Calmodulin D133H Disrupts Cav1.2 and Kv7.1 Regulation to Prolong Cardiac Action Potentials in Long QT Syndrome

Figure 1. LQTS-associated CaM variant D133H prolongs APD in mouse ventricular myocytes. (A) Exemplar APs recorded at 1 Hz in cells dialysed with either CaM-WT (black line) or D133H (green line). (B) APD90 in CaM-WT and D133H cells during steady pacing at different cycle lengths. APD90 was longer in D133H cells at cycle lengths of 500 and 1000 ms. * p < 0.05 CaM-WT vs. D133H, a p < 0.05 vs. 166 ms, b p < 0.05 vs. 250 ms. Mixed-effects model with Šídák’s multiple comparisons test. (C) Exemplar APs recorded during pacing at 166 ms cycle length in a CaM-WT and D133H cells. (D) Poincaré plots showing the relationship between APD (APDn), and the APD of the next beat (APDn+1) constructed from the data shown in panel C. Two clusters of APD values occur in the D133H cell, indicating beat-to-beat variation in repolarisation. (E) The absolute difference in APD between consecutive beats during pacing at 166 ms cycle length was around 2× greater in D133H cells; however, this was not statistically significant (p = 0.08; Mann–Whitney test). (B + E) n/N = 13/4 CaM-WT cells/hearts and n/N = 11/4 D133H cells/hearts.

Figure 2. Effects of arrhythmogenic CaM variant D133H on ICa in mouse ventricular myocytes. (A) ICa current–voltage (I–V) relationship in patch-clamped ventricular myocytes dialysed with either 1 µM CaM-WT (white symbols) or D133H (green symbols). Top panel illustrates the voltage clamp protocol used. Peak ICa at +10 mV was not different between CaM-WT and D133H cells (p = 0.30; unpaired t-test). (B) Exemplar normalised current recordings in a CaM-WT (left) and D133H (right) cells during a 300 ms step to +10 mV, with 2 mM Ca2+ or 2 mM Ba2+ as the divalent cation. (C) Quantification of CDI from the proportion of remaining current at 50 ms in Ca2+ and Ba2+. n/N = 9/4 CaM-WT cells/hearts and n/N = 9/4 D133H cells/hearts.

Figure 3. Arrhythmogenic CaM variant D133H disrupts CDI of Cav1.2 in HEK293 cells. (A) Voltage step protocol for Cav1.2 activation. Cells were depolarised from −40 to +60 mV in 10 mV increments for 300 ms from a holding potential of −60 mV. Representative traces from HEK293-Cav1.2 cells expressing CaM variants are shown. (B) Current–voltage (I–V) relationships and (C) activation curves for Cav1.2 with CaM variants. Data were normalised to the peak current density for each cell. Conductance, G, was normalised to peak conductance, Gmax, to give average activation curves. Half-maximal activation voltages, V50, were determined from Boltzmann fits (CaM-WT, n = 8; D133H, n = 8). (D) Voltage step protocol for inactivation. Cells were subjected to 1000 ms conditioning pulses from −60 to +40 mV in 10 mV increments, followed by a 300 ms test pulse to +10 mV. Representative traces from CaM-variant expressing cells are shown. (E) Steady-state inactivation curves of Cav1.2 (CaM-WT, n = 5; D133H, n = 5). (F,G) (Left panels) Representative Ca2+ and Ba2+ current traces, normalised to their respective peak current, in response to a 300 ms pulse to +10 mV. (Right panels) Fractional residual Ca2+ and Ba2+…

Figure 4. LQTS-associated CaM variant D133H reduces IKs densities and shifts voltage sensitivity at resting intracellular [Ca2+]. HEK293 cells were transiently co-transfected with KCNQ1, KCNE1, and CaM variants. Whole-cell voltage clamp recordings were performed by holding cells at −80 mV and applying 4 s depolarising steps from −60 to +100 mV in 20 mV increments, followed by repolarisation to −40 mV. Currents were recorded at two intracellular Ca2+ concentrations: 100 nM and 1 μM. (A,D) Representative IKs current traces at (A) 100 nM and (D) 1 µM [Ca2+]int. (B,E) Current–voltage (I–V) relationships of IKs at (B) 100 nM and (E) 1 μM [Ca2+]int. (C,F) Activation kinetics at (C) 100 nM and (F) 1 μM [Ca2+]int. Left panels: Data were normalised to the peak current density for each cell. Conductance, G, was normalised to peak conductance, Gmax, to give average activation curves. Right panels: half-maximal activation voltage (V50) derived from Boltzmann fits. Data are mean ± s.e.m. (CaM-WT, n = 5; D133H, n = 6). Differences between groups were determined using two-way ANOVA (I–V) or one-way ANOVA (activation kinetics) with Dunnett’s multiple comparisons test (* p < 0.05, *** p < 0.001, and **** p…

Figure 5. Arrhythmia-associated mutation D133H does not alter apo/CaM structure. (A) Cartoon representation of apo/CaM (PDB 1CFC), highlighting the D133H mutation (green) within the EF-hand motif. (B) Overlay of 1H-15N HSQC NMR spectra for apo/CaM-WT and D133H variants. Each cross-peak represents an amide proton–nitrogen pair, with positions defined by chemical shifts along the 1H (x-axis) and 15N (y-axis) dimensions. (C) Far-UV CD spectra and (D) estimated secondary structure content from the CDSSTR algorithm (reference data set 7). Data are mean ± s.e.m. (CaM-WT, n = 5; D133H, n = 5), and statistical differences were assessed by two-way ANOVA followed by Dunnett’s multiple comparisons test.

Figure 6. LQTS-associated CaM variant D133H exhibits decreased Ca2+ binding and altered Ca2+/CaM structure. (A) Cartoon representation of Ca2+/CaM (PDB 1CLL) highlighting the D133H mutation (green) in the Ca2+-coordinating EF-hand. Dashed grey lines indicate interactions between residues and Ca2+. (B) C-lobe Ca2+ affinity of CaM measured by intrinsic tyrosine fluorescence. Data were fitted using a specific binding model with Hill slope to calculate Kd values (CaM-WT, n = 7; D133H, n = 5). (C) Overlay of 1H-15N HSQC NMR spectra of Ca2+/CaM-WT and D133H variant. Each cross-peak corresponds to an amide proton–nitrogen pair positioned by its chemical shift in both 1H and 15N dimensions. (D) Chemical shift analysis of Ca2+/CaM-WT and D133H variant. Histograms show chemical shift difference for each residue; unassigned residues were given a uniform value of −0.01. The position of the mutation is indicated by a green dotted line. (E) Far-UV CD spectra for Ca2+/CaM variants and (F) secondary structure estimations using the CDSSTR algorithm (reference data set 7). Data are mean ± s.e.m. (CaM-WT, n = 5; D133H, n = 5), and differences between groups were determined using a two-tailed unpaired t-test…

Figure 7. LQTS-associated mutation D133H decreases CaM stability. (A) Limited proteolysis of CaM variants in (left) EGTA (10 mM) or (right) CaCl2 (5 mM). Purified CaM proteins were incubated with increasing trypsin concentrations for 30 min at 37 °C. Remaining intact CaM was quantified via SDS-PAGE and densitometry (Fiji), normalised to control. Data are mean ± s.e.m. (apo/CaM-WT, n = 6; apo/D133H, n = 7; Ca2+/CaM-WT, n = 7; Ca2+/D133H, n = 6). Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons test. (B) Thermal denaturation of apo/CaM proteins monitored by circular dichroism spectroscopy (λ = 222 nm). (left) Average unfolding curves were fitted to the Boltzmann sigmoid equation. (right) Melting temperatures (Tm) were derived from half-maximal unfolding. Data are mean ± s.e.m (CaM-WT, n = 6; D133H, n = 6). Statistical difference between groups was assessed with a two-tailed unpaired t-test (** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 8. Arrhythmia-associated CaM variant D133H shows altered binding to Cav1.2 IQ and NSCaTE domains. (A,D) Example ITC titration profiles illustrating raw heat signals (upper panels) and integrated isotherms after baseline correction (lower panels) for interactions with (A) Cav1.2-IQ1665−1685 and (D) Cav1.2-NSCaTE51−67. (B,E) Binding affinity of Ca2+/CaM-WT and D133H to Cav1.2-IQ1665−1685 and Cav1.2-NSCaTE51−67, respectively. (C,F) Thermodynamic profile of these interactions, including Gibbs free energy change (ΔG), enthalpy change (ΔH), and entropy contribution (-TΔS). DP, differential power. Data are mean ± s.e.m. (IQ: CaM-WT, n = 4, D133H, n = 5; NSCaTE: CaM-WT, n = 6, D133H, n = 6). Differences between groups were determined using a two-tailed unpaired t-test (affinity) and two-way ANOVA with Dunnett’s multiple comparisons test (thermodynamics) (** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 9. Apo/CaM binding to Kv7.1-HB507−536is decreased for LQTS-associated variant D133H. (A) Example ITC titration profiles illustrating raw heat signals (upper panel) and the corresponding binding isotherms after baseline correction (lower panels) for the interaction of apo/CaM with Kv7.1-HB507−536. (B) Binding affinity and (C) thermodynamic parameters for apo/CaM interaction with Kv7.1-HB507−536 obtained by fitting the data to a single-site binding model. (D) Representative ITC titration curves (upper panel) and binding isotherms (lower panel) for the interaction between Ca2+/CaM and Kv7.1-HB507−536. (E) Affinity and (F) thermodynamic profile of the binding of Ca2+/CaM to Kv7.1-HB507−536 obtained by fitting to a two-site binding model. Data are means ± s.e.m. N, stoichiometry; n, number of experimental replicates. The change in free energy (ΔG) was calculated as ΔH − TΔS, where ΔH represents enthalpy change, ΔS is entropy change, and T is absolute temperature. DP, differential power. Statistical analyses were performed using a two-tailed unpaired t-test for affinity data and two-way ANOVA with Dunnett’s multiple comparisons for thermodynamic parameters (*** p < 0.001 and **** p <…