Ion Channel Dysfunction Associated With Arrhythmia, Ventricular Noncompaction, and Mitral Valve Prolapse: A New Overlapping Phenotype

In this issue of the Journal, two elegant papers report the association of arrhythmia, primarily sinus bradycardia, the myocardial disease called left ventricular noncompaction (LVNC), and mitral valve prolapse (MVP) (1, 2) and show that the underlying cause is mutations and dysfunction of the hyperpolarization-activated cyclic nucleotide channel 4 (HCN4), a major constituent of the pacemaker current (If) in the sinoatrial node (SAN) (3). The authors demonstrate abnormalities in channel function consistent with the arrhythmia phenotype and speculate as to the underlying pathogenesis that leads to LVNC, a heterogeneous myocardial phenotype associated with abnormal trabeculation of the left ventricle (LV) (4). Uncertainty, however, belies the question of how do mutations in this ion channel cause the combined phenotype. To develop a plausible hypothesis, understanding the data reported by these two studies, as well as a review of prior studies is required.

Schweizer et al. (1) report identification of HCN4 mutations in two unrelated families and an additional unrelated proband with sinus node dysfunction/bradycardia (SND), LVNC, and MVP. Using a candidate gene approach, they identified a novel HCN4-G482R loss-of-function mutation located within the highly conserved GYG motif of the channel pore domain that segregated with all affected members in the 4-generation index family (5). The common W4R variant in the cysteine and glycine-rich protein 3 (CSRP3) gene encoding a Z-disk protein, previously reported in patients with dilated (DCM) and hypertrophic cardiomyopathy (HCM) and healthy subjects was also identified (6, 7). In the other family and unrelated proband, truncation (HCN-695X) and missense (HCN-P883R) HCN4 mutations were identified with no mutations identified in CSRP3. Family members and probands of all three families had severe SND with or without atrial or ventricular arrhythmias, syncope or sudden death and normal QTc. Noninvasive imaging demonstrated biventricular hypertrabeculation/LVNC, and MVP as well. Patch-clamp studies demonstrated no hyperpolarization-activated inward currents in mutant HCN4-G482R subunits, consistent with loss-of-function. Homozygous HCN4-G482R channels were nonfunctional, and heteromeric mutant and wild-type (WT) HCN4 channel subunits had 65% current reduction, consistent with a dominant-negative mechanism, resulting in If current reduction in heterozygotes and lower current densities.

Milano and colleagues (2) report four families with SND with or without syncope/cardiac arrest, ventricular and atrial arrhythmias and echocardiography demonstrating LVNC with or without MVP. HCN4 mutations were identified in all families (Tyr481His in two families, Gly482Arg and Ala414Gly in one family each). All mutations affected conserved residues with two mutations (Tyr481His, Gly482Arg) affecting highly conserved residues within the pore domain of HCN4 and the other (Ala414Gly) affecting the cytoplasmic S4-S5 linker of HCN. Heterologous expression studies with theTyr481His and Gly482Arg mutations demonstrated a large negative shift of the voltage-dependency of activation compared to expression of WT channels, indicating the importance of the pore region for the voltage-dependency of activation. All mutations resulted in significantly lower HCN4 current density.

Together, these studies demonstrate that HCN4 mutations result in loss of function and significantly reduced If current density associated with bradycardia, arrhythmias, and LVNC with or without MVP. Conceptually, these findings are consistent with the “final common pathway” hypothesis proposed nearly 15 years ago which suggests that mutations in genes encoding proteins within the same pathway (or secondary disturbance of protein function due to binding partner abnormalities, drugs, etc) leads to a common phenotype (8). This hypothesis enabled successful targeted candidate gene screening for arrhythmias, cardiomyopathies, and congenital heart disease (CHD) leading to the current understanding that arrhythmias are caused by disturbed ion channel function (“ion channelopathies”), HCM by disturbed sarcomere function, DCM by disturbed sarcomere and cytoskeleton function, and arrhythmogenic right ventricular cardiomyopathy (ARVC) by disturbed desmosome function (9). For LVNC, the picture is less clear; mutations most commonly occur in sarcomere-encoding genes, but animal and human data suggests a central role of signaling pathways. In the cardiomyopathies,, ion channel gene mutations have also been implicated but the causative mechanism(s) remain unclear.

HCN channels, found in SAN cells and neurons, are responsible for hyperpolarization-activated currents, called If in the heart (3, 5). The HCN channel characteristic distinguishing them from other currents is its unique ion selectivity and gating properties. The HCN channel family has four distinct members (HCN1-4), with HCN4 being the prominent cardiac form. Native If current, as well as the currents induced by heterologously expressed HCN channels, have four hallmark properties: 1) channel activation by membrane hyperpolarization, 2) channel activation by direct interaction with cAMP, 3) Na⁺ and K⁺ permeability, and 4) a specific pharmacological profile. HCN channels consist of four subunits arranged around the centrally located pore forming four different homotetramers with distinct biophysical properties. Each channel subunit consists of: 1) the transmembrane core harboring the gating machinery and ion-conducting pore, 2) the cytosolic NH2-terminal domain, and 3) the COOH-terminal domain with the cyclic nucleotide binding domain (CNBD) and the peptide connecting the CNBD with the transmembrane core (the “C-linker”) that confers modulation by cyclic nucleotides. If current is important in the initiation and regulation of the heart beat and therefore is called the “pacemaker current”. Mutation in the HCN4 gene, located on chromosome 15q24.1, was first reported by Schulze-Bahr et al. in a patient with SND, atrial fibrillation, and chronotropic incompetence (10). The 1 bp deletion mutation (HCN4-573X) resulted in a premature stop codon and a C-terminus lacking the CNBD domain. In vitro heterologous expression revealed a dominant-negative loss of cAMP modulation. Several other publications demonstrating SND with severe bradycardia, with or without atrial fibrillation or ventricular arrhythmias, have now been reported. These findings with HCN4 mutations would be predicted by the “final common pathway” hypothesis: ion channels cause rhythm disturbance. However, Schweizer et al and Milano and colleagues report the additional phenotypes of LVNC and MVP that would not be predicted to result solely from an ion channel mutation (1, 2). Schweizer et al reported one family with a CSRP3 variant which is more in line with the causes of myocardial disease but this was not seen in other gene-positive families (1). So how does LVNC occur?

Neither publication presents mechanistic data but the authors speculate on how LVNC and MVP develop. Schweizer et al. noted that HCN4 is involved in early embryonic heart development, helping to form myocardium and the conduction system (1). During later development HCN4 is down-regulated in the myocardium, with abundant expression restricted to SAN and conduction system. They hypothesize that HCN4 loss of function interferes with molecular mechanisms required during cardiac development, resulting in LVNC. Samsa et al previously suggested that Notch-pathway disturbance causes LVNC with CHD while sarcomere, cytoskeletal, and Z-disk mutations cause myocardial disease-only phenotypes (11). Based on this, Schweizer et al. suggested signaling pathway involvement in ventricular wall maturation and compaction (i.e. Notch, Neuregulin, Ephrin or Bone morphogenic protein), could be involved (1). Milano et al, on the other hand, suggested that since primary channelopathies are associated with myocardial structural abnormalities such as DCM, this also occurs with HCN4 (2). Their second hypothesis was that LVNC is an acquired adaptive remodeling feature in response to sinus bradycardia.

One feature of the “final common pathway” hypothesis that may be at play here is the concept of secondary disruption of the pathway via binding partner abnormalities or other secondary causes. Examples exist where a mutation in a non-ion channel encoding gene, such as caveolin-3 (Cav3) or α-syntrophin 1 (SNTA1), disturbs the function of an ion channel binding partner protein such as the cardiac sodium channel gene, SCN5A, resulting in an SCN5A form of long QT (LQT) syndrome (LQT3) (12, 13). We termed these non-ion channel proteins ChIPs (Channel Interacting Proteins). Many other similar examples exist. Using this example, we could hypothesize that HCN4 mutations cause the arrhythmia phenotype, and also disturb downstream binding partners that cause LVNC (sarcomere, Z-disk, cytoskeletal proteins, or signaling pathways). There is a relative paucity of information regarding HCN4 binding partners but these include Cav3, MiRP1 (encoded by the gene KCNE and shown to be an auxiliary subunit of HERG channel), KCR1 (plasma membrane associated protein that associates with HERG), SAP97 (membrane-associated guanylate kinase scaffold protein), and cyclic AMP. Several of these binding partners are interesting as potential ChIPs-like proteins. For instance, mutated Cav3 disrupts SCN5A function causing LQT3 and arrhythmias (12). In some patients with SCN5A disruption, an arrhythmogenic DCM phenotype develops. Cav3, SNTA1, and SCN5A also bind to dystrophin, the protein that causes Duchenne and Becker muscular dystrophy with DCM or LVNC, as does SAP97 (4, 14). Could a mutation in HCN4 disrupt the binding of Cav3, SNTA1 or SAP97 and dystrophin and be the cause of LVNC in these patients? Another possibility is disturbance of signaling pathways leading to an overlapping phenotype. Notch signaling promotes expression of conduction system–specific genes in neonatal cardiomyocytes, reprogramming them into cells with conduction system characteristics (15). Human and animal studies suggest Notch pathway is involved in development of LVNC with or without CHD. Kuratomi et al demonstrated that HCN4 enhancer function is dependent on myocyte enhancer factor-2 (MEF2) binding sequences located in the regulatory region of HCN4 (16). Overexpression of a dominant-negative MEF2 mutant inhibits enhancer activity, decreases HCN4 mRNA expression and decreases If current amplitude, suggesting MEF2 may play a critical role in HCN4 transcription. MEF2 signaling pathway molecules interact with Notch pathway molecules including Hey2 and Tbx20, which are important in the development of the myocardial compact and noncompact layers and are directly affected in some forms of LVNC (17). It is possible that the relationship of mutant HCN4 and MEF2 triggers a downstream spiral that disturbs Notch pathway function and results in LVNC with or without MVP while bradycardia occurs due to the primary HCN4 mutation.

In any case, the findings reported by the authors are intriguing and potentially paradigm-shifting. If they or others can determine the pathogenic mechanism(s) responsible for this overlapping phenotype, it would enhance our knowledge and enable targeted treatment development, especially since LVNC is commonly associated with arrhythmias (18).