CASQ2

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Calsequestrin-2 precursor (Calsequestrin, cardiac muscle isoform)

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Single delivery of an adeno-associated viral construct to transfer the CASQ2 gene to knock-in mice affected by catecholaminergic polymorphic ventricular tachycardia is able to cure the disease from birth to advanced age.

Catecholaminergic polymorphic ventricular tachycardia is an inherited arrhythmogenic disorder characterized by sudden cardiac death in children. Drug therapy is still insufficient to provide full protection against cardiac arrest, and the use of implantable defibrillators in the pediatric population is limited by side effects. There is therefore a need to explore the curative potential of gene therapy for this disease. We investigated the efficacy and durability of viral gene transfer of the calsequestrin 2 (CASQ2) wild-type gene in a catecholaminergic polymorphic ventricular tachycardia knock-in mouse model carrying the CASQ2(R33Q/R33Q) (R33Q) mutation. We engineered an adeno-associated viral vector serotype 9 (AAV9) containing cDNA of CASQ2 wild-type (AAV9-CASQ2) plus the green fluorescent protein (GFP) gene to infect newborn R33Q mice studied by in vivo and in vitro protocols at 6, 9, and 12 months to investigate the ability of the infection to prevent the disease and adult R33Q mice studied after 2 months to assess whether the AAV9-CASQ2 delivery could revert the catecholaminergic polymorphic ventricular tachycardia phenotype. In both protocols, we observed the restoration of physiological expression and interaction of CASQ2, junctin, and triadin; the rescue of electrophysiological and ultrastructural abnormalities in calcium release units present in R33Q mice; and the lack of life-threatening arrhythmias. Our data demonstrate that viral gene transfer of wild-type CASQ2 into the heart of R33Q mice prevents and reverts severe manifestations of catecholaminergic polymorphic ventricular tachycardia and that this curative effect lasts for 1 year after a single injection of the vector, thus posing the rationale for the design of a clinical trial.

MeSH Terms

  • Aging
  • Animals
  • Calcium-Binding Proteins
  • Calsequestrin
  • Carrier Proteins
  • Dependovirus
  • Disease Models, Animal
  • Female
  • Genetic Therapy
  • Male
  • Membrane Proteins
  • Mice
  • Mice, Knockout
  • Mixed Function Oxygenases
  • Muscle Proteins
  • Mutation
  • Tachycardia, Ventricular
  • Treatment Outcome

Keywords

  • arrhythmias, cardiac
  • calsequestrin
  • death, sudden
  • genetic therapy
  • recovery of function


The role of mutant protein level in autosomal recessive catecholamine dependent polymorphic ventricular tachycardia (CPVT2).

Humans and genetically engineered mice with recessively inherited CPVT develop arrhythmia which may arise due to malfunction or degradation of calsequestrin (CASQ2). We investigated the relation between protein level and arrhythmia severity in CASQ2(D307H/D307H) (D307H), compared to CASQ2(Δ/Δ) (KO) and wild type (WT) mice. CASQ2 expression and Ca²⁺ transients were recorded in cardiomyocytes from neonatal or adult mice. Arrhythmia was studied in vivo using heart rhythm telemetry at rest, exercise and after epinephrine injection. CASQ2 protein was absent in KO heart. Neonatal D307H and WT hearts expressed significantly less CASQ2 protein than the level found in the adult WT. Adult D307H expressed only 20% of CASQ2 protein found in WT. Spontaneous Ca²⁺ release was more prevalent in neonatal KO cardiomyocytes (89%) compared to 33-36% of either WT or D307H, respectively, p<0.001. Adult cardiomyocytes from both mutant mice had more Ca²⁺ abnormalities compared to control (KO: 82%, D307H 63%, WT 12%, p<0.01). Calcium oscillations were most common in KO cardiomyocytes. We then treated mice with bortezomib to inhibit CASQ2(D307H) degradation. Bortezomib increased CASQ2 expression in D307H hearts by ∼50% (p<0.05). Bortezomib-treated D307H mice had lower CPVT prevalence and less premature ventricular beats during peak exercise. No benefit against arrhythmia was observed in bortezomib treated KO mice. These results indicate that the mutant CASQ2(D307H) protein retains some of its physiological function. Its expression decreases with age and is inversely related to arrhythmia severity. Preventing the degradation of mutant protein should be explored as a possible therapeutic strategy in appropriate CPVT2 patients.

MeSH Terms

  • Aging
  • Animals
  • Animals, Newborn
  • Boronic Acids
  • Bortezomib
  • Calcium
  • Calsequestrin
  • Cells, Cultured
  • Gene Expression
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mutation
  • Myocytes, Cardiac
  • Pyrazines
  • Sarcoplasmic Reticulum
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases
  • Severity of Illness Index
  • Tachycardia, Ventricular

Keywords

  • Arrhythmia
  • Bortezomib
  • Calcium transients
  • Calsequestrin
  • Mouse model
  • Protein degradation


ANKRD1 specifically binds CASQ2 in heart extracts and both proteins are co-enriched in piglet cardiac Purkinje cells.

It has been suggested that the cardiac ankyrin repeat domain 1 protein (ANKRD1), also known as CARP, can play a pathophysiological role in the contractile responsiveness of myocardium. Here, we study the potential functional roles of ANKRD1 by searching for endogenous cardiac proteins that interact preferentially with ANKRD1 in the heart-tissue extract from neonatal piglets, using non-biased pull-down approaches. These approaches identified, for the first time, a selective interaction between ANKRD1 and endogenous cardiac calsequestrin-2 (CASQ2) that is important for Ca2 release and excitation-contraction coupling. Blot-overlay and co-immunoprecipitation assays provided further confirmation of the direct and specific interaction between the two proteins. Mapping of the peptides involved in the interaction revealed five non-overlapping binding sequences for CASQ2 on ANKRD1, as well as, three binding peptides for ANKRD1 in CASQ2. For the first time, we show by immunohistochemistry that endogenous ANKRD1 and CASQ2 are co-enriched in piglet cardiac Purkinje cells. Collectively, the results provide the first sing of a possible functional interaction between ANKRD1 and CASQ2 and suggest a potentially novel role for both proteins in cardiac Purkinje fibers.

MeSH Terms

  • Aging
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Calcium
  • Calsequestrin
  • Cell Extracts
  • Dogs
  • Heart
  • Humans
  • Immunohistochemistry
  • Molecular Sequence Data
  • Myocardium
  • Nuclear Proteins
  • Protein Binding
  • Purkinje Cells
  • Repressor Proteins
  • Substrate Specificity
  • Swine
  • Tissue Extracts