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Pflugers Arch
2020 Dec 01;47212:1733-1742. doi: 10.1007/s00424-020-02481-3.
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Disease-associated HCN4 V759I variant is not sufficient to impair cardiac pacemaking.
Erlenhardt N
,
Kletke O
,
Wohlfarth F
,
Komadowski MA
,
Clasen L
,
Makimoto H
,
Rinné S
,
Kelm M
,
Jungen C
,
Decher N
,
Meyer C
,
Klöcker N
.
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The hyperpolarization-activated cation current If is a key determinant for cardiac pacemaker activity. It is conducted by subunits of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family, of which HCN4 is predominant in mammalian heart. Both loss-of-function and gain-of-function mutations of the HCN4 gene are associated with sinus node dysfunction in humans; however, their functional impact is not fully understood yet. Here, we sought to characterize a HCN4 V759I variant detected in a patient with a family history of sick sinus syndrome. The genomic analysis yielded a mono-allelic HCN4 V759I variant in a 49-year-old woman presenting with a family history of sick sinus syndrome. This HCN4 variant was previously classified as putatively pathogenic because genetically linked to sudden infant death syndrome and malignant epilepsy. However, detailed electrophysiological and cell biological characterization of HCN4 V759I in Xenopus laevis oocytes and embryonic rat cardiomyocytes, respectively, did not reveal any obvious abnormality. Voltage dependence and kinetics of mutant channel activation, modulation of cAMP-gating by the neuronal HCN channel auxiliary subunit PEX5R, and cell surface expression were indistinguishable from wild-type HCN4. In good agreement, the clinically likewise affected mother of the patient does not exhibit the reported HCN4 variance. HCN4 V759I resembles an innocuous genetic HCN channel variant, which is not sufficient to disturb cardiac pacemaking. Once more, our work emphasizes the importance of careful functional interpretation of genetic findings not only in the context of hereditary cardiac arrhythmias.
Fig. 1. Detection of the disease-associated HCN4 V759I variant in a 49-year-old patient with sick sinus syndrome. a Baseline 12-lead electrocardiogram of the patient, who suffered from dizziness and nausea, showing sinus bradycardia (37 bpm, cycle length 1600 ms) without any evidence for additional disturbances of cardiac depolarization or repolarization (PR-interval, 140 ms; QRS, 100 ms; QT-interval, 440 ms (QTc, 348 ms)). b Patient’s heart rates during bicycle exercise before pacemaker implantation. Predicted maximum heart rate (reference interval, 90–110%) and predicted maximum workload corrected for age are indicated by horizontal and vertical dotted lines, respectively [13]. c Atrial and ventricular frequency histograms from pacemaker interrogation two months after implantation showing a proportion of 42.3% atrial and < 0.1% ventricular pacing (lower intervention frequency: 45 bpm). d Direct sequencing of all exons of the HCN4 gene revealed heterozygous variance in exon 8, which results in the single amino acid exchange V759I
Fig. 2. The biophysical properties of the disease-associated HCN4 V759I variant are indistinguishable from wild-type HCN4. a Representative currents recorded in Xenopus laevis oocytes expressing wild-type (WT), variant (V759I), and co-expressing both wild-type and variant HCN4 channels (WT/V759I) in response to voltage steps to potentials between − 20 and − 150 mV in 10 mV increments (holding potential − 20 mV, tail potential − 150 mV). Scale bar: 5 μA and 2 s. b Steady-state activation curves of wild-type and variant HCN4 channels as indicated. Data points are mean ± SEM of n = 12–17 oocytes of N = 2 experiments (individual batches of oocytes). Lines are fits of a Boltzmann function to the data with values for V1/2 of − 100.4 ± 2.0 mV (slope factor 14.0 ± 0.3 mV) for WT (n = 12), − 101.5 ± 1.9 mV (slope factor 14.6 ± 0.4 mV) for V759I (n = 12), and − 101.2 ± 1.5 mV (slope factor 12.5 ± 0.2 mV) for co-expressed WT/V759I channels (n = 17). c Representative currents recorded from inside-out patches from X. laevis oocytes expressing wild-type (WT) and variant (V759I) HCN4 channels in response to voltage steps to potentials between − 60 and − 160 mV in − 10 mV increments (holding potential 0 mV, tail potential + 50 mV). Traces in red are current responses to a step potential of − 150 mV. Scale bars: 75 pA, 200 ms. d Quantification of cAMP-mediated modulation of voltage-dependent activation of wild-type (WT) and variant (V759I) HCN4 channels in inside-out patches (Materials and Methods). Left, half-maximal activation voltages (V1/2) prior cAMP application for wild-type (WT) and variant (V759I) HCN4 channels; right, shifts of V1/2 (ΔV1/2) by application of cAMP (100 μM) in HCN4 channels as indicated. Box plots display median and 25/75 percentiles, whiskers indicate outliers. n.s., not significant (n = 8). e Steady-state activation curves of wild-type (WT) and variant (V759I) HCN4 channels with (open symbols) and without (filled symbols) co-expression of the brain HCN channel auxiliary subunit PEX5R. Data points are mean ± SEM of n = 6–10 oocytes of N = 2 TEVC experiments (individual batches of oocytes). Lines are fits of a Boltzmann function to the data with values for V1/2 of − 100.9 ± 1.6 mV (slope factor 12.3 ± 0.1 mV) for WT (n = 8), − 108.9 ± 1.2 mV (slope factor 10.5 ± 0.3 mV) for WT + PEX5R (n = 6), − 99.6 ± 1.2 mV (slope factor 13.3 ± 0.2) for V759I channels (n = 9), and − 109.1 ± 0.9 mV (slope 11.0 ± 0.4) for V759I + PEX5R co-expressed channels (n = 10). f Activation kinetics of wild-type (WT) and variant (V759I) HCN4 channels with (open symbols) and without (filled symbols) co-expression of the brain HCN channel auxiliary subunit PEX5R. Time constants (τ activation) were calculated from mono-exponential fits of current activation at indicated voltage steps (holding potential − 20 mV; n = 4–21 oocytes)
Fig. 3. Surface trafficking of disease-associated HCN4 V759I variant is not disturbed. a Representative micrographs of embryonic rat cardiomyocytes (E18), 16 h after viral transduction with wild-type (WT) or variant (V759I) HCN4 with an extracellular hemagglutinin (HA) tag. Surface and intracellular populations of channels were detected by monoclonal anti-HA immunocytochemistry without and after plasma membrane permeabilization, respectively. Scale bar: 20 μM. b Quantification of wild-type (WT) and variant (V759I) HCN4 channel surface expression [18]. Data are given as mean ± SEM calculated as surface/(surface + intracellular) fluorescence intensities normalized to wild-type control (relative (rel) surface expression, n = 22 cells of N = 3 experiments)
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