Myosin II ATPase Activity Mediates the Long-Term Potentiation-Induced Exodus of Stable F-Actin Bound by Drebrin A from Dendritic Spines

<div><p>The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca²⁺ influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.</p></div

The DA-actin exodus is not blocked by inhibitors of myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK).

<p>Neurons (21 DIV) were preincubated with 10 µM of ML-7, an inhibitor of MLCK (A) or 1 µM H-1152, an inhibitor of ROCK (B) for 30 min, and then stimulated with 100 µM glutamate for 10 min. Neither ML-7 (n = 30 cells; drebrin SDR p<0.01, actin SDR p<0.01, Scheffe's test) nor H1152 (n = 30 cells; drebrin SDR p<0.01, actin SDR p<0.01, Scheffe's test) blocked the DA-actin exodus. F-actin images indicate that spines kept their structure during the experiment although their shapes were changed. Scale bars, 5 µm.</p

Increase in surface GluR1 immunostaining after chemical LTP (cLTP) stimulation.

<p>Neurons (21 DIV) were stimulated with buffer containing 0 µM Mg²⁺, 200 µM glycine, 20 µM bicuculline, 1 µM strychnine and 0.5 µM TTX (cLTP stimulation) for 3 min. (<b>A</b>) Surface GluR1 was labeled before the stimulation (top panel; Con) or 30 min after the stimulation (middle panel; cLTP). Note that cLTP stimulation remarkably increased surface GluR1 immunostaining. The increase was completely blocked by APV (bottom panel; cLTP APV). Scale bar, 7 µm. (<b>B</b>) Quantitative analysis of surface GluR1 cluster density along dendrites. Data are expressed as percentages relative to the average of control neurons. In the absence of APV, cLTP stimulation significantly increased the density of surface GluR1 clusters (n = 21 cells; p<0.01, Scheffe's test). In contrast, in the presence of APV, no increase in surface GluR1 cluster density was observed following cLTP stimulation (cLTP APV). Error bars represent s.e.m.</p

Effects of various excitatory stimulations on DA-actin distribution.

<p>Images were obtained from neurons (21 DIV) double-labeled for drebrin and F-actin. Bar graphs represent the spine-dendrite ratios (SDRs) for drebrin and actin. (<b>A–C</b>) Neurons were stimulated with 100 µM glutamate for 10 min (A), 50 µM bicuculline and 500 µM 4-aminopyridine (Bic/4-AP) for 10 min (B), or 90 mM KCl in Tyrode's solution for 5 min (C). F-actin images indicate that spines kept their structure during the experiment although their shapes were changed. After stimulation, the drebrin and F-actin clusters in the spines disappeared, and a linear staining pattern appeared along the dendrite. Both the drebrin and actin SDRs were significantly decreased (glutamate, n = 170 cells; Bic/4-AP, n = 30 cells; KCl, n = 30 cells; p<0.01, Student's <i>t</i> test). Note that the control drebrin and actin SDRs in (C) were greater than the other SDRs because Tyrode's solution was used instead of normal medium. Scale bars, 5 µm. Error bars represent s.e.m.</p

Effects of various inhibitors of Ca²⁺ entry on DA-actin distribution.

<p>Neurons (21 DIV) were incubated in normal medium containing 50 µM APV (A), 20 mM EGTA (B), 20 µM nifedipine (C), or 1 µM thapsigargin (D) for 30 min. The neurons were then stimulated with 100 µM glutamate for an additional 10 min. F-actin images indicate that spines kept their structure during the experiment although their shapes were changed. Scale bars, 5 µm. (<b>A</b>) APV pretreatment significantly increased both the drebrin and actin SDRs (n = 30 cells; p<0.01, Scheffe's test). In the presence of APV, glutamate stimulation significantly decreased the actin SDR (n = 30 cells; p<0.01, Student's <i>t</i> test) but not the drebrin SDR (n = 30 cells; p = 0.52, Student's <i>t</i> test). (<b>B</b>) EGTA significantly increased the drebrin and actin SDRs (n = 30 cells; p<0.01, Scheffe's test), and blocked the glutamate-induced decreases in drebrin and actin SDRs (n = 30 cells; Student's <i>t</i> test). (<b>C</b>) Nifedipine significantly increased the drebrin and actin SDRs (n = 30; p<0.01, Scheffe's test), but did not block the glutamate-induced decrease in drebrin and actin SDRs (n = 30; p<0.01, Scheffe's test). (<b>D</b>) Thapsigargin neither increased the drebrin and actin SDRs (n = 30; Student's <i>t</i> test) nor blocked the glutamate-induced decreases in drebrin and actin SDRs (n = 30; p<0.01, Scheffe's test). Error bars represent s.e.m.</p

Electrophysiological Characteristics of Human iPSC-Derived Cardiomyocytes for the Assessment of Drug-Induced Proarrhythmic Potential

<div><p>The aims of this study were to (1) characterize basic electrophysiological elements of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that correspond to clinical properties such as QT-RR relationship, (2) determine the applicability of QT correction and analysis methods, and (3) determine if and how these in-vitro parameters could be used in risk assessment for adverse drug-induced effects such as Torsades de pointes (TdP). Field potential recordings were obtained from commercially available hiPSC-CMs using multi-electrode array (MEA) platform with and without ion channel antagonists in the recording solution. Under control conditions, MEA-measured interspike interval and field potential duration (FPD) ranged widely from 1049 to 1635 ms and from 334 to 527 ms, respectively and provided positive linear regression coefficients similar to native QT-RR plots obtained from human electrocardiogram (ECG) analyses in the ongoing cardiovascular-based Framingham Heart Study. Similar to minimizing the effect of heart rate on the QT interval, Fridericia’s and Bazett’s corrections reduced the influence of beat rate on hiPSC-CM FPD. In the presence of E-4031 and cisapride, inhibitors of the rapid delayed rectifier potassium current, hiPSC-CMs showed reverse use-dependent FPD prolongation. Categorical analysis, which is usually applied to clinical QT studies, was applicable to hiPSC-CMs for evaluating torsadogenic risks with FPD and/or corrected FPD. Together, this results of this study links hiPSC-CM electrophysiological endpoints to native ECG endpoints, demonstrates the appropriateness of clinical analytical practices as applied to hiPSC-CMs, and suggests that hiPSC-CMs are a reliable models for assessing the arrhythmogenic potential of drug candidates in human.</p></div

Relationship between field potential duration (FPD)/corrected FPD (FPDc) and interspike interval (ISI) of 96 samples of hiPSC-CM.

<p>The data in sham treatment from 4 facilities were plotted (n = 96) in Fig 2a (FPD-ISI), Fig 2b (FPDcF-ISI) and Fig 2c (FPDcB-ISI), respectively. The solid and dashed lines indicate the linear regression line and 95% confidence bands, respectively. The equation, R² value, and root mean squared prediction error (RMSE) are shown in the figures.</p

Effects of <i>I</i>_Kr and <i>I</i>_Ks inhibitors on field potential duration (FPD)-interspike interval (ISI) plots.

<p>FPD-ISI plots are shown before and after compound application both in terms of absolute values (left) and % change (right), for DMSO (Fig 3a), E-4031 (Fig 3b), cisapride (Fig 3c) and chromanol 293B (Fig 3d). The solid lines and dashed lines indicate the linear regression lines and 95% confidence bands, respectively. Data for the middle concentrations are omitted for clarity. ‘Slope’ refers to the slope of the regression line.</p

Categorical analyses of absolute FPD values after E-4031 application.

<p>Categorical analyses of absolute FPD values after E-4031 application.</p

Categorical analyses of absolute FPD values after cisapride application.

<p>Categorical analyses of absolute FPD values after cisapride application.</p