Search Results (15)

The junctional sarcoplasmic reticulum (jSR) is a critical organelle in cardiomyocytes, regulating calcium homeostasis and Excitation–Contraction Coupling (ECC). A quantitative understanding of its protein composition is essential for investigating cardiac physiology and related pathologies. However, isolating intact jSR vesicles, particularly those enriched in membrane proteins, remains a challenging task. Here, we describe our optimized protocol for reproducible enrichment of jSR vesicles from a single murine heart, without the use of antibodies. The protocol enables the recovery of low-abundance membrane proteins while preserving their native interactions with partners. This strategy facilitates the straightforward identification by Mass Spectrometry of highly relevant yet challenging jSR proteins, including the cardiac Ryanodine Receptor and calsequestrin. Our protocol provides a robust tool for studying the structural and stoichiometric organization of the cardiac jSR components in a widely used animal model. Full article

While calcium (Ca²⁺) is a universal cellular messenger, the ionic properties of magnesium (Mg²⁺) make it less suited for rapid signaling and more for structural integrity. Still, besides being a passive player, Mg²⁺ is the only active Ca²⁺ antagonist, essential for tuning the efficacy of Ca²⁺-dependent cardiac excitation–contraction coupling (ECC) and for ensuring cardiac function robustness and stability. This review aims to provide a comprehensive framework to link the structural and molecular mechanisms of Mg²⁺/Ca²⁺ antagonistic binding across key proteins of the cardiac ECC machinery to their physiopathological relevance. The pervasive “dampening” effect of Mg²⁺ on ECC activity is exerted across various players and mechanisms, and lies in the ions’ physiological competition for multiple, flexible binding protein motifs across multiple compartments. Mg²⁺ profoundly modulates the cardiac action potential waveform by inhibiting the L-type Ca²⁺ channel Cav1.2, i.e., the key trigger of cardiac ryanodine receptor (RyR2) opening. Cytosolic Mg²⁺ favors RyR2 closed or inactive conformations not only through physical binding at specific sites, but also indirectly through modulation of RyR2 phosphorylation by Camk2d and PKA. RyR2 is also potently inhibited by luminal Mg²⁺, a vital mechanism in the cardiac setting for preventing excessive Ca²⁺ release during diastole. This mechanism, able to distinguish between Ca²⁺ and Mg²⁺, is mediated by luminal partners Calsequestrin 2 (CASQ2) and Triadin (TRDN). In addition, Mg²⁺ favors a rearrangement of the RyR2 cluster configuration that is associated with lower Ca²⁺ spark frequencies. Full article

Heart-specific overexpression of transcriptional regulator JDP2 (jun dimerization protein 2) for 5 weeks provokes paroxysmal atrial fibrillation (AF) in mice. We now investigated whether AF and atrial remodeling will be reversible upon termination of JDP2 overexpression, and whether paroxysmal AF converts to permanent AF in the presence of maintained JDP2 overexpression. Cardiac-specific JDP2 overexpression for 5 weeks, resulting in paroxysmal AF, was either continued or repressed via a tet-off system for another 5 weeks. ECGs were recorded weekly. Thereafter, heart and lung weights, and atrial mRNA and protein expression were determined. Extending JDP2 overexpression did not aggravate the AF phenotype, still paroxysmal AF, prolongation of PQ intervals, and atrial hypertrophy were present. This phenotype was completely reversible upon cessation of JDP2 overexpression. A massive downregulation of connexin40 and calcium handling proteins, including SERCA2a, calsequestrin, and ryanodine receptor, was observed in atria after prolonged JDP2 overexpression. In conclusion, atrial remodeling and paroxysmal AF under JDP2 overexpression are not sufficient to maintain or aggravate AF in the absence of JDP2. The comparison of the two groups indicates that the downregulation of calcium proteins and connexins is an important factor in the maintenance of the disease. Full article

The electrophysiological mechanisms underlying melatonin’s actions and the electrophysiological consequences of superimposed therapeutic hypothermia (TH) in preventing cardiac ischemia-reperfusion (IR) injury-induced arrhythmias remain largely unknown. This study aimed to unveil these issues using acute IR-injured hearts. Rabbits were divided into heart failure (HF), HF+melatonin, control, and control+melatonin groups. HF was induced by rapid right ventricular pacing. Melatonin was administered orally (10 mg/kg/day) for four weeks, and IR was created by 60-min coronary artery ligation and 30-min reperfusion. The hearts were then excised and Langendorff-perfused for optical mapping studies at normothermia, followed by TH. Melatonin significantly reduced ventricular fibrillation (VF) maintenance. In failing hearts, melatonin reduced the spatially discordant alternans (SDA) inducibility mainly by modulating intracellular Ca²⁺ dynamics via upregulation of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) and calsequestrin 2 and attenuating the downregulation of phosphorylated phospholamban protein expression. In control hearts, melatonin improved conduction slowing and reduced dispersion of action potential duration (APD_dispersion) by upregulating phosphorylated connexin 43, attenuating the downregulation of SERCA2a and phosphorylated phospholamban and attenuating the upregulation of phosphorylated Ca²⁺/calmodulin-dependent protein kinase II. TH significantly retarded intracellular Ca²⁺ decay slowed conduction, and increased APD_dispersion, thereby facilitating SDA induction, which counteracted the beneficial effects of melatonin in reducing VF maintenance. Full article

Cardiovascular disease is a leading cause of death worldwide. The differentiation of human pluripotent stem cells (hPSCs) into functional cardiomyocytes offers significant potential for disease modeling and cell-based cardiac therapies. However, hPSC-derived cardiomyocytes (hPSC-CMs) remain largely immature, limiting their experimental and clinical applications. A critical challenge in current in vitro culture systems is the absence of standardized metrics to quantify maturity. This study presents a data-driven pipeline to quantify hPSC-CM maturity using gene expression data across various stages of cardiac development. We determined that culture time serves as a feasible proxy for maturity. To improve prediction accuracy, machine learning algorithms were employed to identify heart-related genes whose expression strongly correlates with culture time. Our results reduced the average discrepancy between predicted and observed culture time to 4.461 days and CASQ2 (Calsequestrin 2), a gene involved in calcium ion storage and transport, was identified as the most critical cardiac gene associated with culture duration. This novel framework for maturity assessment moves beyond traditional qualitative methods, providing deeper insights into hPSC-CM maturation dynamics. It establishes a foundation for developing advanced lab-on-chip devices capable of real-time maturity monitoring and adaptive stimulus selection, paving the way for improved maturation strategies and broader experimental/clinical applications. Full article

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. Full article

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe hereditary arrhythmia syndrome predominantly affecting children and young adults. It manifests through bidirectional or polymorphic ventricular arrhythmia, often culminating in syncope triggered by physical exertion or emotional stress which can lead to sudden cardiac death. Most cases stem from mutations in the gene responsible for encoding the cardiac ryanodine receptor (RyR2), or in the Calsequestrin 2 gene (CASQ2), disrupting the handling of calcium ions within the cardiac myocyte sarcoplasmic reticulum. Diagnosing CPVT typically involves unmasking the arrhythmia through exercise stress testing. This diagnosis emerges in the absence of structural heart disease by cardiac imaging and with a normal baseline electrocardiogram. Traditional first-line treatment primarily involves β-blocker therapy, significantly reducing CPVT-associated mortality. Adjunctive therapies such as moderate exercise training, flecainide, left cardiac sympathetic denervation and implantable cardioverter-defibrillators have been utilized with reasonable success. However, the spectrum of options for managing CPVT has expanded over time, demonstrating decreased rates of arrhythmic events. Furthermore, ongoing research into potential new therapies including gene therapies has the potential to further enhance treatment paradigms. This review aims to succinctly encapsulate the contemporary understanding of the clinical characteristics, diagnostic approach, established therapeutic interventions and the promising future directions in managing CPVT. Full article

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. Full article

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). CASQ2^G112+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 β-adrenergic stimulation or in the slowing of heart rate afterward once β-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 β-adrenergic stimulation in the CPVT mutant but not in the wild-type nor in the non-β-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. Full article

Calsequestrin 1 (CASQ1) in skeletal muscle buffers and senses Ca²⁺ in the sarcoplasmic reticulum (SR). CASQ1 also regulates store-operated Ca²⁺ entry (SOCE) by binding to stromal interaction molecule 1 (STIM1). Abnormal SOCE and/or abnormal expression or mutations in CASQ1, STIM1, or STIM2 are associated with human skeletal, cardiac, or smooth muscle diseases. However, the functional relevance of CASQ1 along with STIM2 has not been studied in any tissue, including skeletal muscle. First, in the present study, it was found by biochemical approaches that CASQ1 is bound to STIM2 via its 92 N-terminal amino acids (C1 region). Next, to examine the functional relevance of the CASQ1-STIM2 interaction in skeletal muscle, the full-length wild-type CASQ1 or the C1 region was expressed in mouse primary skeletal myotubes, and the myotubes were examined using single-myotube Ca²⁺ imaging experiments and transmission electron microscopy observations. The CASQ1-STIM2 interaction via the C1 region decreased SOCE, increased intracellular Ca²⁺ release for skeletal muscle contraction, and changed intracellular Ca²⁺ distributions (high Ca²⁺ in the SR and low Ca²⁺ in the cytosol were observed). Furthermore, the C1 region itself (which lacks Ca²⁺-buffering ability but has STIM2-binding ability) decreased the expression of Ca²⁺-related proteins (canonical-type transient receptor potential cation channel type 6 and calmodulin 1) and induced mitochondrial shape abnormalities. Therefore, in skeletal muscle, CASQ1 plays active roles in Ca²⁺ movement and distribution by interacting with STIM2 as well as Ca²⁺ sensing and buffering. Full article

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare and potentially lethal inherited arrhythmia disease characterized by exercise or emotion-induced bidirectional or polymorphic ventricular tachyarrhythmias. The median age of disease onset is reported to be approximately 10 years of age. The majority of CPVT patients have pathogenic variants in the gene encoding the cardiac ryanodine receptor, or calsequestrin 2. These lead to mishandling of calcium in cardiomyocytes resulting in after-depolarizations, and ventricular arrhythmias. Disease severity is particularly pronounced in younger individuals who usually present with cardiac arrest and arrhythmic syncope. Risk stratification is imprecise and long-term prognosis on therapy is unknown despite decades of research focused on pediatric CPVT populations. The purpose of this review is to summarize contemporary data on pediatric CPVT, highlight knowledge gaps and present future research directions for the clinician-scientist to address. Full article

Ryanodine receptor 2 (RyR2) and SERCA2a are two major players in myocyte calcium (Ca) cycling that are modulated physiologically, affected by disease and thus considered to be potential targets for cardiac disease therapy. However, how RyR2 and SERCA2a influence each others’ activities, as well as the primary and secondary consequences of their combined manipulations remain controversial. In this study, we examined the effect of acute upregulation of SERCA2a on arrhythmogenesis by conditionally overexpressing SERCA2a in a mouse model featuring hyperactive RyR2s due to ablation of calsequestrin 2 (CASQ2). CASQ2 knock-out (KO) mice were crossbred with doxycycline (DOX)-inducible SERCA2a transgenic mice to generate KO-TG mice. In-vivo ECG studies have shown that induction of SERCA2a (DOX+) overexpression markedly exacerbated both ventricular and atrial arrhythmias in vivo, compared with uninduced KO-TG mice (DOX-). Consistent with that, confocal microscopy in both atrial and ventricular myocytes demonstrated that conditional upregulation of SERCA2a enhanced the rate of occurrence of diastolic Ca release events. Additionally, deep RNA sequencing identified 17 downregulated genes and 5 upregulated genes in DOX+ mice, among which Ppp1r13l, Clcn1, and Agt have previously been linked to arrhythmias. Our results suggest that conditional upregulation of SERCA2a exacerbates hyperactive RyR2-mediated arrhythmias by further elevating diastolic Ca release. Full article

Increases in the prevalence of obesity, insulin resistance, and metabolic syndrome has led to the increase of atrial fibrillation (AF) cases in the developed world. These AF risk factors are associated with mitochondrial dysfunction, previously modelled using peroxisome proliferator activated receptor-γ (PPARγ) coactivator-1 (Pgc-1)-deficient murine cardiac models. We explored gene and protein expression profiles of selected molecular targets related to electrophysiological function in murine Pgc-1α^−/− atria. qPCR analysis surveyed genes related to Na⁺-K⁺-ATPase, K⁺ conductance, hyperpolarisation-activated cyclic nucleotide-gated (Hcn), Na⁺ channels, Ca²⁺ channels, and indicators for adrenergic and cholinergic receptor modulation. Western blot analysis for molecular targets specific to conduction velocity (Na_v1.5 channel and gap junctions) was performed. Transcription profiles revealed downregulation of molecules related to Na⁺-K⁺-ATPase transport, Hcn-dependent pacemaker function, Na⁺ channel-dependent action potential activation and propagation, Ca²⁺ current generation, calsequestrin-2 dependent Ca²⁺ homeostasis, and adrenergic α_1D dependent protection from hypertrophic change. Na_v1.5 channel protein expression but not gap junction expression was reduced in Pgc-1α^−/− atria compared to WT. Na_v1.5 reduction reflects corresponding reduction in its gene expression profile. These changes, as well as the underlying Pgc-1α^−/− alteration, suggest potential pharmacological targets directed towards either upstream PGC-1 signalling mechanisms or downstream ion channel changes. Full article

Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc₂Man₁), while cardiac calsequestrin had five additional mannoses (GlcNAc₂Man₆). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca²⁺-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic “guiderail”-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca²⁺ concentrations, and proper glycosylation, in turn, guarantees both effective Ca²⁺ storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site. Full article

Calsequestrin (CASQ) is a major Ca²⁺-storage/buffer protein present in the sarcoplasmic reticulum of both skeletal (CASQ1) and cardiac (CASQ2) muscles. CASQ has significant affinity for a number of pharmaceutical drugs with known muscular toxicities. Our approach, with in silico molecular docking, single crystal X-ray diffraction, and isothermal titration calorimetry (ITC), identified three distinct binding pockets on the surface of CASQ2, which overlap with 2-methyl-2,4-pentanediol (MPD) binding sites observed in the crystal structure. Those three receptor sites based on canine CASQ1 crystal structure gave a high correlation (R² = 0.80) to our ITC data. Daunomycin, doxorubicin, thioridazine, and trifluoperazine showed strong affinity to the S1 site, which is a central cavity formed between three domains of CASQ2. Some of the moderate-affinity drugs and some high-affinity drugs like amlodipine and verapamil displayed their binding into S2 sites, which are the thioredoxin-like fold present in each CASQ domain. Docking predictions combined with dissociation constants imply that presence of large aromatic cores and less flexible functional groups determines the strength of binding affinity to CASQ. In addition, the predicted binding pockets for both caffeine and epigallocatechin overlapped with the S1 and S2 sites, suggesting competitive inhibition by these natural compounds as a plausible explanation for their antagonistic effects on cardiotoxic side effects. Full article