Complex Arrhythmia Syndrome in a Knock-In Mouse Model Carrier of the N98S Calm1 Mutation

Abstract

Background: Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in CALM1 resulting in a p.N98S substitution in a patient with sinus bradycardia and stress-induced bidirectional ventricular ectopy. The objectives of the present study were to determine if mice carrying the N98S mutation knocked into Calm1 replicate the human arrhythmia phenotype and to examine arrhythmia mechanisms.

Methods: Mouse lines heterozygous for the Calm1^N98S allele (Calm1^N98S/+) were generated using CRISPR/Cas9 technology. Adult mutant mice and their wildtype littermates (Calm1^+/+) underwent electrocardiographic monitoring. Ventricular de- and repolarization was assessed in isolated hearts using optical voltage mapping. Action potentials and whole-cell currents and [Ca²⁺]_i, as well, were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was used for in situ membrane voltage monitoring of ventricular conduction fibers.

Results: Two biologically independent knock-in mouse lines heterozygous for the Calm1^N98S allele were generated. Calm1^N98S/+ mice of either sex and line exhibited sinus bradycardia, QT_c interval prolongation, and catecholaminergic bidirectional ventricular tachycardia. Male mutant mice also showed QRS widening. Pharmacological blockade and activation of β-adrenergic receptors rescued and exacerbated, respectively, the long-QT phenotype of Calm1^N98S/+ mice. Optical and electric assessment of membrane potential in isolated hearts and single left ventricular myocytes, respectively, revealed β-adrenergically induced delay of repolarization. β-Adrenergic stimulation increased peak density, slowed inactivation, and left-shifted the activation curve of I_Ca.L significantly more in Calm1^N98S/+ versus Calm1^+/+ ventricular myocytes, increasing late I_Ca.L in the former. Rapidly paced Calm1^N98S/+ ventricular myocytes showed increased propensity to delayed afterdepolarization-induced triggered activity, whereas in situ His-Purkinje fibers exhibited increased susceptibility for pause-dependent early afterdepolarizations. Epicardial mapping of Calm1^N98S/+ hearts showed that both reentry and focal mechanisms contribute to arrhythmogenesis.

Conclusions: Heterozygosity for the Calm1^N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QTc interval prolongation, and bidirectional ventricular tachycardia. β-Adrenergically induced I_Ca.L dysregulation contributes to the long-QT phenotype. Pause-dependent early afterdepolarizations and tachycardia-induced delayed afterdepolarizations originating in the His-Purkinje network and ventricular myocytes, respectively, constitute potential sources of arrhythmia in Calm1^N98S/+ hearts.

Keywords: arrhythmias, cardiac; calcium channels; calmodulin; heart diseases; model, animal.

Figure 1.. Calm1^N98S/+ mice exhibit sinus bradycardia, QRS widening, QT_c prolongation and catecholaminergic ventricular tachycardia.

A, Top two rows show representative ECG traces recorded during the dark and light cycle in a littermate control (blue) and a Calm1^N98S/+ mouse (red) from line 2. Traces in the third and fourth row show signal-averaged ECGs obtained by overlaying all consecutive cycles recorded within a 5- to 10-s interval, using QRS maximum or QRS minimum for the alignment. Lower row shows superimposed QRS complexes at expanded time scales. B, Dot plots of RR, QRS and QT_c intervals from littermate Calm1^+/+ and Calm1^N98S/+ mice of line 2 and line 18. Each dot represents the 24-h average in a single animal. Horizontal lines superimposed on dots depict mean and SEM. P values by One Way ANOVA followed by Student-Newman-Keuls Method for multiple comparisons (RR and QT_c) or Kruskal-Wallis One Way ANOVA on Ranks followed by Dunn’s Method for multiple comparisons (QRS). Line 2: 8 Calm1^+/+and 11 Calm1^N98S/+mice; Line 18: 9 Calm1^+/+ and 10 Calm1^N98S/+ mice. C, Calm1^N98S/+ mice display bidirectional ventricular tachycardia (BVT). Representative telemetric ECG recordings from a conscious Calm1^+/+ and Calm1^N98S/+ mouse obtained before and shortly after intraperitoneal injection of epinephrine (2 mg per kg body weight) and caffeine (120 mg per kg body weight). In the Calm1^N98S/+ mouse, an episode of sustained BVT was preceded by ventricular bigeminy (*) and spontaneously reverted to sinus rhythm. No ventricular arrhythmias were observed in the Calm1^+/+ mouse. D, Complete dissociation of P waves (arrowheads) from QRS complexes during an episode of induced BVT. E, Polymorphic ventricular tachycardia in a male Calm1^N98S/+ mouse. * marks transition from BVT into polymorphic VT and ^# denotes conversion to sinus rhythm. F, Prevalence of sustained (>15 s) BVT induced by co-administration of epinephrine and caffeine. * P < 0.001 versus Calm1^+/+ by Fisher’s Exact test. G, Incidence, cumulative duration, cycle length and CI/RR ratio of episodes of sustained (≥ 15 s) BVT during a 30-min period of post-injection telemetric ECG recordings in conscious Calm1^N98S/+ mice from line 2 and line 18. Dot plots with mean ± SEM shown as horizontal lines. N = 7, line 2; N = 8, line 18; *P=0.041 by t test. VT indicates ventricular tachycardia. CI indicates coupling interval and RR indicates RR interval.

Figure 2.. Delayed repolarization in ventricles of Calm1^N98S/+ mice.

A, Isochrone maps of epicardial activation during atrial pacing at a cycle length of 120 ms. Asterisks denote epicardial breakthrough sites on the anterior surface near the apex of the left (LV) and right ventricle (RV). Depolarization spread from the apex to the base. Sequential depolarizations are depicted in different colors according to the color legend shown in the left. Isochronal lines are 1 ms apart. B, Epicardial ventricular conduction velocities. Ventricular activation isochrone maps of the anterior aspect of a Calm1^+/+ and Calm1^N98S/+ heart. The anisotropic propagation in the ventricle enables measurements of maximal and minimal conduction velocities (CV_max and CV_min). C, Dot plots of CVs. Numbers indicate mean and SEM in m/s from 6 Calm1^+/+ and 4 Calm1^N98S/+ hearts. No statistically significant differences between the two genotypes were found. D, Left: Representative optical action potentials from the anterior left ventricular epicardium of littermate Calm1^+/+ and Calm1^N98S/+ mice during atrial pacing at a cycle length of 120 ms. Middle three panels: exemplar maps of the repolarization time points at APD₃₀, APD₅₀ and APD₈₀. Colored scale bars are shown in the inserts. Right: superimposition of the same optical action potentials shown in the left panels. E, Dot plots showing APD₃₀, APD₅₀ and APD₈₀ of littermate control and Calm1^N98S/+ mice. Black horizontal lines depict mean ± SEM. * P ≤ 0.02 by t-test. N = 11 per genotype. F, Exacerbation of long QT phenotype upon exposure to epinephrine (E) and caffeine (C) combined. Exemplar ventricular repolarization (APD₈₀) isochrone maps of the anterior aspect of a Calm1^+/+ and a Calm1^N98S/+ heart at baseline, during E and C exposure, and following washout. Pacing cycle length, 120 ms. G, Scatter plots of APD₈₀. Black lines are mean ± SEM from 8 Calm1^+/+ and 6 Calm1^N98S/+ hearts. P values by Repeated Measures ANOVA followed by Tukey test for intragroup analyses; for intergroup analyses, a t-test (for comparisons at baseline and during washout) and Mann-Whitney Rank Sum test (for comparison during epinephrine) were used.

Figure 3.. β-adrenergic stimulation induces a long QT phenotype in Calm1^N98S/+ mice.

A, Left panels: superimpositions of representative action potentials electrically recorded from single myocytes isolated from the left ventricular free wall of a Calm1^+/+ (blue) and a Calm1^N98S/+ (red) heart. Cells were paced at 1 and 7 Hz. Right panels: single action potentials at 1 and 7 Hz pacing at expanded time scales. B, Dot plots of APD₃₀, APD₅₀ and APD₉₀ in single ventricular myocytes paced at 1 and 7 Hz. Black lines denote mean ± SEM; 11 cells each from 3 Calm1^+/+ mice and from 4 Calm1^N98S/+ mice. * P = 0.004 vs. Calm1^N98S/+ 1 Hz by paired t-test. C, Superimpositions of representative action potentials recorded from a Calm1^+/+ and a Calm1^N98S/+ ventricular myocyte at baseline, during isoproterenol (50 nM; Iso) treatment, and during washout. Action potentials were evoked by 2-ms square current injections delivered at 1 Hz. D, Summary of the effect of isoproterenol (Iso) on APD₉₀ in Calm1^+/+ and Calm1^N98S/+ ventricular myocytes; 14 cells from 5 Calm1^+/+ mice and 10 cells from 4 Calm1^N98S/+ mice. * P < 0.01 by Repeated Measures ANOVA followed by Tukey test for pairwise multiple comparisons. Numbers denote mean ± SEM in ms. Base: baseline. E, Original recordings of a delayed afterdepolarization (*) and delayed afterdepolarization-triggered action potentials (arrows) in a Calm1^+/+ and a Calm1^N98S/+ ventricular cardiomyocyte, respectively. Traces show the terminal 6 paced action potentials from a 10-s train at 7 Hz in the presence of 50 nM isoproterenol. F, Summary of the effects of propranolol (1 mg/kg) or isoproterenol (0.17 mg/kg) on heart rate. Horizontal black lines denote mean ± SEM. Propranolol (Prop): N = 8, Calm1^+/+ mice; N = 14, Calm1^N98S/+ mice. Isoproterenol (Iso): N = 7, Calm1^+/+; N = 7, Calm1^N98S/+. P values by unpaired and paired t-test. Base: baseline. G, Representative QT interval changes in conscious male Calm1^+/+ and Calm1^N98S/+ mice following intraperitoneal injection of propranolol (1 mg/kg) or isoproterenol (0.17 mg/kg). Shown are signal-averaged ECGs obtained by overlaying all consecutive cycles recorded within a 5- to 10-s interval, ca. 20 min after injection. Arrows mark QT intervals. H, Summary of the effects of propranolol and isoproterenol on QT_c interval. Horizontal black lines denote mean ± SEM. Propanolol (Prop): N = 8, Calm1^+/+ mice; N = 14, Calm1^N98S/+ mice. Isoproterenol (Iso): N = 7, Calm1^+/+; N = 7, Calm1^N98S/+. P values by unpaired and paired t-test. Base: baseline.

Figure 4.. Ventricular arrhythmia in a Calm1^N98S/+ heart.

A, Volume-conducted ECG and epicardial activation map in sinus rhythm. Asterisks denote concentric breakthroughs on the anterior right and left ventricular free walls. B, Volume-conducted ECGs and epicardial voltage maps in the presence of 100 nM isoproterenol and 3.6 mmol/L extracellular Ca²⁺. QRS complex numbers on ECG correspond to voltage map sequences with the same numbers. The ECG shows a junctional escape rhythm with AV dissociation and a QRS morphology similar to that during SR, a single premature ventricular complex (PVC) and 4 episodes of non-sustained VT (NSVT). Maps show epicardial breakthrough pattern during junctional escape rhythm (sequence #1), RV focal discharge (white arrowheads) coinciding with the emergence of an R-on-T ectopic ventricular beat (sequence #2) in the ECG, RV focal discharge (green arrowhead) initiating NSVT with a large wave front traveling across the epicardial surface (sequences #3 - #5), followed by an RV focal discharge (yellow arrowhead) coinciding with the last beat of the arrhythmia episode (sequence #6). Map sequences of the other 3 VT episodes similarly showed RV focal discharges initiating NSVT. Times denote intervals after the onset of epicardial breakthroughs.

Figure 5.. Calm1^N98S heterozygosity potentiates β-adrenergic receptor-mediated effects on cardiac I_Ca.L.

A and C, Exemplar whole-myocyte I_Ca.L traces evoked by 300-ms step depolarizations to voltages from −50 to +60 mV from a holding potential of −70 mV in control (A) and during exposure to isoproterenol (C). Interpulse interval was 5 s. Top: voltage-clamp protocol. B and D, Peak I_Ca.L density – voltage relations in Calm1^+/+ and Calm1^N98S/+ ventricular myocytes in the absence (B) and presence of isoproterenol (D). Values are mean ± SEM. Control: 11 cells each from 5 Calm^+/+ mice and 5 Calm1^N98S/+ mice. Isoproterenol: 24 cells from 5 Calm1^+/+ mice and 29 cells from 5 Calm1^N98S/+ mice. * P < 0.03 versus Calm1^+/+ by t-test. E and F, Voltage-dependence of steady-state I_Ca.L activation and inactivation in the absence (E) and presence (F) of isoproterenol (50 nM). Values are mean ± SEM. Solid lines are best fits of the data to a Boltzmann function (fit parameters in Supplemental Table 6). Half-activation voltage in the presence of isoproterenol was significantly more negative in Calm1^N98S/+ versus Calm1^+/+ myocytes (−16.8 ± 0.8 mV versus −13.9 ± 0.8 mV; P = 0.018 by t-test). Insert: Calculation of the predicted window I_Ca.L density as a function of voltage. Amplitude of window I_Ca.L density was estimated as SS-act x SS-inact x I_Ca.L (at V_peak). Shaded area marks the range of membrane potential typically occurring during the action potential plateau of isoproterenol-treated Calm1^N98S/+ ventricular myocytes. SS-act and SS-inact indicate steady state activation and steady state inactivation, respectively. G, Exemplar whole-myocyte I_Ca.L traces elicited by 300-ms step depolarizations to +10 mV in the absence (top) and presence of 50 nM isoproterenol (bottom). Solid black lines are best fits of I_Ca.L decay to a double exponential: y(t) = A_fast exp(−t/τ_fast) + A_slow exp(−t/τ_slow) + y₀. H, Summary of I_Ca.L inactivation time constants, τ_fast and τ_slow, as a function of test potential. Values are mean ± SEM. Control: 11 cells from 5 Calm1^+/+ mice and 12 cells from 4 Calm1^N98S/+ mice; isoproterenol: 23 cells from 5 Calm1^+/+ mice and 25 cells from 6 Calm1^N98S/+ mice. Numbers above brackets indicate P values by One way ANOVA followed by multiple comparisons using the Bonferroni t-test (for τ_slow at 10 and 25 mV) or Kruskal Wallis One Way ANOVA on Ranks followed by Dunn’s Method for multiple comparisons (for all other voltages).

Figure 6.. β-adrenergic receptor stimulation enhances peak and late I_Ca.L during the action potential in Calm1^N98S/+ ventricular myocytes.

A and B, L-type Ca²⁺ currents (lower panels) elicited by “typical” action potential waveforms (upper panels) that had been pre-recorded from isoproterenol (50 nM) - treated Calm1^+/+ and Calm1^N98S/+ ventricular myocytes. Action potential waveforms were delivered at 1 Hz steady-state frequency in the presence of isoproterenol (50 nM). Each data point in an I_Ca.L trace represents the mean ± SEM of 20 and 22 cells isolated from 4 Calm1^+/+ and 4 Calm1^N98S/+ hearts, respectively. C and D, The same action potential waveforms and I_Ca.L traces shown in A and B at expanded time scales. E and F, Dot plots of total I_Ca.L (∫I_Ca.L; E) normalized to cell capacitance and peak I_Ca.L density during the ventricular action potential in isoproterenol-treated cardiomyocytes. AP: action potential. Horizontal lines indicate mean ± SEM; 20 cells from 4 Calm1^+/+ mice and 22 cells from 4 Calm1^N98S/+ mice. P values by ANOVA followed by Bonferroni test for post hoc comparison.

Figure 7.. Resting Ca²⁺ spark properties in Calm1^+/+ and Calm1^N98S/+ ventricular myocytes.

A, Confocal line-scan images of diastolic Ca²⁺ sparks recorded in a Calm1^+/+ and a Calm1^N98S/+ cell in the absence (top) and presence (bottom) of 1 μM isoproterenol. B, Box and whisker plots of (B) Ca²⁺ spark occurrence, (C) spark amplitude, (D) full width at half maximum (FWHM), (E) full duration at half maximum (FDHM), and (F) time to peak (TtP). The ends of the box are the upper and lower quartiles. The medians are marked by the horizontal lines inside the boxes, whereas the whiskers extend to the 10% and 90% quartiles. Black dots mark 5% and 95% percentiles. Numbers above horizontal lines denote P values by Kruskal-Wallis One Way ANOVA on Ranks followed by pairwise comparisons using Dunn’s method; Calm1^+/+: 443 sparks in 127 cells from 11 hearts without isoproterenol, 546/32/5 with 1 μM isoproterenol; Calm1^N98S/+: 274/125/9 without isoproterenol, 517/37/4 with 1 μM isoproterenol.

Figure 8.. Pause-dependent early afterdepolarizations in the His-Purkinje network of Calm1^N98S/+ hearts.

A and B Top: original traces of membrane potential obtained from His-Purkinje myocytes in Calm1^N98S/+ hearts. The traces shown include the last 5 action potentials from a train of 30 action potentials at 5 Hz pacing, followed by >1-s pauses and spontaneous action potentials. Middle: Magnified views of the final paced action potential. Bottom: Magnified views of the boxed regions in the top panels, illustrating pause-dependent repetitive early afterdepolarizations (A) and an early afterdepolarization (B) arising from a spontaneous action potential. Neither cell developed diastolic (delayed) afterdepolarizations. Note the presence of automaticity in the fiber shown in B. C, Properties of early afterdepolarizations. N and n are the number of hearts and conduction fibers, respectively. EAD, early afterdepolarization; mean values for take-off potential, EAD amplitude and diastolic interval preceding the EAD-containing cycle were averaged from 202 and 23 EADs in Calm1^N98S/+ and Calm1^+/+ wedges, respectively. * P < 0.001 versus Calm1^+/+ by Chi-square test. ^# P = 0.006 versus Calm1^+/+ by Mann-Whitney Rank Sum test.