Divergent regulation of ryanodine receptor 2 calcium release channels by arrhythmogenic human calmodulin missense mutants

Abstract

Rationale: Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known.

Objective: To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels.

Methods and results: We studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II.

Conclusions: A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.

Keywords: calcium; catecholaminergic polymorphic ventricular tachycardia; long QT syndrome; ryanodine receptor calcium release channel; sarcoplasmic reticulum.

Figure 1. Ca binding affinity of CPVT-CaMs

Shown are Ca titration curves for WT and CPVT-CaMs (N54I, N98S) for the CaM-C and CaM-N domain. Data were used to derive dissociation constants (K_d, in µM) for the each domain. C-domain: 3.3±0.2 (WT), 3.1±0.2 (N54I), 11±1 (N98S). N-domain: 23±3 (WT), 23 ±3 (N54I), 22±2 (N98S). Values are averages of three experiments and error was determined by analysis of the curve fits.

Figure 2. Only CPVT-CaMs promote spontaneous Ca waves in permeabilized myocytes

(A) Representative line-scans (red arrow) from permeabilized mouse ventricular myocytes after 30 min incubation with either WT or mutant CaMs (100nM). CPVT-CaMs promoted higher Ca wave frequency (B) and lower Ca wave amplitude (C). After permeabilization, myocytes were incubated in internal solution composed of 120 nM free [Ca] (calculated using Max Chelator), 100 µM EGTA, and 25 µM Fluo 4. Bars represent mean + SE of values normalized by WT values on each experimental day. WT (white, n=45), D96V (light grey, n=33), D130G (grey, n=15), F142L (dark grey, n=20), N54I (red, n=35), N98S (blue, n=35). Casq2KO: myocytes isolated from Casq2 null mice and incubated with WT-CaM (violet, n=21). *P < 0.05, **P < 0.01 vs WT CaM.

Figure 3. CPVT-CaMs bind with higher affinity to RyR2 Ca release channels in nanomolar Ca conditions

(A) In cardiac SR vesicles stripped of native CaM, RyR2 was pre-labeled with AF488-FKBP (donor), then incubated with 100 nM AF568-CaM (acceptor) and a series of concentrations of WT and mutant (Mut) CaMs (D96V light grey, D130G grey, F142L dark grey, N54I red, N98S blue). AF568-CaM binding to RyR2 was determined from the decrease in AF488-FKBP fluorescence due to FRET. Data are mean ± SE, n=3–4, for each concentration. (B) Mean ratio of mutant and WT IC₅₀ of CaM binding to RyR2. *P < 0.05 vs WT-CaM, n=3–4 experiments per group.

Figure 4. Abnormal regulation of single RyR2 channels by CPVT-associated CaM mutants

(A) Continuous records of RyR2 gating in the absence of CaM. Cytoplasmic bath contained 1 µM [Ca]. Arrows indicate addition of 100nM of WT-CaM, CPVT-CaMs (N54I, N98S) or LQTS-CaM (D96V). Solution changes were achieved within <5 s using local perfusion of the RyR2 (see methods). Average open probability (P_o) values before and after adding CaM are given at the left and right of each trace, respectively. (B) Continuous records of a single RyR2 beginning in the absence of CaM at 0.1 µM [Ca]_cyt. Addition of 100 nM N54I CaM (arrow) increased RyR2 channel activity. Average effect of WT and mutant CaMs on RyR2 P_o, at 1 µM (C) and 0.1 µM cytosolic [Ca] (D). P_o values were obtained from 1–2 min of continuous recording under each condition. Before averaging, each P_o value was normalized to the P_o of the same RyR2 channel obtained before adding CaM. *P < 0.05, **P < 0.01, **P < 0.001 by Mann-Whitney Test, ^#P < 0.05, ^##P < 0.01 by Wilcoxon Signed-Rank Test versus CaM-free RyR2. n=10–14 records per group.

Figure 5. CPVT-CaMs promote Ca sparks and lower SR Ca content

(A) Representative line-scan images of Ca sparks in permeabilized rat ventricular myocytes in CaM-free, WT-CaM, and CPVT-CaMs (N54I, N98S). Line plots of spark events (red arrow) are indicated below their images. (B) Average Ca spark frequency. Bars represent mean + SE. CaM-free (black, n=5), WT (white, n=9), N54I (red, n=8), N98S (blue, n=10).(C) Line-scan (top) and line plot (bottom) examples of SR Ca²⁺ content evaluated by 10 mM caffeine-evoked Ca transient in WT-CaM. (D) Average SR Ca content. CaM-free (black, n=5), WT (white, n=6), N54I (red, n=6), N98S (blue, n=7). [Ca]_i = 50 nM, EGTA = 0.5 mM, [WT] or [mutant CaMs] = 100 nM. **P < 0.01, ***P < 0.001 vs. WT-CaM.

Figure 6. CPVT-CaMs exhibit a dominant effect on Ca waves

(A) Representative line-scans (red arrow) from permeabilized mouse myocytes after 30 min incubation with either WT-CaM or CPVT-CaMs mixed with WT-CaM (0%, 50%, 75%, and 87.5% (total free [CaM] = 100nM) for 30 min. (B) Ca wave frequency and (C) amplitude for each group. Bars represent mean + SE of values normalized by WT values on each experimental day. WT (white, n=39); N54I (red, n=21–37 each), N98S (blue, n=27–37 each)*P < 0.05, **P < 0.01. vs. WT-CaM.

Figure 7. CaMKII activation is not responsible for the effect of CPVT-CaMs on Ca waves

(A), (B) Effect of CaMKII inhibition with KN93 on Ca wave frequency and amplitude. Permeabilized myocytes were incubated with 100nM of CaM mutants in presence or absence of KN93 (1µM, 30 min pre-incubation). Bars represent mean + SE. WT (white, n=40), D96V (light grey, n=20), D130G (grey, n=15), F142L (dark grey, n=20), N54I (red, n=29), N98S (blue, n=12), *P < 0.05 vs + KN93. With the exception of WT-CaM and LQTS-CaM D96V, CaMKII activation by CaM did not contribute to the effect of CaM mutants on Ca waves. (C) CaM-dependent activation of CaMKII (WT-Camui) measured in HEK 293 cell lysate at 37°C with saturating Ca (200 µM). CaM±1mM EGTA (black, n=13 each), N54I (red, n=9), N98S (blue, n=10), D96V (light grey, n=11), F142L (grey, n=9) and D130G (dark grey, n=10). Decreases in FRET were normalized for each mutant (maximal FRET change was similar to WT for all mutants, except F142L and D96V where it was smaller). (D) [CaM] for half-maximal change in FRET (K_0.5) obtained from panel C for WT and mutant CaM.