Silbernagel et al., 2018 - The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function. FASEB journal : official publication of the Federation of American Societies for Experimental Biology   32(11):6159-6173 Full text @ FASEB J.

Figure 3

VAPB determines surface expression and dendritic localization of HCN2. A) Live cell imaging of HeLa cells transfected with an N-terminally EGFP-tagged HCN2 carrying an extracellular HA-epitope (EGFPHCN2HAEx) alone or cotransfected with VAPB or the TM segment of VAPB (TMVAPB). B) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for EGFPHCN2HAEx alone and after cotransfection with VAPB (1.6 ± 0.1). Upper inset illustrates a representative control Western blot showing an unaltered HCN2 protein expression. C) Chemiluminescence surface expression assay as in B, but using TMVAPB (1.6 ± 0.1). D) Immunocytochemistry of HAVAPB transfected cortical neurons. Endogenous HCN2 (green) is colocalizing (white) with HAVAPB (magenta) in the soma and dendrites. Anti–MAP2-staining illustrating an intact neuronal network and dendrites (blue). E) Immunocytochemistry experiment as in D, but transfecting the ALS8 mutation HAVAPBP56S (magenta), leading to an aggregation of VAPBP56S in the soma of the neurons. Also, HCN2 fluorescence (green) was focused in the soma and dendritic localization was lost, despite an intact neuronal network (α-MAP2, blue). Scale bars, 20 µm (A, D, E). All data are presented as means ± sem. The number of experiments (n) is indicated in the respective bar graphs. **P < 0.01 (unpaired Student’s t test).

Figure 4

Codistribution of VAPs with HCN2 and contribution to thalamic Ih. AE), Distribution of HCN2, VAPB, and VAPA mRNA in mouse brain and spinal cord. ISH analysis of HCN2, VAPB, and VAPA using DIG-labeled riboprobes, revealing mRNA expression of VAPB in cortical areas (A), hippocampus (B), thalamus (C), cerebellum (D) (arrows point to interneurons in the granular layer), and spinal cord (E). Note the overlapping distribution of VAPB with HCN2 and VAPA mRNA. Am, amygdala; CA, cornu ammonis; DG, dentate gyrus; DH, dorsal horn; gcl, granule cell layer; Hb, habenulae; ic, internal capsule; LG, lateral geniculate ncl.; m, molecular cell layer; pcl, Purkinje cell layer; RTh, reticular thalamic ncl.; Sth, subthalamic ncl.; VB, ventrobasal thalamus; Th, thalamus; VH, ventral horn. F) Representative current traces elicited in slice patch-clamp experiments of the ventrobasal thalamus (VB) of wild-type animals (control) and VAPB−/− mice. G) The Ih current was significantly reduced in VAPB−/− mice (15.4 ± 1.1 pA/pF) compared with control animals (22.2 ± 2.3 pA/pF). H) Average activation curves of the VB Ih current for control and VAPB−/− mice. V1/2 of activation for control (−91.6 ± 1.3 mV, n = 8) and VAPB−/− (−87.5 ± 1.2 mV, n = 7). Scale bars: 500 µm (A–C, E), 100 µm (D). All data are presented as means ± sem. The number of experiments (n) is indicated in the respective bar graphs. *P < 0.05 (unpaired Student’s t test).

Figure 5

Bradycardia in knock-down zebrafish embryos and VAPB−/− mice. A) Zebrafish embryos at 72 hpf. Control-injected and MO-injected embryos against VAPA (MOVAPA) or VAPB (MOVAPB) exhibit no significant abnormalities (left), particularly no structural heart defects (right). Note a light cardiac edema in MOVAPB and a strong edema in MOVAPA. B) Heart rate in beats per minute (bpm) of control and MOVAPA, MOVAPB, or MOVAPA/B injected zebrafish embryos at 72 hpf. C) Representative examples of calcium transients (relative fluorescence intensity) in the cardiac atrium (black) and cardiac ventricle (gray) of control-injected zebrafish at 72 hpf, displaying a regular atrio-ventricular propagation of excitation from atrium to ventricle in a 1:1-ratio. D, E), Representative calcium transients recorded in MOVAPA/B double knock-down morphants, illustrating strongly reduced heart rates with variable frequency. F) Representative example of atrial and ventricular calcium measurements from a MOVAPA/B morphant with a 2:1 atrio-ventricular block, in which only every second atrial excitation leads to a ventricular excitation. Data were obtained from 3 independent batches of injections (AF). G) Heart rate in beats per minute (bpm) of VAPB−/− mice compared with wild-type littermates (control), analyzed by using tail-cuff measurements. H) Representative surface ECG recordings of VAPB−/− mice and their wild-type littermates (control). ECGs of VAPB−/− mice show bradycardia and an increased T-wave amplitude. IN) Analyses of the ECG parameters of VAPB−/− mice. I) Heart rate in beats per minute (bpm). J) PQ interval (PQ) duration. K) QRS complex (QRS) duration. L) Frequency corrected QT interval (QTc). M) Frequency-corrected Tpeak to Tend duration (Tp-Tec). N) T-wave amplitude (JTp). Scale bars: 500 µm (A); 500 ms (CF), and 100 ms (H). All data are presented as means ± sem. The number of animals (n) is indicated in the respective bar graphs. N.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001 [Student’s t test (G, H, J–N) or Welch’s t test (B, I)].

Figure 6

VAPB modulates If of spontaneously beating cardiac HL-1 cells. A) Immunocytochemistry of VAPB in HL-1 cells. Scale bar, 20 µm B) Western blot illustrating the knock-down of VAPB expression in HL-1 cells by shRNA transfection. Control, HL-1 cells transfected with scrambled shRNA. C) Representative If currents of HL-1 cells under control conditions and after VAPB transfection. D) Percentage of HL-1 cells containing If under control conditions (38%) and after VAPB transfection (58%). E) Beating frequency under control conditions (158 ± 4) and after VAPB transfection (179 ± 4), analyzed by optical counting of contractions in original Claycomb medium containing norepinephrine (60). F) Accelerated activation kinetics of VAPB-transfected HL-1 cells (n = 9–10). G) Activation curves of HL-1 cells under control conditions (n = 10) and after VAPB transfection (n = 11). H) Positive shift in the V1/2 of activation of If recorded in VAPB transfected HL-1 cells. Control (scrambled shRNA), −90.3 ± 3.4 mV (n = 10); VAPB transfected, −79.6 ± 2.7 mV (n = 11). I) Representative action potential measurements of wild-type HL-1 and shRNA transfected cells (shVAPB). J) Analysis of the diastolic depolarization (DD duration). K) Beating frequency of HL-1 cells under control conditions and after VAPB knock-down. L) VAPB knock-down slows the activation kinetics (n = 5) of endogenous If currents. M) Transfection of shRNA (n = 5) shifts the voltage-dependence of activation (V1/2) of If to more negative potentials (n = 6). N) V1/2 values for control (scrambled shRNA) were −88.2 ± 3.1 mV (n = 6) and for shRNA-transfection (shVAPB), −96.2 ± 2.3 mV (n = 5), respectively. (I, J), Scrambled shRNA was used as control. All data are presented as means ± sem. The number of experiments (n) is indicated in the bar graphs. N.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001 [unpaired Student’s t test (D, G, H, M) or Mann-Whitney U test (E, F, JL, N)

Figure S8

Cardiac expression of VAPB in embryonic zebrafish hearts, rescue experiments after MO-VAPB knock-down and overexpression of VAPBP56SA) Immunostaining with an α-VAPB antibody (see Supplementary Methods) showing an uniform expression of VAPB in embryonic zebrafish hearts at 72 hours post fertilization. A, atrium. V, ventricle.B) Two representative embryonic zebrafish hearts stained against VAPB, either after injection with control morpholinos or with the VAPB knock-down morpholino, respectively. The embryonic zebrafish heart was co-stained against DAPI to show the cell nuclei. The experiments illustrate an efficient knock-down of VAPB and specificity of the antibody. C) After morpholino-antisense mediated VAPB knock-down (MOVAPB), the injection of cRNA encoding for either VAPB or D) VAPA rescued the reduction in embryonic heart rates. Data were obtained from four and three independent batches of injections, respectively. E) The injection of cRNA for the human VAPA or VAPB did not alter embryonic heart rates. Moreover, injection of VAPBP56S cRNA is likely to not act in a dominant-negative manner on zebrafish Ichannels, as the heart rates are not altered. Data were obtained from three independent batches of injections. All data are presented as mean ± s.e.m.. The number of experiments (n) are indicated in the bar graphs. ***, P < 0.001 using an unpaired Welch́s T-test.

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ZFIN wishes to thank the journal FASEB journal : official publication of the Federation of American Societies for Experimental Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ FASEB J.