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 it gradually to M199 medium supplemented with 15% fetal bovine serum and 1% streptamycin and 0.5% gentamicin and incubate at 37 &deg;C in 5% CO2.</li>
 <li>Incubate cells with calcium green AM (1-5 &mu;M; Molecular Probes) or any desired calcium indicator for 30-45 min at room temperature. Cover the dish with aluminum foil to avoid photobleaching of the dye.</li>
 <li>Wash the cells three times with Leibovitz15 medium with 14mM EGTA (if performing experiments in low calcium) or any appropriate viewing media.</li>
 <li>Incubate for another 30-45 minutes in Leibovitz15 medium containing14mM EGTA.</li>
 <li>If testing effects of inhibitors (i.e. 10&mu;M U73122 PLC inhibitor) add them during the second incubation.</li>
</ol>
2. Microinjecting cells
<ol>
 <li>Culture cardiomyocytes in glass bottom MatTek dishes (or other viewing chambers) for 24h.</li>
 <li>Change the medium to phenol-free Leibovitz-15 with 14 mM EGTA (cardiomyocytes have to be injected in calcium free medium, other cells can be injected in medium containing calcium).</li>
 <li>Prepare microinjection solution - our studies used a peptide that eliminates G&alpha;q - caveolae interactions. Keep in mind that only a small amount of volume is injected into the cell (&tilde;fL) and so reasonably concentrated stocks must be used. We used 2 &mu;M of this peptide, Cav3-scaffold, with DAPI in calcium-free medium. DAPI is a blue dye that stains the nucleus and allows the identification of cells that have been microinjected. DAPI does not interfere with the fluorescence of many calcium indicators. The amount of DAPI is variable and we generally use enough to visualize the nucleus (0.5 and 2 &mu;M)</li>
 <li>Prepare control solution containing DAPI - the dye tracer alone.</li>
 <li>For microinjections use self-pulled or commercially available needles. Practice with dye injection to determine proper needle parameters. We note that different cells might require different needle diameters and microinjection pressures.</li>
 <li>Use automated microinjection system, for example an InjectMan NI2 with a FemtoJet pump from Eppendorf. Set the injection pressure Pi to 90 hPa, and the compensation pressure Pc to 45 hPa. Set the injection time t to 0.7 s. Readjust parameters of injection as need.</li>
 <li>Perform microinjections on an inverted microscope (e.g. Zeiss Axiovert 200M) equipped with long working distance phase contrast objective (for example air 40x phase 2 objective).</li>
 <li>Inject as many cells as possible in a rapid fashion. Examine injected cells using the phase contrast to select viable cells.</li>
</ol>
3. Co-immunofluorescence studies
<ol>
 <li>Wash cells twice with PBS and fix with 3.7% paraformaldehyde for 30 minutes. If required, cells can be stimulated before fixation.</li>
 <li>Wash fixed cells three times with PBS, and incubate with 0.2% NP40 in PBS for 5minutes.</li>
 <li>Block using PBS containing 4% goat serum (or other appropriate serum) for 1hour.</li>
 <li>Incubate cells with primary antibody at appropriate dilution with 1% goat serum in PBS for 1hour.</li>
 <li>Wash cells three times for 10 minutes with 150 mM NaCl, 25 mM Tris, pH 7.6 followed by addition of fluorescent-labeled secondary antibody, such as Alexa-Fluor-488 anti-rabbit or Alexa-Fluor-647 anti-mouse secondary antibody diluted to 1:1000 1% goat serum in PBS (note that when probing for two different proteins the primary antibodies must be raised in different species and the secondary must be against corresponding species).</li>
 <li>Incubate at 37&deg;C for 1hour, wash three times with TBS.</li>
 <li>View cells in TBS buffer or another appropriate viewing medium.</li>
</ol>
4. Fast calcium imaging
<ol>
 <li>Use laser scanning confocal microscope (for example Fluoview 1000 Olympus).</li>
 <li>Use high numerical aperture objective.</li>
 <li>Set the acquisition parameters. For calcium green use 488 nm excitation and long pass 515 emission filters.</li>
 <li>Adjust the pixel time - for fast calcium measurements set it to 2 &mu;s/pixel.</li>
 <li>Set the image zoom to achieve pixel size of 0.05 - 0.3 &mu;m.</li>
 <li>Select a line from a cell of choice in a particular region.</li>
 <li>In the time controller window select time acquisition for at least 1500 scans (to get desired time frame of the response - 1.3 ms/line would yield 2 seconds).</li>
 <li>Record background reading prior to the stimulation.</li>
 <li>Stimulate cells with 5 &mu;M carbachol and immediately start recording data. Keep cells in 1 mL of Leibovitz15 medium, prepare 10 &mu;M carbachol in Leibovitz15 and add gently 1 mL into the dish</li>
</ol>
5. Data analysis
<ol>
 <li>Extract the intensities for each pixel from recorded image. Save the intensity data as a table using Fluoview 1000 software.</li>
 <li>Import intensity data into Sigmaplot or a similar data analysis program and bin the data into &tilde;9-40 bins. Compare to control experiments and electronic noise.</li>
</ol>
6. Representative Results:
We imaged cells on laser scanning confocal microscope LSM510 (Zeiss, Jena, Germany) equipped with multi-line laser excitation and a 40x/1.2 NA apochromat water immersion objective. In Fig. 1 (red) we show the distribution of caveolae in canine adult ventricular cardiomyocytes that have been fixed and stained with an antibody to caveolin-3 which is the major structural caveolar protein in these cells 9. Caveolin-3 was immunolabelled with Alexa-647 and excited with the 633-nm line from a He:Ne laser. Images were recorded using LP650 nm emission filter. The distribution of caveolin-3 was compared to G&alpha;q that was immunolabeled with Alexa-488 (green). Alexa-488 was excited with 488nm Argon-ion laser line and the image was recorded using BP505-530nm emission filter. In these colocalization measurements, the two fluorophores were excited in a sequential manner using Multi Track acquisition. This procedure minimizes channel cross-talk. Data analysis for colocalization was performed using AIM software provided with Zeiss LSM510-Meta. As can be seen, the two proteins are colocalized.
To determine how the coupling between caveolin-3 and G&alpha;q affect calcium signals, we carried out calcium imaging studies on a confocal scanning laser microscope system (Fluoview, Olympus, Tokyo, Japan) coupled to an inverted microscope (IX70, Olympus) with a 40x objective (UPlanApo, Olympus, numerical aperture 1.3) although these measurements could also be performed on the Zeis systems described above. For these measurements, cells were loaded with Ca2+ Green. We excited the dye with a 488nm Argon ion laser and collected images through an LP515 emission filter. Image frames were collected at 500 ms intervals. Intensity values for each pixel were extracted using Olympus software and ImageJ. The line scan mode was used for quantitative analysis of faster Ca2+ transients and Ca2+ sparks. The pixel size used in the present study was 0.1-0.3 &mu;m. The line-scan rate was about several hundred of &mu;s per line. In Fig. 2 we show a line scan of a Ca2+ Green-loaded cardiomyocyte where the pixel dwell time was 2 &mu;s, the line time was 142 &mu;s. The image is composed of 1200 lines.
Each line of the scan corresponds to the intensity fluctuation of Ca2+ Green consisting of 71 points taken over a 142 &mu;s range, and can be used as a rapid read-out for calcium responses. For example, in Fig. 3 we show the Ca2+ activity of a single line of an individual cardiomyocyte, as measured by Ca2+ Green fluorescence intensity as a function of time in seconds before (top) and after (bottom) the addition of 5 &mu;M carbachol which enhances the level of intracellular Ca2+ through G&alpha;q-PLC&beta; activity. When many lines are averaged, carbachol stimulation produces an &tilde;1.8 fold increase in Ca2+ Green intensity. Due to variations in experimental conditions (room temperature, laser power, detector response, dye distributions, etc), this increase may not be seen in individual line scans. Even though the intensity values (y axis values) are similar, it is clear that the carbachol-stimulated plot on the bottom has much broader peaks, corresponding to more sustained calcium levels, than the narrow fluctuation peaks seen before stimulation. This broadening and relative rise in intensity obscures small, slower oscillations. Our goal is to better discriminate between these different apparent types of calcium fluctuations.
Another example of these fast calcium fluctuations is shown in Fig. 4. The top graph shows raw line data for stimulated ventricular cardiomyocytes (red) and an average of 3 line traces is shown in black. To determine the extent that these calcium fluctuations were mediated by G&alpha;q - caveoline3 interactions, we microinjected a peptide that destabilizes caveolin-3 - G&alpha;q interactions (i.e. Cav3 peptide) which eliminates the longer Ca2+ current and these data are shown in blue (we note that we subtracted 500 from the Cav3 peptide intensities so that the two traces can be better discerned).
We imported the columns of raw data from our excel files into Sigmaplot (Jandel, Inc.) and carried out a histogram analysis. In this analysis, the number of events is grouped into an equal number of boxes (bins) to produce a histogram. The width of bin represents an equal interval of time. The intensities are inserted into the different time binds so that the height of a particular bin represents the number of times that intensity occurs in the particular time window. Since this type of analysis only depends on collecting data at the same scan rate, many traces can be analyzed simultaneously. Additionally, this analysis is not sensitive to differences in dye levels, laser power, etc., and electronic and background noise occurs at time scales very fast and are only seen in the first 1-2 bins.
The corresponding histograms of the raw data in Fig. 4 are shown on the bottom of the page. The histogram for the control cells are spread over a large number of bins corresponding to a broad time distribution, as depicted by the red line (the line is for guiding the eye and no model is assumed). In contrast, the bins for the response of cells where the G&alpha;q -Cav3 interaction was disrupted by peptide are confined to a narrow bin distribution suggesting that the presence of peptide eliminates longer duration fluxes. Microinjection of a control peptide that does not disrupt G&alpha;q-Cav3 produces a histogram similar to un-injected cells8.
In Fig. 5 we compare the fast intensity fluctuations of Ca2+Green in adult canine ventricular cardiomyocytes (left) and fresh, unstimulated rat neonatal cardiomyocytes that lack caveolae (right). Here, the corresponding histograms of the intensities over a 3 minute period were binned at 35 to view slower calcium events. Note that the neonatal cardiomyocytes display fast fluctuations on a flat baseline showing the lack of slower &nbsp; Ca2+ activity while a more structured baseline is seen for the adult cells. These differences are most easily seen in the histograms.
<img alt="Figure 1" src="/files/ftp_upload/3505/3505fig1.jpg" /> 
Figure 1. Images of canine adult ventricular cardiomyocytes showing the distribution of G&alpha;q immunolabeled with Alexa-488 where Alexa-488 was excited with 488nm Argon-ion laser line and the image was recorded using BP505-530nm emission filter. The top right is the same image viewing the localization of caveolin-3 immunolabeled with Alexa-647, excited with the 633-nm line from a He:Ne laser and recorded using LP650 nm emission filter. The merged image where colocalization is seen in orange is on the bottom left with an expanded image to the right.



<img alt="Figure 2" src="/files/ftp_upload/3505/3505fig2.jpg" /> 
Figure 2. Calcium imaging of a living canine adult ventricular cardiomyocyte loaded with Ca2+ Green. The image was taken on a confocal scanning laser microscope system with a 40x objective using a 488nm Argon ion laser and an LP515 emission filter. The image is composed of 1200 lines, the pixel dwell time was 2 &mu;s, and the pixel size was 0.1 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3505/3505fig3.jpg" /> 
Figure 3. TOP Intensity fluctuations of a line trace from a living canine adult ventricular cardiomyocyte loaded with Ca2+ Green and imaged as described, and BOTTOM the same cell stimulated by the addition of 5 &mu;M carbachol.
<img alt="Figure 4" src="/files/ftp_upload/3505/3505fig4.jpg" /> 
Figure 4. TOP An example of line traces from a living canine adult ventricular cardiomyocyte loaded with Ca2+ Green (red) taken over 180 ms where the average of 3 traces is shown as a black line, and compared to a cell that was microinjected with a peptide that disrupts G&alpha;q-caveolin 3 association (red) where an average of three traces is in black. We note that we subtracted 400 intensity counts from the lower set of data for viewing purposes. The lower panels are the corresponding histograms of the binned data as described in the text, and where the bin number is arbitrary and the bin count (or simply count) corresponds to the total amount of intensity in that particular bin. We note that the line red and blue lines drawn in the control (left) and peptide (right) histograms are to allow for easier identification and comparisons of the data and no model is assumed.
<img alt="Figure 5" src="/files/ftp_upload/3505/3505fig5.jpg" /> 
Figure 5. Intensity fluctuations of Ca2+Green in adult canine ventricular cardiomyocytes (left) and fresh, unstimulated rat neonatal cardiomyocytes that lack caveolae (right) and their corresponding histograms are shown below.

<ol>
	<li>Cheng, H., Lederer, M. R., Lederer, W. J., Cannell, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sparks+and+[Ca2+]i+waves+in+cardiac+myocytes.">Calcium sparks and [Ca2+]i waves in cardiac myocytes.</a> Am. J. Physiol. Cell. Physiol. 270, 148-159 (1996).</li><li>Cheng, H., Lederer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Sparks.">Calcium Sparks.</a> Physiol. Rev. 88, 1491-1545 (2008).</li><li>Rebecchi, M. J., Pentyala, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function,+and+control+of+phosphoinositide-specific+phospholipase.">Structure, function, and control of phosphoinositide-specific phospholipase.</a> C. Physiol.Rev. 80, 1291-1335 (2000).</li><li>Suh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+of+phosphoinositide-specific+phospholipase+C+isozymes.">Multiple roles of phosphoinositide-specific phospholipase C isozymes.</a> BMB reports. 41, 415-434 (2008).</li><li>Schlegel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crowded+little+caves:+structure+and+function+of+caveolae.">Crowded little caves: structure and function of caveolae.</a> Cell. Signal. 10, 457-463 (1998).</li><li>Anderson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+caveolae+membrane+system.">The caveolae membrane system.</a> Annu. Rev. Biochem. 67, 199-225 (1998).</li><li>Sengupta, P., Philip, F., Scarlata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caveolin-1+alters+Ca2++signal+duration+through+specific+interaction+with+the+G{alpha}q+family+of+G+proteins.">Caveolin-1 alters Ca2+ signal duration through specific interaction with the G{alpha}q family of G proteins.</a> J. Cell. Sci. 121, 1363-1372 (2008).</li><li>Guo, Y., Golebiewska, U., Scarlata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Ca2++Activity+in+Cardiomyocytes+through+Caveolae-G[alpha]q+Interactions.">Modulation of Ca2+ Activity in Cardiomyocytes through Caveolae-G[alpha]q Interactions.</a> Biophysical. Journal. 100, 1599-1607 (1016).</li><li>Rybin, V. O., Xu, X., Lisanti, M. P., Steinberg, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+targeting+of+beta+-adrenergic+receptor+subtypes+and+adenylyl+cyclase+to+cardiomyocyte+caveolae.+A+mechanism+to+functionally+regulate+the+cAMP+signaling+pathway.">Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway.</a> J. Biol. Chem. 275, 41447-41457 (2000).</li><li>Tovey, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+puffs+are+generic+InsP3-activated+elementary+calcium+signals+and+are+downregulated+by+prolonged+hormonal+stimulation+to+inhibit+cellular+calcium+responses.">Calcium puffs are generic InsP3-activated elementary calcium signals and are downregulated by prolonged hormonal stimulation to inhibit cellular calcium responses.</a> J. Cell. Sci. 114, 3979-3989 (2001).</li><li>Lohn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ignition+of+Calcium+Sparks+in+Arterial+and+Cardiac+Muscle+Through+Caveolae.">Ignition of Calcium Sparks in Arterial and Cardiac Muscle Through Caveolae.</a> Circ. Res. 87, 1034-1039 (2000).</li></ol>

No conflicts of interest declared.

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...

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<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments </td>
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<tr>
<td>DMEM</td>
<td>Invitrogen</td>
<td>ABCD1234</td>
<td>&nbsp;</td>
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</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 ...

measuring-fast-calcium-fluxes-in-cardiomyocytes

Research

JoVE Journal

Biology

15.1K Aufrufe.  Queensborough Community College. Wir präsentieren eine Methode zur schnellen (Mikrosekunde) Calcium Ereignisse aus langsamer Flüsse in lebenden Zellen mittels Laser-Scanning-konfokalen Mikroskopie zu isolieren. Das Verfahren misst Fluoreszenzintensität Schwankungen des Kalzium-Indikatoren, die von der Aufnahme Line-Scans von mehreren hundert Pixel in einer Zelle. Histogramm-Analyse ermöglicht es uns, die Zeitskalen der verschiedenen Kalzium-Ströme zu isolieren.

Serie: Measuring Fast Calcium Fluxes in Cardiomyocytes

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

Immunology and Infection

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Bioengineering

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Medicine

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Real-Time Measurements of Calcium and Contractility Parameters in hiPSC-CMs

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. In this study, it is demonstrated that this optics-based system is a powerful tool to assess the functional parameters of hiPSC-CMs in 2D. By using this platform, it is possible to perform paired measurements in a well-preserved temperature environment on different plate layouts. Moreover, this system provides researchers with instant data analysis.
This paper describes a method for measuring the contractility of unmodified hiPSC-CMs. Contraction kinetics are measured at 37 &#176;C based on pixel correlation changes relative to a reference frame taken at relaxation at a 250 Hz sampling frequency. Additionally, simultaneous measurements of intracellular calcium transients can be acquired by loading the cell with a calcium-sensitive fluorophore, such as Fura-2. Using a hyperswitch, ratiometric calcium measurements can be performed on a 50 &#181;m diameter illumination spot, corresponding to the area of the contractility measurements.

Author Spotlight: Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes  

Here, an established method is described to perform contractility and calcium measurements in human induced pluripotent stem cell-derived cardiomyocytes using an optics-based platform. This platform enables researchers to study the effect of mutations and the response to various stimuli in a rapid and reproducible manner.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte ...