The expression of lipid-depleting dimerization constructs dramatically reduces <i>I</i>_Ks prior to rapamycin addition.

<p><b>A</b>. Representative traces of currents recorded from HEK-<i>I</i>_Ks cells (control), and HEK-<i>I</i>_Ks cells transiently expressing PJ-DEAD, PJ-SAC, PJ-INPP5E or PJ with LYN₁₁-FRB. Mean CD <b>(B)</b> and PTCD <b>(C)</b> of currents from HEK-<i>I</i>_Ks cells transiently expressing PJ (n = 11), PJ-SAC (n = 23), PJ-INPP5E (n = 24) or PJ-DEAD (n = 16) with LYN₁₁-FRB. <b>D</b>. Representative traces of the effect of PJ expression alone (without LYN₁₁-FRB) on <i>I</i>_Ks in HEK-<i>I</i>_Ks cells. <b>E</b>. Effect of PJ expression alone (without LYN₁₁-FRB) on <i>I</i>_Ks PTCD in HEK-<i>I</i>_Ks cells (PJ-expressing cells, n = 12; control (untransfected cells), n = 12). Data are presented as mean ± S.E.M. * indicates significant difference (<i>P</i> <0.05) from control (untransfected cells) value.</p

Measuring and manipulating the cell-surface and total expression level of KCNQ1.

<p><b>A</b> and <b>B</b>. Representative on-cell [37°C live] and in-cell western assays, enabling quantification of the cell-surface and total channel expression of the VSV-KCNE1-KCNQ1 (VSV-E1-Q1) channel, respectively. The VSV-E1-Q1 construct was co-expressed in HEK293 cells with either pcDNA3.1, SAR1-H79G or KCNQ1-E261D. Cells expressing VSV-E1-Q1 with pcDNA3.1 were incubated with either 10 μM wortmannin (WTM) for 1 hour or 5 μM brefeldin A (Brf A) for 24 hours before the start of the assay. Each condition was performed in triplicate. Mean data, normalised to VSV-E1-Q1 + pcDNA3.1 values (VSV Control), from three independent experiments for cell-surface and total channel expression are shown in <b>C</b> and <b>D</b>, respectively. Data presented as mean ± S.E.M. * indicates significant difference (<i>P</i> <0.05) from control (VSV-E1-Q1 + pcDNA3.1) value.</p

Charge-neutralising mutations in a PIP₂-binding region in the proximal C-terminus of KCNQ1 increase retention of the channel complex in the ER.

<p><b>A</b>. Representative confocal images of the localisation of WT and mutant KCNQ1-GFP channel complexes in CHO-K1 cells. The left panel shows the GFP-tagged KCNQ1 subunit (GFP), the middle panel shows DsRed2-ER, an ER marker, and the right panel shows the merged images. In the merged image panel, the presence of yellow indicates colocalisation between KCNQ1-GFP and DsRed2-ER. Top row: WT KCNQ1-GFP with KCNE1. Lower rows: Localisation of the mutant KCNQ1 channels investigated (all n = 25, except KCNQ1(R360A-GFP), where n = 24). Scale bar indicates 20 μm. <b>B</b>. Quantified data showing the proportion of ER colocalisation of the WT and mutant KCNQ1-GFP subunits (in all cases KCNE1 was co-expressed). Data are presented as mean ± S.E.M. * indicates significant difference (<i>P</i> <0.05) from control (KCNQ1-GFP + KCNE1) value.</p

Depletion of PI(4)P at the Golgi does not affect the cell-surface or total expression level of KCNQ1.

<p><b>A</b> and <b>B</b>. Representative on-cell [4°C fixed] and in-cell western assays, enabling quantification of the cell-surface and total cellular expression, respectively. The VSV-E1-Q1 construct was co-expressed in HEK293 cells with pcDNA3.1, or with Tgn38-FRB and PJ-SAC or PJ-DEAD. Cells expressing Tgn38-FRB with either PJ-SAC or PJ-DEAD were incubated in the absence of 5 μM rapamycin (Rap), or in the presence of Rap for 1 hour or 24 hours as indicated. Each condition was performed in triplicate. Mean data, normalised to VSV-E1-Q1 + pcDNA3.1 values (VSV Control), from three independent experiments for cell-surface and total channel expression are shown in <b>C</b> and <b>D</b>, respectively. Data presented as mean ± S.E.M. * indicates significant difference (<i>P</i> <0.05) from control (VSV-E1-Q1 + pcDNA3.1) values.</p

The localisation of the PIP₂ sensor, Tubby-YFP, in HEK293 cells before and after the rapamycin-induced recruitment of PJ or PJ-DEAD to the PM.

<p>The rapamycin (Rap)-induced dimerization of PJ and PJ-DEAD with PM-localised LYN₁₁-FRB was investigated in transiently transfected HEK293 cells. Tubby-YFP localises to the PM in the presence of a sufficient PIP₂ concentration. <b>A</b> and <b>B</b>. Top panel: Before rapamycin addition. Lower panels: Increasing time after addition of rapamycin showing the increased PM signal of PJ and PJ-DEAD. Upon recruitment of PJ to the PM, Tubby-YFP redistributes from the PM to the cytosol (<b>A</b>). Upon recruitment of PJ-DEAD to the PM, Tubby-YFP remains PM-localised (<b>B</b>). Scale bar indicates 15 μm. <b>C–F</b>. Quantified line (<b>C</b> and <b>E</b>) and box plots (<b>D</b> and <b>F</b>) from the indicated red lines and boxed areas in <b>A</b> and <b>B</b> (located in the top right hand merged panels), highlighting translocation of Tubby-YFP to the cytosol when PJ (<b>C</b> and <b>D</b>) but not PJ-DEAD (<b>E</b> and <b>F</b>) is recruited to the PM. Red arrows indicate rapamycin addition.</p

Intra-individual reproducibility of extreme values.

<p>Intra-individual reproducibility of abnormality, defined as the probability for an individual who was in a given quantile during EST1 to remain in the same quantile or to change of quintile during EST2. For example, intra-individual reproducibility of extreme values for ΔHR_ex (first quintile) and ΔHR_rec (fifth quintile) was 58% and 57%, respectively.</p

Influence of maximum workload differences on intra-individual correlation.

<p>Intra-individual correlation of HR indices was assessed considering individuals who underwent ESTs with maximum work load that did not differ more than |ΔWL|. Bars represent the Spearman’s correlation coefficient. The number of individuals for each group is reported on top of each bar. Horizontal line is the mean Spearman’s correlation coefficient for resting HR.</p

Intra-individual correlation of HR indices.

<p>Scatter-plots showing the correlation between HR indices at first (EST1) and second (EST2) assessment. The Spearman’s correlation coefficient, ρ_sp, and the coefficient of determination, R², quantify the intra-individual correlation for each HR index and are reported in each panel. Dashed grey and red lines represent the identity line and the linear regression line, respectively.</p

Heart rate profile and indices.

<p>A: Definition of heart rate (HR) profile and HR indices during the exercise stress test. The heart rate profile, x_HR(t) (solid dark line) is a function of time obtained by filtering the instantaneous heart rate (dots) with a median filter 15 beats long. HR_rest: Mean x_HR(t) over 15 sec resting pre-test; HR_ex: Maximum x_HR(t) during exercise; HR_rec: minimum x_HR(t) during recovery; ΔHR_ex = HR_ex − HR_rest: HR increase during exercise; ΔHR_rec = HR_rec − HR_ex HR decrease during the 1 min recovery phase. HR_m: Mean HR during the entire test; B: Distribution of the heart rate profile across all participants. Black solid line, dark and light shadowed areas represent median, 25th-75th percentiles and 5th-95th percentile intervals, respectively.</p

Правда коммунизма. 1988. № 106

<div><p>Background</p><p>The heart rate (HR) response to exercise provides useful information about the autonomic function and has prognostic value, but its reproducibility over a long period of time, a critical requirement for using it as a clinical biomarker, is undetermined.</p><p>Aim</p><p>To determine the intra-individual reproducibility of HR dynamics during sub-maximum exercise and one minute recovery.</p><p>Methods</p><p>1187 individuals from the Cardio physical fitness assessment test of the UK Biobank repeated a standard exercise stress test twice (recall time 34.2 ± 2.8 months) and were prospectively studied.</p><p>Results</p><p>821 individuals complied with inclusion criteria for reproducibility analysis, including peak workload differences between assessments ≤10 W. Intra-individual correlation between HR profile during the first and the second assessment was very high and higher than inter-individual correlation (0.92±0.08 vs 0.87±0.11, p<0.01). Intra-individual correlation of indices describing HR dynamics was: ρ = 0.81 for maximum HR during exercise; ρ = 0.71 for minimum HR during recovery; ρ = 0.70 for HR changes during both exercise and recovery; Intra-individual correlation was higher for these indices of HR dynamics than for resting HR (ρ = 0.64). Bland-Altman plots demonstrated good agreement between HR indices estimated during the first and second assessment. A small but consistent bias was registered for all repeated measurements. The intra-individual consistency of abnormal values was about 60–70%.</p><p>Conclusions</p><p>The HR dynamics during exercise and recovery are reproducible over a period of 3 years, with moderate to strong intra-individual reproducibility of abnormal values.</p></div