scholarly journals Aberrant interaction of calmodulin with the ryanodine receptor develops hypertrophy in the neonatal cardiomyocyte

2011 ◽  
Vol 438 (2) ◽  
pp. 379-387 ◽  
Author(s):  
Jaya P. Gangopadhyay ◽  
Noriaki Ikemoto
Keyword(s):  
Ryanodine Receptor ◽  
Domain Interaction ◽  
Activated T Cells ◽  
Calmodulin Binding ◽  
Neonatal Cardiomyocytes ◽  
Signalling Molecules ◽  
Neonatal Cardiomyocyte ◽  
Link Formation ◽  
Cultured Cardiomyocytes ◽  
Transcription Factor Nfat

We have shown previously that the inter-domain interaction between the two domains of RyR (ryanodine receptor), CaMBD [CaM (calmodulin)-binding domain] and CaMLD (CaM-like domain), activates the Ca2+ channel, and this process is called activation-link formation [Gangopadhyay and Ikemoto (2008) Biochem. J. 411, 415–423]. Thus CaM that is bound to CaMBD is expected to interfere the activation-link formation, thereby stabilizing the closed state of the channel under normal conditions. In the present paper, we report that, upon stimulation of neonatal cardiomyocytes with the pro-hypertrophy agonist ET-1 (endothelin-1), CaM dissociates from the RyR, which induces a series of intracellular events: increased frequency of Ca2+ transients, translocation of the signalling molecules CaM, CaMKII (CaM kinase II) and the transcription factor NFAT (nuclear factor of activated T-cells) to the nucleus. These events then lead to the development of hypertrophy. Importantly, an anti-CaMBD antibody that interferes with activation-link formation prevented all of these intracellular events triggered by ET-1 and prevented the development of hypertrophy. These results indicate that the aberrant formation of the activation link between CaMBD and CaMLD of RyR is a key step in the development of hypertrophy in cultured cardiomyocytes.

Interaction of the Lys3614–Asn3643 calmodulin-binding domain with the Cys4114-Asn4142 region of the type 1 ryanodine receptor is involved in the mechanism of Ca2+/agonist-induced channel activation

2008 ◽  
Vol 411 (2) ◽  
pp. 415-423 ◽  
Author(s):  
Jaya P. Gangopadhyay ◽  
Noriaki Ikemoto
Keyword(s):  
Ryanodine Receptor ◽  
Binding Activity ◽  
Significant Inhibition ◽  
Binding Domain ◽  
Domain Pair ◽  
Dependent Manner ◽  
Domain Interaction ◽  
Channel Activation ◽  
Calmodulin Binding

In the present study we show that the interaction of the CaM (calmodulin)-binding domain (Lys3614–Asn3643) with the Cys4114–Asn4142 region (a region included in the CaM-like domain) serves as an intrinsic regulator of the RyR1 (type-1 ryanodine receptor). We tested the effects of antibodies raised against the two putative key regions of RyR1 [anti-(Lys3614–Asn3643) and anti-(Cys4114–Asn4142) antibodies]. Both antibodies produced significant inhibition of [3H]ryanodine-binding activity of RyR1. This suggests that the inter-domain interaction between the two domains, Lys3614–Asn3643 and Cys4114–Asn4142, activates the channel, and that the binding of antibody to either side of the interacting domain pair interfered with the formation of a ‘channel-activation link’ between the two regions. In order to spectroscopically monitor the mode of interaction of these domains, the site of inter-domain interaction was fluorescently labelled with MCA [(7-methoxycoumarin-4-yl)acetyl] in a site-directed manner. The accessibility of the bound MCA to a large molecular mass fluorescence quencher, BSA-QSY (namely, the size of a gap between the interacting domains) decreased with an increase of [Ca2+] in a range of 0.03–2.0 μM, as determined by Stern–Volmer fluorescence quenching analysis. The Ca2+-dependent decrease in the quencher accessibility was more pronounced in the presence of 150 μM 4-CmC (4-chlorometacresol), and was reversed by 1 mM Mg2+ (a well-known inhibitor of Ca2+/agonist-induced channel activation). These results suggest that the Lys3614–Asn3643 and Cys4114–Asn4142 regions of RyR1 interact with each other in a Ca2+- and agonist-dependent manner, and this serves as a mechanism of Ca2+- and agonist-dependent activation of the RyR1 Ca2+ channel.

Abstract 5251: Defective Inter-domain Interaction May Cause Spontaneous Ca 2+ Release Via Reduced Affinity of Calmodulin Binding to RyR2 in Failing Hearts

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Makoto Ono ◽  
Masafumi Yano ◽  
Takeshi Suetomi ◽  
Xiaojuan Xu ◽  
Hitoshi Uchinoumi ◽  
...  
Keyword(s):  
Domain Interaction ◽  
Mutation Site ◽  
Calmodulin Binding ◽  
Large Size ◽  
Rv Pacing ◽  
Co Addition ◽  
Domain Peptide ◽  
Fluorescence Quencher ◽  
A Site

We previously reported that interaction between N-terminal 1–600 and central domains 2000–2500 of ryanodine receptor (RyR2), harboring many mutation sites in CPVT, is defective (i.e. domain unzipping) in failing hearts. Here, we investigated the pathogenic role of calmodulin (CaM), one of the accessory proteins in RyR2, on Ca 2+ release in failing hearts. Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles {normal (N), n=6; 4-weeks rapid RV pacing (HF: n=6). To assess CaM binding to RyR2, SR was mixed with CaM-SANPAH conjugate (16nM-1μM), followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. The affinity of CaM binding to RyR2 was lower in failing SR than normal SR (Kd: 47nM in HF: 19nM in N , p<0.01). To assess the possible relationship between domain unzipping and CaM dissociation from RyR2, RyR2 was also fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460–2495 (DPc10), which harbors a mutation site in CPVT; R2474S, as a site-directing carrier. In failing SR, domain unzipping was already taken place, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. Interestingly, addition of FK506 (10 μM), which was found to dissociate FKBP12.6 from RyR2 and to induce domain unzipping mimicking failing SR, to normal SR indeed reduced the CaM binding affinity to RyR2. In saponin-permeabilized, failing cardiomyocytes ([Ca 2+ ]=buffered at 75 nM), the frequency of Ca 2+ sparks was markedly increased (SpF; s −1 ·100μm −1 : 13.9±0.63 in HF; 7.3±0.6 in N, p<0.01). Addition of CaM (1 μM) in the presence of KN-93 (CaMKII inhibitor) inhibited the increase in SpF (7.9 ±0.41 , p<0.01). This CaM’s effect was, however, markedly inhibited by co-addition of CaM-binding domain peptide within RyR2 (3583–3603) (10.4±0.65, p=ns), strongly suggesting that the re-binding of CaM to RyR2 corrects the abnormal Ca 2+ release in failing cardiomyocytes. In conclusion, the defective inter-domain interaction between N-terminal and central domains within RyR2 seems to increase spontaneous Ca 2+ release events in failing SR, via the reduced CaM-binding to RyR2. Fixing CaM binding to RyR2 may be protective against the diastolic Ca 2+ release in failing hearts.

Abstract 309: MiR-31a-5p Controls Cardiomyocyte Proliferation in Postnatal Hearts

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Hui Liu ◽  
Jing Shi ◽  
Hui Wang ◽  
Qingkao Xuan ◽  
Yihua Bei ◽  
...  
Keyword(s):  
Nuclear Antigen ◽  
Cardiomyocyte Proliferation ◽  
Ki 67 ◽  
Chain Reactions ◽  
Neonatal Cardiomyocytes ◽  
Polymerase Chain Reactions ◽  
Proliferation And Apoptosis ◽  
Neonatal Cardiomyocyte ◽  
Polymerase Chain ◽  
Proliferating Cell

Backgroud: MicroRNAs (miRNAs, miRs) are a class of endogenous non-codingRNAs, participating in a variety of essential biological processes including development, differentiation, proliferation and apoptosis. Rodents have the capacity to regenerate their hearts in response to injury while the capacity would be lost 7 day after birth, suggesting that mammals gradually lose their regenerative potential during postnatal development. The roles of miRNAs in regulating cardiomyocyte proliferation in postnatal hearts are largely unclear. Methods and Results: Cardiomyocytes were isolated from rat at day 0 and day 10. Agilent rat miRNA arrays were performed to determine the dysregulated miRNAs in cardiomyocytes between day 0 and day 10. A total of 32 miRNAs were found to be dysregulated between day 0 and day 10 (Fold change over 2 and P values less than 0.05). As determined by quantitative reverse transcription polymerase chain reactions and functional assays using EdU staining and Ki-67 staining, miR-31a-5p was found to be able to promote neonatal cardiomyocyte proliferation. Moreover, the expression of proliferation maker- Proliferating Cell Nuclear Antigen (PCNA) was also increased in cardiomyocytes transfected with miR-31a-5p mimics as determined by PCRs and Western blotting analysis. Tumor suppressor RhoBTB1 was found to be negatively regulated by miR-31a-5p in cardiomyocytes and also was responsible for the pro-proliferation effects of miR-31a-5p in neonatal cardiomyocytes. Conclusions: These studies demonstrate that miR-31a-5p regulates cardiomyocytes proliferation in postnatal hearts by targeting RhoBTB1. miR-31a-5p represents a therapeutic target for cardiac repair and regeneration.

Abstract 214: TLR3 Mediates Neonatal Heart Repair and Regeneration Through Glycolysis-dependent YAP/TAZ Mediated miR-152 Expression

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Xiaohui Wang ◽  
Yuanping Hu ◽  
Tuanzhu Ha ◽  
John Kalbfleisch ◽  
Race Kao ◽  
...  
Keyword(s):  
Cardiac Metabolism ◽  
Poly I:C ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Neonatal Cardiomyocytes ◽  
Neonatal Heart ◽  
Heart Repair ◽  
Inhibition Of Glycolysis ◽  
Neonatal Cardiomyocyte

The neonatal heart possesses the capability of regenerating and repairing damaged myocardium which is lost when cardiac metabolism switches from predominate glycolysis to oxidative phosphorylation seven days after birth. We have observed that Toll-like receptor 3 (TLR3) deficient neonatal hearts exhibit impaired cardiac function and larger infarct size after myocardial infarction (MI). We also found that stimulation of neonatal cardiomyocytes with the TLR3 ligand, poly (I:C) significantly enhances glycolytic capacity. Our observation suggests that TLR3 is required for neonatal heart repair and regeneration of damaged myocardium. This study investigated the mechanisms by which TLR3 mediates neonatal heart regeneration and repair. Neonatal cardiomyocytes were isolated from one day old WT mice and treated with poly (I:C) (1μg/ml) for 12-36 hours. We observed that poly (I:C) treatment: i) significantly enhances glycolytic metabolism; ii) increases YAP/TAZ activation: iii) increases miR-152 expression; iv) suppresses expression of DNMT1 and p27kip1, and v) promotes cardiomyocyte proliferation. However, inhibition of glycolysis with 2-Deoxyglucose (2-DG) prevented poly (I:C)-induced YAP/TAZ activation and miR-152 expression in neonatal cardiomyocytes. Similarly, inhibition of YAP/TAZ activation with Verteprofin (VP) abolished poly (I:C) induced miR-152 expression and neonatal cardiomyocyte proliferation. To investigate the role of miR-152 in neonatal cardiomyocyte proliferation, we transfected neonatal cardiomyocytes with miR-152 mimics and observed that increased miR-152 levels significantly promotes neonatal cardiomyocyte proliferation. We also observed that transfection of neonatal cardiomyocytes with miR-152 mimics markedly suppresses the expression of DNMT1 and p27kip1. Inhibition of DNMT1 with 5Azcytidine significantly promotes neonatal cardiomyocyte proliferation. Finally, we observed that treatment of neonatal mice (n=6) with 2-DG abolished cardiac functional recovery 3 weeks after MI. We conclude that TLR3 is required for neonatal heart regeneration and repair after MI. The mechanisms involve glycolytic dependent activation of YAP/TAZ mediated by miR-152 which represses DNMT1/p27kip1 expression.

scholarly journals The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor

2020 ◽  
Vol 295 (22) ◽  
pp. 7620-7634
Author(s):  
Christian Holt ◽  
Louise Hamborg ◽  
Kelvin Lau ◽  
Malene Brohus ◽  
Anders Bundgaard Sørensen ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Ryanodine Receptor ◽  
Hydrophobic Surface ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Open Probability ◽  
Calmodulin Binding ◽  
Terminal Domain ◽  
Genes Encoding ◽  
Cardiac Ryanodine Receptor

Mutations in the genes encoding the highly conserved Ca2+-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca2+-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia–causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca2+ coordination and has only a minor impact on binding affinity toward Ca2+ and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca2+ channel, Cav1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca2+-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84–2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.

scholarly journals Suramin and the suramin analogue NF307 discriminate among calmodulin-binding sites

2001 ◽  
Vol 355 (3) ◽  
pp. 827-833 ◽  
Author(s):  
Markus KLINGER ◽  
Elisa BOFILL-CARDONA ◽  
Bernd MAYER ◽  
Christian NANOFF ◽  
Michael FREISSMUTH ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Ryanodine Receptor ◽  
Binding Sites ◽  
Metabotropic Glutamate Receptor ◽  
Target Proteins ◽  
Porcine Brain ◽  
Metabotropic Glutamate ◽  
Calmodulin Binding ◽  
Brain Membranes ◽  
Bacterial Lysates

Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [125I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC50 = 4.9±1.2µM and 19.9±1.8µM, respectively) and blocked the cross-linking of [125I]calmodulin to some, but not all, target proteins in brain membranes by [125I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle, neuronal NO synthase (nNOS) from Sf9 cells, G-protein βγ dimers (Gβγ) from porcine brain and a glutathione S-transferase-fusion protein comprising the C-terminal calmodulin-binding domain of the metabotropic glutamate receptor 7A (GST-CmGluR7A) from bacterial lysates]. Three of the proteins employed (Gβγ, GST-CmGluR7A and RyR1) display a comparable affinity for calmodulin (in the range of 50–70nM). Nevertheless, suramin and NF307 only blocked the binding of Gβγ and RyR1 to calmodulin–Sepharose. In contrast, the association of GST-CmGluR7A and nNOS was not impaired, whereas excess calmodulin uniformly displaced all proteins from the matrix. Thus suramin and NF307 are prototypes of a new class of calmodulin antagonists that do not interact directly with calmodulin but with calmodulin-recognition sites. In addition, these compounds discriminate among calmodulin-binding motifs.

scholarly journals Malignant hyperthermia mutation sites in the Leu2442–Pro2477 (DP4) region of RyR1 (ryanodine receptor 1) are clustered in a structurally and functionally definable area

2006 ◽  
Vol 401 (1) ◽  
pp. 333-339 ◽  
Author(s):  
Mark L. Bannister ◽  
Tomoyo Hamada ◽  
Takashi Murayama ◽  
Peta J. Harvey ◽  
Marco G. Casarotto ◽  
...  
Keyword(s):  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Structural Studies ◽  
Amino Acid Residues ◽  
Domain Interaction ◽  
Closed State ◽  
Interdomain Interaction ◽  
Ryanodine Receptor 1 ◽  
Domain Peptide ◽  
Interdomain Interactions

To explain the mechanism of pathogenesis of channel disorder in MH (malignant hyperthermia), we have proposed a model in which tight interactions between the N-terminal and central domains of RyR1 (ryanodine receptor 1) stabilize the closed state of the channel, but mutation in these domains weakens the interdomain interaction and destabilizes the channel. DP4 (domain peptide 4), a peptide corresponding to residues Leu2442–Pro2477 of the central domain, also weakens the domain interaction and produces MH-like channel destabilization, whereas an MH mutation (R2458C) in DP4 abolishes these effects. Thus DP4 and its mutants serve as excellent tools for structure–function studies. Other MH mutations have been reported in the literature involving three other amino acid residues in the DP4 region (Arg2452, Ile2453 and Arg2454). In the present paper we investigated the activity of several mutants of DP4 at these three residues. The ability to activate ryanodine binding or to effect Ca2+ release was severely diminished for each of the MH mutants. Other substitutions were less effective. Structural studies, using NMR analysis, revealed that the peptide has two α-helical regions. It is apparent that the MH mutations are clustered at the C-terminal end of the first helix. The data in the present paper indicates that mutation of residues in this region disrupts the interdomain interactions that stabilize the closed state of the channel.

scholarly journals Molecular Basis of Calmodulin Binding to Cardiac Muscle Ca2+Release Channel (Ryanodine Receptor)

2003 ◽  
Vol 278 (26) ◽  
pp. 23480-23486 ◽  
Author(s):  
Naohiro Yamaguchi ◽  
Le Xu ◽  
Daniel A. Pasek ◽  
Kelly E. Evans ◽  
Gerhard Meissner
Keyword(s):  
Cardiac Muscle ◽  
Ryanodine Receptor ◽  
Molecular Basis ◽  
Release Channel ◽  
Calmodulin Binding

scholarly journals Calmodulin Binding and Inhibition of Cardiac Muscle Calcium Release Channel (Ryanodine Receptor)

2001 ◽  
Vol 276 (23) ◽  
pp. 20144-20153 ◽  
Author(s):  
David M. Balshaw ◽  
Le Xu ◽  
Naohiro Yamaguchi ◽  
Daniel A. Pasek ◽  
Gerhard Meissner
Keyword(s):  
Cardiac Muscle ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Calcium Release Channel ◽  
Release Channel ◽  
Calmodulin Binding ◽  
Muscle Calcium
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