Name: measuring fast calcium fluxes in cardiomyocytes
Uploaded: 2011-11-29T13:00:00
Description: Watch this Scientific Journal Video about Measuring Fast Calcium Fluxes in Cardiomyocytes at JoVE.com

This work was supported by the National Institutes of Health grants 2R01 GM53132 and P50 GM071558.

1. Loading cells with calcium indicators
<ol>
 <li>Plate cells in 35mm glass bottom MatTek dishes (or any other glass bottom viewing chambers).</li>
 <li>If using primary myocytes coat the chambers 1 day before with 10&mu;g/ml laminin. Plate cardiac myocytes (isolated from adult dogs, neonatal rats, or other organism) in KB buffer (K-reversal Tyrode buffer 35 mmol/L of HEPES, 140 mmol/L of KCl, 8 mmol/L of KHCO3, 2 mmol/L of MgCl2, and 0.4 mmol/L of KH2PO4, pH 7.5) and change...

<ol>
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We have developed a method to view and isolate rapid calcium signals in living cardiomyocytes. While the experimental conditions of these measurements have previously been applied to other cell systems10 as well as cardiomyocytes11, the use of histograms and bin analysis allows data from many Ca2+ events to be acquired. Faster events can be readily isolated from slower ones and models adapted to understand their underlying mechanisms. Culturing cardiomyocytes, as many primary cell line...

<table border="1">
<tbody>
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments </td>
</tr>
<tr>
<td>DMEM</td>
<td>Invitrogen</td>
<td>ABCD1234</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

measuring fast calcium fluxes in cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

Watch this Scientific Journal Video about Measuring Fast Calcium Fluxes in Cardiomyocytes at JoVE.com

Measuring Fast Calcium Fluxes in Cardiomyocytes

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

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