-1 (CASQ1) ablation induces a compensatory constitutive assembly of CEUs in EDL fibers, resulting in enhanced constitutive and maximum SOCE that counteracts SR Ca2+ depletion during repetitive activity. However, whether CEUs form in slow-twitch fibers, which express both the skeletal CASQ1 and the cardiac CASQ2 isoforms, is unknown. Herein, we compared the structure and function of soleus muscles from WT and knockout mice that lack either CASQ1 (CASQ1-null) or both CASQs (dCASQ-null). Ultrastructural analyses showed that SR/T-tubule junctions at the I band, virtually identical to CEUs in EDL muscle, were present and more frequent in CASQ1-null than WT mice, with dCASQ-null exhibiting the highest incidence. The greater incidence of CEUs in soleus from dCASQ-null mice correlated with increased specific force production during repetitive, high-frequency stimulation, which depended on Ca2+ entry. Consistent with this, Orai1 expression was significantly increased in soleus of CASQ1-null mice, but even more in dCASQ-null mice, compared with WT. Together, these results strengthen the concept that CEU assembly strongly depends on CASQ expression and provides an alternative source of Ca2+ needed to refill SR Ca2+ stores to maintain specific force production during sustained muscle activity.

Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene (CASQ2), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2-K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known. METHODS: A K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators. RESULTS: K180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness. CONCLUSIONS: CASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

Oxygen Consumption and Basal Metabolic Rate as Markers of Susceptibility to Malignant Hyperthermia and Heat Stroke.

Calsequestrin 1 (CASQ1) and Ryanodine receptor 1 (RYR1) are two of the main players in excitation-contraction (EC) coupling. CASQ1-knockout mice and mice carrying a mutation in RYR1 (Y522S) linked to human malignant hyperthermia susceptibility (MHS) both suffer lethal hypermetabolic episodes when exposed to halothane (MHS crises) and to environmental heat (heat stroke, HS). The phenotype of Y522S is more severe than that of CASQ1-null mice. As MHS and HS are hypermetabolic responses, we studied the metabolism of adult CASQ1-null and Y522S mice using wild-type (WT) mice as controls. We found that CASQ1-null and Y522S mice have increased food consumption and higher core temperature at rest. By indirect calorimetry, we then verified that CASQ1-null and Y522S mice show an increased oxygen consumption and a lower respiratory quotient (RQ). The accelerated metabolism of CASQ1-null and Y522S mice was also accompanied with a reduction in body fat. Moreover, both mouse models displayed increased oxygen consumption and a higher core temperature during heat stress. The results collected suggest that metabolic rate, oxygen consumption, and body temperature at rest, all more elevated in Y522S than in CASQ1-null mice, could possibly be used as predictors of the level of susceptibility to hyperthermic crises of mice (and possibly humans).

Generation of Atrial-Specific Construct Using Sarcolipin Promoter-Associated CRM4 Enhancer.

Cardiac gene therapy has been hampered by off-target expression of gene of interest irrespective of variety of delivery methods. To overcome this issue, cardiac-specific promoters provide target tissue specificity, although expression is often debilitated compared to that of ubiquitous promoters. We have previously shown that sarcolipin promoter with an enhancer calsequestrin cis-regulatory module 4 (CRM4) combination has an improved atrial specificity. Moreover, it showed a minimal extra-atrial expression, which is a significant advantage for AAV9-mediated cardiac gene therapy. Therefore, it can be a useful tool to study and treat atrial-specific diseases such as atrial fibrillation. In this chapter, we introduce practical and simple methodology for atrial-specific gene therapy using sarcolipin promoter with an enhancer CRM4.

Mice lacking MBNL1 and MBNL2 exhibit sudden cardiac death and molecular signatures recapitulating myotonic dystrophy.

Myotonic dystrophy (DM) is caused by expansions of C(C)TG repeats in the non-coding regions of the DMPK and CNBP genes, and DM patients often suffer from sudden cardiac death due to lethal conduction block or arrhythmia. Specific molecular changes that underlie DM cardiac pathology have been linked to repeat-associated depletion of Muscleblind-like (MBNL) 1 and 2 proteins and upregulation of CUGBP, Elav-like family member 1 (CELF1). Hypothesis solely targeting MBNL1 or CELF1 pathways that could address all the consequences of repeat expansion in heart remained inconclusive, particularly when the direct cause of mortality and results of transcriptome analyses remained undetermined in Mbnl compound knockout (KO) mice with cardiac phenotypes. Here, we develop Myh6-Cre double KO (DKO) (Mbnl1-/-; Mbnl2cond/cond; Myh6-Cre+/-) mice to eliminate Mbnl1/2 in cardiomyocytes and observe spontaneous lethal cardiac events under no anesthesia. RNA sequencing recapitulates DM heart spliceopathy and shows gene expression changes that were previously undescribed in DM heart studies. Notably, immunoblotting reveals a nearly 6-fold increase of Calsequestrin 1 and 50% reduction of epidermal growth factor proteins. Our findings demonstrate that complete ablation of MBNL1/2 in cardiomyocytes is essential for generating sudden death due to lethal cardiac rhythms and reveal potential mechanisms for DM heart pathogenesis.

Purification of Therapeutic Antibodies Using the Ca2+-Dependent Phase-Transition Properties of Calsequestrin.

Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding ∼20-fold less DNA and ∼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.

Effects of acute ischemia and hypoxia in young and adult calsequestrin (CSQ2) knock-out and wild-type mice.

Calsequestrin (CSQ2) is the main Ca2+-binding protein in the sarcoplasmic reticulum of the mammalian heart. In order to understand the function of calsequestrin better, we compared two age groups (young: 4-5 months of age versus adult: 18 months of age) of CSQ2 knock-out mice (CSQ2(-/-)) and littermate wild-type mice (CSQ2(+/+)). Using echocardiography, in adult mice, the basal left ventricular ejection fraction and the spontaneous beating rate were lower in CSQ2(-/-) compared to CSQ2(+/+). The increase in ejection fraction by ß-adrenergic stimulation (intraperitoneal injection of isoproterenol) was lower in adult CSQ2(-/-) versus adult CSQ2(+/+). After hypoxia in vitro (isolated atrial preparations) by gassing the organ bath buffer with 95% N2, force of contraction in electrically driven left atria increased to lower values in young CSQ2(-/-) than in young CSQ2(+/+). In addition, after global ischemia and reperfusion (buffer-perfused hearts according to Langendorff; 20-min ischemia and 15-min reperfusion), the rate of tension development was higher in young CSQ2(-/-) compared to young CSQ2(+/+). Finally, we evaluated signs of inflammation (immune cells, autoantibodies, and fibrosis). However, whereas no immunological alterations were found between all investigated groups, pronounced fibrosis was found in the ventricles of adult CSQ2(-/-) compared to all other groups. We suggest that in young mice, CSQ2 is important for cardiac performance especially in isolated cardiac preparations under conditions of impaired oxygen supply, but with differences between atrium and ventricle. Lack of CSQ2 leads age dependently to fibrosis and depressed cardiac performance in echocardiographic studies.

The Purkinje-myocardial junction is the anatomic origin of ventricular arrhythmia in CPVT.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 Ca release channels hyperactive, provoking spontaneous Ca release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs: Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to WT mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje-myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.

Pacing Dynamics Determines the Arrhythmogenic Mechanism of the CPVT2-Causing CASQ2G112+5X Mutation in a Guinea Pig Ventricular Myocyte Computational Model.

Calsequestrin Type 2 (CASQ2) is a high-capacity, low-affinity, Ca2+-binding protein expressed in the sarcoplasmic reticulum (SR) of the cardiac myocyte. Mutations in CASQ2 have been linked to the arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT2) that occurs with acute emotional stress or exercise can result in sudden cardiac death (SCD). CASQ2G112+5X is a 16 bp (339-354) deletion CASQ2 mutation that prevents the protein expression due to premature stop codon. Understanding the subcellular mechanisms of CPVT2 is experimentally challenging because the occurrence of arrhythmia is rare. To obtain an insight into the characteristics of this rare disease, simulation studies using a local control stochastic computational model of the Guinea pig ventricular myocyte investigated how the mutant CASQ2s may be responsible for the development of an arrhythmogenic episode under the condition of ß-adrenergic stimulation or in the slowing of heart rate afterward once ß-adrenergic stimulation ceases. Adjustment of the computational model parameters based upon recent experiments explore the functional changes caused by the CASQ2 mutation. In the simulation studies under rapid pacing (6 Hz), electromechanically concordant cellular alternans appeared under ß-adrenergic stimulation in the CPVT mutant but not in the wild-type nor in the non-ß-stimulated mutant. Similarly, the simulations of accelerating pacing from slow to rapid and back to the slow pacing did not display alternans but did generate early afterdepolarizations (EADs) during the period of second slow pacing subsequent acceleration of rapid pacing.

Multiple regions within junctin drive its interaction with calsequestrin-1 and its localization to triads in skeletal muscle.

Junctin is a transmembrane protein of striated muscles, located at the junctional sarcoplasmic reticulum (SR). It is characterized by a luminal C-terminal tail, through which it functionally interacts with calsequestrin and the ryanodine receptor (RyR). Interaction with calsequestrin was ascribed to the presence of stretches of charged amino acids (aa). However, the regions able to bind calsequestrin have not been defined in detail. We report here that, in non-muscle cells, junctin and calsequestrin assemble in long linear regions within the endoplasmic reticulum, mirroring the formation of calsequestrin polymers. In differentiating myotubes, the two proteins colocalize at triads, where they assemble with other proteins of the junctional SR. By performing GST pull-down assays with distinct regions of the junctin tail, we identified two KEKE motifs that can bind calsequestrin. In addition, stretches of charged aa downstream these motifs were found to also bind calsequestrin and the RyR. Deletion of even one of these regions impaired the ability of junctin to localize at the junctional SR, suggesting that interaction with other proteins at this site represents a key element in junctin targeting.

Calsequestrin 2 overexpression in breast cancer increases tumorigenesis and metastasis by modulating the tumor microenvironment.

The spatial tumor shape is determined by the complex interactions between tumor cells and their microenvironment. Here, we investigated the role of a newly identified breast cancer-related gene, calsequestrin 2 (CASQ2), in tumor-microenvironment interactions during tumor growth and metastasis. We analyzed gene expression and three-dimensional tumor shape data from the breast cancer dataset of The Cancer Genome Atlas (TCGA) and identified CASQ2 as a potential regulator of tumor-microenvironment interaction. In TCGA breast cancer cases containing information of three-dimensional tumor shapes, CASQ2 mRNA showed the highest correlation with the spatial tumor shapes. Furthermore, we investigated the expression pattern of CASQ2 in human breast cancer tissues. CASQ2 was not detected in breast cancer cell lines in vitro but was induced in the xenograft tumors and human breast cancer tissues. To evaluate the role of CASQ2, we established CASQ2-overexpressing breast cancer cell lines for in vitro and in vivo experiments. CASQ2 overexpression in breast cancer cells resulted in a more aggressive phenotype and altered epithelial-mesenchymal transition (EMT) markers in vitro. CASQ2 overexpression induced cancer-associated fibroblast characteristics along with increased hypoxia-inducible factor 1α (HIF1α) expression in stromal fibroblasts. CASQ2 overexpression accelerated tumorigenesis, induced collagen structure remodeling, and increased distant metastasis in vivo. CASQ2 conferred more metaplastic features to triple-negative breast cancer cells. Our data suggest that CASQ2 is a key regulator of breast cancer tumorigenesis and metastasis by modulating diverse aspects of tumor-microenvironment interactions.

Expression of Genes and Proteins of the Sarcoplasmic Reticulum Са2+-Transport Systems in Cardiomyocytes in Concomitant Coronary Heart Disease and Type 2 Diabetes Mellitus.

We compared the expression of Са2+-ATPase (SERCA2a), calsequestrin (CASQ2), ryanodine receptors (RyR2) proteins and their genes (ATP2A2, CASQ2, and RYR2) in coronary heart disease (CHD) patients with and without comorbid type 2 diabetes mellitus. All studies were performed on the right atrial appendages resected during coronary bypass surgeries. Expression of SERCA2a and RyR2 proteins and their ATP2A2 (p=0.046) and RYR2 genes in comorbid pathology was significantly (p=0.042) higher (by 1.2 and 2 times; p=0.025). The expression of CASQ2 protein and its gene did not differ significantly between the groups (p=0.82 and p=0.066, respectively). It was concluded that the expression of SERCA2a and RyR2 proteins and their genes (but not CASQ2 and its gene) is elevated in CHD associated with type 2 diabetes mellitus. Expression of the studied proteins correlated with the expression of their genes. Increased expression of CASQ2 protein and its gene can probably prevent imbalance of the Ca2+-transporting systems in cardiomyocytes and contractile dysfunction of the myocardium, even in CHD associated with type 2 diabetes mellitus.

Functional Calsequestrin-1 Is Expressed in the Heart and Its Deficiency Is Causally Related to Malignant Hyperthermia-Like Arrhythmia.

BACKGROUND: Calsequestrins (Casqs), comprising the Casq1 and Casq2 isoforms, buffer Ca2+ and regulate its release in the sarcoplasmic reticulum of skeletal and cardiac muscle, respectively. Human inherited diseases associated with mutations in CASQ1 or CASQ2 include malignant hyperthermia/environmental heat stroke (MH/EHS) and catecholaminergic polymorphic ventricular tachycardia. However, patients with an MH/EHS event often experience arrhythmia for which the underlying mechanism remains unknown. METHODS: Working hearts from conventional (Casq1-KO) and cardiac-specific (Casq1-CKO) Casq1 knockout mice were monitored in vivo and ex vivo by ECG and electric mapping, respectively. MH was induced by 2% isoflurane and treated intraperitoneally with dantrolene. Time-lapse imaging was used to monitor intracellular Ca2+ activity in isolated mouse cardiomyocytes or neonatal rat ventricular myocytes with knockdown, overexpression, or truncation of the Casq1 gene. Conformational change in both Casqs was determined by cross-linking Western blot analysis. RESULTS: Like patients with MH/EHS, Casq1-KO and Casq1-CKO mice had faster basal heart rate and ventricular tachycardia on exposure to 2% isoflurane, which could be relieved by dantrolene. Basal sinus tachycardia and ventricular ectopic electric triggering also occurred in Casq1-KO hearts ex vivo. Accordingly, the ventricular cardiomyocytes from Casq1-CKO mice displayed dantrolene-sensitive increased Ca2+ waves and diastole premature Ca2+ transients/oscillations on isoflurane. Neonatal rat ventricular myocytes with Casq1-knockdown had enhanced spontaneous Ca2+ sparks/transients on isoflurane, whereas cells overexpressing Casq1 exhibited decreased Ca2+ sparks/transients that were absent in cells with truncation of 9 amino acids at the C terminus of Casq1. Structural evaluation showed that most of the Casq1 protein was present as a polymer and physically interacted with ryanodine receptor-2 in the ventricular sarcoplasmic reticulum. The Casq1 isoform was also expressed in human myocardium. Mechanistically, exposure to 2% isoflurane or heating at 41 °C induced Casq1 oligomerization in mouse ventricular and skeletal muscle tissues, leading to a reduced Casq1/ryanodine receptor-2 interaction and increased ryanodine receptor-2 activity in the ventricle. CONCLUSIONS: Casq1 is expressed in the heart, where it regulates sarcoplasmic reticulum Ca2+ release and heart rate. Casq1 deficiency independently causes MH/EHS-like ventricular arrhythmia by trigger-induced Casq1 oligomerization and a relief of its inhibitory effect on ryanodine receptor-2-mediated Ca2+ release, thus revealing a new inherited arrhythmia and a novel mechanism for MH/EHS arrhythmogenesis.

Calsequestrin, a key protein in striated muscle health and disease.

Calsequestrin (CASQ) is the most abundant Ca2+ binding protein localized in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. The genome of vertebrates contains two genes, CASQ1 and CASQ2. CASQ1 and CASQ2 have a high level of homology, but show specific patterns of expression. Fast-twitch skeletal muscle fibers express only CASQ1, both CASQ1 and CASQ2 are present in slow-twitch skeletal muscle fibers, while CASQ2 is the only protein present in cardiomyocytes. Depending on the intraluminal SR Ca2+ levels, CASQ monomers assemble to form large polymers, which increase their Ca2+ binding ability. CASQ interacts with triadin and junctin, two additional SR proteins which contribute to localize CASQ to the junctional region of the SR (j-SR) and also modulate CASQ ability to polymerize into large macromolecular complexes. In addition to its ability to bind Ca2+ in the SR, CASQ appears also to be able to contribute to regulation of Ca2+ homeostasis in muscle cells. Both CASQ1 and CASQ2 are able to either activate and inhibit the ryanodine receptors (RyRs) calcium release channels, likely through their interactions with junctin and triadin. Additional evidence indicates that CASQ1 contributes to regulate the mechanism of store operated calcium entry in skeletal muscle via a direct interaction with the Stromal Interaction Molecule 1 (STIM1). Mutations in CASQ2 and CASQ1 have been identified, respectively, in patients with catecholamine-induced polymorphic ventricular tachycardia and in patients with some forms of myopathy. This review will highlight recent developments in understanding CASQ1 and CASQ2 in health and diseases.