A cardiac arrhythmia syndrome caused by loss of ankyrin-B function

Abstract

220-kDa ankyrin-B is required for coordinated assembly of Na/Ca exchanger, Na/K ATPase, and inositol trisphosphate (InsP(3)) receptor at transverse-tubule/sarcoplasmic reticulum sites in cardiomyocytes. A loss-of-function mutation of ankyrin-B identified in an extended kindred causes a dominantly inherited cardiac arrhythmia, initially described as type 4 long QT syndrome. Here we report the identification of eight unrelated probands harboring ankyrin-B loss-of-function mutations, including four previously undescribed mutations, whose clinical features distinguish the cardiac phenotype associated with loss of ankyrin-B activity from classic long QT syndromes. Humans with ankyrin-B mutations display varying degrees of cardiac dysfunction including bradycardia, sinus arrhythmia, idiopathic ventricular fibrillation, catecholaminergic polymorphic ventricular tachycardia, and risk of sudden death. However, a prolonged rate-corrected QT interval was not a consistent feature, indicating that ankyrin-B dysfunction represents a clinical entity distinct from classic long QT syndromes. The mutations are localized in the ankyrin-B regulatory domain, which distinguishes function of ankyrin-B from ankyrin-G in cardiomyocytes. All mutations abolish ability of ankyrin-B to restore abnormal Ca(2+) dynamics and abnormal localization and expression of Na/Ca exchanger, Na/K ATPase, and InsP(3)R in ankyrin-B(+/-) cardiomyocytes. This study, considered together with the first description of ankyrin-B mutation associated with cardiac dysfunction, supports a previously undescribed paradigm for human disease due to abnormal coordination of multiple functionally related ion channels and transporters, in this case the Na/K ATPase, Na/Ca exchanger, and InsP(3) receptor.

Fig. 1.

Identification of ankyrin-B mutations in human probands with cardiac arrhythmia. (A) Domain organization of 220-kDa ankyrin-B with previously identified E1425G mutation and L1622I, T1626N, R1788W, and E1813K mutations denoted. Yellow box denotes death domain, red box denotes C-terminal domain. (B) ECG recordings from European proband with ankyrin-B R1788W mutation. ECG recordings show normal resting ECG and polymorphic ventricular tachycardia induced by exercise.

Fig. 2.

Pedigrees of probands with Ank2 mutations. Females are denoted by circles, and males are denoted by squares. Hatched symbols represent humans with uncertain or unknown LQT phenotype. Open symbols represent individuals without LQT phenotype at rest. Filled symbols represent individuals diagnosed with LQT phenotype. Mutation carriers are denoted by an asterisk. Probands are marked by a star. Sudden death is denoted by SD, and death is indicated by a diagonal slash. Roman numerals indicate generations in the respective families.

Fig. 3.

Human ankyrin-B mutations do not restore abnormal spontaneous contractions or abnormal Ca²⁺ dynamics in ankyrin-B^+/- cardiomyocytes. Compared to wild-type cardiomyocytes, ankyrin-B^+/- cardiomyocytes display decreased spontaneous contraction rates (A) and abnormal calcium dynamics (B). Abnormal ankyrin-B^+/- cardiomyocyte contraction rates and calcium dynamics are rescued by transfection of GFP-220-kDa ankyrin-B, but not GFP-220-kDa ankyrin-B mutants E1425G, L1622I, T1626N, R1788W, or E1813K (n > 200 cells; P < 0.05). In B, calcium dynamics (expressed in Hz) were measured by using Fluo-3 AM. (C) Raw data of Ca²⁺ levels in cardiomyocytes (F/F_o). Graphs represent untransfected wild-type and ankyrin-B^+/- neonatal cardiomyocytes and GFP-220-kDa ankyrin-B and GFP-220-kDa ankyrin-B R1788W transfected ankyrin-B^+/- myocytes.

Fig. 4.

Human mutations are ankyrin-B loss-of-function mutations in mouse cardiomyocytes. (A) Immunolocalization of endogenous ankyrin-B (Left) and InsP₃ receptor (Right) in wild-type neonatal cardiomyocytes. (Scale bar, 10 μm.) (B) Immunolocalization of ankyrin-B (Left) and InsP₃ receptor (Right) in ankyrin-B^+/- neonatal mouse cardiomyocytes. Reduction in ankyrin-B levels leads to loss of InsP₃ receptor expression and organization (compare InsP₃ receptor staining in A and B). (Scale bar, 10 μm.) (C) Abnormal localization of InsP₃ receptor in ankyrin-B^+/- cardiomyocytes is rescued by transfection of GFP-220-kDa ankyrin-B. GFP-220-kDa ankyrin-B was immunolocalized by using affinity-purified GFP antibody. (Scale bar, 10 μm.) (D) R1788W is an ankyrin-B loss-of-function mutation. Expression of GFP-220-kDa ankyrin-B R1788W does not rescue abnormal expression or localization of InsP₃ receptor in ankyrin-B^+/- neonatal cardiomyocytes. GFP-220-kDa ankyrin-B was immunolocalized by using affinity-purified GFP antibody. (Scale bar, 10 μm.) GFP-220-kDa ankyrin-B, GFP-220-kDa ankyrin-B R1788W, and endogenous ankyrin-B (in +/+ cell) are localized in the same striated pattern. The perimeter of the cell has been outlined in B and D.

Fig. 5.

GFP-220-kDa ankyrin-B, but not GFP-220-kDa ankyrin-B R1788W, rescues abnormal Na/Ca exchanger and Na/K ATPase localization in ankyrin-B^+/- cardiomyocytes. (A) Immunolocalization of endogenous ankyrin-B with Na/Ca exchanger and Na/K ATPase in wild-type neonatal cardiomyocytes. (Scale bar, 10 μm.) (B) Immunolocalization of endogenous ankyrin-B with Na/Ca exchanger and Na/K ATPase in ankyrin-B^+/- neonatal mouse cardiomyocytes. Reduction in ankyrin-B levels leads to loss of Na/Ca exchanger and Na/K ATPase expression and organization (compare Na/Ca exchanger and Na/K ATPase staining in A and B). (Scale bar, 10 μm.) (C) Abnormal expression and localization of Na/Ca exchanger and Na/K ATPase in ankyrin-B^+/- neonatal cardiomyocytes is rescued by transfection of GFP-220-kDa ankyrin-B. GFP-220-kDa ankyrin-B was immunolocalized by using affinity-purified GFP antibody. (Scale bar, 10 μm.) (D) R1788W is an ankyrin-B loss-of-function mutation. Expression of GFP-220-kDa ankyrin-B R1788W does not rescue abnormal expression or localization of Na/Ca exchanger or Na/K ATPase in ankyrin-B^+/- neonatal cardiomyocytes. (Scale bar, 10 μm.) GFP-220-kDa ankyrin-B, GFP-220-kDa ankyrin-B R1788W, and endogenous ankyrin-B (in +/+ cell) are localized in the same striated pattern. The perimeter of the cardiomyocyte in D is outlined.

Fig. 6.

Reduction of ankyrin-B does not affect Na_v1.5 expression or localization in ankyrin-B^+/- cardiomyocytes. Immunolocalization of endogenous ankyrin-B and Na_v1.5 in wild-type (A) and ankyrin-B^+/- (B) neonatal cardiomyocytes using affinity-purified antibodies. We observed no difference in Na_v1.5 expression or localization between wild-type and ankyrin-B^+/- cardiomyocytes. The perimeter of the cell is outlined in B. Distance between ankyrin-B striations equals 1.8 μm. (C and D) Quantitative Western blot analysis of Na_v1.5 expression in wild-type and ankyrin-B^+/- adult (C) and neonatal (D) cardiomyocytes using ¹²⁵I-labeled protein A. Protein levels were quantitated by PhosphorImaging. Reduction of ankyrin-B levels in ankyrin-B^+/- cardiomyocytes has no affect on Na_v1.5 expression (n = 3, P > 0.05; not significant).