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 lines, can be difficult. While it is generally thought that measurements should be taken soon after the cells are acquired, we have found that the adult canine cardiomyocytes we used in this study only begin to lose their specific properties after 3 days. This longer time frame allows the cells to firmly attach on to the substrate insuring that the cells will survive potentially lethal treatments such as microinjection. Firm attachment is critical for the success of microinjection.
We verified the role of G&alpha;q-caveolin 3 association in calcium fluxes by microinjecting a peptide that disrupts their interaction. We would like to stress that microinjection must be done with great care to minimize damaging the cell and leakage of cellular contents. However, these risks must be balanced by the use of larger diameter needles that are needed to deliver an adequate amount of material into the relatively large cardiomyocytes. The inclusion of a dye such as DAPI that allows one to track which cells have been microinjection is important. As mentioned, it is important that for the microinjection myocytes are bathed in calcium free medium and that the solutions injected is also free of calcium. Influx of extracellular calcium initiates contractions and subsequent death of cells
The measurements themselves can be adapted to other cell types, dyes and confocal laser systems to follow other rapid cell events such as insoitol 1,4,5 trisphospate and cAMP fluxes, although the Ca2+ fluxes are certainly one the most complex cellular messengers. One should be cautious about choosing fluorescent indicators that are not easily photobleached and have a robust and dynamic fluorescence response so that low laser powers can be used. Low laser power will also avoid local heating which could give rise to unwanted cellular effects.

<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 ...

measuring-fast-calcium-fluxes-in-cardiomyocytes

Nanyang Technological University

Research

JoVE Journal

Biology

15.1K Views.  Queensborough Community College. 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.

Video: Measuring Fast Calcium Fluxes in Cardiomyocytes

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...