CPVT-associated calmodulin variants N53I and A102V dysregulate Ca2+ signalling via different mechanisms

Figures

• 

  Fig. 1.. Ca2+/CaM binding to RyR2 is decreased for the CPVT-associated variants N53I and A102V. (A,B) Representative ITC titration curves (upper panels) and binding isotherms (lower panels) for CaM interaction with RyR23583-3603 in the absence (A) and presence (B) of Ca2+. DP, differential power. (C,D) Affinity (left) and thermodynamic profile (right) of the binding of apo-CaM (C) and Ca2+/CaM proteins (D) to RyR23583-3603 obtained by fitting to a one-site binding model. Data were processed using the MicroCal PEAQ-ITC software. Data are mean±s.e.m. N, stoichiometry; n, number of experimental replicates. The sum of the change in enthalpy (ΔH) and the change in entropy (ΔS) multiplied by the absolute temperature (T) gives the change in free energy (ΔG). Experiments were performed in the presence of 5 mM EGTA or 5 mM CaCl2 at 25°C. Number of replicates (n) for each condition is shown in the lower panels of A and B. ***P<0.001; ****P<0.0001 (versus CaM-WT; differences between three groups were determined using one-way ANOVA with Dunnett's post-hoc test).

• 

  Fig. 2.. CPVT-associated CaM variants N53I and A102V do not show altered secondary structure but induce local structural changes in CaM. Analysis of the secondary structures of CaM-WT and mutants (5 µM) using circular dichroism spectroscopy. (A) Circular dichroism spectra were obtained in the presence of 1 mM EGTA (left panel) or 1 mM CaCl2 (right panel). Data are representative traces from experiments performed in triplicate. (B) Protein secondary structure content estimated using the CDSSTR method (Dichroweb, reference set 3). Data are mean±s.e.m. Experiments were performed at 20°C, in triplicate. Differences between the three groups were determined using two-way ANOVA with the Sidak's post-hoc test and CaM-WT as control. No significant differences were found. (C,D) Histogram of chemical shift differences observed between CaM-WT and CaM-N53I (C) or CaM-A102V (D) (left panels). Data shown are from single NMR experiments. Chemical shift data were normalised between minimum and maximum values, and converted into a colour spectrum of five unique colours (key, blue for smallest shifts through to red for largest shifts; shown as dashed lines in histograms), which were then mapped onto the CaM…

• 

  Fig. 3.. The arrhythmogenic N53I mutant causes subtle changes in the three-dimensional structure of Ca2+/CaM–RyR23583-3603. (A-C) Cartoon representation of the crystal structures of Ca2+/CaM proteins in complex with RyR2 peptide. (A) Ca2+/CaM-WT–RyR23583-3603 (PDB 6XXF). (B) Ca2+/CaM-N53I–RyR23583-3603 (PDB 6XY3). (C) Ca2+/CaM-A102V–RyR23583-3603 (PDB 6XXX). (D,E) Alignments of Ca2+/CaM-WT–RyR23583-3603 with Ca2+/CaM-N53I–RyR23583-3603 (D) or Ca2+/CaM-A102V–RyR23583-3603 (E) complex structures. Ca2+ ions are shown as orange spheres and water molecules as olive spheres. The WT and mutant residues are shown in stick representation. CaM-WT is displayed in beige, CaM-N53I in salmon, CaM-A102V in green and RyR23583-3603 peptide in blue. Images were generated using PyMOL software.

• 

  Fig. 4.. Ca2+/CaM–RyR23583-3603 peptide complexes show differences in the H-bond network at the site of mutation, and increased hydrophobicity. (A,B) Ca2+/CaM-WT–RyR23583-3603 with Ca2+/CaM-WT in beige (A) and Ca2+/CaM-N53I–RyR23583-3603 with Ca2+/CaM-N53I in salmon (B). Ca2+ ions are shown as orange spheres and water molecule as a blue sphere. Interactions are represented by black dashed lines, with residues represented as sticks and electron density as mesh. (C,D) Comparison of the hydrophobicity of the surface areas of Ca2+/CaM-WT–RyR23583-3603 (C) and Ca2+/CaM-A102V–RyR23583-3603 (D). CaM is shown in surface representation, with surface coloured from white to red based on the Eisenberg hydrophobicity scale (Eisenberg, 1984). White is less hydrophobic than red. Arrows indicate the areas of the Ala102 and Val102 residues. Images were generated using PyMOL software.

• 

  Fig. 5.. Arrhythmogenic variant A102V increases CaMKIIδ substrate phosphorylation activity, whereas T287 CaMKIIδ autophosphorylation levels remain unchanged. (A) Quantification of phosphorylation activity of CaMKIIδ using radiolabelled ATP. CaMKIIδ activity measurements were performed using the SignaTECT Calcium/Calmodulin-Dependent Protein Kinase Assay System (Promega) on freshly isolated mice hearts. CaM-WT, CaM-N53I or CaM-A102V recombinant proteins (1 µM) were used as CaMKIIδ activators. [γ-32P]-syntide-2 was used as an indicator of CaMKIIδ phosphorylation activity and quantified using a scintillation counter. The numbers of experimental replicates were as follows: n=12 for CaM-WT, n=5 for CaM-N53I and n=9 for CaM-A102V, from three independent mice heart lysate preparations. Data are mean±s.e.m. **P<0.01 (versus CaM-WT; differences between the three groups were determined using one-way ANOVA with Dunnett's post-hoc test). (B) Measurement of the relative levels of CaMKIIδ Thr287 autophosphorylation. GST-CaMKIIδ was incubated with calmodulin variants and ATP for 0 s, 15 s, 30 s, 60 s, 120 s and 300 s at room temperature. CaM-WT, CaM-N53I or CaM-A102V recombinant proteins were used as…

• 

  Fig. 6.. CPVT-associated mutations N53I and A102V affect spontaneous Ca2+ transients in cells. HEK293T cells transfected with RyR2 and CaM variants were loaded with Calbryte 520 to monitor intracellular Ca2+ concentration changes. Live cells were imaged on a 3i spinning-disk confocal microscope. (A-D) Representative fluorescence signals in HEK293T cells expressing RyR2 (A) with CaM-WT (B), CaM-N53I (C) and CaM-A102V (D). (E-J) Analysis of the Ca2+ transients kinetic parameters using Analyse Spikes applet for Matlab and Fiji (Schindelin et al., 2012). Data are mean±s.e.m. The numbers of experimental replicates for the oscillations experiments (N=dishes, n=fields of view) were as follows: N=6, n=20 for CaM-WT; N=6, n=16 for CaM-N53I; and N=6, n=19 for CaM-A102V. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (versus CaM-WT; differences between the groups were determined using one-way ANOVA with Dunnett's post-hoc test). F.U., fluorescence unit; ΔF, difference between the maximum fluorescence and the initial fluorescence; F0, initial fluorescence.

• 

  Fig. 7.. Proposed regulatory mechanisms for CPVT-associated CaM variants. (1) Upon stimulation (action potential), Ca2+ enters the cell through L-type Ca2+ channels (Cav1.2) and (2) triggers release from the RyR2 by a process known as CICR. (3) Cytosolic Ca2+ diffuses to myofibrils and promotes interaction between actin and myosin that leads to cardiac muscle contraction. (A) In normal conditions, Ca2+/CaM modulates RyR2 activity through direct binding (inhibition) and phosphorylation (P) via CaMKII (activation). A fine-tuned balance between these two processes is required to control cardiac muscle contraction. (B) CaM-N53I: a moderate reduction in affinity for RyR2 and altered CaM conformation reduce the inhibition of RyR2 by Ca2+/CaM. This leads to an increase of Ca2+ release from the SR, promoting arrhythmia. (C) CaM-A102V: a large reduction in affinity for RyR2 reduces the inhibition of RyR2 by Ca2+/CaM, and is combined with an increase in CaMKIIδ phosphorylation activity, which promote the opening of the RyR2 channel. Both phenomena lead to an increase in Ca2+ release from the SR, promoting arrhythmia.