Unexpected Structural and Functional Consequences of the R33Q Homozygous Mutation in Cardiac Calsequestrin

Abstract

Abstract-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disordercharacterized by life threatening arrhythmias elicited by physical and emotional stress in young individuals. The recessive form of CPVT is associated with mutation in the cardiac calsequestrin gene (CASQ2). We engineered and characterized a homozygous CASQ2 R33Q/R33Q mouse model that closely mimics the clinical phenotype of CPVT patients. CASQ2R33Q/R33Q mice develop bidirectional VT on exposure to environmental stress whereas CASQ2 R33Q/R33Q myocytes show reduction of the sarcoplasmic reticulum (SR) calcium content, adrenergically mediated delayed (DADs) and early (EADs) afterdepolarizations leading to triggered activity. Furthermore triadin, junctin, and CASQ2-R33Q proteins are significantly decreased in knock-in mice despite normal levels of mRNA, whereas the ryanodine receptor (RyR2), calreticulin, phospholamban, and SERCA2a-ATPase are not changed. Trypsin digestion studies show increased susceptibility to proteolysis of mutant CASQ2. Despite normal histology, CASQ2 R33Q/R33Q hearts display ultrastructural changes such as disarray of junctional electron-dense material, referable to CASQ2 polymers, dilatation of junctional SR, yet normal total SR volume. Based on the foregoings, we propose that the phenotype of the CASQ2 R33Q/R33Q CPVT mouse model is portrayed by an unexpected set of abnormalities including (1) reduced CASQ2 content, possibly attributable to increased degradation of CASQ2-R33Q, (2) reduction of SR calcium content, (3) dilatation of junctional SR, and (4) 4 The identification of the genes underlying CPVT has had implications that extend beyond those impacting clinical management of patients inasmuch as it stimulated fundamental research targeted to understand the links between intracellular calcium regulation and arrhythmogenesis. We recently developed 5 a knock-in mouse model carrying the R4496C RyR2 mutation identified in the first genotyped CPVT family and demonstrated that the RyR2 R4496C mice develop bidirectional and polymorphic VT similar to those observed in patients. In this model we also demonstrated 6 the occurrence of delayed after depolarizations (DADs) induced by adrenergic stimulation in isolated myocytes from the heart of heterozygous mice, suggesting that arrhythmias are elicited by triggered activity. Recently 2 mutants CASQ2 knock-in mice models were developed by Song et al 7 : the first strain carries the homozygous point mutation discovered by Lahat et al 3 in the first recessive CPVT family (D307H), and the second strain carries a homozygous deletion ⌬E9/⌬E9; in analogy with RyR2 mice, 5 both models develop bidirectional-polymorphic Materials and MethodsDetailed methods for mouse generation, electrophysiological measurements, immunofluorescence, real-time PCR, microarray, protein and electron microscopy analysis are reported in the online supplement (available online at http://circres.ahajournals.org). Generation of Knock-In of R33Q CASQ2 in Mouse ModelThe kno...

Unfolded Protein Response Is Activated in the Hearts of Catecholaminergic Polymorphic Ventricular Tachycardia (Cpvt) Mice

“…The CSQ2 mutation knock-in mouse (9) was characterised in vitro and in vivo and was demonstrated to be an identical copy of the patient phenotype. CSQ2 and triadin protein levels were reduced, while the corresponding mRNAs levels remained unchanged (9). The CSQ2-R33Q protein levels found in this study confirmed the results of Rizzi and colleagues (9), who reported that CSQ2-R33Q proteins levels were significantly lower than wild-type protein levels.…”

“…CSQ2 and triadin protein levels were reduced, while the corresponding mRNAs levels remained unchanged (9). The CSQ2-R33Q protein levels found in this study confirmed the results of Rizzi and colleagues (9), who reported that CSQ2-R33Q proteins levels were significantly lower than wild-type protein levels. Comparison of the results reported in Figures 2A and 2B suggested that TD32 levels decreased due to the lack of functional CSQ2 from birth in the CSQ2-R33Q/R33Q mice.…”

“…In mammals, there are two CSQ isoforms encoded by different genes: the skeletal isoform CSQ1 is expressed in fast-and slow-twitch skeletal muscle, while the cardiac isoform CSQ2 is expressed in both cardiac and skeletal muscle. Some CSQ2 mutations seem to affect protein synthesis, leading to the reduction or absence of CSQ2 in the heart (9), while other mutations seem to induce the expression of defective proteins, which show altered abilities to bind Ca 2+ and/or to perform their proper function (9). All experimental evidence has implicated mechanisms of posttranscriptional regulation.…”

Gene Transfer of Engineered Calmodulin Alleviates Ventricular Arrhythmias in a Calsequestrin‐Associated Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

“…CASQ2 R33Q22 and knock out (KO)23 mice (3–6 months old, males) in the C57BL/6 genetic background were utilized in this study. All animal procedures were approved by The Ohio State University Institutional Animal Care and Use Committee.…”

Regulation of the Cardiac Sodium/Bicarbonate Cotransporter by Angiotensin II: Potential Contribution to Structural, Ionic and Electrophysiological Myocardial Remodelling

“…The increase in [Ca 2+ ] i enhance the sarcoplasmic reticulum (SR) calcium load, exceeding the ryanodine receptor channel (RyR) threshold necessary to be opened and finally leading to spontaneous diastolic calcium release. The transient increase in citosolic Ca 2+ (waves) activates an inward (depolarizing) current (I ti ), mediated by the forward mode of NCX [99,100]. I ti is responsible for the generation of DADs which, when are sufficiently large to achieve the threshold, generate spontaneous CAP, leading to triggered activity [101].…”