Arrhythmogenic calmodulin E105A mutation alters cardiac RyR2 regulation leading to cardiac dysfunction in zebrafish

Calmodulin (CaM) is a universal calcium (Ca2+)‐binding messenger that regulates many vital cellular events. In cardiac muscle, CaM associates with ryanodine receptor 2 (RyR2) and regulates excitation–contraction coupling. Mutations in human genes CALM1, CALM2, and CALM3 have been associated with life‐threatening heart disorders, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia. A novel de novo LQTS‐associated missense CaM mutation (E105A) was recently identified in a 6‐year‐old boy, who experienced an aborted first episode of cardiac arrest. Herein, we report the first molecular characterization of the CaM E105A mutation. Expression of the CaM E105A mutant in zebrafish embryos resulted in cardiac arrhythmia and increased heart rate, suggestive of ventricular tachycardia. In vitro biophysical and biochemical analysis revealed that E105A confers a deleterious effect on protein stability and a reduced Ca2+‐binding affinity due to loss of cooperativity. Finally, the CaM E105A mutation resulted in reduced CaM–RyR2 interaction and defective modulation of ryanodine binding. Our findings suggest that the CaM E105A mutation dysregulates normal cardiac function by a complex mechanism involving alterations in both CaM–Ca2+ and CaM–RyR2 interactions

Biophysical and biochemical study of the role of calmodulin and its mutants in calcium-signaling pathways: molecular mechanisms of pathogenesis

Many studies the last years have associated numerous mutations in all three genes (CALM1, CALM2, CALM3) that encode human calmodulin (CaM) with pathological phenotypes of fatal arrhythmogenic syndromes, mainly CPVT and LQTS. CaM is a multifunctional Ca2+ - binding protein that, among other things, is an important regulator of the ryanodine receptor 2 (RyR2) and plays an important role in Ca2+ signaling during the excitation contraction coupling (ECC) mechanism in cardiomyocytes. The exact mechanism of the interaction between CaM and RyR2 is still not fully understood, which makes it difficult to determine the mechanism that may lead in the development of calmodulinopathies. The present study contributes in better understanding of the underlying mechanism of the interaction between CaM and RyR2, through the study of the effect of CaM-mutations, which have been associated with fatal arrhythmogenic heart syndromes, both on the CaM molecule itself and also on the interaction with RyR2.In the present thesis, is being studied the effect of certain CaM mutations on the wild type CaM molecule (CaMwt) in terms of structure, thermal stability and Ca2+ - binding affinity using the techniques of circular dichroism spectroscopy (CD) and fluorescence spectroscopy, respectively. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM- binding region (peptide B), as well other potential CaM-binding region of human RyR2. No gross changes observed in tertiary structure of the mutant CaM proteins, either in absence or presence of Ca2+. Decrease in thermal stability compared to CaMwt, is a feature of most of the studied CaM mutations, with. Also, none of the CaM mutations, affected Ca2+ - binding to the N-lobe of CaM but nearly all of them, with exception of one, resulted in decrease Ca2+ - binding in the C-lobe to varying degrees. The two sequences of human RyR2 (peptides B and F) interact with significant affinity with CaMwt, in the presence and absence of Ca2+, indicating that peptide F is a potential CaM-binding region of RyR2. The results of the interaction of mutant CaMs with peptides B and F gave a general framework for small changes in binding affinity relative to CaMwt that do not affect the ability of CaM to bind to the receptor in the presence of Ca2+. The absence of Ca2+ precludes the binding of the mutant forms of CaM to both peptides. The present study is divided into two parts. In the first part, the theoretical background on which this work is based is thoroughly analyzed, while in the second part, all the experimental methods used in this study, the experimental results and finally the conclusions are presented in detail.Τα τελευταία χρόνια έρευνες έχουν συσχετίσει πληθώρα μεταλλάξεων και στα τρία γονίδια (CALM1,CALM2,CALM3) που κωδικοποιούν την καλμοδουλίνη (CaM) με παθολογικούς φαινότυπους θνησιγενών κολπικών αρρυθμιών, κυρίως CPVT και LQTS. Η CaM είναι μία πολυλειτουργική πρωτεϊνη δέσμευσης Ca2+ που μεταξύ άλλων αποτελεί σημαντικό ρυθμιστή των υποδοχέων ρυανοδίνης 2 (RyR2) και κατέχει σημαντικό ρόλο στη σηματοδότηση του Ca2+, κατά το μηχανισμό διέγερσης-συστολής των καρδιομυοκυττάρων (ECC). Ο ακριβής μηχανισμός της αλληλεπίδρασης μεταξύ CaM και RyR2 δεν είναι πλήρως γνωστός, γεγονός που προκαλεί δυσκολία στη μελέτη και τον προσδιορισμό του μηχανισμού που μπορεί να οδηγεί σε καλμοδουλινοπάθειες. Η παρούσα διατριβή συμβάλλει στην καλύτερη κατανόηση του μηχανισμού αλληλεπίδρασης μεταξύ CaM-RyR2, μέσω της μελέτης της επίδρασης CaM-μεταλλάξεων, που έχουν συσχετισθεί με θνησιγενή αρρυθμιογενή καρδιακά σύνδρομα, τόσο στο ίδιο το μόριο της CaM όσο και στην αλληλεπίδραση με τον RyR2. Αρχικά, ερευνάται η επίδραση που επιφέρουν ορισμένες CaM μεταλλάξεις στο μόριο της CaM αγρίου τύπου (CaMwt) απο πλευράς δομής, θερμικής σταθερότητας και ικανότητας δέσμευσης Ca2+ με τις τεχνικές της φασματοπολωσιμετρίας κυκλικού διχροϊσμού (CD) και της φασματοσκοπίας φθορισμού. Στη συνέχεια, μελετάται η αλληλεπίδραση της CaMwt και ορισμένων εκ των CaM μεταλλαγμένων μορφών με συνθετικά πεπτίδια που αφορούν στην κύρια περιοχή πρόσδεσης της CaM στον RyR2 (πεπτίδιο Β) και σε μία δεύτερη περιοχή του RyR2, που πιθανώς αποτελεί και αυτή περιοχή πρόσδεσης της CaM (πεπτίδιο F). Η θερμοδυναμική μελέτη της εν λόγω αλληλελεπίδρασης πραγματοποιείται με χρήση της θερμιδομετρίας ισόθερμης τιτλοδοτησης (ITC). Όλες οι μεταλλαγμένες μορφές της CaM που μελετήθηκαν στην παρούσα εργασία διατηρούν την τριτοταγή τους διαμόρφωση, τόσο παρουσία όσο και απουσία Ca2+. Κατά πλειονότητα οι μεταλλάξεις προκαλούν σε διαφορετικό βαθμό απώλεια στη θερμοσταθερότητα του μορίου της CaM, που σχετίζεται με τη δέσμευση Ca2+ και προκαλούν μείωση στη συγγένεια σύνδεσης του Ca2+ στο C-λοβό. Οι δύο αλληλουχίες του ανθρώπινου RyR2 (πεπτίδια Β και F) βρέθηκε να αλληλεπιδρούν με υψηλή συγγένεια με την CaMwt, τόσο παρουσία όσο και απουσία Ca2+, γεγονός που υποδεικνύει πως το πεπτίδιο F αποτελεί πιθανή περιοχή πρόσδεσης της CaM στον RyR2. Τα αποτελέσματα της αλληλεπίδρασης των μεταλλαγμένων CaM με τα πεπτίδια Β και F, έδωσαν σε ένα γενικό πλαίσιο μικρές μεταβολές στη συγγένεια σύνδεσης σε σχέση με την CaMwt που δεν επηρεάζουν την ικανότητα πρόσδεσης της CaM στον υποδοχέα, παρουσία Ca2+. Η απουσία Ca2+ αποκλείει την πρόσδεση των μεταλλαγμένων μορφών της CaM και στα δύο πεπτίδια. Η εργασία αυτή χωρίζεται σε δύο μέρη. Στο πρώτο μέρος αναλύεται διεξοδικά το θεωρητικό υπόβαθρο στο οποίο βασίζεται η εν λόγω εργασία, ενώ στο δεύτερο μέρος παρουσιάζονται αναλυτικά όλες οι πειραματικές μέθοδοι που χρησιμοποιήθηκαν σε αυτή τη μελέτη, τα πειραματικά αποτελέσματα και τέλος τα συμπεράσματα που προκύπτουν

Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket

Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca2+) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaMN98I, CaMD132E, CaMD134H and CaMQ136P) and we used a CaM-RyR2 co-immunoprecipitation and a [3H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [3H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca2+, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association.We are grateful to Xuexun Fang (Laboratory of Molecular Enzymology and Enzyme Engineering of the Ministry of Education, Jilin University, China) for providing the pHSIE vector

Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket

Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca2+) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaMN98I, CaMD132E, CaMD134H and CaMQ136P) and we used a CaM-RyR2 co-immunoprecipitation and a [3H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [3H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca2+, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association

Arrhythmogenic calmodulin E105A mutation alters cardiac RyR2 regulation leading to cardiac dysfunction in zebrafish

Calmodulin (CaM) is a universal calcium (Ca2+)‐binding messenger that regulates many vital cellular events. In cardiac muscle, CaM associates with ryanodine receptor 2 (RyR2) and regulates excitation–contraction coupling. Mutations in human genes CALM1, CALM2, and CALM3 have been associated with life‐threatening heart disorders, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia. A novel de novo LQTS‐associated missense CaM mutation (E105A) was recently identified in a 6‐year‐old boy, who experienced an aborted first episode of cardiac arrest. Herein, we report the first molecular characterization of the CaM E105A mutation. Expression of the CaM E105A mutant in zebrafish embryos resulted in cardiac arrhythmia and increased heart rate, suggestive of ventricular tachycardia. In vitro biophysical and biochemical analysis revealed that E105A confers a deleterious effect on protein stability and a reduced Ca2+‐binding affinity due to loss of cooperativity. Finally, the CaM E105A mutation resulted in reduced CaM–RyR2 interaction and defective modulation of ryanodine binding. Our findings suggest that the CaM E105A mutation dysregulates normal cardiac function by a complex mechanism involving alterations in both CaM–Ca2+ and CaM–RyR2 interactions