The overall goal of this procedure is to isolate human atrial myocytes suitable for simultaneous measurements of calcium, transients and membrane currents. The right atrial specimen is obtained from patients undergoing open heart surgery and is quickly transported to begin the procedure. The first step of the procedure is to clean the tissue and cut it into small tissue chunks.
The second step is a twofold enzymatic digestion of the tissue with proteolytic enzymes resulting in isolated atrial myocytes. The final step comprises loading of the myocytes with a calcium sensitive fluorescent dye. Ultimately, the combined usage of epi fluorescence microscopy and the patch clamp technique produce simultaneous recordings of calcium transients with membrane currents or action potentials.
This method can help to understand the molecular basis of cellular calcium handling abnormalities underlying cardiac diseases such as atrial fibrillation, demonstrating the method will be Claudia Lira, the senior technician of our laboratory, The human atrial tissue obtained from patients undergoing open heart surgery for coronary bypass grafting, or mitral valve replacement should be quickly transported to the lab in transport solution. Once at the lab, transfer the tissue sample and the solution to a 10 centimeter dish. Then roughly remove the fatty tissue using scissors.
Next, wave the tissue sample. Keep between 200 and 600 milligrams for the cell isolation and freeze the remainder in liquid nitrogen for biochemical analysis. Transfer the tissue for cell isolation into a dish of cool calcium free solution and chop the tissue into one cubic millimeter chunks to wash the tissue.
Transfer it together with the calcium free solution to the jacketed beaker. Heated to 37 degrees. Provide oxygen from a line capped with a pipette tip to prevent strong bubbling.
Stir the tissue carefully for about three minutes with a magnetic stir bar. Then allow the tissue to settle. Now carefully strain the supernatant through a 200 micron nylon mesh.
Most of the tissue should remain in the beaker. Refill the beaker with warmed 37 degree calcium free solution. Then return the strained tissue chunks from the mesh into the beaker using forceps and repeat the washing procedure twice.
Begin this portion of the procedure by preparing 20 milliliters of warmed 37 degrees Celsius enzyme solution E one containing collagenase and protease. Carefully strain the calcium free solution after the final washing step and resuspend the washed tissue in the E one solution. Return the tissue collected on the mesh to the beaker.
Stir the suspension carefully for 10 minutes. Then add 40 microliters of 10 millimolar calcium chloride, and continue stirring for 35 minutes. Next, strain the supernatant carefully through a nylon mesh.
Most of the tissue should remain in the beaker. Prepare 20 milliliters enzyme solution E two containing collagenase one only, and use it to refill the beaker. Return the tissue collected on the mesh to the beaker.
Then immediately add 40 microliters of 10 millimolar calcium chloride solution during the second digestion, occasionally use scissors to chop up tissue clots. After five minutes, take a sample of the suspension to check the dissociation of the cells under a microscope. Keep taking samples every two to three minutes until rod-shaped striated cardiomyocytes are clearly visible.
Then stop the stirring, allowing the tissue to settle. Once settled, carefully strained the supernatant through a nylon mesh and into a plastic 50 milliliter tube. Next, suspend the tissue in 20 milliliters storage solution and return the strained tissue chunks from the mesh to the beaker.
Further dissociate the cells by gentle mechanical tation. With a 20 milliliter pipette, try to avoid bubble formation. Next, carefully strain the supernatant through a nylon mesh after the straining centrifuge, both supernatant collections at 90 5G for 10 minutes.
To pellet the cells, discard the supernatants by aspiration without disturbing the pellet resus. Suspend each pellet in 1.5 milliliters of storage solution at room temperature. Next, add 7.5 microliters of 10 millimolar calcium chloride solution to each tube and wait 10 minutes.
Then add 7.5 more microliters of calcium chloride, and wait another 10 minutes. After the wait, add a third 15 microliter volume of 10 millimolar calcium chloride to bring the final concentration of calcium chloride in solution to 0.2 millimolar. The purpose of this isolation is to achieve cells which are suitable for intracellular calcium measurements.
Therefore, it is a critical requirement to omit any calcium buffers such as EGGA from the storage solution. Transfer 1.5 milliliters of the cell suspension into a two milliliter micro centrifuge tube. The following steps should be executed under consideration of the light sensitivity of the fluorescent calcium indicator.
Flu O three. First, make a one millimolar flu oh 3:00 AM stock solution. This solution can be stored at negative 20 degrees Celsius for up to one week.
Next, add 15 microliters of the flu oh 3:00 AM stock solution to the 1.5 milliliter cell suspension. Agitate the mixture carefully and incubate the suspension for 10 minutes at room temperature in an opaque box. After the incubation, briefly centrifuge the tissue at about 6, 000 RPM.
Then discard the supernatant and resuspend the pellet in 1.5 milliliters of bath solution. Leave the cell suspension for about 30 minutes for d esterification before beginning the experiments. Then proceed with simultaneous patch clamp and epi fluorescent calcium measurements using the outlined protocol.
Myocytes were isolated from atrial tissue and quantified on a cell finder microscope slide. Average cell yields clearly indicate that there is a tendency to lower cell yields in samples from patients with chronic atrial fibrillation. Flu oh three loaded myocytes were stimulated at 0.5 hertz in current clamp configuration to elicit action potentials, about 90%of the investigated cells showed regular contractions.
In response to this stimulation, the switch to fluorescence microscopy visualizes the action potential triggered calcium release from the sarcoplasmic reticulum, which initiates the cellular contraction. Representative recordings from patients with sinus rhythm and atrial fibrillation show that sr. Patient action potentials typically have a spike and dome shape, whereas action potential triangulation and shortening are typical hallmarks of atrial remodeling.
In atrial fibrillation patients simultaneously recorded intracellular calcium transients revealed increased diastolic calcium levels and reduced calcium transient amplitude in myocytes from atrial fibrillation patients to record L type calcium currents. Potassium currents were blocked using four amino paridine and barium chloride. The cells were stimulated at 0.5 hertz in voltage clamp configuration using a holding potential of minus 80 millivolts with a 100 milliseconds ramp to minus 40 millivolts and a 100 millisecond test pulse to 10 millivolts.
Representative recordings show that atrial fibrillation is associated with a reduced L type calcium current. Again, the diastolic calcium level was increased, whereas the calcium transient amplitude was lower. In atrial fibrillation.
Application of the non-selective beta adrenal receptor agonist isoprene increased the amplitudes of L type calcium currents and cytosolic calcium transient. In both groups, this suggests the cells have an intact beta adrenergic signal transduction cascade. The results show that this method provides high quality calcium tolerant myocytes, which are suitable for simultaneous patch clamp and calcium transient measurements.
In addition, the obtained myocytes may be useful for cell shortening measurements, immune staining, or TTU staining.
