Journal: bioRxiv

Article Title: Cardiac electrophysiological remodeling associated with enhanced arrhythmia susceptibilty in a canine model of elite exercise

doi: 10.1101/2022.07.13.499876

Figure Lengend Snippet: Two bar diagrams on the left show the relative protein expression of Kv4.3 and KChiP2 subunits determined by Western blot in sedentary (n=12 dogs) and trained dog (n=12) left ventricular samples. Bottom panels indicates the representative Kv4.3 and KChIP2 bands and their corresponding loading control (GAPDH). Two bar diagrams on the right panels show the relative density of dog cardiomyocytes with Kv4.3 and KChiP2 immunolabelling obtained from the sedentary (n=30 cells/6 dogs) and trained (n=30 cells/6 dogs) groups. Bottom panels on the right show original immunofluorescent images of dog cardiomyocytes with Kv4.3 and KChiP2 immunolabelling. Figure 6–Source Data 1 Protein expression of Kv4.3 subunit determined by Western blot technique in sedentary and trained dogs. Figure 6–Source Data 2 Protein expression of KChiP2 subunit determined by Western blot technique in sedentary and trained dogs. Figure 6–Source Data 3 Relative density of Kv4.3 subunit determined by immunocytochemical technique in sedentary and trained dogs. Figure 6–Source Data 4 Relative density of KChiP2 subunit determined by immunocytochemical technique in sedentary and trained dogs. Figure 6–Source Data 5 Original, unedited membranes of western blots with the relevant bands clearly labelled. Figure 6–Source Data 6 Original files of the full raw unedited membranes of western blots.

Article Snippet: The membrane was blocked with 2.5% non-fat milk for 1 hour at room temperature and immunolabelled overnight at 4°C with anti-KChIP2 (Alomone, #APC-142, RRID:AB_2756744) and anti-Kv4.3 (Alomone, #APC-017, RRID:AB_2040178) primary antibody diluted to 1:1000.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Long-Term Fish Oil Supplementation Induces Cardiac Electrical Remodeling by Changing Channel Protein Expression in the Rabbit Model

doi: 10.1371/journal.pone.0010140

Figure Lengend Snippet: ( A ) – ( D ) Immunoblot images of rabbit and rat hearts (except (A), right lane, rat brain), probed with Abs marked on top. Kv1.4 was detected in whole tissue lysates (WTL), while the other three proteins were detected in membrane-enriched fraction (Memb). ( E ) Lengths, accession numbers and molecular sizes (in kDa) of rat and rabbit channel subunit orthologs currently available in the NCBI protein database. The Kv1.4 sizes are those of unglycosylated forms, which are smaller than the N-glycosylated forms shown in (A). Kv4.2 and Kv4.3 do not have N-glycosylation signals. The rabbit Kv4.3 we detected (∼60 kDa, panel (C), left lane) was likely the short isoform (62.4 kDa). KChIP2 is a cytosolic protein, and thus is not glycosylated.

Article Snippet: After fractionation, the proteins were blotted to PVDF membranes (Amersham), and probed with the following antibodies: Cav1.2 mAb (NeuroMabs), Cav1.1 mAb (Abcam), Kv4.2 pAb (Sigma), Kv4.3 pAb (Alomone), KChIP2 mAb (NeuroMabs), Kv1.4 mAb (NeuroMabs) and hERG pAb (Alomone).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Long-Term Fish Oil Supplementation Induces Cardiac Electrical Remodeling by Changing Channel Protein Expression in the Rabbit Model

doi: 10.1371/journal.pone.0010140

Figure Lengend Snippet: ( A ) Immunoblot images of Kv4.2, Kv4.3, and KChIP2 (∼80 ug/lane). ( B ) Immunoblot images of Kv1.4 (∼160 ug/lane). Size marker bands (in kDa) are listed on the left. Sizes for proteins of interest are marked on the right. Note that for panel (A) here and , each channel subunit immunoblot was corrected by its own α-actin immunoblot for loading variations, although only one representative α-actin immunoblot is shown. Loading variation in Fig. 6B was further checked by coomassie blue (CB) stain. ( C ) Data summary: background-subtracted band intensities were divided by corresponding α-actin band intensity, and then normalized by the mean value of control lanes.

Article Snippet: After fractionation, the proteins were blotted to PVDF membranes (Amersham), and probed with the following antibodies: Cav1.2 mAb (NeuroMabs), Cav1.1 mAb (Abcam), Kv4.2 pAb (Sigma), Kv4.3 pAb (Alomone), KChIP2 mAb (NeuroMabs), Kv1.4 mAb (NeuroMabs) and hERG pAb (Alomone).

Techniques: Western Blot, Marker, Staining

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: A. Plasmid vector for bicistronic for Kv4.3 and KChIP2.1 with amino acids shown in black (linkers in brown). A CMV or cardiac-specific troponin T promoter (cTnT) was used in different cell types. B. Western blot of KChIP2.1 expression by the bicistronic transgene in HEK293 cells. A single band corresponding to the estimated molecular weight of KChIP2.1 or KChIP2.1-Amcyan was detected in lysates from bicistronic transgene transfected cells but not from cells transfected with Kv4.3-mCherry alone. C Expression of Kv4.3-mCherry upstream of the P2A sequence was detected at an apparent molecular weight of ~80 kDa while expression of Kv4.3 alone was detected at ~60 kDa. These data are consistent with the expected weights of Kv4.3 (71 kDa) and mCherry (20 kDa), given a − 20% gel shift for K channel protein . Additional higher molecular weight bands possibly reflect multimeric forms of Kv4.3. Lysates of cells transfected with untagged Kv4.3 and Amcyan-P2A-mCherry were used as positive and negative controls respectively and SYPRO® staining was used as internal loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

Techniques: Plasmid Preparation, Western Blot, Expressing, Molecular Weight, Transfection, Sequencing, Electrophoretic Mobility Shift Assay, Staining

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: Biophysical properties of I to,f currents produced by Kv4.3 alone (-KChIP2.1) and with KChIP2.1 added via the bicistronic vector in HEK293 cells. A. Exemplar whole-cell currents elicited by 500 ms-depolarization pulses from −60 mV to +40 mV in 10 mV increments from a holding potential of −80 mV. B. The peak current at all potentials was increased by bicistronic addition of KChIP2.1 and the current activated at ~ − 30 mV as expected for Kv4.3 in the presence of KChIP2.1. C. The fast time constant of I to,f inactivation as a function of test potential (Kv4.3 alone: 7.66 ± 1.5 ms, n = 5 vs. Bicistronic Kv4.3/KChIP2.1: 21.2 ± 1.6 ms, n = 18 at +40 mV; p < 0.002 Students t -test). D. Time-dependent recovery from inactivation at −80 mV. To calculate the recovery rate from inactivation, normalised tail peak current amplitudes were recorded at +40 mV and plotted as a function of the inter-pulse interval (Δt) (protocol illustrated in the inset). Error bars show mean ± s.e.m.

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

Techniques: Produced, Plasmid Preparation

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: Subcellular colocalization of Kv4.3 and KChIP2.1. A. Exemplar confocal images of living HEK293 cells expressing bicistronic Kv4.3-mCherry and KChIP2.1-AmCyan. B. Representative confocal images of a transduced rabbit ventricular cardiomyocytes expressing Kv4.3-mCherry and KChIP2 (see also Fig. S2A). Surface membranes were labelled with an anti-Cav3 antibody (top). C shows an enlarged view of the boxed regions in B and the fluorescence intensity profile from each label at bottom. D Co-localization between Kv4.3 and KChIP2.1 in different subcellular compartments (sarcolemma -SL; t-tubules TT; and remaining cytoplasm -Cyt.) measured by unbiased Pearson's correlation coefficient (n/ N = 8/3). Error bars show s.e.m.

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

Techniques: Expressing, Fluorescence

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: Characterization of I to,f currents encoded by the bicistronic Kv4.3/KChIP2.1construct in rabbit ventricular cardiomycytes. A. Typical patch-clamp recordings of I to currents from freshly isolated cardiomyocytes and 40 h after transduction. Currents were evoked by step depolarizations to potentials between −30 and + 40 mV (in 10 mV increments). B. The left panel shows current-voltage relationships of intrinsic I to (n/ N = 7/3) and exogenous I to,f (n/ N = 16/8) and the fast time constant of I to,f inactivation (right hand panel). C. Concentration-dependence of the I to,f block by 4-AP block (n/N = 7/4). Exemplar recordings of the I to,f currents at +30 mV before and after application of 1 mmol/l of 4-AP are shown in the inset. D. Exemplar I to,f traces recorded from a transduced myocyte under basal conditions and in the presence of NS5806 (5 μmol/l). The right hand panel shows the augmentation of the mean peak I to,f current-voltage relationship by 5 μmol/l NS5806 ( p < 0.001, n/N = 7/3, matched pair t-test). The cells used for these experiments were cultured for 48 h (40 h in A,B) to further increase peak I to,f to minimise the contribution from NS5806-augmented native I to (typically 3 pA/pF ). Error bars show mean ± s.e.m.

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

Techniques: Patch Clamp, Isolation, Transduction, Concentration Assay, Blocking Assay, Cell Culture

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: Electrophysiological effects of culture and viral bicistronic transduction in cardiomyocytes.

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

Techniques: Transduction, Isolation

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: Increasing I to,f density with bicistronic Kv4.3/KChIP2.1 expression affects cardiomyocyte AP waveform. Panel A shows APs recorded from 2 day cultured (untransduced black), and transduced myocytes with increasing I to,f current densities at a cycle length of 1 s. Introduction of I to,f (lower panel traces show exemplar records for pulses to −30, +10 and + 40 mV) resulted in a prominent AP phase 1 in all transduced myocytes. As I to,f density increased, there was a progressive change in AP morphology that caused the AP to resemble that recorded in other species. B. Increasing density of I to,f (at +40 mV) decreased APD20. APD90 (right panel) decreased approximately exponentially (solid line) with increasing I to,f except when I to,f was between ~12 and ~ 17 pA/pF (deviation highlighted as dashed line) where emergence of a marked ‘spike-and-dome’ morphology developed (as shown center right in A). All trend curves drawn by eye. C. Representative traces of I to,f recorded in untransduced (Control) iPSC-CMs and iPSC-CMs expressing bicistronic Kv4.3-mCherry/KChIP2.1 under a cTnT promoter. After a brief step to −40 mV to inactivate Na + channels, subsequent membrane depolarizations to −30 mV, −10 mV, +10 mV, +30 mV and + 50 mV were used to elicit I to in control and transduced iPSC-CMs. D. Three examplar AP waveforms of iPSC-CMs without (black traces) or with expressed I to,f currents (green lower traces). I to,f densities in the exemplar transduced iPSC-CMs were 5–10 pA/pF at +40 mV. APs were elicited at a frequency of 1 Hz. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

Techniques: Expressing, Cell Culture

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Inward Rectifier K + Currents Contribute to the Proarrhythmic Electrical Phenotype of Atria Overexpressing Cyclic Adenosine Monophosphate Response Element Modulator Isoform CREM‐IbΔC‐X

doi: 10.1161/JAHA.119.016144

Figure Lengend Snippet: Experimental animals involved wild‐type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM‐IbΔC‐X, and were distributed according to age in 6‐week‐old (WT 6w and TG 6w ) and 12‐week‐old (WT 12w and TG 12w ) groups. A , Representative transient outward K + current (I to ) traces recorded as −40 mV sensitive currents from atrial cardiomyocytes of WT 12w and TG 12w . For clarity, current density (expressed in picoamperes/picofarads [pA/pF]) traces from −40 mV with +20 mV increments are displayed. B , Mean±SE of current–voltage plots of the peak current density (I to ). For WT 6w , n/N are 41/15, for TG 6w n/N are 22/14, for WT 12w n/N are 35/19, and for TG 12w n/N are 20/16. Ca through d , Representative immunoblots and quantification of protein expression of calsequestrin (CSQ; Ca ) normalized to Ponceau and of K + voltage‐gated channel subfamily D member 2 (Kv4.2, Cb ) and 3 (Kv4.3, Cc ), and K + channel interacting protein 2 (KChIP2) ( Cd ) normalized to CSQ. Data show mean±SE of N=7 mice per group, except for Kv4.3, where N=6 as the result of an undetectable band in TG 12w . * P <0.05 TG 6w vs WT 6w (gray), TG 12w vs WT 12w (black), # P <0.05 WT 12w vs WT 6w (gray), TG 12w vs TG 6w (black) from 2‐way repeated‐measures analysis of variance with Bonferroni post hoc test ( B ) and parametric or nonparametric 1‐way analysis of variance with the corresponding post hoc test ( C ).

Article Snippet: The following rabbit polyclonal primary antibodies were used against K + voltage‐gated channels (Kv) subunits Kv4.2 (1:200, APC 023, Alomone Labs, Jerusalem, Israel), Kv4.3 (1:200, APC 017, Alomone Labs), K + channel interacting protein 2 (KChIP2) (1:200, sc‐25685, Santa Cruz Biotechnology Inc, Santa Cruz, CA), and K + inwardly rectifying channel (Kir) subunits Kir2.1 (1:200, APC 159, Alomone Labs), Kir2.3 (1:1000, APC 032, Alomone Labs), Kir3.1 (1:200, APC 005, Alomone Labs), Kir3.4 (1:200, APC 027, Alomone Labs), and as loading control calsequestrin (1:2500, PA1‐913, Thermo Fisher Scientific).

Techniques: Transgenic Assay, Expressing, Western Blot