A method for isolating rat cardiomyocytes by acute enzymatic hydrolysis

Bo Liang,Hui-Ling Liao,*

1School of Integrated Traditional Chinese and Western Medicine,Luzhou,China.2Cardiovascular Department,Hospital(TCM)Affiliated to Southwest Medical University,Luzhou,China.

Introduction

With the development and progress of medical electrophysiology,patch clamp has received more and more attention.Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function[1].And then,there are mainly two kinds of cells used for patch clamp experiments,acutely isolated cells 2-4 and cultured cells[2].As for the former,each laboratory has its own isolation methods,the results are not always same.However,for cardiomyocytes patch clamp,the isolation of good state of the cardiomyocytes is a major prerequisite for experimental success.In this paper,through the continuous exploration,we explore an effective method ofacute enzymatic separation ofrat cardiomyocytes.

All methods described here have been approved by the Institutional Animal Care and Use Committee(IACUC)of Southwest Medical University.

Materials and methods

Preparation

Autoclave all instruments,including but not limited to surgical instruments,measuring cylinders,bakers and pipette tips,to be used at 120°C for 30 min.Prepare buffers according to Table 1.This can be done up to a week and stored at 4°C.All reagents are purchased from Sigma-Aldrich.

Update the Langendroff perfusion system(Figure 1).Connect the needle of syringe and the low outlet of the system.This tip of needle should be flat enough to hang the heart though the aorta.Turn on the system,and set the input temperature to make sure that the output temperature was reach 37°C.Clean the system by flushing with 75%ethanol.Then clean the remaining ethanol by flushing with sterile water.Perfuse the system with Ca2+free Tyrode solution(Table 1)to fill the system and ensure no air bubbles are present.Adjust the follow rate to 12 ml/min,and then suspend the perfusion system.

Prepare a surgical suture to facilitate the posterior fixation of the aorta.Filled with Ca2+free Tyrode solution and digestion solution with oxygen.Some Ca2+free Tyrode solution is frozen into ice.

Heart extraction

The ratwas weighed to calculate the dose.Administer heparin(4 IU/g)via intraperitoneal injection.Wait 20 min to allow the heparin to circulate to the heart.Anesthetize the ratby intraperitoneal injection of 3%pentobarbital sodium(0.2 ml/100g).(Deep anesthesia is confirmed by a lack of both toe pinch and corneal reflexes.If the rat has not reached deep anesthesia by 8-10min,then administeran additionaldose ofpentobarbital sodium.)Immobilize the rat to the surgery platform by securing each limb with the needle of syringe.Disinfect the incision area with 75%ethanol.Lift the skin with tissue forceps just below the sternum.Make a T-shaped incision with scissors,cutting through the skin and abdomen.Use hemostats to gently lift the ribcage via the sternum.Then extend the T-shaped incision to the chest and exposure the heart.Take care not to puncture the heart.Free the heart bluntly and remove the pericardium.Transect the veins and arteries attached to the heart after the heart is lifted using curved forceps gently.Put the heart into the ice-cold solution in order to let muscle contraction.Trim any excess non-cardiac tissue,such as adipose tissue,lungs and excess blood vessels,using fine iris scissors.Use caution when cutting near the aorta.Transect the aorta near the brachiocephalic artery and take care not to let bubbles or tissue debris enter the opening.Hang the aorta onto the system such that the tip is just distal to the aortic valve.(The time from the heart is taken out to be hanged should be minimized to reduce hypoxic exposure and maximize the survival and health of the isolated cardiomyocytes.This time should be less than 2min ideally).The aorta is ligated on the tip of needle with a pre-prepared surgical suture.(Ensure the tip of the needle is still above the aortic valve before and after tightening.)

Perfusion

Return on the perfusion system.Perfuse the heart 5 min with Ca2+free Tyrode solution to ensure the remaining blood in the heart.The color of solution gradually changes from bright red to translucent will be observed due to the reduction of remaining blood.Change the perfusion solution from Ca2+free Tyrode solution to digestion solution,and perfuse 17-20 min.As the last of the Ca2+free Tyrode solution leaves the heart and the digestion solution begins circulating through the heart,the color changes from clear to light brown will be observed due to the presence of collagenase.The solution also becomes filamentous.(When changing to the perfusion solution,the system can be suspended.Try to avoid the occurrence of air embolism.Theconcentration ofcollagenasein perfusion solution is important.)

The heart will begin slouching as the collagen is degraded and it loses mechanical support.

Table 1 The relevant solution of this treatment

Figure 1 Langendroff perfusion system

Mechanical dissociation and purification

Remove the heart from the system quickly and place in abeakerfilled with KB solution.Quickly dissociate mechanically the atrial and ventricular tissue with fine forceps,so that most pieces are smaller than 1 mm3.Gently blow the pieces with a straw and filter cells with cell sieve.Store at room temperature for 1 h before medical electrophysiology experiment.Handle animal carcasses properly.

The slowly recovery of calcium

Take four 20 ml test tube and mark 1-4 respectively.Put 20 ml of cell-containing liquid in tube 1,let stand for 10min after the supernatant,to retain 10 ml containing cell liquid.Put 10 ml KB solution and 50 μl 100 mM CaCl2in tube 2.Put the solution of tube 2 into tube 1,mix gently and let stand for 10min after the supernatant,to retain 10 ml containing cell liquid.The first re-calcium has been completed.Put 10 ml KB solution and 100 μl 100 mM CaCl2in tube 3.The second recalcification method is same as the first time.Put 10 ml KB solution and 200 μl 100 mM CaCl2in tube 4.The third recalcification method is same as the first time.According to the above method recalcification,the final recalcification is completed.

Representative results

Though the microscope,good-state cardiomyocytes with clear stripes and strong sense of three-dimensional can be observed(Figure 2).Those cells can be used to undergo medical electrophysiology experiment,including the study of iron channel.

Figure 2 The good-state cardiomyocytes with clear stripes and strong sense of three-dimensional can be observed

Discussion

From its invention in the 1970s,the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels,which are responsible for action potentials.The conventional patch-clamp remains unmatched in fundamental research due to its versatility.Nowadays,the patch-clamp technique is the major technique in electrophysiology to record action potentials or membrane current from isolated or cultured cells,using a patch pipette to gain electrical access to the cell.Current clamp and voltage clamp are the common recording modes of the patch-clamp technique.In the current clamp mode,the current injected through the patch pipette is under control while the free-running membrane potential of the cell is recorded.Current clamp allows for measurements of action potentials that may either occur spontaneously or in response to an injected stimulus current.In voltage clamp mode,the membrane potential is held at a set level through a feedback circuit,which allows for the recording of the net membrane current at a given membrane potential.By coupling computational models to biological systems,dynamic clamp has become a proven tool in electrophysiology with many applications,such as generating hybrid networks in neurons or simulating channelopathies in cardiomyocytes.However,the dynamic clamp shares a limitation with current and voltage-clamp techniques:artifacts of electrode resistance and capacitance.Ossola merged the patch clamp and atomic force microscope(AFM)techniques,thus equipping the patch-clamp with the sensitive AFM force control.

The current and voltage-clamp techniques require higher cell state,although cultured cells have an artificially controllable state,acutely isolated cells are more in line with human cell environment because they come from animals.In a word,the cell state of acutely isolated cells is vital for electrophysiological experiment.A critical step is the speed with which the isolated heart is hung up on the perfusion system.The length of the enzymatic digestion period may be a little different for each rat.The adjustment depends on how soft the heart becomes after the regular period of digestion.The slowly recovery of Ca2+after enzyme digestion is essential for obtaining Ca2+tolerant healthy cells.Here,we described an improved method to acutely isolation of rat cardiomyocytes,good-state cardiomy oc ytes with clear stripes and strong sense of three dimensional can be got and usedtoundergomedical electrophysiology experiment,including thestudyof iron channel.

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