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Case Reports
. 2005 Jun 7;102(23):8089-96; discussion 8086-8.
doi: 10.1073/pnas.0502506102. Epub 2005 Apr 29.

Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations

Igor Splawski  1 , Katherine W Timothy, Niels Decher, Pradeep Kumar, Frank B Sachse, Alan H Beggs, Michael C Sanguinetti, Mark T Keating
Affiliations
Case Reports

Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations

Igor Splawski et al. Proc Natl Acad Sci U S A. .

Abstract

Timothy syndrome (TS) is a multisystem disorder that causes syncope and sudden death from cardiac arrhythmias. Prominent features include congenital heart disease, immune deficiency, intermittent hypoglycemia, cognitive abnormalities, and autism. All TS individuals have syndactyly (webbing of fingers and toes). We discovered that TS resulted from a recurrent, de novo cardiac L-type calcium channel (CaV1.2) mutation, G406R. G406 is located in alternatively spliced exon 8A, encoding transmembrane segment S6 of domain I. Here, we describe two individuals with a severe variant of TS (TS2). Neither child had syndactyly. Both individuals had extreme prolongation of the QT interval on electrocardiogram, with a QT interval corrected for heart rate ranging from 620 to 730 ms, causing multiple arrhythmias and sudden death. One individual had severe mental retardation and nemaline rod skeletal myopathy. We identified de novo missense mutations in exon 8 of CaV1.2 in both individuals. One was an analogous mutation to that found in exon 8A in classic TS, G406R. The other mutation was G402S. Exon 8 encodes the same region as exon 8A, and the two are mutually exclusive. The spliced form of CaV1.2 containing exon 8 is highly expressed in heart and brain, accounting for approximately 80% of CaV1.2 mRNAs. G406R and G402S cause reduced channel inactivation, resulting in maintained depolarizing L-type calcium currents. Computer modeling showed prolongation of cardiomyocyte action potentials and delayed afterdepolarizations, factors that increase risk of arrhythmia. These data indicate that gain-of-function mutations of CaV1.2 exons 8 and 8A cause distinct forms of TS.

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Figures

Fig. 1.
Fig. 1.
Dysmorphic facial features, marked QT interval prolongation, arrhythmia, and skeletal muscle defects in affected individuals. (A–C) Individual 1 exhibiting dysmorphic facial features including flat nasal bridge, thin upper lip, and protruding tongue. (D) Electrocardiogram shows severe QT interval prolongation, causing 2:1 atrio-ventricular block seen as two atrial beats (P-waves) for each ventricular beat (QRS complex). (E) Electrocardiogram shows polymorphic ventricular tachycardia for individual 1. (F) Frozen section of left quadriceps biopsy stained with modified Gomori trichrome showing rare nemaline rods (indicated by arrows) in scattered myofibers (scale bar, 20 μm). (G) Electron micrograph of subsarcolemmal collections of electron dense rods (×17,800). (H) Electron micrograph illustrating normal sarcomeric architecture for comparison with G (×17,800).
Fig. 2.
Fig. 2.
De novo Cav1.2 mutations cause TS2. (A) Pedigree shows sporadic occurrence of the disease phenotype and de novo G1216A missense mutation in individual 1. This mutation leads to the substitution of glycine 406 with arginine (G406R) in exon 8. Circles and squares indicate females and males, respectively. Filled and empty symbols denote affected and unaffected individuals, respectively. The individual with a slash is deceased. Sequence tracings were derived from blood DNA samples unless otherwise indicated. (B) Pedigree shows sporadic occurrence of the disease phenotype and de novo G1204A missense mutation in individual 2. This mutation leads to the substitution of glycine 402 with serine (G402S) in exon 8. The mutant peak (green, arrow) is present in sequence from blood DNA but only a small peak is detected in oral mucosa DNA, indicating mosaicism. Mosaicism may explain the milder phenotype in this individual. (C) Amino acid sequence alignment showing conservation of glycine at positions 402 and 406 from multiple species. Bracket indicates the end of the sixth transmembrane segment of domain I (DI/S6). (D)(Upper) Predicted topology of CaV1.2, showing the location of the mutations. (Lower) Homology of the amino acid sequence of exons 8 and 8A. DI/S6 is underlined. G402 and G406, the amino acids mutated in TS2, are both in exon 8 and are shown in red. G406, mutated in TS, is in exon 8A and is shown in blue. These data indicate that de novo missense mutations in CaV1.2 exon 8 cause severe long QT syndrome and other phenotypic abnormalities.
Fig. 3.
Fig. 3.
The CaV1.2 exon 8 splice variant is highly expressed in heart and brain. (A) Human Northern blot analyses show expression of CaV1.2 exon 8 in brain, heart, and other tissues. (B) mRNA dot blot demonstrates expression of CaV1.2 exon 8 in multiple fetal and adult tissues, including many regions of the brain.
Fig. 4.
Fig. 4.
Mutations of CaV1.2 exon 8 cause nearly complete absence of voltage-dependent channel inactivation. (A–C) WT (A), G402S (B), and G406R (C) CaV1.2 channel currents were recorded from Xenopus oocytes in response to voltage pulses applied in 10-mV increments from -70 to +40 mV, second pulse to +10 mV. External solution contained 40 mM BaCl2 to eliminate Ca2+-dependent inactivation of CaV1.2 channels. Note the lack of current inactivation for mutant channels compared with WT channel currents. (D) G406R channels have similar Ba2+ current–voltage (I–V) relationship compared with WT. (E) Voltage dependence of Ba2+ current activation for WT (V1/2 = 4.5 ± 0.1 mV; k = 5.5 ± 0.1 mV; n = 7), G402S (V1/2 = 6.2 ± 0.1 mV; k = 7.5 ± 0.1 mV; n = 14), and G406R (V1/2 = -4.4 ± 0.3 mV; k = 5.4 ± 0.2 mV; n = 14). (F) Voltage dependence of Ba2+ current inactivation for WT (V1/2 = -6.6 ± 0.4 mV; k = 7.6 ± 0.4 mV; n = 8) and mutant CaV1.2 channels. The minimum value for relative inactivation was 0.10 ± 0.01 for WT (n = 8), but 0.88 ± 0.03 for G406R (n = 15) and 0.91 ± 0.04 for G402S (n = 6), indicating nearly complete absence of voltage dependence of inactivation.
Fig. 5.
Fig. 5.
Glycine is a critical amino acid at position 406 of CaV1.2. Voltage dependence of inactivation for WT and mutant exon 8 Cav1.2 channels were determined in Xenopus oocytes by measuring the peak amplitude of current evoked during a pulse to +10 mV that followed a 2-s conditioning pulse applied to a variable potential. Currents were normalized and plotted as a function of the conditioning potential. External solution contained 40 mM BaCl2 to eliminate Ca2+-dependent inactivation. WT, n = 6; G406R, n = 10; G406E, n = 11; G406K, n = 9; G406P, n = 8; G406S, n = 8; G406V, n = 6; G406A, n = 11.
Fig. 6.
Fig. 6.
Computer modeling revealed prolonged action potentials, altered intracellular calcium handling, and DADs induced by CaV1.2 mutations. (A) Simulated voltage dependence of L-type calcium channel current inactivation for WT (black), G406R (green), WT/exon 8 G406R heterozygotes (red), and WT/exon 8A G406R heterozygotes (blue). The relative voltage-dependent inactivation was plotted as a function of Vm. (B) Intracellular calcium transients during a simulated action potential. The peak of the transient was increased by 29% in WT/exon 8 G406R heterozygotes compared with 7% in WT/exon 8A G406R, because the exon 8 splice variant expression was assumed to predominate in the heart with a ratio ≈3.5:1. (C) Cardiac action potentials were prolonged by 30% in WT/exon 8 G406R compared with 8% prolongation in WT/exon 8A G406R heterozygotes. (D–F) Transients of [Ca2+]JSR (D), [Ca2+]i (E), and Vm (F) were calculated for a stimulus frequency of 3 Hz. The last three action potentials of 3-Hz pacing were followed by a pause. During this pause, exon WT/exon 8A G406R led to a DAD after 509 ms with a peak voltage of -54 mV; WT/exon 8 G406R led to a triggered action potential after 423 ms with a peak voltage of 26 mV. In this model, DADs result from spontaneous release of Ca2+ from the sarcoplasmic reticulum. Experimental studies of cardiac myocytes have shown that DADs result from Cai overload and subsequent activation of the Na/Ca exchanger, and to a lesser extent, a Cai-activated cation-nonselective conductance and a Cai-activated Cl- conductance (–25).
Fig. 7.
Fig. 7.
Transient changes in stimulation frequency lead to electrophysiological instabilities in myocytes harboring CaV1.2 channel mutations. In this simulation, the stimulus frequency was increased from 2.5 to 3 Hz at time 0 s. The myocyte was then stimulated at 3 Hz for a variable duration (t), followed by a pause. The peak values of [Ca2+]JSR (A), [Ca2+]i (B), and Vm (C) as well as the start time of spontaneous calcium release (D) were determined during the pause. The start time is specified relative to the time of the last stimulus. In the transitional phase, myocytes with WT/exon 8A G406R CaV1.2 showed subthreshold DADs between t = 9 and 77 s. With longer times (t >77 s) the DAD triggered an action potential. Myocytes with WT/exon 8 G406R CaV1.2 also exhibited DADs, which were sporadic for t <82 s. Only some of these DADs triggered action potentials (e.g., at t = 67 s). WT myocytes never showed DADs. Together, these simulations predict that the CaV1.2 mutations promote electrophysiological instability that could induce lethal arrhythmia in response to relatively minor changes in heart rate.
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