Stimulus protocols

Dear All
We are using a rat cardiomyocyte prep on a MEA60-INV-BC.

We usually don't have any problems getting the preps to spontaneously beat but we would be interested in standardising beat rate etc. to allow easier comparison between different plates and batches. Therefore we have been looking at the possibility of using a stimulator to "drive" the plate at the desired rate.

We are using a STG1008 stimulus generator and our current stimulus protocol is a rectangular biphasic pulse (+250mV for 100uS and -250mV for 100uS) with a gap of 800uS.

This protocol however is only a first "guestimate" and may not be the optimium protocol to use.

Does anyone have a cardiomyocyte stimulation protocol they know works well? Thank you.
Steve

(My colleagues having some problems posting so I posted this on his behalf..Scott)

Dear Steve,

you might want to have alook at the paper from Meiry et al from the group of Ofer Binah, where stimulation of this cells is described. The actual protocol of course varies by size of the electrodes and cell density and size of the plated area.
Assuming you are using standard 30um TiN electrodes I would increase all parameters of your protocol : We use for HL-1 cells for example a biphasic pulse of +2V for 500-800us, then -2V for 500-800us. Due to these large pulses blanking circuit helps a lot to obtain easier to analyze signals.

Thomas

Meiry G, Reisner Y, Feld Y, Goldberg S, Rosen M, Ziv N & Binah O. (2001). Evolution of action potential propagation and repolarization in cultured neonatal rat ventricular myocytes. J Cardiovasc Electrophysiol 12, 1269-1277.

Hello All,

I'm also interested in hearing about the current ranges/electrode configuration (monopolar vs. bipolar, monophasic vs. biphasic) used by people working with in vitro cardiac tissue preperations.

Steve did your protocol work??

I am working with in vitro sinus node and atrial preperations (about 1cm^2 in area) excised from rabbit hearts. I'm trying to figure out a stimulus protocol.

From the literature, most people report using x2 or x1.5 threshold values to stimulate cardiac tissue, however no mention is made of what the threshold stimulus is. Which provides little information for people trying to setup an experiment for the first time or replicate their methodology. I understand many factors play a role in determining the magnitude of the threshold stimulus (tissue size, electrode type size and configuration) but reporting a mean or range of values will be very helpful indeed.

After a few experiments I found out that monophasic pulses (0.8mA, 2ms using 0.25um diameter Stainless steel electrodes in a monopolar configuration) is sufficient to entrain the spontaneous activity of the sinus node pacemaker.

I have also tried using platinum electrodes (0.125um diameter, monopolar configuration) to stimulate right atrial muscular preperations. I found I needed monophasic pulses of at least 2mA for 2ms to excite the tissue and generate action potentials.

I am planning to buy a STG1002 and was wondering if the current/voltage outputs are sufficient to excite such preperations? (the standard current output of 0.8mA seems a bit low and I'm thinking of upgrading it to 3.2mA).

If anybody is working with in vitro tissue prep. what sort of stimulus current/voltage are they using.

I have looked at the paper Meiry et al but couldn't find anything on stimulus magnitude and besides that they are using cultured myocytes.

cheers,
Amr

Dear Amr,

I cotancted a colleague at the NMI Reutlingen who is working with cardiac slice preparations and paces them to have a look at your questions. Maybe just one technical note: compliance voltage (if you stimulate with current) of our STG is 120V. If you require higher voltage output then 8V you can combine channels and make use of the +/- U output on every channels.
So we never had problems to pace ventricular slices with an external bipolar electrode using a standard STG2004.

Thomas

Dear Amr,

I guess I am the guy Thomas mentioned... :-)
As he stated correctly we are doing cardiac slice preparations here at the NMI in Reutlingen, Germany. For this kind of preparations we mainly work on ventricular slices of the adult mouse. For stimulation we use concentric bipolar electrodes with a tip diameter of 2-3 µm, the base of the tip has 0.126 µm and the "height" of the tip is <0.2 mm (according to the manufacturer). We use bipolar electrodes rather than monopolar since this minimizes the stimulation artefact (even though we are using the 1060BC blanking circuit amplifier). As for the stimulation protocol we found using a rectangular +1 V pulse with a duration of 1 ms at an interval of 800 ms to be sufficient (ground being the outer shaft of the concentric bipolar electrode). This is our standard stimulus, we do not adjust the pulse according to thresholds. This works reliably for > 1 h in more than 60% of our slices (quality check is a reliable circular signal propagation throughout the entire slice). Unfortunately I cannot tell how much current flows at this potential since the manufacturer of the electrodes does not provide resistance values. The stimulus generator is a regular STG2004.

Hope this helps
Cheers
Udo

Dear Thomas and Udo,

Thanks very much for the tips. The technical information is really helpful.

Udo, I did an experiment using bipolar electrodes (in house built - two wires seperated by a small distance) and, as you mentioned, that reduced the stimulus artefact considerably. I have just asked for a quote for the concentric electrodes and hopefully that will reduce the stimulus noise even further (and speed up data analysis!!!).

I also found that 3-4V ,1ms pulses were sufficient to excite the atrial tissue. I guess I needed to use a higher voltage than you because my electrodes are of larger diameters (I think the charge densities would be very similar).

Cheers

Amr