Giorgia Valle

Clinical Phenotype and Functional Characterization of CASQ2 Mutations Associated With Catecholaminergic Polymorphic Ventricular Tachycardia

Background— Four distinct mutations in the human cardiac calsequestrin gene (CASQ2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). The mechanisms leading to the clinical phenotype are still poorly understood because only 1 CASQ2 mutation has been characterized in vitro. Methods and Results— We identified a homozygous 16-bp deletion at position 339 to 354 leading to a frame shift and a stop codon after 5aa (CASQ2G112+5X) in a child with stress-induced ventricular tachycardia and cardiac arrest. The same deletion was also identified in association with a novel point mutation (CASQ2L167H) in a highly symptomatic CPVT child who is the first CPVT patient carrier of compound heterozygous CASQ2 mutations. We characterized in vitro the properties of CASQ2 mutants: CASQ2G112+5X did not bind Ca2+, whereas CASQ2L167H had normal calcium-binding properties. When expressed in rat myocytes, both mutants decreased the sarcoplasmic reticulum Ca2+-storing capacity and reduced the amplitude of ICa-induced Ca2+ transients and of spontaneous Ca2+ sparks in permeabilized myocytes. Exposure of myocytes to isoproterenol caused the development of delayed afterdepolarizations in CASQ2G112+5X. Conclusions— CASQ2L167H and CASQ2G112+5X alter CASQ2 function in cardiac myocytes, which leads to reduction of active sarcoplasmic reticulum Ca2+ release and calcium content. In addition, CASQ2G112+5X displays altered calcium-binding properties and leads to delayed afterdepolarizations. We conclude that the 2 CASQ2 mutations identified in CPVT create distinct abnormalities that lead to abnormal intracellular calcium regulation, thus facilitating the development of tachyarrhythmias.

Abnormal Interactions of Calsequestrin With the Ryanodine Receptor Calcium Release Channel Complex Linked to Exercise-Induced Sudden Cardiac Death

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca2+-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2R33Q in adult rat myocytes led to an increase in excitation–contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca2+-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.

Luminal Ca2+ Regulation of Single Cardiac Ryanodine Receptors: Insights Provided by Calsequestrin and its Mutants

The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 muM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below approximately 0.5 mM.

Modulation of SR Ca Release by Luminal Ca and Calsequestrin in Cardiac Myocytes: Effects of CASQ2 Mutations Linked to Sudden Cardiac Death

Cardiac calsequestrin (CASQ2) is an intrasarcoplasmic reticulum (SR) low-affinity Ca-binding protein, with mutations that are associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). To better understand how CASQ2 mutants cause CPVT, we expressed two CPVT-linked CASQ2 mutants, a truncated protein (at G112+5X, CASQ2(DEL)) or CASQ2 containing a point mutation (CASQ2(R33Q)), in canine ventricular myocytes and assessed their effects on Ca handling. We also measured CASQ2-CASQ2 variant interactions using fluorescence resonance transfer in a heterologous expression system, and evaluated CASQ2 interaction with triadin. We found that expression of CASQ2(DEL) or CASQ2(R33Q) altered myocyte Ca signaling through two different mechanisms. Overexpressing CASQ2(DEL) disrupted the CASQ2 polymerization required for high capacity Ca binding, whereas CASQ2(R33Q) compromised the ability of CASQ2 to control ryanodine receptor (RyR2) channel activity. Despite profound differences in SR Ca buffering strengths, local Ca release terminated at the same free luminal [Ca] in control cells, cells overexpressing wild-type CASQ2 and CASQ2(DEL)-expressing myocytes, suggesting that a decline in [Ca](SR) is a signal for RyR2 closure. Importantly, disrupting interactions between the RyR2 channel and CASQ2 by expressing CASQ2(R33Q) markedly lowered the [Ca](SR) threshold for Ca release termination. We conclude that CASQ2 in the SR determines the magnitude and duration of Ca release from each SR terminal by providing both a local source of releasable Ca and by effects on luminal Ca-dependent RyR2 gating. Furthermore, two CPVT-inducing CASQ2 mutations, which cause mechanistically different defects in CASQ2 and RyR2 function, lead to increased diastolic SR Ca release events and exhibit a similar CPVT disease phenotype.

Ryanodine Receptor Luminal Ca2+ Regulation: Swapping Calsequestrin and Channel Isoforms

Sarcoplasmic reticulum (SR) Ca(2+) release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca(2+) channel and the intra-SR Ca(2+) buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca(2+) regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca(2+) dependence of open probability (Po) over the physiologically relevant range (0.05-1 mM Ca(2+)) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca(2+) sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca(2+) sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca(2+) regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca(2+) regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca(2+) buffer.

Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

Release of Ca2+ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca2+ regulation of SR Ca2+ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca2+ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca2+ regulates ryanodine receptor (RyR)2-mediated SR Ca2+ release through mechanisms localized inside the SR; one of these involves luminal Ca2+ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function. CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca2+] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca2+ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca2+ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca2+ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.

Vesicle budding from endoplasmic reticulum is involved in calsequestrin routing to sarcoplasmic reticulum of skeletal muscles.

CS (calsequestrin) is an acidic glycoprotein of the SR (sarcoplasmic reticulum) lumen and plays a crucial role in the storage of Ca2+ and in excitation-contraction coupling of skeletal muscles. CS is synthesized in the ER (endoplasmic reticulum) and is targeted to the TC (terminal cisternae) of SR via mechanisms still largely unknown, but probably involving vesicle transport through the Golgi complex. In the present study, two mutant forms of Sar1 and ARF1 (ADP-ribosylation factor 1) were used to disrupt cargo exit from ER-exit sites and intra-Golgi trafficking in skeletal-muscle fibres respectively. Co-expression of Sar1-H79G (His79-->Gly) and recombinant, epitope-tagged CS, CSHA1 (where HA1 stands for nine-amino-acid epitope of the viral haemagglutinin 1), barred segregation of CSHA1 to TC. On the other hand, expression of ARF1-N126I altered the subcellular localization of GM130, a cis -medial Golgi protein in skeletal-muscle fibres and myotubes, without interfering with CSHA1 targeting to either TC or developing SR. Thus active budding from ER-exit sites appears to be involved in CS targeting and routing, but these processes are insensitive to modification of intracellular vesicle trafficking and Golgi complex disruption caused by the mutant ARF1-N126I. It also appears that CS routing from ER to SR does not involve classical secretory pathways through ER-Golgi intermediate compartments, cis -medial Golgi and trans -Golgi network.

Post-natal heart adaptation in a knock-in mouse model of calsequestrin 2-linked recessive catecholaminergic polymorphic ventricular tachycardia

Cardiac calsequestrin (CASQ2) contributes to intracellular Ca(2+) homeostasis by virtue of its low-affinity/high-capacity Ca(2+) binding properties, maintains sarcoplasmic reticulum (SR) architecture and regulates excitation-contraction coupling, especially or exclusively upon β-adrenergic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with cardiac arrest in children or young adults. Recessive CPVT variants are due to mutations in the CASQ2 gene. Molecular and ultra-structural properties were studied in hearts of CASQ2(R33Q/R33Q) and of CASQ2(-/-) mice from post-natal day 2 to week 8. The drastic reduction of CASQ2-R33Q is an early developmental event and is accompanied by down-regulation of triadin and junctin, and morphological changes of jSR and of SR-transverse-tubule junctions. Although endoplasmic reticulum stress is activated, no signs of either apoptosis or autophagy are detected. The other model of recessive CPVT, the CASQ2(-/-) mouse, does not display the same adaptive pattern. Expression of CASQ2-R33Q influences molecular and ultra-structural heart development; post-natal, adaptive changes appear capable of ensuring until adulthood a new pathophysiological equilibrium.

Characterization of fast-twitch and slow-twitch skeletal muscles of calsequestrin 2 (CASQ2)-knock out mice: unexpected adaptive changes of fast-twitch muscles only

This study investigates the functional role of calsequestrin 2 (CASQ2) in both fast-twitch and slow-twitch skeletal muscles by using CASQ2−/− mice; CASQ2 is expressed throughout life in slow-twitch muscles, but only in the developmental and neonatal stages in fast-twitch muscles. CASQ2−/− causes increase in calsequestrin 1 (CASQ1) expression, but without functional changes in both muscle types. CASQ2−/− mice have ultrastructural changes in fast-twitch muscles only, i.e., formation of pentads and stacks in the sarcoplasmic reticulum.