TITLE: Isolation of cardiac myocytes and measurement of myocyte shortening
AUTHORS: Gustavo Marchini, Ismar N. Cestari, Idágene A. Cestari

[STEPS]
?. [Prepare solutions]
Prepare 400 mL of buffer solution. Adjust the pH to 7.4 with NaOH.  ABCD1       Concentration
  [mM]    Molar
  Mass [g/mol]    Mass
  [mg]  2  NaCl    134    58,44    3132,38  3  KCl    4,0    74,56    119,30  4  NaH2PO4    1,2    156,01    74,88  5  HEPES    10    238,30    953,20  6  MgSO4    0,5    120,40    24,08  7  D-Glucose    11    180,16    435,99
ABCD1       Concentration
  [mM]    Molar
  Mass [g/mol]    Mass
  [mg]  2  NaCl    134    58,44    3132,38  3  KCl    4,0    74,56    119,30  4  NaH2PO4    1,2    156,01    74,88  5  HEPES    10    238,30    953,20  6  MgSO4    0,5    120,40    24,08  7  D-Glucose    11    180,16    435,99
?. [Prepare solutions]
Prepare 10 mL of 500 mM CaCl2 solution.  ABCD1       Concentration
  [mM]    Molar
  Mass [g/mol]    Mass
  [g]  2  CaCl2    500    147,02    0,74
ABCD1       Concentration
  [mM]    Molar
  Mass [g/mol]    Mass
  [g]  2  CaCl2    500    147,02    0,74
?. [Prepare solutions]
Prepare 50 mL of 100 mM CaCl2 solution.  ABCD1       Concentration
  [mM]    Molar
  Mass [g/mol]    Mass
  [g]  2  CaCl2    100    147,02    0,74
ABCD1       Concentration
  [mM]    Molar
  Mass [g/mol]    Mass
  [g]  2  CaCl2    100    147,02    0,74
?. [Prepare solutions]
Separate 200 mL of buffer solution and add 0,5 mL of 500 mM CaCl2 solution to obtain a buffer solution with 1,25 mM of Ca2+.
?. [Prepare solutions]
Separate 50 mL of buffer solution and add 25 µL of 100 mM CaCl2 solution to obtain a buffer solution with 50 µM of Ca2+.
?. [Prepare solutions]
Separate 15 mL of buffer solution and add 15 µL of 100 mM CaCl2 solution to obtain a buffer solution with 100 µM of Ca2+.
?. [Prepare solutions]
Separate 15 mL of buffer solution and add 30 µL of 100 mM CaCl2 solution to obtain a buffer solution with 200 µM of Ca2+.
?. [Prepare solutions]
Separate 15 mL of buffer solution and add 75 µL of 100 mM CaCl2 solution to obtain a buffer solution with 500 µM of Ca2+.
?. [Prepare solutions]
Separate 15 mL of buffer solution and add 150 µL of 100 mM CaCl2 solution to obtain a buffer solution with 1 mM of Ca2+.
?. [Experimental setup]
Assemble a constant flow Langendorff apparatus using a peristaltic pump, tubing, cannula and a heat exchanger coil. Connect the exchanger coil to a circulating water bath temperature set at 37 °C.
?. [Experimental setup]
Connect a thermometer to the outflow of the Langendorff apparatus, to measure the temperature of the heated solution.
?. [Experimental setup]
Fill the Langendorff apparatus with pre-heated buffer solution with 1,25 mM of Ca2+. Eliminate possible air bubbles. Adjust the peristaltic pump to a flow rate of 5,5 mL/min at outflow from the cannula.
?. [Myocyte isolation]
Inject mouse with heparin solution (5000 U/Kg), via intraperitoneal injection.
?. [Myocyte isolation]
After 30 min, anesthetize rat with a single intraperitoneal injection of sodium thiopental (50 mg/kg). When fully anesthetized (no response to strong foot pinch), euthanize the animal by cervical dislocation.
?. [Myocyte isolation]
Open the chest and identify the aorta. Excise the heart, being careful not to damage the aorta. Place the heart in 4 °C, pre-oxygenated buffer solution with 1,25 mM Ca2+.
?. [Myocyte isolation]
Cannulate the heart by the aorta in the cannula attached to the Langendorff system. Secure the aorta to the cannula by tying a loop of silk suture.
?. [Myocyte isolation]
Perfuse the heart with a buffer solution with 1,25 mM Ca2+ at a flow of 5,5 ml/min for 10 min.
?. [Myocyte isolation]
Switch the solution to Ca2+ free buffer and perfuse the heart with this solution at a flow of 7,5 ml/min for 5 min.
?. [Myocyte isolation]
Switch the solution to digestion buffer and perfuse the heart with this solution at a flow of 7,5 ml/min for 5 min. Recirculate the solution with the enzyme. Keep the perfusion until the heart become flaccid and pale (20 – 30 minutes approximately).
?. [Myocyte isolation]
Remove heart from cannula and place in petri dish with buffer solution with 50 µM Ca2+. Cut the heart into small chunks and pipette several times with a transfer pipette to further disperse cells.
?. [Myocyte isolation]
Filter the cell suspension into two 15 ml conical tube through a 250 μm nylon mesh filter.
?. [Myocyte isolation]
Centrifuge the cell suspension for 3 minutes, 35g.
?. [Myocyte isolation]
Aspirate the supernatant, and resuspend the pellet to buffer solution with 100 µM Ca2+.
?. [Myocyte isolation]
Allow cells to pellet for 10 min. Aspirate the supernatant, and resuspend the pellet to buffer solution with 200 µM Ca2+.
?. [Myocyte isolation]
Repeat last step, until cells are in the buffer solution with 1 mM Ca2+.
?. [Measuring contractility]
Add some drops of buffer solution with cells to C-Stim chamber (IonOptix, USA).
?. [Measuring contractility]
Adjust syringe pump to perfuse (1 ml/min) buffer solution with 1 mM Ca2+ into the chamber. Adjust the temperature controller to 37 °C to pre-heat the solution in the chamber inlet and attach vacuum aspiration to chamber outlet.
?. [Measuring contractility]
Use 40X objective to locate desired cell. A healthy cell should be rod shaped and not spontaneously contracting.
?. [Measuring contractility]
Set the electrical stimulator for bipolar pulses with 5 ms pulse duration, 25 V pulse amplitude and 1 Hz frequency.
?. [Measuring contractility]
Adjust camera rotation so cell is aligned horizontally on the screen.
?. [Measuring contractility]
In the software Ionwizard (IonOptix, USA), align edge detection bars to each end of myocyte and adjust threshold for contractility measurement with myocyte length variation.
?. [Measuring contractility]
Still in the software, align sarcomere detection on an area of cell with uniform sarcomeres. Choose a longer bar in the longitudinal axis to include as many sarcomeres as possible and improve contractility measurement with sarcomere length variation.
?. [Measuring contractility]
Turn the electrical stimulator on and pace cells. Record tracing for posterior analysis.
?. [On the day of the experiment]
Separate 100 mL of buffer solution with 1,25 mM of Ca2+, oxygenate the solution and keep it in the fridge at 4 °C.
?. [On the day of the experiment]
Heat and maintain the remaining 100 mL of buffer solution with 1,25 mM of Ca2+ at 37 °C.
?. [On the day of the experiment]
Heat and maintain the buffers solutions with different Ca2+ concentrations (50 µM, 200 µM, 500 µM, 1 mM) at 37 °C.
?. [On the day of the experiment]
Prepare the digestion buffer. Add 10 mg of type II collagenase in 40 mL buffer solution with 20 µM of Ca2+. Maintain the digestion buffer in the fridge at 4 °C.