Journal: Theranostics

Article Title: Stress-induced premature senescence is associated with a prolonged QT interval and recapitulates features of cardiac aging

doi: 10.7150/thno.70884

Figure Lengend Snippet: Alterations of I Kr , I NaL and Ca 2+ handling in SenCMs. (A) E-4031 (3µM)-sensitive currents (I Kr ) activated according to the protocol shown on top and relative I/V relationships in iCMs (N = 21) and SenCMs (N = 26) 5-7 days after treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (B) HERG (KCNH2) protein expression levels in iCMs and SenCMs. Quantitative data of four independent experiments ± SEM (densitometric values for the protein of interest normalized for histone H3). ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (C) TTX (2 µM)-sensitive current (I TTX ) activated during slow voltage ramps (28 mV/sec) from a holding potential of -100 mV. Mean ± SEM I/V relationships for iCMs (N = 12) and SenCMs (N = 13) are shown. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. Statistics of I TTX at 0 mV, representative of I NaL , are reported on the right. (D) pThr286 CAMKII:CAMKII total protein expression levels in SenCMs versus iCMs. Quantitative data are from four independent experiments ± SEM (densitometric values for the proteins of interest normalized for GAPDH). * P < 0.05, ** P < 0.01 vs iCMs. (E) Membrane currents and Ca 2+ transients (CaT) were recorded simultaneously according to the voltage clamp protocol shown on top in Fluo4-loaded iCMs. Examples (left panel) and statistics (right panel) of CaL influx, CaT amplitude and caffeine-induced CaT amplitude (estimating CaSR) in iCMs (N = 22) and SenCMs (N= 14) 5-7 days after Dox treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (F) SERCA2 and monomeric (m) PLN protein expression levels in iCMs and SenCMs. Quantitative data of five independent experiments ± SEM (densitometric values for the protein of interest normalized for actin). (G) Statistics of Ca 2+ spark rate and characteristics in SenCMs (N=410) vs iCMs (N=266). Line scan (xt) images are shown on the left (time bar: 100 ms). * P < 0.05, ** P < 0.01 vs iCMs.

Article Snippet: The membranes were blocked for 1 h with Intercept (TBS) Blocking Buffer (Licor) or milk and incubated with the primary Abs at 4 °C overnight (anti-p16, 1:1'000, Proteintech 10883; anti-p21, 1:1'000 eBiosciences 14-6715-81; anti-KCNH2, 1:200, Alomone APC-109; anti-P AMPK, 1:1'000, Cell Signaling 2537; anti-CAMKII, 1:2'000, Abcam 92332; anti-P CAMKII, 1:2'000, Abcam 171095; anti-SERCA2, 1:1000, N-19 Santa Cruz Biotechnology; anti-PLN 1:1000, 2D12, Abcam; anti-actin 1:5000, Merck).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Mutagenesis, Binding Assay, Sequencing

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Western Blot, Transfection, Mutagenesis, Produced, Molecular Weight, Expressing, Cotransfection, Immunoprecipitation, Construct, Binding Assay

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Dominant Negative Mutation, Construct, Transfection, Activation Assay

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Staining, Transfection, Immunocytochemistry, Confocal Microscopy, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Expressing, Transfection, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Incubation, Western Blot, Cotransfection

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Incubation, Expressing